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Vertebrate reproductive science and technology

Reproduction, Fertility and Development

Reproduction, Fertility and Development

Reproduction, Fertility and Development publishes research and reviews on all aspects of vertebrate reproduction and developmental biology. Read more about the journalMore

Editor-in-Chief: Graeme Martin

Current Issue

Reproduction, Fertility and Development

Volume 29 Number 5 2017

Glucocorticoids (GCs) sustain life and the development of human embryo; however, fetoplacental exposure to high maternal GC levels may lead to placental and fetal abnormalities, with the adverse consequences of GC excess manifested later in life. The mechanisms through which GC excess alters placental and fetal development are yet to be identified; however, changes in placental GR levels and subcellular localization and ratios may modify placental function, in utero growth of the fetus and lifelong health.

RD15358Dietary-induced gestational iron deficiency inhibits postnatal tissue iron delivery and postpones the cessation of active nephrogenesis in rats

Mary Y. Sun, Joseph C. Woolley, Sharon E. Blohowiak, Zachary R. Smith, Ashajyothi M. Siddappa, Ronald R. Magness and Pamela J. Kling
pp. 855-866

Gestational iron deficiency can cause developmental programming of adult hypertension, but the critical developmental window during nephrogenesis has not been studied. The present study examined renal development during perinatal iron deficiency in rats and found altered timing of kidney maturation and histological renal adaptations that may contribute to dysfunctional glomeruli and renal hyperfiltration, which themselves contribute to hypertension.

RD15402Preantral follicle density in ovarian biopsy fragments and effects of mare age

K. A. Alves, B. G. Alves, G. D. A. Gastal, K. T. Haag, M. O. Gastal, J. R. Figueiredo, M. L. Gambarini and E. L. Gastal
pp. 867-875

Information about follicle density is critical to determine the quantity of fragments during biopsy procedures in women and domestic animals. This study evaluated the effects of age and determined the minimum number of ovarian fragments and histological sections required to estimate equine follicle density in ovarian biopsy fragments. The results show that follicle density is negatively influenced by increasing age and, independent of follicle heterogeneity, three to four ovarian fragments combined with 65 histological sections are required to accurately determine follicle density in equine ovarian tissue.

RD15315Insulin exposure during in vitro bovine oocyte maturation changes blastocyst gene expression and developmental potential

Denise Laskowski, Ylva Sjunnesson, Patrice Humblot, Marc-André Sirard, Göran Andersson, Hans Gustafsson and Renée Båge
pp. 876-889

Metabolic imbalance impairs female fertility. We wanted to find out how metabolism affects bovine oocyte quality, and demonstrated that insulin exposure during in vitro oocyte maturation affects embryo development, morphology and gene expression. Even short-term exposure to a metabolic imbalance has lasting effects on embryo development, and this metabolic programming may have consequences later in life.

RD15319Fateful triad of reactive oxygen species, mitochondrial dysfunction and lipid accumulation is associated with expression outline of the AMP-activated protein kinase pathway in bovine blastocysts

S. Prastowo, A. Amin, F. Rings, E. Held, D. Salilew Wondim, A. Gad, C. Neuhoff, E. Tholen, C. Looft, K. Schellander, D. Tesfaye and M. Hoelker
pp. 890-905

In vitro-derived bovine embryos frequently exhibit lipid accumulation, mitochondrial dysfunction and enhanced levels of reactive oxygen species. With the aim of unravelling the role of the AMP-activated protein kinase cytalytic subunit 1alpha (AMPKα), the present study identified AMPKα activity and distinct expression patterns of AMPKα-related genes to be correlated with that phenotype. These results prove the important role of AMPKα in embryonic energy metabolism.

RD15282MicroRNA indicators of follicular steroidogenesis

F. X. Donadeu, S. D. Sontakke and J. Ioannidis
pp. 906-912

Finding novel biomarkers of ovarian follicle function would be of benefit. This study investigated the follicle biomarker potential of microRNAs (miRNAs) and found that levels of miR-873 and miR-202 can be used as indicators of steroidogenic capacity in cattle. These miRNAs, in addition or alternatively to commonly used steroid level measurements, could be used to accurately classify follicles based on steroidogenic capacity.

Bone marrow chimerism is a unique and normal feature of Callithrichidae development that originates through shared placental circulation. A new X and Y chromosome-specific fluorescent in situ hybridisation protocol for Callithrix jacchus has been developed and validated to detect and investigate chimerism between male and female littermates. One of the strengths of this method is that it is useful in interphase cells, and allows cell morphology to remain intact to facilitate identification of cell type.

Most species of Australasian old endemic rodents have a highly complex sperm head morphology that contains an apical hook together with two ventral processes of variable length and orientation. This study tested whether this variation is a sexually selected trait using relative testes mass as a proxy for the intensity of inter-male sperm competition and controlling for phylogeny. The angle of these processes, but not length, was positively correlated with relative testes mass; its possible functional significance is briefly discussed.

RD15435Serial transvaginal ultrasound-guided biopsy of the porcine corpus luteum in vivo

S. Björkman, J. Yun, M. Niku, C. Oliviero, N. M. Soede and O. A. T. Peltoniemi
pp. 931-939

For the first time, a minimal invasive method that allows ovarian tissue collection from sows is described in detail. This method, a transvaginal ultrasound-guided biopsy, is quick, can be performed by one person, and has no major effects on the sow’s reproductive performance. It can be applied on the farm to study effects of housing or nutrition on luteal function.

RD15424Lower dietary n-6 : n-3 ratio and high-dose vitamin E supplementation improve sperm morphology and oxidative stress in boars

Qing Liu, Yuanfei Zhou, Runjia Duan, Hongkui Wei, Siwen Jiang and Jian Peng
pp. 940-949

Dietary PUFA and vitamin E are known to influence sperm morphology and oxidative stress. The effects of dietary n-6 : n-3 and vitamin E on the sperm morphology and oxidative stress in boars were investigated. An n-6 : n-3 ratio of 6 :1 and 400 mg kg–1 vitamin E have beneficial effects on sperm morphology by improving oxidative stress.

RD15215Morphometric developmental pattern of bovine handmade cloned concepti in late pregnancy

R. P. C. Gerger, F. C. Zago, E. S. Ribeiro, S. Gaudencio Neto, L. T. Martins, L. H. Aguiar, V. H. V. Rodrigues, F. H. Furlan, I. Ortigari, R. D. Sainz, C. L. Ferrell, M. A. Miglino, C. E. Ambrósio, J. L. Rodrigues, R. Rossetto, F. Forell, L. R. Bertolini and M. Bertolini
pp. 950-967

Cloned concepti on Day 225 of gestation were characterised by lower pregnancy rates, high gestational losses, a distinctive biphasic growth pattern, with early growth restriction followed by accelerated growth and foetal weight on Day 225, and significant morphological changes in placental mass and shape when compared with in vivo-derived pregnancies. Conversely, Day-225 IVF-derived concepti were widely similar to controls.

RD15306Increased frequency of chromosome congression defects and aneuploidy in mouse oocytes cultured at lower temperature

Jitka Danadova, Natalie Matijescukova, Anna Mac Gillavry Danylevska and Martin Anger
pp. 968-974

In vitro manipulation of oocytes is a standard procedure used in assisted reproduction. The aim of the present study was to analyse the effect of suboptimal culture temperatures on various aspects of meiotic division, including chromosomal congression and segregation defects. The results revealed that even a small deviation from optimal culture temperature has a marked effect on chromosome congression and aneuploidy.

The uterus produces steroids and primary steroidogenic enzymes are expressed in the uterus of pregnant and cyclic pigs. In this study we examined 17β-hydroxysteroid dehydrogenase expression and the effects of LH, FSH and prolactin on oestrone and 17β-oestradiol release in vitro in endometrial explants. We show that LH, FSH and prolactin may regulate oestrogen release by the endometrium.

RD15214Prion protein 2 (dublet) gene (PRND): role in ovine semen capacitation, cryopreservation and fertility

L. M. Ferreira, M. Garcia-Herreros, A. Domingos, C. C. Marques, P. Mesquita, J. P. Barbas, M. C. Baptista, J. Pimenta, A. E. M. Horta, J. A. M. Prates and R. M. L. N. Pereira
pp. 985-997

The physiological functions of prion and prion-like proteins are overlapped by their pathological features, namely in the invariably fatal neurodegenerative diseases of both humans and animals, such as Kuru, Creutzfeldt-Jakob disease and new variant, bovine spongiform encephalopathy and sheep scrapie. The aim of the present study was to investigate the role of the prion-like Doppel (PRND) protein and different PRND genotypes in the sperm capacitation process, cryoresistance, and fertility potential. Both factors were crucial for sperm capacitation potential, cryoresistance, and embryo production. Thus, an important physiological function of Doppel protein and genotype in male fertility has been demonstrated.

We are in need of safe, effective and affordable male contraceptives. Indazole carboxylic acids such as lonidamine and adjudin disrupt adhesion between Sertoli and germ cells in the rat testis, resulting in infertility. One of the key findings of this study was that lonidamine-ethyl ester also displayed contraceptive effects. Additional studies are required to determine whether indazole carboxylic acids can enter the clinical pipeline

RD15361Oestrogenic action of neonatal tamoxifen on the hypothalamus and reproductive system in female mice

Rahmatollah Parandin, Morteza Behnam-Rassouli and Nasser Mahdavi-Shahri
pp. 1012-1020

Tamoxifen is widely used in the treatment of breast cancer and can interfere with the reproductive system. The present study showed that tamoxifen administration during the neonatal period has oestrogenic actions on oestrogen-sensitive nuclei in the brain controlling the reproductive axis in female mice. Therefore, tamoxifen may have adverse effects on brain differentiation and reproductive function.

RD15409Local effect of lysophosphatidic acid on prostaglandin production in the bovine oviduct

Yuka Yoshimoto, Yoshihiko Kobayashi, Izabela Woclawek-Potocka, Emilia Sinderewicz, Yuki Yamamoto, Koji Kimura and Kiyoshi Okuda
pp. 1021-1028

Prostaglandins induce contraction and relaxation of oviductal smooth muscle, which are essential for transport of gametes and embryo. The present results demonstrate that lysophosphatidic acid significantly stimulated prostaglandin production by cultured isthmic stromal cells. It is suggested that locally produced lysophosphatidic acid contributes to oviductal motility in the isthmus by regulating prostaglandin production in cows.

The hormone oestrogen is essential for testis development and its synthesis is dependent on the enzyme aromatase. However, the molecular mechanism of aromatase regulation is still poorly understood. This study describes for the first time the cellular localisation of proteins important for aromatase regulation, which are well known in breast tissue, in the normal testis. This new finding may help develop new therapies for oestrogen-mediated infertility.

Stallion spermatozoa lose viability and fertilising capability relatively quickly when cooled; robust spermatozoa can be selected by colloid centrifugation. In the present study, selected spermatozoa were shown to metabolise differently to controls, producing less hydrogen peroxide, which is known to damage spermatozoa. These results indicate that colloid centrifugation could be used to improve the quality of stallion semen for AI.

RD15521Cerium oxide nanoparticles (CeO2 NPs) improve the developmental competence of in vitro-matured prepubertal ovine oocytes

F. Ariu, L. Bogliolo, A. Pinna, L. Malfatti, P. Innocenzi, L. Falchi, D. Bebbere and S. Ledda
pp. 1046-1056

Among the family of engineered nanostructures, cerium dioxide nanoparticles (CeO2 NPs) are receiving particular attention for their antioxidant properties with potential applications in biomedical therapies. This study examined the effect of supplementation with different doses of CeO2 NPs during in vitro maturation of prepubertal ovine oocytes on their embryonic development. Low concentration of CeO2 NPs increased development to blastocyst stage and embryo quality.

Online Early

The peer-reviewed and edited version of record published online before inclusion in an issue

Published online 28 April 2017

RD16366Development of pre-implantation porcine blastocysts cultured within alginate hydrogel systems either supplemented with secreted phosphoprotein 1 or conjugated with Arg-Gly-Asp Peptide

Taylor D. Laughlin, Jeremy R. Miles, Elane C. Wright-Johnson, Lea A. Rempel, Clay A. Lents and Angela K. Pannier

Deficiencies in the initiation of embryo elongation play a major role in early embryonic mortality in the pig. This study demonstrates that embryos encapsulated and cultured in alginate hydrogels with the integrin-binding Arg-Gly-Asp (RGD) sequence had increased embryonic survival and modulation of gene expression to support increased steroid production and adequate immune responsiveness, with a larger number exhibiting the morphological changes compared to other in vitro treatments in this study. These results illustrate the importance of the integrin-binding RGD peptide sequence for stimulating the initiation of embryo elongation and improving embryo survival.

Published online 26 April 2017

RD16378Neuromodulatory effect of oestradiol in the metabolism of ovarian progesterone and oestradiol during dioestrus II: participation of the superior mesenteric ganglion

Adriana Vega Orozco, Cynthia Bronzi, Sandra Vallcaneras, Zulema Sosa and Marilina Casais

This work demonstrates that estradiol through the peripheral nervous system modulates the ovarian physiology, favoring the physiological luteolysis. The objective of this work was to demonstrate whether estradiol in superior mesenteric ganglion modifies the release of ovarian progesterone, estradiol, nitric oxide and noradrenaline on DII day. The ganglionic stimulation produces a decrease in the ovarian release of progesterone and estradiol by an increase of nitric oxide and decrease of noradrenaline.

Published online 26 April 2017

RD17046Protective effects of polydatin on experimental testicular torsion and detorsion injury in rats

Huilian Qiao, He Ma, Wanjun Cao, Hao Chen, Jinhua Wei and Zhen Li

The notable pathophysiological event that occurs in testicular torsion–detorsion (T/D) is ischaemia–reperfusion (I/R), which lacks efficient clinical treatment. Herein we describe, for the first time, the protective effects of polydatin, a traditional Chinese medicine, against testicular T/D injury in the rat. Our findings may provide new clues for male infertility associated with testicular torsion.

Published online 26 April 2017

RD16428Intracytoplasmic oxidative stress reverses epigenetic modifications in polycystic ovary syndrome

Fatemeh Eini, Marefat Ghaffari Novin, Khojasteh Joharchi, Ahmad Hosseini, Hamid Nazarian, Abbas Piryaei and Arash Bidadkosh

In PCOS, elevated levels of plasma androgens affects the quality of oocytes and ovulation rate, and causes anovulation in women of reproductive age. We studied the mechanism in a mouse model of PCOS, and showed that intracytoplasmic oxidative stress in oocyte via increase in histone acetylation and decrease in DNA methylation and histone dimethylation alters epigenetic modification. Consistently, mRNA expression of DNA methyltransferase-1 and histone deacetylase-1 was decreased with PCOS. There was a significant correlation between ROS production and increased histone acetylation. We concluded that antioxidant therapy in PCOS is a viable approach to reduce the adverse effects of epigenetic alterations.

Published online 19 April 2017

RD16343Andrographolide disrupts meiotic maturation by blocking cytoskeletal reorganisation and decreases the fertilisation potential of mouse oocytes

Hong-xing Liang, Sheng-sheng Lu, Zheng Yan, Yan-ping Kuang, Xiang-xing Zhu, Zhi-guang Yan, Tong Du, Wei-ran Chai, Hui Long and Qi-feng Lyu

Andrographolide should be prudently consumed by women attempting to conceive. The aim of the present study was to investigate the effects of andrographolide on oocytes. The findings indicate that andrographolide may disrupt mouse oocyte meiotic maturation and fertilisation potential by blocking cytoskeletal reorganisation. This study provides an important platform for understanding the potential adverse effects of andrographolide on female fertility.

Published online 19 April 2017

RD16459Comparative analysis of granulosa cell gene expression in association with oocyte competence in FSH-stimulated Holstein cows

David A. Landry, Chloé Fortin, Anne-Marie Bellefleur, Rémi Labrecque, François-Xavier Grand, Christian Vigneault, Patrick Blondin and Marc-André Sirard

In cattle, a major limitation of the success of IVF is the developmental competence that oocytes acquire during ovarian stimulation. The aim of the present study was to better understand the follicular conditions associated higher versus lower embryo yields. We revealed that the level of follicular differentiation (timing) is the main cause of failure and provide new biomarkers to identify such follicular conditions to adapt the ovarian stimulation protocol to individual animals.

Published online 18 April 2017

RD16435Proteomic analysis of the endometrium during early pregnancy in the domestic pig

Justyna Kolakowska, Serhiy Souchelnytskyi, Ravi Kanth Rao Saini and Anita Franczak

High embryo loss during early pregnancy in pigs is still an unresolved issue. Comparing the proteomic profile of the endometrium harvested from crucial stages of early pregnancy (Days 12–13 and 15–16) with the proteomic profile of tissue from corresponding days of the oestrous cycle revealed the presence of proteins unique for early pregnancy that are involved in adhesion, angiogenesis and cytoskeletal organization. The results of the present study may help us understand the causes of early pregnancy loss in pigs.

Binding of spermatozoa to the egg coat is a key step in fertilisation and provides a mechanism for reproductive isolation and sexual selection. To test this theory we investigated the molecular evolution of the ZP3 gene and its encoded sperm-binding region in a diverse murine rodent group. Our analyses provides evidence for positive selection on the sperm-binding region in several murine lineages, but the nature of the selective force remains unresolved.

Published online 12 April 2017

RD16498Endo-siRNA deficiency results in oocyte maturation failure and apoptosis in porcine oocytes

Wanxin Liu, Qi Zhao, Shanhua Piao, Chunsheng Wang, Qingran Kong and Tiezhu An

Endo-siRNAs play an important role in mouse oocyte meiosis but the function of endo-siRNAs in other species is unknown. In this study, we found that an endo-siRNA deficiency resulted in oocyte spindle abnormalities and apoptosis, suggesting that endo-siRNAs are indispensible for oocyte maturation in pigs. This study unequivocally demonstrates an essential function for endo-siRNAs in mammals.

Published online 10 April 2017

RD16267Pannexin channels increase propidium iodide permeability in frozen–thawed dog spermatozoa

J. L. Torres, J. Palomino, R. D. Moreno and M. De los Reyes

Pannexins (Panx) are proteins that form functional single membrane channels that have not yet been described in dogs. The aim of the present study was to detect Panx1, Panx2 and Panx3 in frozen–thawed dog semen. All three Panx channels were present in dog spermatozoa and increased propidium iodide (PI) permeability in frozen–thawed dog spermatozoa, suggesting that the percentage of PI-positive spermatozoa used as an indicator of non-viable cells may lead to overestimation of non-viable cells.

Published online 10 April 2017

RD16474Differential effects of high and low glucose concentrations during lipolysis-like conditions on bovine in vitro oocyte quality, metabolism and subsequent embryo development

J. De Bie, W. F. A. Marei, V. Maillo, L. Jordaens, A. Gutierrez-Adan, P. E. J. Bols and J. L. M. R. Leroy

Maternal metabolic disorders (e.g. obesity and diabetes in women or a negative energy balance in dairy cows) are associated with altered metabolic profiles, which can impact oocyte quality and thus fertility. We showed that altered glucose can differentially affect oocyte quality in the presence of elevated free fatty acids. These results further highlight the importance of maternal metabolic health for fertility.

Published online 10 April 2017

RD16442Supplementation of culture medium with L-carnitine improves the development and cryotolerance of in vitro-produced porcine embryos

J. L. Lowe, L. K. Bartolac, R. Bathgate and C. G. Grupen

Porcine embryos contain substantial amounts of lipid, with little known regarding its metabolic role during development. This study investigated the association of lipid metabolism with carbohydrate substrates in the culture medium and revealed positive effects of adding L-carnitine, a limiting co-factor required for lipolysis, on early cleavage and blastocyst quality. Supplementing medium with L-carnitine is a useful strategy to improve the success of porcine blastocyst vitrification.

Published online 07 April 2017

RD16432Low doses of bisphenol A can impair postnatal testicular development directly, without affecting hormonal or oxidative stress levels

Fernanda M. Ogo, Glaucia E. M. L. Siervo, Géssica D. Gonçalves, Rubens Cecchini, Flavia A. Guarnier, Janete Ap. Anselmo-Franci and Glaura S. A. Fernandes

The peripubertal period is considered a critical stage of reproductive development and more vulnerable to the action of toxic agents, such as BPA, which can lead to impaired sexual development. We investigated the exposure to BPA during the peripubertal period and eEvidencing a reduction in the number of Sertoli cells, histopathological abnormalities and changes in sperm morphology. Therefore, BPA may silently impair testicular postnatal development in rats.

Published online 06 April 2017

RD16265Sex-specific differences and developmental programming for diseases in later life

Deepali P. Sundrani, Suchitra S. Roy, Anjali T. Jadhav and Sadhana R. Joshi

Sex-specific differences have been reported in most of the diseases. This review provides data on differential adaptation of male and female fetuses in utero in response to maternal nutritional, physiological and environmental insults resulting in sex-specific differences. Because sex differences are associated with fetal outcome and survival, it is of importance to study the underlying biological mechanisms to improve both pre-natal and neonatal care.

Published online 05 April 2017

RD16235Endogenous lysophosphatidic acid participates in vascularisation and decidualisation at the maternal–fetal interface in the rat

Micaela S. Sordelli, Jimena S. Beltrame, Elsa Zotta, Natalia Gomez, Ganna Dmytrenko, María Elena Sales, Sandra M. Blois, Carlos Davio, Silvina Perez Martinez, Ana M. Franchi and María L. Ribeiro

Decidualisation and vascular remodelling are fundamental for placentation and pregnancy success. Disruption of endogenous lysophosphatidic acid signalling modified the development of uterine vessels with consequences in the formation of the decidua and placenta, compromising the growth of embryos. Defects in these mechanisms could appear as obstetric complications, like implantation failure, positioning lysophosphatidic acid as a regulatory key lipid at implantation.

Published online 03 April 2017

RD16513Melatonin potentially acts directly on swine ovary by modulating granulosa cell function and angiogenesis

Giuseppina Basini, Simona Bussolati, Roberta Ciccimarra and Francesca Grasselli

The reproductive effects of melatonin have been extensively studied, but its involvement in the local control of ovarian physiology has not yet been clarified. The present research, which was undertaken to study the effects of physiological levels of melatonin on the main parameters of swine granulosa cell function, documents that the hormone is involved in the modulation of ovarian follicle growth and development.

Successful artificial fertilisation primarily depends on two characteristics of male quality: 1) the maintenance of high sperm viability during storage and 2) the effective activation of sperm motility. This study identified optimal conditions for sperm motility activation in the critically endangered booroolong frog. Results reported make a significant contribution to the growing field of amphibian reproduction technologies.

Published online 30 March 2017

RD16320Steroid hormones interact with natriuretic peptide C to delay nuclear maturation, to maintain oocyte–cumulus communication and to improve the quality of in vitro-produced embryos in cattle

Ana Caroline S. Soares, Valentina Lodde, Rodrigo G. Barros, Christopher A. Price, Alberto M. Luciano and José Buratini

This study provides evidence that natriuretic peptide C and steroids interact to slow oocyte nuclear maturation and to enhance oocyte-cumulus communication in cattle. Moreover, it indicates that exposure of the cumulus–oocyte complex to physiological concentrations of these hormones is beneficial to oocyte developmental competence. Therefore, the dataset is of relevance for the progress of oocyte biology and for the improvement of in vitro maturation outcomes.

Published online 30 March 2017

RD16511Cryopreservation of saltwater crocodile (Crocodylus porosus) spermatozoa

S. D. Johnston, E. Qualischefski, J. Cooper, R. McLeod, J. Lever, B. Nixon, A. L. Anderson, R. Hobbs, J. Gosálvez, C. López-Fernández and T. Keeley

A combination of permeating and non-permeating cryoprotectants and freezing rates was used to successfully cryopreserve saltwater crocodile spermatozoa. Although glycerol concentrations >1.35 M had an inhibitory effect on motility before freezing, 0.68 M glycerol combined with 0.2 M sucrose and post-thaw washing of spermatozoa in capacitating media resulted in motility of between 12% and 14.2%, the highest ever recorded for this species.

Published online 30 March 2017

RD16475Placentation in the plains zebra (Equus quagga)

W. R. (Twink) Allen, Fiona Stansfield and Sandra Wilsher

This paper describes findings during a unique opportunity to monitor placental structure and function throughout pregnancy in the under-researched zebra. Zebras are more donkey-like than horse-like in placental structure, rate of fetal development and gestation length. The results highlight the retention of unique reproductive mechanisms across the Genus Equus despite many other evolutionary changes within the member species.

Published online 27 March 2017

RD16289Successful chimera production in the Hungarian goose (Anser anser domestica) by intracardiac injection of blastodermal cells in 3-day-old embryos

Nikoletta Sztán, Bence Lázár, Nóra Bodzsár, Barbara Végi, Krisztina Liptói, Bertrand Pain and Eszter Patakiné Várkonyi

The aim of this study was to develop a new genome preservation technique for goose species. According to our results, goose primordial germ cells (PGCs) migrate between 69 and 84 h of development. The goose blastoderm contains PGCs and by isolating and injecting these blastodermal cells we were able to generate chimeras. Altogether, this method could contribute to the cryopreservation of this species.

Published online 27 March 2017

RD17020Glutamine protects rabbit spermatozoa against oxidative stress via glutathione synthesis during cryopreservation

Zhendong Zhu, Xiaoteng Fan, Yinghua Lv, Yan Lin, De Wu and Wenxian Zeng

The addition of 20 mM glutamine to the freezing extender significantly improved post-thaw sperm motility, acrosome integrity, membrane integrity and mitochondrial activity. The exposure of spermatozoa to glutamine enhanced their γ-GCS activity and GSH synthesis, while decreasing ROS accumulation, LPO and DNA damage. Therefore, glutamine protects spermatozoa against ROS-induced cryodamage by enhancing GSH synthesis.

Published online 16 March 2017

RD16144Prostaglandin F–PTGFR signalling activation, growth factor expression and cell proliferation in bovine endometrial explants

Shuangyi Zhang, Bo Liu, Long Gao, Wei Mao, Changqi Fu, Duritahala, Nan Zhang, Ying Zhang, Yuan Shen and Jinshan Cao

The association between bovine endometrial growth and prostaglandin (PG) F–PGF receptor (PTGFR) signalling activation is unknown. In the present study, cell proliferation and the expression of growth factors essential for endometrial growth were upregulated by PGF-PTGFR signalling activation by fluprostenol in bovine endometrial explants in vitro. These results indicate that PGF-PTGFR signalling activation could be involved in endometrial growth.

uPA shRNA lentivirus system induced by Dox inhibited the expression of uPA in TM4 cells. Through injecting the lentivirus into mice testis, we found that downregulation of uPA in mice testis decreased the fertility of male mice, which may be caused by a reduction in sperm motility. The inducible uPA shRNA system is safe and reversible to regulate male fertility.

Live cell RNA imaging in mammalian embryos would represent a quantum leap in understanding cell differentiation and early embryonic development. Applicability of RNA imaging by SmartFlare technology was tested in equine embryos surrounded by an embryonic capsule and/or the zona pellucida, nude trophoblastic vesicles and fibroblast cell cultures. Encapsulated embryos block SmartFlare uptake, but stages surrounded by the zona pellucida only and nude vesicles incorporate the probes, leading to specific fluorescence suitable for tracing gene expression cascades necessary for further embryo development.

Published online 28 February 2017

RD16426Effects of antifreeze glycoprotein 8 (AFGP8) supplementation during vitrification on the in vitro developmental capacity of expanded bovine blastocysts

Shuang Liang, Bao Yuan, Yong-Xun Jin, Jia-Bao Zhang, Jeong Kyu Bang and Nam-Hyung Kim

Antifreeze glycoprotein 8 (AFGP8) supplementation during vitrification beneficially protects bovine blastocysts against chill-induced injury. AFGP8 supplementation during vitrification enhances the cryosurvival of bovine blastocysts. AFGP8 supplementation during vitrification improves the subsequent in vitro developmental capacity of bovine blastocysts.

Published online 28 February 2017

RD16478Expression of protocadherin 11Yb (PCDH11Yb) in seminal germ cells is correlated with fertility status in men

Thottathil R. Anilkumar, Anandavalli N. Devi, Sathy M. Pillai, Krishnapillai Jayakrishnan, Oommen V. Oommen and Pradeep G. Kumar

Knowledge of molecular defects in spermatozoa becomes critical during assisted reproduction to treat male factor infertility. Herein we show that the absence or low expression of protocadherin 11Yb (PCDH11Yb) in spermatozoa is associated with male factor infertility. Profiling of a panel of differentially expressed sperm proteins, including PCDH11Yb, in men with infertility may be of diagnostic and therapeutic value in infertility management.

Published online 24 February 2017

RD16383Localisation of stem cell factor, stanniocalcin-1, connective tissue growth factor and heparin-binding epidermal growth factor in the bovine uterus at the time of blastocyst formation

M. Muñoz, D. Martin, S. Carrocera, M. Alonso-Guervos, M. I. Mora, F. J. Corrales, N. Peynot, C. Giraud-Delville, V. Duranthon, O. Sandra and E. Gómez

Limited information is available about the role of growth factors in very early embryonic development. This study demonstrates the presence of stem cell factor, stanniocalcin-1, connective tissue growth factor and heparin-binding epidermal growth factor-like growth factor in bovine uterine fluid, endometrium and embryos during blastocyst formation. The results suggest a role for these proteins in early embryo development in cattle.

Published online 14 February 2017

RD16186Identification of differentially expressed placental transcripts during multiple gestations in the Eurasian beaver (Castor fiber L.)

A. Lipka, L. Paukszto, M. Majewska, J. P. Jastrzebski, K. Myszczynski, G. Panasiewicz and B. Szafranska

It is still unspecified if and how the number of embryos or fetal sex affect pregnancy outcome. The present study determined whether there are differences in transcriptome profiles of the beaver placenta as a result of fetal sex or multiplicity of gestation. The results indicate that the number of fetuses affects the expression profile in the subplacental transcriptome. Enhancement of transcriptome resources will improve our understanding of the pathways relevant to proper placental development and successful reproduction in general.

Published online 14 February 2017

RD16302Polydatin improves the developmental competence of bovine embryos in vitro via induction of sirtuin 1 (Sirt1)

Imran Khan, Sung Woo Kim, Kyung-Lim Lee, Seok-Hwan Song, Ayman Mesalam, M. M. R. Chowdhury, Zia Uddin, Ki Hun Park and Il-Keun Kong

Polydatin (PD) has antioxidant activity and is useful for embryo development in vitro. Supplementation of IVM medium with PD scavenged reactive oxygen species and improved embryo developmental competence via induction of sirtuin 1 (Sirt1).

Published online 13 February 2017

RD16294Sperm gamma-aminobutyric acid type A receptor delta subunit (GABRD) and its interaction with purinergic P2X2 receptors in progesterone-induced acrosome reaction and male fertility

Wenming Xu, Ke Wang, Yan Chen, Xiao Tong Liang, Mei Kuen Yu, Huanxun Yue and M. Louise Tierney

The mechanism underlying the non-genomic action of progesterone in sperm functions and related Ca2+ mobilisation remains elusive. Herein we report the expression of gamma-aminobutyric acid type A receptor delta subunit (GABRD) in human and rodent spermatozoa and its involvement in mediating the progesterone-induced acrosome reaction through its interaction with purinergic P2X2 receptors. GABRD represents a novel progesterone receptor or modulator in sperm responsible for the progesterone-induced Ca2+ influx required for the acrosome reaction.

Published online 10 February 2017

RD16333Restoration of immune and renal function in aged females by re-establishment of active ovarian function

Rhett L. Peterson, Kate C. Parkinson and Jeffrey B. Mason

Immune and renal function, both critical for reproductive success, were evaluated to determine if exposure of postreproductive female mice to young ovaries would influence these non-reproductive physiological functions. Postreproductive mice received new (60-day-old) ovaries at 12 months of age and were evaluated at 16 months of age. Age-related declines in immune and renal function in 16-month-old control mice were restored to levels found in 6-month-old mice by ovarian transplantation. Re-establishment of reproductive function in aged female mice positively influenced non-reproductive functions.

Published online 08 February 2017

RD16473In vitro addition of docosahexaenoic acid improves the quality of cooled but not frozen–thawed stallion semen

D. M. Silva, S. A. Holden, A. Lyons, J. C. Souza and S. Fair

Improvements in the quality of stallion semen are required to increase the success of artificial insemination. The aim of this study was to assess the effects of the addition of docosahexaenoic acid to semen diluents on the quality of stallion semen. Docosahexaenoic acid increased spermatozoa motility and membrane fluidity in cooled semen and thus can be used to increase the quality of spermatozoa.

Published online 03 February 2017

RD16327Gene-specific profiling of DNA methylation and mRNA expression in bovine oocytes derived from follicles of different size categories

F. Mattern, J. Heinzmann, D. Herrmann, A. Lucas-Hahn, T. Haaf and H. Niemann

The present study investigated DNA methylation profiles and mRNA expression of imprinted and non-imprinted candidate genes in bovine oocytes from antral follicles of three different size classes (≤2 mm, 3–5 mm, ≥6 mm) to unravel the epigenetic contribution to follicular and oocyte growth. We observed an increased number of aberrantly methylated alleles in bH19, bSNRPN and bDNMT3 Lo of oocytes from small antral follicles and an increased frequency of CpG sites with an unclear methylation status for DNMT3 Ls in large follicles. Results indicate that a follicle diameter of ~2 mm is critical for establishing DNA methylation profiles.

Published online 02 February 2017

RD16193Microtubule stabilisers docetaxel and paclitaxel reduce spindle damage and maintain the developmental competence of in vitro-mature bovine oocytes during vitrification

Jakkhaphan Pitchayapipatkul, Tamás Somfai, Satoko Matoba, Rangsan Parnpai, Takashi Nagai, Masaya Geshi and Thevin Vongpralub

The aim of this study was to compare the efficacy of paclitaxel (PT) and docetaxel (DT) in reducing spindle damage in bovine oocytes during vitrification. The important finding of the study was that pretreatment of bovine oocytes with 0.05 µM DT for 30 min before vitrification reduced spindle damage to a greater degree than pretreatment with 1.0 µM PT. This finding will be useful in improving oocyte cryopreservation in the future.

Published online 01 February 2017

RD16233Extracellular-like matrices and leukaemia inhibitory factor for in vitro culture of human primordial follicles

Assiel J. Younis, Galit Lerer-Serfaty, Dana Stav, Bethsabee Sabbah, Tzippy Shochat, Gania Kessler-Icekson, Muayad A. Zahalka, Michal Shachar-Goldenberg, Avi Ben-Haroush, Benjamin Fisch and Ronit Abir

The possibility of growing immature human ovarian follicles in laboratories will assist fertility restoration. The aim of the present study was to investigate whether growth of immature follicles can be promoted by using different culture surfaces and supplementation with a growth factor. It was unclear whether any of the culture surfaces had an advantage over the others. In addition, the growth factor did not promote growth. Further studies should test other culture surfaces and identify additional fluid supplements, so that an optimal system can be developed.

Poly(A)-binding proteins function in the timely regulation of gene expression during oocyte maturation, fertilisation and early embryo development in vertebrates. In this review, we comprehensively evaluate and discuss the expression patterns and particular functions of the embryonic poly(A)-binding protein (EPAB) and poly(A)-binding protein, cytoplasmic 1 (PABPC1) genes, especially in mouse and human oocytes and early embryos.

Published online 12 January 2017

RD16276Epigenetic changes of hepatic glucocorticoid receptor in sheep male offspring undernourished in utero

Stella Chadio, Basiliki Kotsampasi, Stylliani Taka, Emmanouil Liandris, Nikolaos Papadopoulos and Elias Plakokefalos

Evidence exists that early life nutritional insults can predispose to metabolic disorders, like Type 2 diabetes, for which excessive hepatic glucose production is a contributing factor. Maternal undernutrition alters the epigenetic status of the glucocorticoid receptor and increases the expression of gluconeogenic enzymes in the liver of male offspring. Undernutrition in the womb programs long-lasting changes in specific gene expression that leads to persistent metabolic abnormalities.

Published online 09 January 2017

RD16277Maternal obesity in the rat impairs male offspring aging of the testicular antioxidant defence system

Claudia J. Bautista, Guadalupe L. Rodríguez-González, Angélica Morales, Consuelo Lomas-Soria, Fabiola Cruz-Pérez, Luis A. Reyes-Castro and Elena Zambrano

Maternal obesity leads to premature aging of male offspring reproductive capacity and increases testes oxidative stress in male rat offspring. Our results show that maternal obesity modifies the antioxidant enzyme system in the testes of offspring, which, in turn, affects testicular physiological functions and leads to premature aging of male reproductive capacity.

Published online 09 January 2017

RD16348Mitochondrial ferritin deficiency reduces male fertility in mice

Federica Maccarinelli, Maria Regoni, Fernando Carmona, Maura Poli, Esther G. Meyron-Holtz and Paolo Arosio

Mitochondrial ferritin is an iron-storage protein highly expressed in human and mouse spermatozoa. This work showed that male mice, but not female, in which mitochondrial ferritin was genetically deleted have reduced fertility without significant alterations in sperm parameters. These data show that mitochondrial ferritin has a role in male fertility.

Published online 21 December 2016

RD16198Ontogeny of clock and KiSS-1 metastasis-suppressor (Kiss1) gene expression in the prepubertal mouse hypothalamus

Cassandra C. Yap, Peter J. Mark, Brendan J. Waddell and Jeremy T. Smith

Kisspeptin neurons in the brain are crucial for the hormone surge required for ovulation, but are established before puberty. This study examined kisspeptin and brain clock gene rhythms (known to restrict kisspeptin action) in young mice. The data show that the kisspeptin peak is not seen prior to puberty and there is incomplete development of clock gene rhythmicity at this time.

Published online 21 December 2016

RD16284Intrafollicular oestradiol production, expression of the LH receptor (LHR) gene and its isoforms, and early follicular deviation in Bos indicus

S. Wohlres-Viana, E. K. N. Arashiro, M. A. Machado, L. S. A. Camargo, L. G. B. Siqueira, M. P. Palhao and J. H. M. Viana

In this study we investigated the expression of the LH receptor (LHR) gene during dominant follicle selection in Bos indicus cattle. The main finding was that the early deviation of the dominant follicle in this subspecies is driven by the differential expression of full-length LHR and its isoforms in small follicles. This finding will contribute to explaining the differences in ovarian physiology between B. indicus and Bos taurus.

Published online 16 December 2016

RD16087Gel-coated tubes extend above-freezing storage of honey bee (Apis mellifera) semen to 439 days with production of fertilised offspring

Brandon K. Hopkins, Susan W. Cobey, Charles Herr and Walter S. Sheppard

Honey bees, which are vital agricultural pollinators, are at risk from numerous factors. The ability to artificially inseminate these species provides a means to strengthen them genetically by collecting semen from bees of different regions throughout the world and then inseminating bees at distant locations. Our study aimed to substantially improve the viability of spermatozoa between collection and insemination.

Preferential transportation of essential fatty acids over non-essential fatty acids occurs from the mother to fetus in humans. However, little is known about placental transfer of fatty acids in the bovine. In the present study, prepartum dietary fat supplementation of cows reduced maternal long-chain polyunsaturated fatty acids, neonatal total omega-3 fatty acids, eicosapentaenoic and docosahexaenoic acids and total, fat possibly through reduced expression of certain placental fatty acid transporter genes.

Published online 12 December 2016

RD16321Presence of vascular endothelial growth factor during the first half of IVM improves the meiotic and developmental competence of porcine oocytes from small follicles

Tra M. T. Bui, Tra M. T. Bui, Khánh X. Nguy?n, Khánh X. Nguy?n, Asako Karata, Asako Karata, Pilar Ferré, Pilar Ferré, Minh T. Tr?n, Minh T. Tr?n, Takuya Wakai, Takuya Wakai, Hiroaki Funahashi and Hiroaki Funahashi

Ovaries contain a large number of small follicles, but the meiotic and developmental competence of oocytes from these follicles is quite low. In the present study we demonstrated that the addition of 200 ng mL–1 vascular endothelial growth factor to the IVM medium increased the maturation rate of porcine oocytes from small follicles and that blastocyst formation following parthenogenetic activation also increased. These findings may contribute to efficient animal production and human assisted reproductive technology.

Published online 12 December 2016

RD16181Modulation of adiponectin system expression in the porcine uterus during early pregnancy by prostaglandin E2 and F2?

Kamil Dobrzyn, Nina Smolinska, Karol Szeszko, Marta Kiezun, Anna Maleszka and Tadeusz Kaminski

Adiponectin is a hormone perceived as the link between energy metabolism and the reproductive system, but its interactions with factors responsible for regulation of uterine homeostasis during pregnancy remain unknown. Herein we demonstrated the effects of prostaglandins E2 and F on adiponectin and its receptors during early gestation. Recognition of these interactions will contribute to our understanding of the physiology of early pregnancy.

Published online 06 December 2016

RD15542Cholesterol metabolism in rabbit blastocysts under maternal diabetes

S. Mareike Pendzialek, Maria Schindler, Torsten Plösch, Jacqueline Gürke, Elisa Haucke, Stefanie Hecht, Bernd Fischer and Anne Navarrete Santos

A diabetic pregnancy leads to distinct changes in maternal cholesterol metabolism, whereas embryonic cholesterol concentrations and cholesterol metabolism genes are not affected. The trophoblast : embryoblast cholesterol ratio suggests a higher metabolic activity of the embryoblast in diabetes. The findings of the present study imply independent and functional cholesterol metabolism in the mammalian embryo before implantation.

Published online 06 December 2016

RD16382The current state of reproductive biology research in Australia and New Zealand: core themes from the Society for Reproductive Biology Annual Meeting, 2016

L. K. Akison, P. H. Andraweera, M. J. Bertoldo, H. M. Brown, J. S. M. Cuffe, T. Fullston, O. Holland and J. E. Schjenken

Reproductive science is central to our understanding of biology, translating to biological processes across many organ systems. The Society for Reproductive Biology conference review provides a focused summary of symposia at the 2016 annual meeting. The presentations highlight the breadth of reproductive research in Australia and New Zealand and its potential to affect the fertility and health of future generations.

Published online 28 November 2016

RD16213Protein in culture and endogenous lipid interact with embryonic stages in vitro to alter calf birthweight after embryo vitrification and warming

E. Gómez, S. Carrocera, S. Uzbekova, D. Martín, A. Murillo, M. Alonso-Guervós, F. Goyache and M. Muñoz

Intracellular lipid is involved in long-term blastocyst competence to survive vitrification. We studied how protein removal affects bovine embryo development in vitro, lipid content and pregnancy outcomes, and found that calf weight at birth was altered depending on specific embryonic kinetics within vitrified–warmed embryos. Therefore, it is possible to modify calf phenotypes by introducing simple changes in culture conditions combined with selection of embryos.

Published online 23 November 2016

RD16174Rosmarinic acid reverses the effects of metronidazole-induced infertility in male albino rats

Zina M. Al-Alami, Ziad A. Shraideh and Mutasem O. Taha

Metronidazole, an essential medicine, adversely affects male fertility. We aimed at investigating the capability of Rosmarinic acid to reverse metranidazole induced male infertility. We found that certain doses of Rosmarinic acid could reverse metronidazole’s effects on sperm count, motility, morphology and testis ultrastructure. Our findings have great clinical implications where Rosmarinic acid might have a potential use in reversing Metronidazole induced male infertility.

Published online 17 November 2016

RD15504Regulation of heat-inducible HSPA1A gene expression during maternal-to-embryo transition and in response to heat in in vitro-produced bovine embryos

Jean-Marc Lelièvre, Nathalie Peynot, Sylvie Ruffini, Ludivine Laffont, Daniel Le Bourhis, Pierre-Marie Girard and Véronique Duranthon

Early mammalian embryos are sensitive to heat stress (HS) despite abundant oocytic iHSPA protein chaperones and mRNAs typically induced by stress. In this work, bovine HSPA1A gene transcription was detectable during embryo genome activation but induced by HS only after this stage, which coincides with better embryonic tolerance. Absence of stress-induced gene expression may therefore contribute to embryo sensitivity.

Published online 11 November 2016

RD16297Effect of nutritionally induced hyperlipidaemia on in vitro bovine embryo quality depends on the type of major fatty acid in the diet

Waleed F. A. Marei, Maria Arias Alvarez, Veerle Van Hoeck, Alfonso Gutierrez-Adan, Peter E. J. Bols and Jo L. M. R. Leroy

Hyperlipidemia associated with unbalanced diet and obesity is directly linked with reduced fertility. This study shows that substituting saturated fat with omega-3 fat in high caloric diets can alleviate detrimental effects of hyperlipidemic serum on bovine early embryo development and quality in vitro. This suggests that dietary manipulation of serum fatty acid profile may enhance fertility in patients with metabolic disorders.

Published online 02 November 2016

RD16154Gene expression profile in heat-shocked Holstein and Nelore oocytes and cumulus cells

J. S. Ticianelli, I. P. Emanuelli, R. A. Satrapa, A. C. S. Castilho, B. Loureiro, M. J. Sudano, P. K. Fontes, R. F. P. Pinto, E. M. Razza, R. S. Surjus, R. Sartori, M. E. O. A. Assumpção, J. A. Visintin, C. M. Barros and F. F. Paula-Lopes

The deleterious effects of heat stress on fertility are more pronounced in dairy cows than beef cows. The transcriptome profile in Nelore and Holstein oocytes subjected to heat shock during IVM and the mRNA abundance of selected genes in cumulus cells were determined. The analyses indicated that the deficiency in dairy cattle in terms of cellular protection against heat stress is regulated at the post-transcriptional level.

Published online 26 October 2016

RD15535Sleep restriction in Wistar rats impairs epididymal postnatal development and sperm motility in association with oxidative stress

Gláucia E. M. L. Siervo, Fernanda M. Ogo, Aline D. Valério, Thamara N. X. Silva, Larissa Staurengo-Ferrari, Tathiana A. Alvarenga, Rubens Cecchini, Waldiceu A. Verri, Flávia A. Guarnier, Monica L. Andersen and Glaura S. A. Fernandes

Sleep alterations are a public health problem worldwide that affect adolescents during puberty because of early school hours and interactive activities. In the present study, sleep restriction during peripuberty in rats affected epididymal postnatal development and sperm motility, in association with oxidative stress and a decrease in the epithelial compartment in the cauda epididymidis. The results of this study suggest a possibility for improving fertility after sleep restriction.

Published online 26 October 2016

RD16223Effect and possible mechanisms of melatonin treatment on the quality and developmental potential of aged bovine oocytes

Shuang Liang, Jing Guo, Jeong-Woo Choi, Nam-Hyung Kim and Xiang-Shun Cui

This research expands our understanding of the regulators that delay oocyte aging. Treatment of aged oocytes with exogenous melatonin improves oocyte quality, rearranges the cytoskeleton and improves developmental capacity. Melatonin is a good agent for delaying oocyte aging in vitro and preventing poor developmental competence of bovine oocytes in assisted reproductive technologies.

Published online 19 October 2016

RD16183Induced sub-lethal oxidative damage affects osmotic tolerance and cryosurvival of spermatozoa

Franziska Ertmer, Harriëtte Oldenhof, Saskia Schütze, Karl Rohn, Willem F. Wolkers and Harald Sieme

If the physiological balance between production and scavenging of reactive oxygen species (ROS) is shifted towards production of ROS this may result in accumulation of cell damage. The aim of this study was to establish whether sub-lethal oxidative damage negatively affects cryosurvival and pre-freeze sperm osmotic tolerance. Generation of defined levels of intracellular ROS can be used for testing strategies designed to counteract oxidative damage.

Published online 19 October 2016

RD16310Reproductive dysfunction after mercury exposure at low levels: evidence for a role of glutathione peroxidase (GPx) 1 and GPx4 in male rats

Caroline S. Martinez, Franck M. Peçanha, Daniela S. Brum, Francielli W. Santos, Jeferson L. Franco, Ana Paula P. Zemolin, Janete A. Anselmo-Franci, Fernando B. Junior, María J. Alonso, Mercedes Salaices, Dalton V. Vassallo, Fábio G. Leivas and Giulia A. Wiggers

Mercury has been identified as a risk factor for declines in sperm quality and infertility in humans. The present study delves further into the mechanisms of action of mercury, showing a role for glutathione peroxidase (GPx) 1 and GPx4 in the reproductive toxicity of mercury. The involvement of the glutathione system highlights this antioxidant system as a potential target of mercury after its accumulation in reproductive organs.

Published online 14 October 2016

RD16037Ovariectomy increases the phenotypic plasticity of the female prostate epithelium in the Mongolian gerbil (Meriones unguiculatus)

Marianna Zanatelli, Ana M. G. Custodio, Manoel F. Biancardi, Fernanda C. A. Santos, Rejane M. Góes, Luiz R. Falleiros-Jr, Patricia S. L. Vilamaior and Sebastião R. Taboga

After hormonal deprivation the female gerbil prostate undergoes several morphophysiological changes. This work identified and characterised the cellular heterogeneity of the female prostate epithelium in normal conditions and after ovariectomy. Secretory cells of the female prostate are not morphologically and functionally uniform, presenting a phenotypical plasticity according to the hormonal environment in which they operate.

The current literature does not provide a comprehensive profile of the factors involved in placental development in rats during intrauterine trophoblast migration. In this study we evaluated the gene and/or protein expression of several placental factors during the second half of pregnancy in rats and demonstrated that the expression of proteases and immunological, angiogenic, hormonal and apoptotic mediators is variable and depends on the stage of trophoblast migration. These changes in the expression profile are probably related to alterations in placental morphogenesis and function.

Published online 11 October 2016

RD16232Influence of hyaluronan on endometrial receptivity and embryo attachment in sheep

Waleed F. A. Marei, D. Claire Wathes, Kabir A. Raheem, Omnia Mohey-Elsaeed, Fataneh Ghafari and Ali A. Fouladi-Nashta

Infusion of the ovine uterus with hyaluronan (HA) during the pre-attachment period changes the expression of some adhesion markers at the embryo–maternal interface, resulting in reduced endometrial receptivity and failure of embryo attachment. In contrast, local inhibition of HA production in the uterus enhances embryo attachment.

Published online 05 October 2016

RD15537Circulating anti-Müllerian hormone concentrations in relation to age and season in male and female beluga (Delphinapterus leucas)

Gisele A. Montano, Todd R. Robeck, Karen J. Steinman and Justine K. O'Brien

Anti-Müllerian hormone (AMH) has been proposed as a biomarker of ovarian reserve in women and has been detected in many species; however, this is the first characterisation of AMH in a cetacean. AMH was described in males and females from the year of birth to over 30 years of age during different reproductive seasons, and a decrease in AMH in older animals (>30 years old) was detected. AMH may be of value for reproductive health monitoring of the species and as a biomarker of reproductive senescence in both sexes.

Published online 05 October 2016

RD16200Non-surgical sterilisation methods may offer a sustainable solution to feral horse (Equus caballus) overpopulation

Sally Elizabeth Hall, Brett Nixon and R. John Aitken

Culling feral horses is currently met with public criticism, which has created an urgent need to develop new methods of population control. Delivery of a contraceptive agent that causes sterility, without the need for surgery, is currently under investigation. These novel techniques may provide a sustainable and effective alternative for feral horse control.

Sertoli cells, which support and coordinate developing sperm in the testes, were isolated from 8-week-old Holstein bull calves. A combination of two hormones (IGF-I and FSH) enhanced multiplication of Sertoli cells. We concluded that increased IGF-I concentrations in bull calves fed high-levels of nutrition during early life increases Sertoli cell number, supports testicular development and results in earlier puberty.

Published online 03 October 2016

RD16190Effects of freezing and activation on membrane quality and DNA damage in Xenopus tropicalis and Xenopus laevis spermatozoa

S. Morrow, J. Gosálvez, C. López-Fernández, F. Arroyo, W. V. Holt and M. J. Guille

Xenopus frogs are key organisms for studying basic biology and for modelling human disease. Genetically altered lines of frogs are therefore made, and it is important for animal welfare and cost-effectiveness to store these as frozen spermatozoa. In this study we show that Xenopus sperm cryopreservation damages the DNA and plasma membrane of spermatozoa and start to identify factors affecting damage levels.

Published online 28 September 2016

RD15553In vivo and in vitro maturation of rabbit oocytes differently affects the gene expression profile, mitochondrial distribution, apoptosis and early embryo development

M. Arias-Álvarez, R. M. García-García, J. López-Tello, P. G. Rebollar, A. Gutiérrez-Adán and P. L. Lorenzo

Understanding the cellular and molecular mechanisms implicated in oocyte competence during rabbit in vivo and in vitro maturation is necessary to improve IVM systems in these species. This study showed for the first time that gene expression profile of in vivo-matured rabbit COCs is different from that of IVM COCs and these changes are clearly accompanied by stage-specific cytoplasmic remodelling of mitochondria, a lower rate of apoptosis of in vivo-matured CCs and better early embryo development, indicating that oocyte quality is compromised during IVM.

The antioxidant defences of epididymal fluid and spermatozoa are modulated from the caput to cauda epididymidis in a region-specific manner in the goat (Capra hircus), and loss of some of the antioxidant enzymes from spermatozoa during their epididymal transit is compensated for through a simultaneous increase in the expression and/or activity of these enzymes in the epididymal fluid of the respective segments.

Published online 28 September 2016

RD16164Testicular parameters and spermatogenesis in different birthweight boars

P. A. Auler, G. H. F. A. Moreira, C. O. Hogg, C. J. Ashworth, F. P. Bortolozzo, H. Chiarini-Garcia and F. R. C. L. Almeida

Reports of the effects of birthweight on testicular development and implication for sperm production in boars are scarce. These effects were investigated in this study. It seems that high birthweight boars have the potential to produce more spermatozoa and semen doses per ejaculate. Hence, the selection of those boars would be predictive of better lifetime productivity in the boar stud.

Diabetes has a profound impact on women’s reproductive health. We aimed to investigate the protective effect of fish oil and wheat-germ oil supplementation on ovarian function in streptozotocin-diabetic rats. These natural supplements counteracted oxidative stress and inflammation induced by diabetes and regained ovarian function by increasing FSH, E2, LH and AMH levels and restoring follicle counts.

Published online 28 September 2016

RD16075Targeted deletion of the Kv6.4 subunit causes male sterility due to disturbed spermiogenesis

Glenn Regnier, Elke Bocksteins, Waleed F. Marei, Isabel Pintelon, Jean-Pierre Timmermans, Jo L. M. R. Leroy and Dirk J. Snyders

Several voltage-gated K+ (Kv) channel subunits are expressed in testis, but their contribution to testicular function remains largely unknown. We provide direct evidence of the relevance of Kv6.4 for male fertility since the targeted deletion of this gene in mice disrupted spermiogenesis, resulting in male sterility. This makes Kv6.4 a potentially interesting pharmacological target for the development of non-hormonal male contraceptives.

Published online 28 September 2016

RD16063Proliferation and apoptosis processes in the seasonal testicular development of the wild Daurian ground squirrel (Citellus dauricus Brandt, 1844)

Yingying Han, Jinqi Zhan, Ying Xu, Fengwei Zhang, Zhengrong Yuan and Qiang Weng

Male seasonal breeders show synchronised cycles of testicular growth and involution throughout the breeding cycle. The aim of the present study was to elucidate the regulatory role of cell proliferation and apoptosis in this dynamic testicular development. The study demonstrates, for the first time, that both cell proliferation and apoptosis are stimulated during the prehibernation period in wild Daurian ground squirrels. These findings advance our current understanding of the regulatory mechanisms of seasonal reproduction.

Published online 22 September 2016

RD15524Analysis of in vitro follicle development during the onset of premature ovarian insufficiency in a mouse model

Heidy Kaune, Sairah Sheikh and Suzannah A. Williams

The developmental potential of follicles from a mouse model of premature ovarian insufficiency (POI) was assessed using follicle culture. Although, follicles cultured from 3- and 6-week-old mice had decreased survival compared to Controls, a population of follicles retain the potential of full development in vitro. These results indicate that in vitro follicle culture is a promising method to rescue follicle development after the onset of POI in this model.

Published online 21 September 2016

RD15486Discs large homologue 1 (Dlg1) coordinates mouse oocyte polarisation during maturation

Jun-Chao Wang, Hong Lv, Ke-Liang Wu, Yun-Shan Zhang, Hai-Ning Luo and Zi-Jiang Chen

Mouse oocyte maturation is a unique asymmetric division and a lot of polarity proteins may play vital roles during this progress. Our study described an evolutionarily conserved polarity protein Discs-large homologue 1 becomes highly polarized by associating with cytoskeleton and lose-of-function of Dlg1 compromises oocyte maturation. It may provide a new theoretic framework for mammalian meiosis research.

Published online 21 September 2016

RD16238Imprinted and DNA methyltransferase gene expression in the endometrium during the pre- and peri-implantation period in cattle

A. M. O'Doherty, L. C. O'Shea, O. Sandra, P. Lonergan, T. Fair and N. Forde

A comprehensive understanding of biological processes in the endometrium is vital for identifying the key factors required for successful embryo recognition and implantation. The aim of the present study was to compare the expression of imprinted genes and DNA methyltransferases in the endometrium of cyclic and pregnant beef heifers. Results from this investigation provide new evidence that, in addition to their role in fetal and placental development, imprinted genes and DNA methyltransferases may have a role in establishing uterine receptivity.

Published online 21 September 2016

RD15489Expression of C-type lectin receptors and Toll-like receptors in decidua of patients with unexplained recurrent spontaneous abortion

Liang Xu, Tian Qiu, Yudong Wang, Yan Chen and Weiwei Cheng

This study aimed to explore the mechanisms underlying the pathogenesis of unexplained recurrent spontaneous abortion (URSA). The results suggest that the balance between C-type lectin-like receptors and Toll-like receptors (TLRs) was tilted towards a TLR-dominant response in URSA patients, which may disrupt maternal–fetal immune tolerance, resulting in spontaneous abortion.

Published online 13 September 2016

RD16110Profiling bovine blastocyst microRNAs using deep sequencing

R. Pasquariello, B. Fernandez-Fuertes, F. Strozzi, F. Pizzi, R. Mazza, P. Lonergan, F. Gandolfi and J. L. Williams

MicroRNAs (miRNAs) control several reproductive functions, including oocyte maturation, implantation and early embryonic development. We developed a method for accurate miRNA identification in a small sample of bovine blastocysts. Using pools of only 30 embryos, we identified miRNAs that regulate target genes and metabolic pathways that had not been associated with early embryogenesis before, and are potential biomarkers of developmental competence.

Published online 13 September 2016

RD15514Role of insulin-like growth factor 1 on cross-bred Bos indicus cattle germinal vesicle oocytes exposed to heat shock

Rafaela S. Lima, Pedro H. B. Risolia, Jéssica Ispada, Mayra E. O. A. Assumpção, José A. Visintin, Cássia Orlandi and Fabíola F. Paula-Lopes

Germinal vesicle oocytes are susceptible to heat stress. The present study demonstrated that the physiological IGF-I concentration of 12.5 ng/ml was able to revert the deleterious effects of heat shock on bovine GV oocyte cellular function and ability to became a blastocyst. This thermoprotective role of IGF1 warrants further in vivo IGF1 manipulation to improve dairy cow fertility during summer.

Published online 05 September 2016

RD15488In vitro oocyte maturation in a medium containing reduced sodium chloride improves the developmental competence of pig oocytes after parthenogenesis and somatic cell nuclear transfer

Joohyeong Lee, Hanna Lee, Yongjin Lee, Bola Park, Fazle Elahi, Seung Tae Lee, Choon-Keun Park, Sang-Hwan Hyun and Eunsong Lee

The quality of oocytes is a crucial factor in the field of reproductive biotechnology. We observed that maturation of pig oocytes in hypotonic medium with reduced NaCl improves the developmental competence of oocytes by affecting the cytoplasmic microenvironment. An oocyte maturation system using a medium with reduced NaCl could potentially improve the efficiency of reproduction in livestock species after further optimisation.

Published online 29 August 2016

RD16119Consumption of a high-fat diet alters the seminal fluid and gut microbiomes in male mice

Angela B. Javurek, William G. Spollen, Sarah A. Johnson, Nathan J. Bivens, Karen H. Bromert, Scott A. Givan and Cheryl S. Rosenfeld

We recently discovered that the male seminal vesicles harbour a novel seminal fluid microbiome (SFM). The present study shows that short-term feeding of a high-fat diet (HFD) changes the bacterial composition of the seminal fluid and fecal microbiomes. HFD-induced SFM alterations might lead to detrimental health consequences in males, their reproductive partners and resulting offspring.

Published online 24 August 2016

RD15512Effects of fetal calf serum on cGMP pathway and oocyte lipid metabolism in vitro

Kátia R. L. Schwarz, Ramon Cesar Botigelli, Maite Del Collado, Fernanda Cavallari de Castro, Hugo Fernandes, Daniela M. Paschoal and Cláudia Lima Verde Leal

Serum affects lipid metabolism in oocytes and embryos, and cGMP has been shown to affect lipolysis in adipocytes. The present study shows that serum in the maturation medium interferes with lipid metabolism in oocytes and cumulus cells and these effects depend, at least in part, on the cGMP pathway. Evidence of involvement of this pathway in lipolysis in cumulus–oocyte complexes could contribute to the development of better culture systems for the in vitro production of oocytes and embryos with lower lipid accumulation and better cryotolerance.

Published online 22 August 2016

RD15530Thawing boar semen in the presence of seminal plasma improves motility, modifies subpopulation patterns and reduces chromatin alterations

Rocío Fernández-Gago, Manuel Álvarez-Rodríguez, Marta E. Alonso, J. Ramiro González, Beatriz Alegre, Juan C. Domínguez and Felipe Martínez-Pastor

Seminal plasma could benefit boar semen when added during thawing. This study shows that that the addition of 10% or 50% seminal plasma during post-thawing incubation improved total motility and the motility pattern (motile subpopulations) of spermatozoa. Chromatin changes were almost non-existent for samples from some male boars, both after thawing and during the incubation, but in males susceptible to these sorts of chromatin changes, seminal plasma reduced or abolished them.

Published online 18 August 2016

RD16124Periconceptional undernutrition affects in utero methyltransferase expression and steroid hormone concentrations in uterine flushings and blood plasma during the peri-implantation period in domestic pigs

A. Franczak, K. Zglejc, E. Waszkiewicz, B. Wojciechowicz, M. Martyniak, W. Sobotka, S. Okrasa and G. Kotwica

A restricted diet applied to females during the peri-conceptional period may have consequences during the peri-implantation period. In this study we examined DNA methyltransferase 1 (DNMT1) and DNMT3a expression in endometrium and myometrium and oestradiol 17β (E2) and progesterone concentrations in the uterus and blood plasma in pigs during the peri-implantation period. Undernutrition during the peri-conceptional period affects maintenance and de novo DNA methylation in the endometrium, de novo DNA methylation in the myometrium and intrauterine E2 concentrations during the peri-implantation period.

Published online 11 August 2016

RD16205Changes in testicular function proteins and sperm acrosome status in rats treated with valproic acid

Wannisa Sukhorum and Sitthichai Iamsaard

The mechanism by which valproic acid (VPA) causes male infertility is not known. In the present study, testicular proteins and acrosome status were investigated in VPA-treated rats. Acrosome reacted spermatozoa and the expression of Ki-67, cholesterol side-chain cleavage enzyme (P450scc) and tyrosine phosphorylated proteins, except for steroidogenic acute regulatory protein, were decreased in VPA-treated rats. These changes to testicular proteins responsible for spermatogenesis result in male infertility after VPA treatment.

Published online 10 August 2016

RD16163Potential risks to offspring of intrauterine exposure to maternal age-related obstetric complications

Juan J. Tarín, Miguel A. García-Pérez and Antonio Cano

Several hypotheses have been proposed to explain the negative effects of delayed motherhood on an offspring’s morbidity later in life, but they are not supported by clinical and epidemiological evidence. In the present study, we show that the negative effects on offspring of delayed motherhood may be induced by embryo or fetal epigenetic DNA reprogramming associated with maternal age-related obstetric complications. This outcome has direct clinical implications for those women wanting to enrol in a fertility preservation program to offset age-related declines in fertility.

Published online 04 August 2016

RD16056Evidence of proteinuria, but no other characteristics of pre-eclampsia, in relaxin-deficient mice

Kelly P. O'Sullivan, Sarah A. Marshall, Scott Cullen, Tahnee Saunders, Natalie J. Hannan, Sevvandi N. Senadheera and Laura J. Parry

This paper reports that pregnant mice deficient in relaxin (Rln–/–) did not exhibit characteristics associated with preeclampsia; plasma sFlt-1 concentrations and blood pressure in pregnant Rln–/– mice were no different compared with age-matched wildtype mice. Although there was evidence of proteinuria in older pregnant Rln–/– mice, this was also observed in non-pregnant mice and was not a consequence of pregnancy. Therefore, pregnant Rln–/– mice are not a suitable animal model to study preeclampsia.

Published online 03 August 2016

RD16007Circumventing the natural, frequent oestrogen waves of the female cheetah (Acinonyx jubatus) using oral progestin (Altrenogest)

Adrienne E. Crosier, Pierre Comizzoli, Diana C. Koester and David E. Wildt

We found that cheetahs do not display typical estrous cycles, but rather frequent waves of estrogenic activity every 7 to 10 days. For effective ovulation induction and the most mature oocytes, gonadotropin treatments had to be given at least 3 days after an estrogen peak. Follicular activity can be suppressed by progestin supplementation, thereby providing a way to quiesce the ovary beforehand.

Published online 01 August 2016

RD15463Beneficial effects of diazepin-quinazolin-amine derivative (BIX-01294) on preimplantation development and molecular characteristics of cloned mouse embryos

Yanfang Huang, Xiaohong Jiang, Miao Yu, Rongfu Huang, Jianfeng Yao, Ming Li, Fangfang Zheng and Xiaoyu Yang

Somatic cell nuclear transfer is frequently associated with abnormal epigenetic modifications. The present study investigated the effects of BIX-01294 on cloned mouse preimplantation embryos and the results indicated that BIX-01294 treatment of cloned embryos has beneficial effects in terms of correcting abnormal epigenetic modifications. It will be investigated further whether BIX may affect postimplantation development by correcting epigenetic modifications.

Macrophage migration inhibitory factor (MIF) may be the molecular link between maternal obesity and the quality of early embryos. This study revealed that MIF expression in the oviduct, where mammalian embryos spend their first 3 days, is higher during the postovulatory phase than during oestrus and the luteal phase in heifers. Therefore, oviduct MIF may be essential for producing good-quality embryos.

The aim of this study was to explore the mechanism of action of stathmin 1 (Stmn1), a protein identified by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) in a previous proteomic analysis, in regulating embryo implantation, a critical process in pregnancy. Stmn1 was found to play a role in decidualisation and embryo implantation by inhibiting the expression of phosphorylated Akt, hypoxia-inducible factor-1α and vascular endothelial growth factor. These findings contribute to a better understanding of the potential role of Stmn1 during embryo implantation.

Published online 26 July 2016

RD16091Role of the Na+/K+-ATPase ion pump in male reproduction and embryo development

D. R. Câmara, J. P. Kastelic and J. C. Thundathil

This review describes the involvement of the sodium pump Na+/K+-ATPase in various aspects of animal reproduction, including critical roles in embryonic development and specific sperm functions. In addition to regulating ion concentrations, Na+/K+-ATPase participates in several cell signalling pathways. Understanding the role of this molecule creates new opportunities to enhance or suppress various aspects of reproductive biology.

Published online 21 July 2016

RD16031Methylated oligonucleotide (MON)-induced promoter hypermethylation is associated with repression of CDH1 expression and contributes to the migration and invasion of human trophoblast cell lines

Xi Lan, Li-Juan Fu, Zhuo-Ying Hu, Qian Feng, Xue-Qing Liu, Xue Zhang, Xue-Mei Chen, Jun-Lin He, Ying-Xiong Wang and Yu-Bin Ding

Aberrant DNA methylation has been linked to deficiencies in trophoblast motility and invasion in pregnancy complications. We increased trophoblast cell motility and invasiveness through inducing site-specific hypermethylation and repressing gene expression of CDH1 by sense-methylated oligonucleotides (MONs). The results indicate that MON-mediated DNA methylation on the CDH1 promoter may alter gene expression and contribute to trophoblast motility and invasion.

Published online 04 July 2016

RD16086In vitro characterisation of fresh and frozen sex-sorted bull spermatozoa

Shauna A. Holden, Craig Murphy, Juan F. Moreno, Stephen T. Butler, Andrew R. Cromie, Patrick Lonergan and Sean Fair

The use of sex-sorted fresh spermatozoa has the potential to yield higher pregnancy rates than sex-sorted frozen spermatozoa. This study sought to characterise the in vitro quality of X and Y sex-sorted bull spermatozoa and identified the need for tailored diluents for fresh sex-sorted spermatozoa to avoid head-to-head agglutination. The use of fresh sex-sorted spermatozoa, when managed correctly, has enormous potential in the dairy industry.

Published online 01 July 2016

RD16225Thiols of flagellar proteins are essential for progressive motility in human spermatozoa

María Eugenia Cabrillana, María de los Ángeles Monclus, Tania Estefania Sáez Lancellotti, Paola Vanina Boarelli, Amanda Edith Vincenti, Miguel Matias Fornés, Eduardo Alfredo Sanabria and Miguel Walter Fornés

This work demonstrates that it is necessary for progressive sperm motility that flagellar proteins, like ODF1, maintain reduced cysteines after epididymal transit. To determine how oxidative changes in flagellar proteins affect sperm motility, a sulfhydryl blocker was used; sperm progressive motility was abolished without affecting the level of ATP. Incomplete oxidation in flagellar proteins of asthenozoospermic samples was also observed. Inadequate maturation or an unbalanced thiol status leads to non-progressive motility.

Published online 27 June 2016

RD16104GRIM-19, a gene associated with retinoid-interferon-induced mortality, affects endometrial receptivity and embryo implantation

Yang Yang, Yanyan Sun, Laiyang Cheng, Anna Li, Yanjun Shen, Ligang Jiang, Xiaohui Deng and Lan Chao

We present a new hypothesis of the molecular mechanisms responsible for implantation, which could provide an important basis for further understanding of embryo implantation. Our study shows that spontaneous elimination of GRIM-19 is required for endometrial receptivity and the embryo implantation process. Our results demonstrate the involvement of GRIM-19 in the embryo implantation process by regulating adhesion, apoptosis and immune tolerance.

Published online 22 June 2016

RD15404Reproductive competency and mitochondrial variation in aged Syrian hamster oocytes

Fang Li, Frank J. Castora, Wentia Ford, Khalid Alarid, Howard W. Jones and R. James Swanson

Using a hamster model, we found significant changes in mitochondrial number, structure and function as well as fecundity, ova production and follicular number in aged compared to younger animals. Old hamsters had decreased fertility rates, smaller litters, and decreased numbers of preantral follicles and MII oocytes. In addition, old ova contained large areas of collapsed, non-luminal cytoplasmic lamellae. Finally, old hamster ova contained higher levels of reactive oxygen species, lower mitochondrial membrane potential and decreased mitochondrial numbers, all of which could be contributing to the age-related decline in mammalian fertility rates.

Published online 22 June 2016

RD16022Subfertility in androgen-insensitive female mice is rescued by transgenic FSH

K. A. Walters, M. C. Edwards, M. Jimenez, D. J. Handelsman and C. M. Allan

Androgens (male steroids) interact with the key regulator of female reproduction, FSH, but the nature of their interaction is not clear. Examination of mutant androgen-insensitive mice with and without the expression of human transgenic FSH (Tg-FSH) revealed that subfertility in androgen-insensitive mice is rescued by the presence of Tg-FSH. These findings demonstrate that androgens and FSH actions interact in a synergistic manner to maintain optimal female fertility.

Published online 20 June 2016

RD16061Putrescine supplementation during in vitro maturation of aged mouse oocytes improves the quality of blastocysts

Dandan Liu, Guolong Mo, Yong Tao, Hongmei Wang and X. Johné Liu

Poor egg quality underlies reproductive aging in mice and in women. Oocyte maturation is a critical stage in determining the quality of eggs and hence embryonic potential. We demonstrated that putrescine supplementation during in vitro maturation of aged mouse oocytes resulted in healthier embryos. Peri-ovulatory putrescine supplementation may be an effective therapy in reproductive aging.

Stem cell factor (SCF) and epidermal growth factor (EGF) play key roles in ovarian folliculogenesis. Herein we report that SCF promotes cat follicle growth by upregulating c-kit mRNA expression and AKT phosphorylation. EGF suppresses the stimulating effect of SCF, leading to a downregulation of FSH receptor mRNA expression. The ability to grow premature follicles in vitro could become a pivotal tool for preserving fertility in animal disease models and endangered felids.

Published online 09 June 2016

RD15391Iloprost supports early development of in vitro-produced porcine embryos through activation of the phosphatidylinositol 3-kinase/AKT signalling pathway

Pil-Soo Jeong, Seung-Bin Yoon, Seon-A Choi, Bong-Seok Song, Ji-Su Kim, Bo-Woong Sim, Young-Ho Park, Hae-Jun Yang, Seong-Eun Mun, Young-Hyun Kim, Philyong Kang, Kang-Jin Jeong, Youngjeon Lee, Yeung Bae Jin, Jae-Won Huh, Sang-Rae Lee, Deog-Bon Koo, Young Il Park, Sun-Uk Kim and Kyu-Tae Chang

Despite evidence of the presence of PGI2 in mammalian oviducts, its role in development of early embryos is largely unknown. We revealed that supplementation of PGI2 analog enhanced early development of IVP porcine embryos through activation of AKT cascade. These results indicate that iloprost can be a useful IVC supplement for production of IVP early porcine embryos with high quality.

Published online 06 June 2016

RD15475High glucose levels affect spermatogenesis: an in vitro approach

Renata S. Tavares, Joana M. D. Portela, Maria I. Sousa, Paula C. Mota, João Ramalho-Santos and Sandra Amaral

Diabetes mellitus (DM) has been associated with male reproductive impairment but the underlying mechanisms are poorly understood owing to the diversity of factors involved in the disease. In this study we focused on the in vitro effects of high glucose levels, the primary recognised signature of DM, on spermatogenesis. We found that while mouse testicular luminal area was decreased, Sertoli cell number was increased. These findings suggest an impairment of Sertoli cell function in high-glucose conditions that may impact spermatogenesis.

Published online 02 June 2016

RD16098Sex of co-twin affects the in vitro developmental competence of oocytes derived from 6- to 8-week-old lambs

Jennifer M. Kelly, David O. Kleemann, Hayley McGrice, Jose A. Len, Karen L. Kind, Will H. E. J. van Wettere and Simon K. Walker

The effect of birth type (single, twin, triplet) and co-twin sex on in vitro developmental competence of oocytes from 6 to 8-week old lambs significantly increased blastocyst development rates for lambs born with a female co-twin compared with a male co-twin. This indicates a female co-twin enhancement effect on oocyte development. Selection of lambs for embryo production programs based on co-twin sex is warranted.

Published online 01 June 2016

RD16145Role of Ca2+ in the IVM of spermatozoa from the sterlet Acipenser ruthenus

Olga Bondarenko, Borys Dzyuba, Marek Rodina and Jacky Cosson

Spermatozoa maturation is a necessary step for the best success in artificial reproduction. This study focused on the role of calcium ions in the acquisition of testicular sturgeon sperm to be able to swim: our results show that an influx of Ca2+ ions is required for finalisation of this maturation process.

Published online 31 May 2016

RD15309Maternal food restriction in rats of the F0 generation increases retroperitoneal fat, the number and size of adipocytes and induces periventricular astrogliosis in female F1 and male F2 generations

A. O. Joaquim, C. P. Coelho, P. Dias Motta, L. F. Felício, E. F. Bondan, E. Teodorov, M. F. M. Martins, T. B. Kirsten, L. V. Bonamin and M. M. Bernardi

Transgenerational food restriction and overweightAdverse environmental factors during gestation, such as under- or overnutrition, may predispose individuals to certain diseases later in life by altering their fetal programming. The present study investigated whether prenatal 40% food restriction was able to induce weight, metabolic and neuroimmune disturbances in the next two generations, even if they were normally fed. We showed a transgenerational transmission effect for overweight over two generations, associated with changes in immune and inflammatory patterns, revealing the severe impact of undernutrition over generations.

Mammalian males do not produce equal numbers of X- and Y-chromosome-bearing sperm, but factors influencing the variation are unknown. We show that sperm sex ratios and ejaculate traits vary with mating rate in laboratory mice, and hypothesise that males that mate frequently are usually more attractive, and would be advantaged by producing sons that inherit their attractiveness.

Published online 26 May 2016

RD15520Temporary undernutrition during early gestation, corpora lutea morphometrics, ovarian progesterone secretion and embryo survival in gilts

P. Langendijk, E. G. Bouwman, T. Y. Chen, R. E. Koopmanschap and N. M. Soede

This study looked at how off-feed incidents or competition for feed can affect hormonal regulation of luteal function and early pregnancy in pigs. We found no effect of a day’s fasting on LH secretion, but progesterone secretion was reduced and fewer piglets were born at subsequent farrowing. Malnutrition in group-housed sows or in periods of hot weather can potentially reduce ovarian progesterone output and fertility.

Published online 26 May 2016

RD15357Non-genomic action of vitamin D3 on N-methyl-D-aspartate and kainate receptor-mediated actions in juvenile gonadotrophin-releasing hormone neurons

Pravin Bhattarai, Janardhan P. Bhattarai, Min Sun Kim and Seong Kyu Han

Vitamin D deficiency has been linked to impaired pubertal development affecting reproductive physiology. The effects of vitamin D3 were investigated on the juvenile gonadotrophin-releasing hormone (GnRH) neuron, a central regulator of the hypothalamic–pituitary–gonadal axis. Exposure to vitamin D3 suppressed the N-methyl-D-aspartate and kainate-mediated responses of juvenile GnRH neurons. We suggest that vitamin D is essential for normal and timely functioning of pubertal development.

Published online 25 May 2016

RD16077Aquaglyceroporins 3 and 7 in bull spermatozoa: identification, localisation and their relationship with sperm cryotolerance

Noelia Prieto-Martínez, Roser Morató, Rodrigo Muiño, Carlos O. Hidalgo, Joan E. Rodríguez-Gil, Sergi Bonet and Marc Yeste

For the first time the presence and localisation of AQP3 and AQP7 in bull spermatozoa was determined, together with their relationship to sperm cryotolerance. Our findings show that neither the relative amounts nor the localisation of these proteins was altered by cryopreservation procedures. Furthermore, the abundance of AQP7 in fresh semen was found to predict bull sperm cryotolerance.

Published online 25 May 2016

RD15414Membrane lipid profile of in vitro-produced embryos is affected by vitrification but not by long-term dietary supplementation of polyunsaturated fatty acids for oocyte donor beef heifers

Beatriz C. S. Leão, Nathália A. S. Rocha-Frigoni, Ériklis Nogueira, Elaine C. Cabral, Christina R. Ferreira, Marcos N. Eberlin, Mônica F. Accorsi, Thiago V. Neves and Gisele Z. Mingoti

In this study we evaluated the effect of feeding oocyte donor beef heifers with a protected fat enriched in linoleic acid on embryonic developmental rates after IVF, embryo cryotolerance and the membrane lipid composition of fresh and vitrified–warmed embryos. Although embryo yield, cryotolerance and membrane lipid profile were unaffected by dietary supplementation, the membrane lipid profile was affected by the cryopreservation process. Because the lipid ion abundances of fresh and vitrified–warmed embryos were distinct, they can be used as potential markers of post-cryopreservation embryonic survival.

Published online 23 May 2016

RD15330The effects of sildenafil citrate on feto–placental development and haemodynamics in a rabbit model of intrauterine growth restriction

Jorge López-Tello, María Arias-Álvarez, Maria-Ángeles Jiménez-Martínez, Alicia Barbero-Fernández, Rosa María García-García, María Rodríguez, Pedro L. Lorenzo, Laura Torres-Rovira, Susana Astiz, Antonio González-Bulnes and Pilar G. Rebollar

Current treatments for intrauterine growth restriction are not particularly effective. Sildenafil citrate (SC), a vasodilator and angiogenic molecule, could be an option to enhance conceptus growth. Here we studied, in a rabbit model, whether the administration of SC could improve or ameliorate diet-induced defects in conceptus development and offspring outcome. SC seems to benefit pregnancies complicated by placental insufficiency; however, further research is needed.

Published online 23 May 2016

RD15483Variation among individual bulls in the distribution of acrosomal tyrosine-phosphorylated proteins in epididymal and ejaculated spermatozoa

Miyuki M. Arai, Kenta Minami, Yukari Ogura, Nagisa Otsuka, Shohei Hama, Hiroshi Harayama, Mitsuhiro Sakase and Moriyuki Fukushima

A valid biomarker for sperm molecular characterisation was found in Japanese black cattle. The distribution of acrosomal tyrosine-phosphorylated proteins largely differs among individual bulls. The grades of acrosomal freezability and results of AI with cryopreserved spermatozoa can be predicted by assessing the distribution of acrosomal tyrosine-phosphorylated proteins in spermatozoa before cryopreservation.

Published online 17 May 2016

RD15538Expressions of lipoprotein receptors and cholesterol efflux regulatory proteins during luteolysis in bovine corpus luteum

Kei Horihata, Shin Yoshioka, Masahiro Sano, Yuki Yamamoto, Koji Kimura, Dariusz J. Skarzynski and Kiyoshi Okuda

Progesterone, which is essential for pregnancy in mammals, is synthesised from cholesterol in the corpus luteum (CL). We investigated the involvement of cholesterol uptake receptors and efflux transporters in bovine CL regression (luteolysis), and found that the expression of scavenger receptor B1, a cholesterol uptake receptor, decreased during luteolysis. The decline in cholesterol uptake may reduce progesterone production in the CL during luteolysis.

Published online 17 May 2016

RD16090The effects in vitro of TNF-α and its antagonist ‘etanercept’ on ejaculated human sperm

Nicola A. Pascarelli, Antonella Fioravanti, Elena Moretti, Giacomo M. Guidelli, Lucia Mazzi and Giulia Collodel

The effect of cytokines on human male fertility is attracting more attention. In this in vitro study, the effects of TNF-α and its antagonist, etanercept, in ejaculated human spermatozoa were analysed. The negative influence of TNF-α on sperm function and the positive action of etanercept were demonstrated. This data may have clinical relevance for the management of chronic inflammatory diseases related to male reproduction.

Published online 17 May 2016

RD15385Effects of fetal genotype and sex on developmental response to maternal malnutrition

Laura Cogollos, Consolacion Garcia-Contreras, Marta Vazquez-Gomez, Susana Astiz, Raul Sanchez-Sanchez, Ernesto Gomez-Fidalgo, Cristina Ovilo, Beatriz Isabel and Antonio Gonzalez-Bulnes

The occurrence of intrauterine growth restriction is linked to a plethora of non-communicable disorders during postnatal life. Our study, using a translational swine model, provides evidence that prenatal developmental is strongly modulated by fetal genotype and sex. Such findings stress the necessity for considering ethnicity and sex effects in the study of the developmental origins of adult health and disease.

The reproductive tract secretes molecules collectively known as embryokines that regulate embryonic growth and development. In this study we tested four growth factors expressed in the endometrium for actions on the bovine preimplantation embryo. All four molecules affected embryonic development or gene expression and may therefore function in the endometrium as embryokines.

High temperatures are correlated with reduced fertility. Ovarian function in pseudopregnant rats was determined in a laboratory setting of repeated heat stress. Heat stress altered the activity of mitogen-activated protein kinases and the expression of heat shock protein 70 and activating transcription factor 3 in the corpus luteum. A deeper understanding of the effects of heat stress on ovarian function will enable better approaches to promote female fecundity and sustainable animal production.

Published online 11 May 2016

RD15209Roles of interferon-stimulated gene 15 protein in bovine embryo development

Shuan Zhao, Yi Wu, Hui Gao, Alexander Evans and Shen-Ming Zeng

ISG15 mRNA exists in bovine embryo from one cell stage, which is earlier than IFNT mRNA. When the expression of ISG15 was knockdown, the development of blastocyst was negatively affected, , and the expressions of Ets2, IFNT and connexion 43 were inhibited in bovine blastocysts.

Published online 11 May 2016

RD16026p21-activated kinase 1 activity is required for histone H3 Ser10 phosphorylation and chromatin condensation in mouse oocyte meiosis

Nana Zhang, Xiuhong Li, Xiaoyun Liu, Yan Cao, Dandan Chen, Xiaoyu Liu, Qian Wang, Juan Du, Jing Weng and Wei Ma

Histone H3 phosphorylation is essential for chromatin condensation, which is a prerequisite for accurate chromosome separation in mitosis or meiosis. The present study demonstrates that p21-activated kinase 1 (Pak1) activity is required for histone H3 phosphorylation and consequent chromatin condensation in mouse oocytes upon the resumption of meiotic progression.

Feasibility of luteal phase ovarian stimulation has already be certified in poor ovarian responders. Embryos produced during the luteal phase resulted in higher implantation rates compared with those produced during the follicular phase. Luteal phase ovarian stimulation should be recommended for poor ovarian responders.

Published online 11 May 2016

RD15517Perinatal exposure to insecticide fipronil: effects on the reproductive system in male rats

Aline L. de Barros, Julie H. Bae, Cibele S. Borges, Josiane L. Rosa, Marilia M. Cavariani, Patrícia V. Silva, Patricia F. F. Pinheiro, Janete A. Anselmo-Franci and Arielle C. Arena

Fipronil, an insecticide widely used in agriculture, veterinary medicine and public health, has been listed as a potential endocrine disrupter. The present study showed that fipronil can affect the sperm motility and compromise sperm quality of the animals. The perinatal exposure to fipronil has long-term effects on sperm parameters, and the epididymis can be a target organ.

Progesterone is a steroid present in the female reproductive tract that aids sperm motility. In the present study we identified human sperm proteins that are modified in response to progesterone and propose a mechanism by which progesterone would aid motility. This information may be useful for the development of drugs to improve sperm motility and contraceptives.

Published online 11 May 2016

RD15432Prominin-1 glycosylation changes throughout early pregnancy in uterine epithelial cells under the influence of maternal ovarian hormones

Samson N. Dowland, Romanthi J. Madawala, Connie E. Poon, Laura A. Lindsay and Christopher R. Murphy

The prominin-1 protein has previously been broadly investigated for its potential role in cancer and stem cell biology. This is the first investigation into the role of prominin-1 in early pregnancy. The study localises prominin-1 to the apical region of uterine epithelial cells and shows that maternal ovarian hormones significantly affect the glycosylation and abundance of this protein.

Published online 11 May 2016

RD15290Anti-Müllerian hormone reduces growth rate without altering follicular survival in isolated caprine preantral follicles cultured in vitro

R. M. P. Rocha, L. F. Lima, I. R. Brito, G. M. Silva, H. H. V. Correia, N. A. Ribeiro de Sá, A. C. A. Ferreira, A. D. Sales, C. H. Lobo, C. C. Campello, J. Smitz, M. B. Wheeler and J. R. Figueiredo

Investigations into the relationship between FSH and anti-Müllerian hormone (AMH) during in vitro culture of isolated preantral follicles are lacking. The aim of the present study was to evaluate the effect of AMH, with and without FSH, on the in vitro development of isolated caprine follicles. We demonstrated that AMH reduces follicular growth without altering follicular survival. Thus, AMH may play an important role in regulating the growth of caprine preantral follicles.

A unique approach to managing coyote (Canis latrans) depredation is the development of a one-time deliverable chemical sterilant. We hypothesised that exposure to a high dose of a gonadotrophin-releasing hormone (GnRH) agonist would cause prolonged suppression of the reproductive axis. The results of the present study suggest that a plasma concentration of 100 pg mL–1 deslorelin must be maintained and that elevations beyond this threshold are likely to be unimportant.

Sperm freezing has advanced animal breeding and genetics in numerous species but is not successful in alpacas due to the gel-like nature of the semen. This research aimed to reduce the viscosity of alpaca semen without affecting sperm function. The enzyme papain (0.1 mg mL–1 for 20 min at 37°C) followed by the papain inhibitor E-64 (10 µm for 5min at 37°C) eliminated viscosity and aided cryopreservation of alpaca spermatozoa. These findings will help to advance the use of reproductive technologies in camelids.

Preventing spontaneous preterm birth, which is the leading cause of perinatal mortality and morbidity, is one of the most important issues facing perinatal medicine today. In this study, we found that spontaneous labour is associated with an increase in optineurin (OPTN) expression in fetal membranes and the myometrium, where it functions as a negative regulator of prolabour mediators. Elucidating the role of OPTN in labour processes enhances our understanding of the mechanisms that drive preterm birth and could perhaps lead to the development of a clinical therapeutic strategy.

Published online 28 April 2016

RD15481Proteome of equine oviducal fluid: effects of ovulation and pregnancy

Katrien Smits, Hilde Nelis, Katleen Van Steendam, Jan Govaere, Kim Roels, Cyrillus Ververs, Bart Leemans, Eline Wydooghe, Dieter Deforce and Ann Van Soom

Early development of the equine embryo occurs in the oviduct, and information on the composition of this oviducal environment is very valuable. We identified the proteins in the oviducal fluid of the horse, and proteins that are important for pregnancy were found in higher amounts when an equine embryo was present. These findings provide a solid basis for further studies on embryo–maternal interactions in the horse.

Published online 26 April 2016

RD15474Sperm function during incubation with oestrus oviductal fluid differs in bulls with different fertility

A. Kumaresan, A. Johannisson and A.-S. Bergqvist

Prediction of breeding bull fertility is of major economic value to the cattle-breeding industry. We proved that spermatozoa from high-fertile bulls showed a low degree of tyrosine phosphorylation and high degree of capacitation and acrosome reaction upon exposure to oviductal fluid. These findings may help in developing tools for assessing fertility of bulls, once validated in more animals.

Because carnitine treatment is used in many cases of male infertility, the aim of the present study was to evaluate the mechanism by which carnitine improves spermatogenesis in mice. In the study, carnitine treatment of mice stimulated peroxisome proliferator-activated receptor-γ coactivator-1α expression, which, in turn, regulated steroidogenesis by enhancing expression of Bcl2 and antioxidant enzymes in the testis.

Published online 06 April 2016

RD15485Short- and long-term outcomes of the absence of protein during bovine blastocyst formation in vitro

A. Murillo-Ríos, V. Maillo, M. Muñoz, A. Gutiérrez-Adán, S. Carrocera, D. Martín-González, A. Fernandez-Buznego and E. Gómez

Removal of protein from culture medium improves analytical reliability, although bovine embryo development in vitro could be affected. However, protein removal over a 24-h period in culture led to embryos with reduced miscarriage rates without affecting birthweight, gestation length or calf morphology. This finding represents direct access to culture medium as a substrate for non-invasive analysis, leading to potential biomarker discovery without compromising embryonic viability.

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