First insight into the regulatory role of the glycoprotein hormones GPA2 and GPB5 in the small-spotted catshark (Scyliorhinus canicula L.) during spermiogenesis.

Glycoprotein hormones alpha 2 (GPA2) and beta 5 (GPB5) are considered to be ancestral members of the glycoprotein hormone (GPH) family. Despite their potential roles in regulating thyroxine production, the immune response, and ovarian function, their roles in the testis remains largely unknown. To further explore this regulation, catshark testicular explants containing late spermatid stages were treated in vitro with recombinant GPA2, GPB5 and single-chain GPB5#GPA2. The comparative proteomic analysis of the treated explants revealed that ScGPB5 and ScGPB5#ScGPA2 were the most effective, modulating abundance of 449 and 525 proteins, respectively. In contrast, ScGPA2 only modulated 212 proteins. Among the differentially abundant proteins (DAPs), 103 showed substantial abundance changes in response to at least one treatment, with mean Log₂ratios of 2.5 and - 2.5. Manual functional annotation linked these proteins to spermiogenesis and immunity. Key proteins and candidates involved in spermiogenesis were highlighted, including SPINK2, which is involved in acrosome protection, and LRRC69, which is related to human spermatogenic failure. These results reinforce the hypothesis of a GPA2/GPB5 paracrine regulation of catshark spermiogenesis. They also underscore the necessity of functional and comparative studies to elucidate the physiological functions of GPA2/GPB5 across a broad spectrum of taxa, from non-vertebrates to mammals. SIGNIFICANCE: To our knowledge, this study is the first to report the effects of stimulation with recombinant ScGPA2, ScGPB5, and single-chain ScGPB5#ScGPA2 on vertebrate testicular tissue in vitro. A large-scale, untargeted proteomic analysis revealed for the first time that all three-ScGPA2, ScGPB5, and single-chain ScGPB5#ScGPA2-can modulate the abundance of 103 of the 7619 identified proteins, including proteins related to spermiogenesis and immunity, as well as others that are less well characterized. This report reinforces the importance of functionally characterizing GPA2 and GPB5 by showing the first results toward the characterization of their spermatogenesis related function in a specie of evolutive interest, the small-spotted catshark. In the future, a complete characterization of GPA2 and GPB5 will further our understanding of the functional evolution of glycoprotein hormones-a keystone group of hormones for vertebrate physiology and critical targets for fertility modulation in both livestock and humans.