FOXL2 and DMRT1L Are Yin and Yang Genes for Determining Timing of Sex Differentiation in the Bivalve Mollusk Patinopecten yessoensis

Abstract

Sex determination and differentiation have long been a research hotspot in metazoans. However, little is known about when and how sex differentiation occurs in most mollusks. In this study, we conducted a combined morphological and molecular study on sex differentiation in the Yesso scallop Patinopecten yessoensis. Histological examination on gonads from 5- to 13-month-old juveniles revealed that the morphological sex differentiation occurred at 10 months of age. To determine the onset of molecular sex differentiation, molecular markers were screened for early identification of sex. The gonadal expression profiles of eight candidate genes for sex determination or differentiation showed that only two genes displayed sexually dimorphic expression, with FOXL2 being abundant in ovaries and DMRT1L in testes. In situ hybridization revealed that both of them were detected in germ cells and follicle cells. We therefore developed LOG10(DMRT1L/FOXL2) for scallop sex identification and confirmed its feasibility in differentiated individuals. By tracing its changes in 5- to 13-month-old juveniles, molecular sex differentiation time was determined: some scallops differentiate early in September when they are 7 months old, and some do late in December when they are 10 months old. Two kinds of coexpression patterns were found between FOXL2 and DMRT1L: expected antagonism after differentiation and unexpected coordination before differentiation. Our results revealed that scallop sex differentiation co-occurs with the formation of follicles, and molecular sex differentiation is established prior to morphological sex differentiation. Our study will assist in a better understanding of the molecular mechanism underlying bivalve sex differentiation.

Phenotypic Stability of Sex and Expression of Sex Identification Markers in the Adult Yesso Scallop Mizuhopecten yessoensis throughout the Reproductive Cycle

“…To describe the process of gonad sex differentiation in the Yesso scallop, molecular markers are crucial for understanding the molecular basis of gonad development, as proposed [23]. In the present study, we aimed to characterize dmrt and foxl2 as markers of testis or ovary differentiation, respectively.…”

“…In addition, sexually dimorphic expression of foxl2 has been reported in bivalves, namely, Chlamys farreri [21] and pearl oyster ( Pinctada margaritifera ) [22]. In the Yesso scallop, Li et al [23] recently developed a method for scallop sex identification during the sex-differentiating phase by using LOG 10 ( DMRT1L / FOXL2 ). Therefore, mechanistically, the involvement of Dmrt and Foxl2 could be critical for testis and ovary differentiation, respectively, after the completion of sex differentiation in the Yesso scallop.…”

Systematic identification and validation of the reference genes from 60 RNA-Seq libraries in the scallop Mizuhopecten yessoensis

“…Examining their expression dynamics during gonadal development is an important task in these studies. However, several traditionally used reference genes, such as EF1A [36,40,61], ACT [62] and GAPDH [63] have been applied, and until now, no study on the transcriptome-wide identification of reference genes in bivalves has been reported. According to our results from RNA-Seq analysis and RT-qPCR validation, ACT is not an appropriate reference gene and may result in misleading conclusions.…”

“…After acclimation, the following tissues were dissected: digestive gland, eyes, foot, gill, gonad, hemocytes, mantle, striated muscle, smooth muscle and nerve ganglia. The ovaries and testes of mollusks at four developmental stages (resting stage, proliferative stage, growing stage and maturation stage) were collected as described in Li et al [40]. Three biological replicates were collected for each tissue, frozen in liquid nitrogen, and then stored at − 80°C prior to RNA isolation.…”

Gonad transcriptome of golden musselLimnoperna fortuneireveals potential sex differentiation genes

“…However, identification and expression of genes related to these processes have been studied in some bivalves, including the vertebrate female-determining gene forkhead box L2 (FoxL2) (19,20,14,21), the male-determining genes double-sex-and mab-3-related transcription factor (Dmrt) (18,20,21), and Sox (Sryrelated HMG box) family genes (11,12,15,16,22,23). FoxL2 and Dmrt are the key genes for sex determination and maintenance, respectively, and their antagonic role in the control of gonadal sex has been characterized in mammals and has been suggested to play similar antagonic roles in bivalve species (12,24). Some of those aforementioned genes usually present sex-biased expression in bivalve gonads, such as SoxH/Sox30 (11,12,22) and Dmrt-like (14,15,23) are male-based and FoxL2 is female-biased (11,14,16,(20)(21)(22)(23).…”