Shuttle Transfer of mRNA Transcripts via Extracellular Vesicles From Male Reproductive Tract Cells to the Cumulus–Oocyte Complex in Rabbits (Oryctolagus cuniculus)

Abstract

Semen is known to contain an ovulation-inducing factor (identified as a nerve growth factor, NGF) that shows a significant increase in ovulation after semen deposition in induced ovulatory species. However, the interplay between the male reproductive tract cells and oocyte maturation through messenger RNA (mRNA) cargo is yet to be investigated. Extracellular vesicles (EVs) from the primary culture of rabbit prostate (pEVs), epididymis (eEVs), and testis (tEVs) were isolated to examine their contents for several mRNA transcripts through relative quantitative PCR (RT-qPCR). The expressions of NGF, neurotrophin (NTF3), vascular endothelial growth factor A (VEGFA), A disintegrin and metalloprotease 17 (ADAM17), midkine (MDK), kisspeptin (KISS1), and gonadotrophin-releasing hormone (GNRH1) were examined in isolated EVs. EVs were characterized through transmission electron microscopy. EV uptake by cumulus cell culture was confirmed through microscopic detection of PKH26-stained EVs. Furthermore, the effects of pEVs, eEVs, and tEVs were compared with NGF (10, 20, and 30 ng/ml) supplementation on oocyte in vitro maturation (IVM) and transcript expression. KISS1, NTF3, MDK, ADAM17, GAPDH, and ACTB 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 ng/ml NGF showed the highest grade of cumulus expansion, followed by tEVs and 10 ng/ml NGF. Control groups and 30 ng/ml NGF showed the least grade of cumulus expansion. Similarly, first polar body (PB) extrusion was significantly increased in oocytes matured with eEVs, pEVs, tEVs, NGF20 (20 ng/ml NGF), NGF10 (10 ng/ml NGF), control, and NGF30 (30 ng/ml NGF). Additionally, the expression of NGFR showed a 1.5-fold increase in cumulus cells supplemented with eEVs compared with the control group, while the expression of PTGS2 (COX2) and NTRK showed 3-fold and 5-fold increase in NGF20-supplemented cumulus-oocyte complexes (COCs), respectively. Oocyte PMP15 expression showed a 1.8-fold increase in IVM medium supplemented with eEVs. Additionally, oocyte NGFR and NTRK expressions were drastically increased in IVM medium supplemented with pEVS (3.2- and 1.6-fold, respectively) and tEVs (4- and 1.7-fold, respectively). This is the first report to examine the presence of mRNA cargo in the EVs of male rabbit reproductive tract cells that provides a model for the stimulation of female rabbits after semen deposition.

Physiological and Functional Roles of Neurotrophin-4 During In Vitro Maturation of Porcine Cumulus–Oocyte Complexes

“…It has been demonstrated that NGF, BDNF, and GDNF enhance oocyte maturation and early embryonic development ( Linher-Melville and Li, 2013 ). NGF plays an essential role in the female and male reproductive systems, and is known to promote ovulation while present in high concentrations in the semen of most species ( Maranesi et al, 2021 ; Abumaghaid et al, 2022 ). Previous studies have shown that BDNF/NT-4-TrkB signaling is required for follicle assembly and growth, as blockade of both BDNF and NT-4 impairs follicular assembly and increases oocyte death ( Ojeda et al, 2000 ; Spears et al, 2003 ).…”

Seminal extracellular vesicles subsets modulate gene expression in cumulus cells of porcine in vitro matured oocytes

“…The functional effect of SP-EVs on the female gamete, nevertheless, has been poorly investigated. Only a study conducted in rabbits reported that EVs isolated from prostate, epididymis and testis primary cultures were able to interact with female gametes during IVM 49 . As the biological activity of EVs relies on the ability of target cells to bind and integrate them 57 , the first experiment aimed to determine the uptake of the two SP-EVs subsets by COCs during IVM.…”

“…Results demonstrated that S-EVs and L-EVs isolated from porcine SP were able to bind CCs, but not oocytes, during IVM. Similarly, Abumaghaid et al (2022) examined the uptake of EV isolated from rabbit epididymal primary cultures by COCs, showing that EVs were able to bind CCs 49 . Several reports described the ability of EVs isolated from other reproductive biofluids, such as the follicular (Porcine 58 , 59 ; Equine 60 ; Bovine 61 ) and oviductal ones (Canine 62 ), to bind COCs.…”

“…Yet, because no macroscopic differences in CCs expansion between the different experimental groups were detected in this study (data not shown), further research to investigate the effect of SP-EVs on CCs expansion is needed. This is of particular relevance if one takes into consideration that the EVs isolated from the primary culture medium of testis, epididymis and prostate enlarge the cumulus in rabbits 49 , and that exosome-like vesicles from follicular fluid also induce the expansion of CCs in porcine 58 . As most of the genes evaluated in this study did not appear to be influenced by SP-EVs, it could be that the EVs isolated from other reproductive biofluids have greater influence on the functional activity of CCs.…”

“…Given the essential role played by both SP and EVs on gamete–female reproductive tract crosstalk 5 , 48 , addressing the role of SP-EVs in this dialog seems imperative. To date, only an in vitro study conducted in rabbit has demonstrated that EVs isolated from cell cultures of testis, epididymis and prostate are able to modulate cumulus cells (CCs) function during in vitro maturation (IVM) 49 . Cumulus cells play an important role during nuclear and cytoplasm maturation of the oocyte, interacting with the female gamete through gap junctions and paracrine factors 50 , 51 .…”