AUTHOR=Abumaghaid Mosleh M. , Abdelazim Aaser M. , Belali Tareg M. , Alhujaily Muhanad , Saadeldin Islam M. 

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

JOURNAL=Frontiers in Veterinary Science

VOLUME=9

YEAR=2022

URL=https://www.frontiersin.org/journals/veterinary-science/articles/10.3389/fvets.2022.816080

DOI=10.3389/fvets.2022.816080

ISSN=2297-1769

ABSTRACT=<p>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 <italic>NGF</italic>, neurotrophin (<italic>NTF3</italic>), vascular endothelial growth factor A (<italic>VEGFA</italic>), A disintegrin and metalloprotease 17 (<italic>ADAM17</italic>), midkine (<italic>MDK</italic>), kisspeptin (<italic>KISS1</italic>), and gonadotrophin-releasing hormone (<italic>GNRH1</italic>) 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 <italic>in vitro</italic> maturation (IVM) and transcript expression. <italic>KISS1, NTF3, MDK, ADAM17, GAPDH</italic>, and <italic>ACTB</italic> were detected in all EV types. <italic>GNRH1</italic> was detected in tEVs. <italic>NGF</italic> was detected in pEVs, whereas <italic>VEGFA</italic> 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 <italic>NGFR</italic> showed a 1.5-fold increase in cumulus cells supplemented with eEVs compared with the control group, while the expression of <italic>PTGS2</italic> (<italic>COX2</italic>) and NTRK showed 3-fold and 5-fold increase in NGF20-supplemented cumulus-oocyte complexes (COCs), respectively. Oocyte <italic>PMP15</italic> expression showed a 1.8-fold increase in IVM medium supplemented with eEVs. Additionally, oocyte <italic>NGFR</italic> and <italic>NTRK</italic> 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.</p>