Progesterone Induces Porcine Sperm Release from Oviduct Glycans in a Proteasome-Dependent Manner

In mammals, the oviduct retains sperm forming a reservoir from which they are released in synchrony with ovulation. However, the mechanisms underlying sperm release are unclear. Herein, we first examined in greater detail the release of sperm from the oviduct reservoir by sex steroids, and second, if the ubiquitin-proteasome system (UPS) mediates this release in vitro. Sperm were allowed to bind to oviductal cells or immobilized oviduct glycans, either bi-SiaLN or a suLeX, and then challenged with steroids in the presence or absence of proteasome inhibitors. Previously, we have demonstrated that progesterone-induced sperm release from oviduct cells and immobilized glycans in a steroid-specific manner. Herein we found that release of sperm from an immobilized oviduct glycan, a 6-sialylated branched structure, and from immobilized fibronectin was inhibited by the CatSper blocker NNC 055-0396, akin to the previously reported ability of NNC 055-0396 to inhibit sperm release from another oviduct glycan, sulfated Lewis X trisaccharide. Thus, CatSper may be required for release from a variety of adhesion systems. One possible mechanism for sperm release is that glycan receptors on sperm are degraded by proteasomes or shed from the sperm surface by proteasomal degradation. Accordingly, the inhibition of proteasomal degradation blocked sperm release from oviduct cell aggregates and both immobilized oviduct glycans as well as fibronectin. In summary, progesterone-induced sperm release required both active CatSper channels and proteasomal degradation, suggesting that hyperactivation and proteolysis are vital parts of the mechanism by which sperm move from the oviduct reservoir to the site of fertilization.

Release of Porcine Sperm from Oviduct Cells is Stimulated by Progesterone and Requires CatSper

Sperm storage in the female reproductive tract after mating and before ovulation is a reproductive strategy used by many species. When insemination and ovulation are poorly synchronized, the formation and maintenance of a functional sperm reservoir improves the possibility of fertilization. In mammals, the oviduct regulates sperm functions, such as Ca2+ influx and processes associated with sperm maturation, collectively known as capacitation. A fraction of the stored sperm is released by unknown mechanisms and moves to the site of fertilization. There is an empirical association between the hormonal milieu in the oviduct and sperm detachment; therefore, we tested directly the ability of progesterone to induce sperm release from oviduct cell aggregates. Sperm were allowed to bind to oviduct cells or an immobilized oviduct glycan and then challenged with progesterone, which stimulated the release of 48% of sperm from oviduct cells or 68% of sperm from an immobilized oviduct glycan. The effect of progesterone on sperm release was specific; pregnenolone and 17α-OH-progesterone did not affect sperm release. Ca2+ influx into sperm is associated with capacitation and development of hyperactivated motility. Progesterone increased sperm intracellular Ca2+, which was abrogated by blocking the sperm–specific Ca2+ channel CatSper with NNC 055-0396. NNC 055-0396 also blocked the progesterone-induced sperm release from oviduct cells or immobilized glycan. An inhibitor of the non-genomic progesterone receptor that activates CatSper similarly blocked sperm release. This is the first report indicating that release of sperm from the sperm reservoir is induced by progesterone action through CatSper channels.

166 Paracrine Regulation of Epidermal Growth Factor-Like Factors in Oviduct Cells and its Effect on Oocyte Maturation and Subsequent Embryo Development

Progesterone (P4) and progesterone receptor signalling appears essential for maintenance of a proper cumulus cell expansion during the oocyte maturation by regulating the epidermal growth factor-like factors (EGF-F) related pathway during the ovulatory process. It is known that expression of EGF-F including amphiregulin (AREG), epiregulin (EREG), and betacellulin (BTC) is critical for cumulus–oocyte complex (COC) expansion and resumption of meiosis. Therefore, we hypothesised that oviduct cells might be involved in nonexclusive mechanisms of actions of P4 that in turn modulate oocyte meiosis resumption by regulating the levels of EGF-F. First, we added different concentrations of P4 (0, 0.5, 1, and 2 μg mL−1) to oviduct cell culture medium and assessed the effect of P4 on expression of AREG, EREG, and BTC in oviduct cells by immunocytochemical analysis. Then, the oviduct cells were used for co-culturing under the proper concentration of P4 with porcine oocytes. The COC were randomly cultured in 3 groups: (1) culturing without oviduct cells, (2) co-culturing with oviduct cells, and (3) co-culturing with oviduct cells treated with P4. After IVM, extrusion of the 1st polar body was observed under the microscope. To evaluate embryo development competence, the matured oocytes were activated with electrical stimulus and parthenotes were cultured in porcine zygote medium-5 for 7 days at 39°C, 5% CO2 and O2 in a humidified atmosphere. The cleavage and blastocyst formation rates were observed under the microscope to evaluate developmental competence. To count the total cell number of blastocysts, they were stained with 5 μg mL−1 of Hoechst 33342 for 10 min. The data were analysed by one-way ANOVA using GraphPad Prism 5.0 (GraphPad Inc., San Diego, CA, USA). Values are means ± standard error of mean (P < 0.05). Significantly higher levels of EGF-F were observed in oviduct cells treated with 1 μg mL−1 progesterone. The oocyte maturation rate of co-culture group treated with P4 (80.7 ± 1.6%) was significantly higher than that of the control (69.7 ± 2.1%). There was a significant difference between co-culture treated with P4 and the control in cleavage rate (67.2 ± 2.4% and 82.0 ± 1.6%). However, no significant difference was observed between the co-culture groups. The co-culture treated with P4 group showed significantly higher rate of blastocyst formation (37.7 ± 0.8%) and total cell number of blastocyst (72.8 ± 1.0) than control and co-culture groups. In conclusion, co-culturing with oviduct cell treated with P4 improved oocyte maturation and subsequent embryo development. Thus, we suggested that oviduct cells induce the expression of EGF-F under the treatment of P4, which has a beneficial effect on porcine oocyte development. This research was supported by NRF-20142A1021187, Korea IPET (#316002-05-2-SB010), RDA (#PJ010928032017) and Research Institute for Veterinary Science, the BK21 plus program.

211 TRANSCRIPTS EXPRESSION RELATED TO MAPK/SMAD2 PATHWAY IN OVIDUCT CELL, CUMULUS CELL, AND OOCYTE DERIVED FROM DIESTRUS BITCHES

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are known to play an essential role in regulating ovarian follicular development and ovulation rate via SMAD pathway in several species and have different functions depending on the species. However, their expression and function have not yet been reported in dogs. Therefore, the aim of present study is to analyse expressions of transcripts related to the MAPK/SMAD2 pathway in oviduct cell, cumulus cell, and oocyte derived from diestrus bitches. Ovariohysterectomy was performed in 5 large, mixed breed dogs 3 days after serum progesterone reached 5–10 ng mL–1. Oviduct cells were obtained by scraping the superficial oviduct tissue and oocytes were obtained by mincing the ovary. Both cumulus cells derived from non-ovulated oocytes (IM-cumulus) and ovulated oocytes (M-cumulus) were obtained. Oviduct, IM-cumulus, and M-cumulus cells were cultured using RCMEP media and cryopreserved using FBS containing 10% DMSO. Total RNA was extracted from Oviduct, IM-cumulus, and M-cumulus cells and oocytes. After cDNA synthesis, transcript expression of β-actin, GDF9, MAPK1, BMP6, and SMAD2 genes were analysed. The data was analysed by one-way ANOVA using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA). The threshold for statistical significance was set at P < 0.05. In results, mRNA expression of MAPK1 was not significantly different among the cells. However, expressions of BMP6, GDF9, and SMAD2 were significantly higher in oocytes compared to oviduct, IM-cumulus, and M-cumulus cells. Although oviduct cells showed increased GDF9 and SMAD2 transcripts similar to that of oocytes, there was no significant difference between them. In conclusion, MAPK/SMAD2 pathway may have important roles in growth of immature oocytes in dogs and oviduct cells may help the maturation of ovulated immature oocytes. Further studies will be conducted to analyse MAPK/SMAD2 pathway in anestrus, proestrus, and oestrus periods.This research was supported by RDA (#PJ008975022014), IPET (#311062-04-3SB010), Research Institute for Veterinary Science, the BK21 plus program, and global PhD Fellowship Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2014H1A2A1021187).