Expression of genes associated with sperm-oocyte interaction in the stallion reproductive tract

Abstract

Sperm-oocyte fusion is an essential function during the fertilization process. Membrane fusion occurs as a result of a complex series of intermolecular interactions between the sperm and the oocyte. Proteins involved in this interaction include izumo sperm-egg fusion 1 (IZUMO1), sperm acrosome associated 6 (SPACA6), transmembrane protein 95 (TMEM95), fertilization influencing membrane protein (FIMP) and sperm-oocyte fusion required 1 (SOF1), ADAM metallopeptidase domain 2 (ADAM2), cysteine rich secretory protein 1-3 (CRISP1-3), cation channel sperm associated 1-4 (CATSPER1-4) and polycystin family receptor for egg jelly (PKDREJ) (Rivera and Swanson, 2022. Front. Cell Dev. Biol., 10:827-827454) on the male gamete, and izumo 1 receptor (JUNO) and cluster of differentiation 9 (CD9) on the female gamete (Inoue et al., 2021. eLife; 10:e66313). The IZUMO1-JUNO interaction is essential for gamete fusion and the block of polyspermy (Bianchi et al., 2014: Nature, 508:483-487). Other proteins called dendrocyte expressed seven transmembrane protein (DC-STAMP) and homologue domain-containing 1 and 2 (DCST1/2) are also required for sperm-egg fusion in mice (Inoue et al., 2021. eLife; 10:e66313). The objective of this study was to identify and quantify the expression of genes associated with sperm-oocyte interaction during the fertilization process in tissue samples from the testis, head, body and tail of the epididymis. Tissues were collected after castration from three light-breed reproductively normal stallions(5-13 years). mRNA was extracted and libraries were generated using Next Generation Sequencing. FPKM of the genes of interest was compared between epididymal head, body and tail, and testis (control tissue) using in silico analysis (online databases). Expression of CRISP3, SPACA6, ADAM2, CATSPER1, CATSPER2, CATSPER3, CATSPER4, GLPR1L1, IZUMO1, PKDREJ, TMEM95, DCST1 and DCST2 genes were identified in the testis and all segments of the epididymis of the horse. All these genes, except for CRISP1, were up-regulated in the testis compared with the three segments of the epididymis, while CRISP1 was down-regulated in the testis compared with the epididymis. In this study, JUNO gene was expressed in the testis (FPKM 48.15), and was downregulated in the head, body and tail of the epididymis (FPKM 2.3, 3.2 and 2.9, respectively). In summary, genes encoding for proteins associated with sperm-oocyte interaction were more strongly expressed within the testis, suggesting that these proteins are acquired during spermatogenesis or spermiogenesis. Meanwhile, CRISP1 may be acquired during sperm transport along the epididymis. The expression of JUNO gene within the stallion reproductive tract is striking since the expression has not been reported in mice (RPKM 0) and human testis (RPKM 0.003). JUNO is a receptor only found on the oocyte membrane. The specific cells that express these genes, and their function within the stallion reproductive tract needs further studies.