Sex is an essential feature for sexual reproduction. Teleost fishes are acquired remarkably diverse sexual patterns (e.g., sex change, hermaphroditism) compared to other vertebrate lineages. However, little is known about how sex changes and intersex traits emerge. This study focused on a marine teleost fish, harlequin sandsmelt Parapercis pulchella. This fish changes sex from female to male after sexual maturation. Also, females possess intersex gonads, called ovotestes, that consist of both ovarian and testicular tissues. Because P. pulchella possesses two interesting features (i.e., sex change and intersex trait), this fish could become an excellent model for studying sexuality in teleost fishes. Nevertheless, the detailed gonadal features and dynamics remain unclear. Therefore, this study aims to uncover the sex change process and ovotestis structure through histological observations and single-cell RNA sequencing.
Histological observations demonstrated that female ovotestes comprised many oocytes and a few cysts of spermatogenic cells. Although early stages of spermatogenic cells (i.e., spermatogonia and spermatocytes) were present, spermatids and sperms were not observed in female ovotestes. In addition, immunohistochemistry using an antibody against Proliferating cell nuclear antigen (Pcna) as a cell proliferation marker revealed that spermatocytes were Pcna negative, suggesting that spermatogenesis is arrested at the spermatocyte stage in female ovotestes.
At the initial phase of sex change, male germ cells increased, and spermatogenesis proceeded. Farther on, oocytes decreased and finally disappeared. The gonads became functional male testes after the sex change. During the sex change, oocytes degenerated through apoptotic cell death (active cleaved-Caspase3a positive by immunohistochemistry). In comparison, some female follicle cells proliferated (Pcna positive), suggesting that such cells were reused in the testes after the sex change.
However, the histological observations alone did not fully identify cell types and functions in female ovotestes. Therefore, single-cell RNA sequencing of female and male gonads was conducted. Because genomic data of this fish is not available, reference transcriptome sequences were assembled and curated. Then, single-cell analyses were performed by mapping to the reference. Based on these analyses, we successfully identified various cell populations, e.g., germ, supporting, and hormone-producing cells, which were essential for spermatogenesis and oogenesis. Such cell atlas and expression data could become valuable resources for understanding ovotestis formation in the focal species and fishes.