The pituitary glycoprotein hormones (GPHs) control several physiological processes in vertebrates such as reproduction and metabolism. They include the luteinizing hormone (LH), the follicle-stimulating hormone (FSH), and the thyroid-stimulating hormone (TSH), which activate their cognate leucine-rich repeat G protein-coupled receptors (LGRs), LHR, FSHR, and TSHR. Each GPH consists of a common α subunit and a specific βFSH, βLH or βTSH subunit. More recently, two supplementary GPH proteins, GPA and GPB, were identified in nearly all bilaterians and are the ancestors of the pituitary GPH α- and β-subunits, respectively. Chondrichthyans (holocephalans and elasmobranchs), the sister group of bony vertebrates, are the most ancient clade to possess diversified GPH subunits. In the present study, GPA2, GPB5, TSHβ2, but not TSHβ1, and TSHR sequences have been identified in several elasmobranch genomes, and their 3D models were analyzed. Functional hormone-receptor interactions were studied in the small-spotted catshark (Scyliorhinus canicula) and showed that conditioned media from cells expressing the recombinant single-chain ScGPB5-ScGPA2 were more effective than independent subunits in activating ScTSHR, ScFSHR, and ScLHR. Expression profiles were analyzed by real-time PCR, in situ hybridization, and immunohistochemistry along the male genital tract, other male and female tissues, and female tissues. A broader tissue distribution expression was observed for tshr and gpa2 than for gpb5, which was mainly observed in the testes. In testis, expression of tshr and gpb5 by Sertoli cells and of gpa2 by germ cells suggested paracrine/autocrine functions of GPA2/GPB5/GPHR signaling during spermatogenesis. This study complements the data on GPA2 and GPB5 by studying a chondrichthyan of phylogenetic interest for understanding the evolution of endocrine regulation in vertebrates.