Groupers (reef fishes of the tribe Epinephelini, subfamily Epinephelinae) are protogynous hermaphrodites: functional females transform into males. A 2021 comparative study of 18 grouper species showed that the ovarian tunica of groupers commonly contains testicular-inducing steroidogenic (TIS) cells — 11-ketotestosterone-producing cells that trigger female-to-male sex change — whereas the protogynous anthiines (Anthiinae, the most closely related subfamily examined) lack TIS cells. The cellular-endocrine account of TIS cells nevertheless remains incomplete: the hormonal pathway of sex change in TIS-lacking anthiines is undetermined; whether Leydig cells degenerated in some grouper lineages and whether TIS cells are precursors of Leydig cells are untested hypotheses; the involvement of TIS and other steroidogenic cells in socially controlled sex change, recently demonstrated in groupers, has not been investigated; and the molecular mechanisms governing TIS cell differentiation, and TIS functions beyond sex change, are unknown.

Background

About 2% of teleost (bony) fish species are sequential hermaphrodites that change sex during life; in protogyny, functional females become males. Groupers — large reef fishes of the tribe Epinephelini in the subfamily Epinephelinae (family Serranidae) — are mostly protogynous and are phylogenetically adjacent to the anthiines (Anthiinae, e.g., Pseudanthias species), which are also protogynous. In vertebrates, androgens are produced in the testis by Leydig cells, interstitial steroidogenic cells located inside the gonad. In the honeycomb grouper (Epinephelus merra), work in the 2000s identified 11-ketotestosterone (11KT), the main teleost androgen, as the key trigger of sex change: 11KT treatment induces female-to-male sex change, and immunohistochemistry against steroidogenic enzymes (Cyp11a, cholesterol side-chain cleavage; Cyp11b, cytochrome P450 11β-hydroxylase, a steroidogenic enzyme involved in 11KT synthesis) localized androgen production to previously unknown cells in the tunica (outer connective sheath) of the ovary — the cells later named TIS cells — while Leydig-like interstitial cells appear separately in the gonadal interior as the testis forms, their lineage relationship to the tunica cells remaining unresolved. Pituitary follicle-stimulating hormone (FSH) was implicated upstream, since FSH beta-subunit expression rises at the onset of sex change. In wrasses — another protogynous lineage, but one lacking such ovarian tunica steroidogenic cells — the initiating endocrine signal appears to be different: a decline in estradiol (E2), the estrogen produced by aromatase (Cyp19a1a) in the ovary, rather than an androgen surge. Murata and colleagues (2021) surveyed 7 genera and 18 grouper species and showed that these tunica androgen-producing cells, named testicular-inducing steroidogenic (TIS) cells, are present across nearly all major grouper clades, leading them to propose that TIS cells are an evolutionary acquisition that made autonomous sex change possible in groupers. Critically, two anthiine species examined (Pseudanthias pleurotaenia and Rabaulichthys suzukii) lacked TIS cells, while the nearest non-grouper relative examined (Diploprion bifasciatum, of the tribe Diploprionini within Epinephelinae) possessed them. Separately, socially controlled sex change — long known in wrasses, where removal of the dominant male triggers the largest female to change sex — was demonstrated in the orange-spotted grouper (Epinephelus coioides) in 2019–2020: social isolation induces adult males to reverse back to females, and removing the males from a female group induces the largest female to become a functional male within weeks. The 2021 paper's own discussion poses the questions that remain: whether anthiine sex change uses a different endocrine pathway (e.g., E2 decline); whether Leydig cells degenerated in some grouper lineages and whether TIS cells are Leydig-cell precursors (the paper notes that testing these hypotheses requires larger samples of male individuals across the grouper phylogeny); whether TIS and other steroidogenic cells participate in socially controlled sex change; and what molecular mechanisms drive TIS cell differentiation or what functions TIS cells have beyond triggering sex change.

Problem Statement

Under the common scientific objective of completing the cellular-endocrine account of TIS cells — the tunica-resident, 11-ketotestosterone-producing cells that trigger protogynous sex change in groupers — the following questions from Murata et al. 2021 remain open; each is independently answerable and can be addressed on its own, and together they would complete this account. (1) TIS-lacking lineages: by what endocrine pathway do protogynous anthiines, whose ovaries contain no TIS cells, initiate and execute sex change, and does this reflect a genuinely different evolutionary pathway of sex change from that of the Epinephelinae? (2) TIS–Leydig lineage relationship: have Leydig cells degenerated in some grouper lineages over evolution, and do TIS cells serve as precursors of Leydig cells during gonadal development? (3) Social control: are TIS cells and other steroidogenic cells involved in the socially controlled sex change recently demonstrated in groupers, and if so, what is their role relative to the brain–pituitary pathways now known to respond to social cues? (4) Differentiation and broader functions: which molecular mechanisms govern the differentiation of TIS cells in the ovarian tunica, and what functions, beyond triggering sex change, do TIS cells perform?

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