112 Shuttle transfer of mRNA transcripts via extracellular vesicles from male cells to the cumulus-oocyte complex in the rabbit

Semen is known to contain an ovulation-inducing factor (identified as a nerve growth factor, NGF) that showed a significant increase in the ovulation after semen deposition in induced-ovulatory species. However, are there any other signals, particularly the mRNA transcript cargo, that can be transferred from males and impact females? We isolated extracellular vesicles (EVs) from the primary culture of rabbit prostate (pEVs), epididymis (eEVs), and testis (tEVs) through a commercial kit (PureExo, 101Bio), and examined their contents for several mRNA transcripts through real-time PCR. Transcripts of nerve growth factor (NGF), neurotrophin (NTF3), vascular endothelial growth factor (VEGFA), A disintegrin and metalloprotease 17 (ADAM17), midkine (MDK), kisspeptin (KISS1), and gonadotrophin-releasing hormone (GnRH1), in addition to the housekeeping genes GAPDH and ACTB, were examined in the isolated EVs. EVs were characterised through transmission electron microscopy and positive immunostaining for CD9. EV uptake by cumulus cell culture was confirmed through microscopic detection of PKH26-stained EVs. Furthermore, effects of pEVs, eEVs, and tEVs were compared with NGF (10, 20 and 30 µM) supplementation on oocyte IVM and transcript expression (ADAM17 and PTGS2 in cumulus cells; NGFR, NTRK, GDF9 and BMP15 in oocytes). Each experiment was repeated five times. Data were expressed as mean ± s.e.m. and compared using one-way ANOVA, with P < 0.05 considered significant. KISS1, NTF3, MDK, and ADAM17, as well as GAPDH and ACTB transcripts, were detected in all EV types. GnRH1 was detected in tEVs. NGF was detected in pEVs, whereas VEGFA was detected in eEVs. pEVs, eEVs, and 20 µM NGF showed the highest grade of cumulus expansion, followed by tEVs and 10 µM NGF. Control groups and 30 µM NGF showed the least grade of cumulus expansion. Similarly, first PB extrusion was significantly increased in oocytes matured with eEVs (54%), pEVs (48%), tEVs (45%), NGF20 (35%), NGF10 (29%), control (23%), and NGF30 (15%; P < 0.05). Additionally, ADAM17 expression showed a 1.5-fold increase in cumulus cells supplemented with tEVs compared with the control group, while PTGS2 (COX2) expression showed a 1.6-fold increase in NGF20-supplemented COCs. Oocyte PMP15 expression showed a 1.8-fold increase in IVM supplemented with eEVs, while GDF9 showed a 1.6-fold increase in IVM supplemented with pEVs. Additionally, oocyte NGFR and NTRK expression were drastically increased in IVM supplemented with pEVS (7.8- and 4.6-fold, respectively) and tEVs (2- and 1.7-fold, respectively). These results suggest the presence of mRNA cargo in the EVs of male reproductive tract cells that provide a novel paradigm for the stimulation of female rabbits after semen deposition. The act of ovulation induction may depend on some neuro-stimulants, such as prostate NGF; other signals, such as KISS1, MDK, and NTF3; and testicular GnRH1.

This research was supported by University of Bisha ISP#48.