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.

Keywords: epididymis; extracellular vesicles; oocyte; prostate; rabbit; testis.

Figure 1

Primary culture of prostate, testis, and epididymis tissues. Asterisk indicates the tissue explants. Outgrowths and cell monolayers were maintained until the 14th day of culture. Scale bar, 500 μm.

Figure 2

Characterization of extracellular vesicles (EVs) from the rabbit prostate (A), testis (B), and epididymis (C). Upper panel shows the TEM images. Scale bars, 100 nm for (A,B) and 200 nm for (C). Lower panel shows the results of nanoparticle tracking analysis (size and concentrations) of the EVs in the corresponding tissue of origin: prostate (A), testis (B), and epididymis (C).

Figure 3

Different degrees of cumulus expansion (A–G) and oocyte maturation (H–O) after in vitro maturation with or without extracellular vesicles (EV) supplementation. (A) Control with no expansion. (B) Prostate-derived EVs (pEVs) with transparency of intercellular spaces between the cumulus cells (grade 4). (C) Testis-derived EVs (tEVs). (D) Epididymis-derived EVs (eEVs). (E–G) Supplementation with 10 ng/ml NGF (E), 20 ng/ml NGF (F), and 30 ng/ml NGF (G). Panels (H–O) show the state of oocyte maturation of the corresponding groups of (A–G). Blue arrows indicate the extrusion of the first polar body. White arrows indicate spontaneously cleaved oocytes. Scale bar, 100 μm.

Figure 4

Comparison between the values of the different degrees of cumulus expansion in experimental groups. Different letters (a–c) indicate a significant difference at p < 0.05.

Figure 5

Comparison between the first polar body extrusion values in the oocytes from different experimental groups. Different letters indicate a significant difference at p < 0.05.

Figure 6

Uptake of PKH26-labeled extracellular vesicles (EVs) by the cultured cumulus cells. (A,B) Control cells cultured in plain culture medium. (C,D) PKH26 dye was applied to the EVs and kept in the culture medium for 22 h. (A,C) Nuclear staining by DAPI. (B,D) Localization of red fluorescence staining of EVs (arrows). Scar bar, 10 μm.

Figure 7

Effects of the prostate, testis, and epididymis extracellular vesicles (EVs) on the gene expression of cumulus cells in comparison to different doses of nerve growth factor (NGF). Different letters (a–c) indicate significant difference at p < 0.05.

Figure 8

Effects of the prostate, testis, and epididymis extracellular vesicles (EVs) on oocyte gene expression compared to different doses of nerve growth factor (NGF). Different letters (a–c) indicate significant difference at p < 0.05.