4.2 Candidate Genes Involved in Sexual Development
Although several sex-related genes have been studied in terms of sex determination and/or differentiation, little information about sex reversal/differentiation cascades is available for G. haimaensis.
Some female-biased genes were expressed more highly in M than F, including β-catenin, fem-1, mPRα and PLAC8. β-catenin, which transduces the canonical Wnt signalling pathway in mammals by promoting stability in the cytoplasm and nuclear entry, is critical in female ovary differentiation . In molluscs, β-catenin was expressed mostly in mature female gonads, which indicates a conserved role in the maintenance of ovaries [11, 39]. The ankyrin repeat protein Fem-1 is a component of the signal transduction pathway controlling sex determination . The fem-1 gene of P. margaritifera was expressed specifically in different reproductive stages (undetermined, sexual inversion, and regression), suggesting that it may be involved in the sperm-oocyte switch . mPRα was primarily localised on the oocyte plasma membrane, where it regulates induction of oocyte maturation by specifically binding progestins . Progestins exert rapid, multifaceted and nongenomic effects on sperm physiology through mPRα in a variety of vertebrate species [42–44], but few on invertebrates have been reported. In Octopus vulgaris, progesterone can also induce activation in spermatozoa via mPRα . However, our qPCR results showed that β-catenin, fem-1 and mPRα were expressed higher in M than F, consistent with the transcriptomic data. The qPCR results differed from the proteomic data, which may be because not all transcripts were translated, and mRNA abundance may not correspond to protein expression due to pre-translational, co-translational, or post-translational modification. Combined with the histological characteristics of gonads, these results implied that they may be involved in oogenesis in intersex gonads. A previous study on Bathymodiolus platifrons revealed low levels of genetic diversity differences between vent and seep populations . Our results also showed low levels of genetic diversity differences for G. haimaensis between vent and seep populations (data not shown, unpublished), which may reflect the survival strategy of sex change that could lead to self-fertilisation to expand the population.
Plac8 is a placental-enriched gene expressed in the spongiotrophoblast layer during mouse development . In mouse, it also plays an important role in spermatocyte differentiation during spermatogenesis . Plac8 has been well studied in vertebrates, yet little is known about its role in invertebrates. In the planarian Dugesia japonica, Plac8 plays essential roles in immune responses and development . In G. haimaensis, Plac8 was expressed higher in M than F, consistent with the transcriptomic data, implying that Plac8 may be involved in spermatocyte differentiation.
Conodipine-M is a novel phospholipase A2 enzyme isolated from the venom of the marine snail Conus magus . Conodipine is secreted by the poison gland in the venom tube and the inner wall of the poison sac. It can specifically act on various ion channels such as potassium, sodium, calcium, and various receptors on the cell membrane, to affect signal transmission in nerve and other cells . Our qPCR results revealed sexual dimorphism for expression of conodipine-M, with significantly higher levels in M than F, suggesting that it may participate in the maintenance of masculinisation. However, the specific role of conodipine-M in testis needs to be clarify in the future.
Some ovary-biased genes reported in other studies and were also identified in our current research. CYP450, EGFR, foxl2, MMP, MPI and vitellogenin were expressed at significantly higher levels in F than M, consistent with the transcriptomic data, and the qPCR results for CYP450 and EGFR were consistent with the proteomic data. In the mammalian female pathway, foxl2 functions by upregulating CYP19A, which encodes the aromatase that converts testosterone into oestrogens . However, CYP19A has been demonstrated to have arisen in the chordate lineage , hence it does not exist in bivalves. This finding suggests that despite the possible deep conservation of sex-determining genes among different clades, their regulatory network may have diverged during evolution. The forkhead box L2 gene (foxl2), which encodes a forkhead class transcription factor, is a key factor in the differentiation and maintenance of ovaries in vertebrates. And in molluscs, foxl2 also showed a sexually dimorphic pattern with higher expression levels in females [10, 13, 14, 17, 53], consistent with our qPCR results, which indicates a conserved structure and function of foxl2. Localisation of the Foxl2 protein in spermatogonia and spermatocytes implies that it may also be involved in spermatogenesis in G. haimaensis. The EGFR system contributes to key stages of reproduction, such as ovulation, fertilisation, embryo implantation, and the attainment of sexual maturity . Most studies have focused on vertebrates, and few findings have been reported for invertebrates. Our results indicate that EGFR may play a conserved role in ovary development.
Matrix metalloproteinases (MMPs) play an important role in the reproductive process by degrading the extracellular matrix and weakening the follicle wall, leading to follicle rupture [55, 56]. MMP2 and MMP9 stimulate luteinising hormone (LH)-induced steroid production by regulating the release of the EGFR ligand . Tissue inhibitors of metalloproteinases (TIMPs) are proteins secreted by a variety of cells that can selectively inhibit the activity of MMPs. The mechanism of action is to form chelates with metal ions necessary for the active sites of MMPs . Under physiological conditions, TIMPs and MMPs jointly maintain the stability of the biological environment in vivo. However, under pathological conditions, due to their direct action on MMPs, TIMPs are an extremely critical factor for maintaining homeostasis when the activity of MMPs activity is imbalanced. In G. haimaensis, MMPs may combine with TIMPs to promote EGFR to stimulate ovary development.
Vitellogenin (VTG), a precursor of yolk protein in oviparous animals, is a molecular carrier that transports nutrients into egg cells. Research on the biological functions of VTG shows that it regulates the osmotic pressure of ovaries [59, 60]. It is also an important immune molecule against pathogenic microorganisms [61–63], and it assists sperm fertilisation [64, 65]. Our transcriptome data and qPCR results showed that vtg was more highly expressed in F than M, indicating an exclusive role in ovaries rather than intersex or testes.
Sox2, a member of the SOX (SRY-related HMG-box) family, is an important transcription factor participating in embryogenesis [66, 67], neurogenesis [68–70], maintenance of stem cells [71–73] and proliferation of primordial germ cells (PGCs) . Also, Sox2 is involved in male germ cell development and stem cell maintenance in fish  and spermatogenesis in Zhikong scallop Chlamys farreri . In medaka, Sox2 is specifically expressed in ovary , and in Paralichthys olivaceus Sox2 is expressed more highly in ovary than testis . Our results showed that sox2 was expressed significantly higher in F than in M, consistent with the transcriptomic data, and localised in both testis and ovary, suggesting that Sox2 may function in male germ cell development and the maintenance of feminisation.
4.3 Putative Mechanism of Sex Change in G. haimaensis
In sex determination, the foxl2-leading pathway and RSPO-1/WNT4/β-catenin signalling pathway act independently and complementary to each other to promote and maintain ovarian development [79–82]. In G. haimaensis, foxl2 was specifically expressed in ovary, while β-catenin was expressed more highly in testis, implying that the foxl2-leading pathway may perform a leading role in the ovary determination/maintenance pathway of G. haimaensis. Also, Foxl2 may upregulate CYP450 to increase estrogens, and MMPs may combine with TIMPs to regulate EGFR and stimulate ovary development. The histological characteristics male gonads resembled intersex gonads, and β-catenin may play a role in intersex gonads to initiate ovary development.
Fem-1, mPRα and PLAC8 are downstream genes of the β-catenin signalling pathway regulated by β-catenin that may be involved in oogenesis in testis. We did not identify a testis-determining factor, such as SRY (Sex Determining Region Y) or DMRT, but there may be other male-determining genes. Sedentary lifestyles, combined with patchy distribution and environmental heterogeneity in darkness and in the presence of high concentrations of heavy metals and other toxic substances may stimulate G. haimaensis to change sex and thereby increase reproductive output.
In conclusion, our sex-biased proteomics and transcriptomics analysis of testes and ovaries in G. haimaensis revealed a strong correlation between mRNA and protein levels of key genes and proteins. Twelve DEGs between sexes were identified, and four ovary-biased genes (β-catenin, fem-1, foxl2 and mPRα) were expressed significantly higher in M than F. Histological characteristics of gonads implied that they may be involved in oogenesis in intersex gonads, further confirming the occurrence of sex change, and gonads might convert from male to female in G. haimaensis. This adaptation may be based on local environmental factors, sedentary lifestyles, and patchy distribution. These findings suggest a deeply conserved function of these genes in sex development, and a diverged regulatory pathway during evolution. This study provides a valuable genetic resource to better understand the mechanisms of sex change and the survival strategies in deep-sea bivalves.