Unveiling the roles of Sertoli cells lineage differentiation in reproductive development and disorders: a review.

In mammals, gonadal somatic cell lineage differentiation determines the development of the bipotential gonad into either the ovary or testis. Sertoli cells, the only somatic cells in the spermatogenic tubules, support spermatogenesis during gonadal development. During embryonic Sertoli cell lineage differentiation, relevant genes, including WT1, GATA4, SRY, SOX9, AMH, PTGDS, SF1, and DMRT1, are expressed at specific times and in specific locations to ensure the correct differentiation of the embryo toward the male phenotype. The dysregulated development of Sertoli cells leads to gonadal malformations and male fertility disorders. Nevertheless, the molecular pathways underlying the embryonic origin of Sertoli cells remain elusive. By reviewing recent advances in research on embryonic Sertoli cell genesis and its key regulators, this review provides novel insights into sex determination in male mammals as well as the molecular mechanisms underlying the genealogical differentiation of Sertoli cells in the male reproductive ridge.

Pivotal temperature is not for everyone: Evidence for temperature-dependent sex determination in three gecko species.

The prevalence of environmental sex determination (ESD) in squamate reptiles is often overestimated in the literature. This is surprising because we have reliable data demonstrating ESD in only a few species. The documentation of ESD in three species of geckos presented here has significantly increased our knowledge, given that satisfactory evidence for ESD existed in only eight other gecko species. For the first time, we document the occurrence of ESD in the family Sphaerodactylidae. Our finding of unexpected variability in the shapes of reaction norms among geckos highlights that traditional descriptions using parameters such as pivotal temperature, that is, temperature producing a 50:50 sex ratio, are unsatisfactory. For example, the gecko Pachydactylus tigrinus lacks any pivotal temperature and its sex ratios are strongly female-biased across the entire range of viable temperatures. We argue for the effective capture of the relationship between temperature and sex ratio using specific nonlinear models rather than using classical simplistic descriptions and classifications of reaction norms.

Inefficient Sox9 upregulation and absence of Rspo1 repression lead to sex reversal in the B6.XYTIR mouse gonad†.

Sry on the Y-chromosome upregulates Sox9, which in turn upregulates a set of genes such as Fgf9 to initiate testicular differentiation in the XY gonad. In the absence of Sry expression, genes such as Rspo1, Foxl2, and Runx1 support ovarian differentiation in the XX gonad. These two pathways antagonize each other to ensure the development of only one gonadal sex in normal development. In the B6.YTIR mouse, carrying the YTIR-chromosome on the B6 genetic background, Sry is expressed in a comparable manner with that in the B6.XY mouse, yet, only ovaries or ovotestes develop. We asked how testicular and ovarian differentiation pathways interact to determine the gonadal sex in the B6.YTIR mouse. Our results showed that (1) transcript levels of Sox9 were much lower than in B6.XY gonads while those of Rspo1 and Runx1 were as high as B6.XX gonads at 11.5 and 12.5 days postcoitum. (2) FOXL2-positive cells appeared in mosaic with SOX9-positive cells at 12.5 days postcoitum. (3) SOX9-positive cells formed testis cords in the central area while those disappeared to leave only FOXL2-positive cells in the poles or the entire area at 13.5 days postcoitum. (4) No difference was found at transcript levels of all genes between the left and right gonads up to 12.5 days postcoitum, although ovotestes developed much more frequently on the left than the right at 13.5 days postcoitum. These results suggest that inefficient Sox9 upregulation and the absence of Rspo1 repression prevent testicular differentiation in the B6.YTIR gonad.

Tenuous transcriptional threshold of human sex determination. II. SRY exploits water-mediated clamp at the edge of ambiguity.

Y-encoded transcription factor SRY initiates male differentiation in therian mammals. This factor contains a high-mobility-group (HMG) box, which mediates sequence-specific DNA binding with sharp DNA bending. A companion article in this issue described sex-reversal mutations at box position 72 (residue 127 in human SRY), invariant as Tyr among mammalian orthologs. Although not contacting DNA, the aromatic ring seals the domain's minor wing at a solvent-exposed junction with a basic tail. A seeming paradox was posed by the native-like biochemical properties of inherited Swyer variant Y72F: its near-native gene-regulatory activity is consistent with the father's male development, but at odds with the daughter's XY female somatic phenotype. Surprisingly, aromatic rings (Y72, F72 or W72) confer higher transcriptional activity than do basic or polar side chains generally observed at solvated DNA interfaces (Arg, Lys, His or Gln). Whereas biophysical studies (time-resolved fluorescence resonance energy transfer and heteronuclear NMR spectroscopy) uncovered only subtle perturbations, dissociation of the Y72F complex was markedly accelerated relative to wild-type. Studies of protein-DNA solvation by molecular-dynamics (MD) simulations of an homologous high-resolution crystal structure (SOX18) suggest that Y72 para-OH anchors a network of water molecules at the tail-DNA interface, perturbed in the variant in association with nonlocal conformational fluctuations. Loss of the Y72 anchor among SRY variants presumably "unclamps" its basic tail, leading to (a) rapid DNA dissociation despite native affinity and (b) attenuated transcriptional activity at the edge of sexual ambiguity. Conservation of Y72 suggests that this water-mediated clamp operates generally among SRY and metazoan SOX domains.

Germline sex determination regulates sex-specific signaling between germline stem cells and their niche.

Establishing germ cell sexual identity is critical for development of male and female germline stem cells (GSCs) and production of sperm or eggs. Germ cells depend on signals from the somatic gonad to determine sex, but in organisms such as flies, mice, and humans, the sex chromosome genotype of the germ cells is also important for germline sexual development. How somatic signals and germ-cell-intrinsic cues combine to regulate germline sex determination is thus a key question. We find that JAK/STAT signaling in the GSC niche promotes male identity in germ cells, in part by activating the chromatin reader Phf7. Further, we find that JAK/STAT signaling is blocked in XX (female) germ cells through the action of the sex determination gene Sex lethal to preserve female identity. Thus, an important function of germline sexual identity is to control how GSCs respond to signals in their niche environment.

Fetal Estrogens are not Involved in Sex Determination But Critical for Early Ovarian Differentiation in Rabbits.

AROMATASE is encoded by the CYP19A1 gene and is the cytochrome enzyme responsible for estrogen synthesis in vertebrates. In most mammals, a peak of CYP19A1 gene expression occurs in the fetal XX gonad when sexual differentiation is initiated. To elucidate the role of this peak, we produced 3 lines of TALEN genetically edited CYP19A1 knockout (KO) rabbits that were devoid of any estradiol production. All the KO XX rabbits developed as females with aberrantly small ovaries in adulthood, an almost empty reserve of primordial follicles, and very few large antrum follicles. Ovulation never occurred. Our histological, immunohistological, and transcriptomic analyses showed that the estradiol surge in the XX fetal rabbit gonad is not essential to its determination as an ovary, or for meiosis. However, it is mandatory for the high proliferation and differentiation of both somatic and germ cells, and consequently for establishment of the ovarian reserve.

Dynamics of epigenetic modifiers and environmentally sensitive proteins in a reptile with temperature induced sex reversal†.

The mechanisms by which sex is determined, and how a sexual phenotype is stably maintained during adulthood, have been the focus of vigorous scientific inquiry. Resources common to the biomedical field (automated staining and imaging platforms) were leveraged to provide the first immunofluorescent data for a reptile species with temperature induced sex reversal. Two four-plex immunofluorescent panels were explored across three sex classes (sex reversed ZZf females, normal ZWf females, and normal ZZm males). One panel was stained for chromatin remodeling genes JARID2 and KDM6B, and methylation marks H3K27me3, and H3K4me3 (Jumonji Panel). The other CaRe panel stained for environmental response genes CIRBP and RelA, and H3K27me3 and H3K4me3. Our study characterized tissue specific expression and cellular localization patterns of these proteins and histone marks, providing new insights to the molecular characteristics of adult gonads in a dragon lizard Pogona vitticeps. The confirmation that mammalian antibodies cross react in P. vitticeps paves the way for experiments that can take advantage of this new immunohistochemical resource to gain a new understanding of the role of these proteins during embryonic development, and most importantly for P. vitticeps, the molecular underpinnings of sex reversal.

Sexual determination in zebrafish.

Zebrafish have emerged as a major model organism to study vertebrate reproduction due to their high fecundity and external development of eggs and embryos. The mechanisms through which zebrafish determine their sex have come under extensive investigation, as they lack a definite sex-determining chromosome and appear to have a highly complex method of sex determination. Single-gene mutagenesis has been employed to isolate the function of genes that determine zebrafish sex and regulate sex-specific differentiation, and to explore the interactions of genes that promote female or male sexual fate. In this review, we focus on recent advances in understanding of the mechanisms, including genetic and environmental factors, governing zebrafish sex development with comparisons to gene functions in other species to highlight conserved and potentially species-specific mechanisms for specifying and maintaining sexual fate.

Relationship between amniotic fluid metabolic profile with fetal gender, maternal age, and gestational week.

BACKGROUND: Amniotic fluid (AF) provides vital information on fetal development, which is also valuable in identifying fetal abnormalities during pregnancy. However, the relationship between the metabolic profile of AF in the second trimester of a normal pregnancy with several maternal-fetal parameters remains poorly understood, which therefore limits its application in clinical practice. The aim of this study was to explore the association between the metabolic profile of AF with fetal gender, maternal age, and gestational week using an untargeted metabolomics method. METHODS: A total of 114 AF samples were analyzed in this study. Clinical data on fetal gender, maternal age, and gestational week of these samples were collected. Samples were analyzed by gas chromatography/time-of-flight-mass spectrometry (GC-TOF/MS). Principal component analysis(PCA), orthogonal partial least square discrimination analysis(OPLS-DA) or partial least square discrimination analysis (PLS-DA) were conducted to compare metabolic profiles, and differential metabolites were obtained by univariate analysis. RESULTS: Both PCA and OPLS-DA demonstrated no significant separation trend between the metabolic profiles of male and female fetuses, and there were only 7 differential metabolites. When the association between the maternal age on AF metabolic profile was explored, both PCA and PLS-DA revealed that the maternal age in the range of 21 to 40 years had no significant effect on the metabolic profile of AF, and only four different metabolites were found. There was no significant difference in the metabolic profiles of AF from fetuses of 17-22 weeks, and 23 differential metabolites were found. CONCLUSIONS: In the scope of our study, there was no significant correlation between the AF metabolic profile and the fetal gender, maternal age and gestational week of a small range. Nevertheless, few metabolites appeared differentially expressed.

Testicular inducing steroidogenic cells trigger sex change in groupers.

Vertebrates usually exhibit gonochorism, whereby their sex is fixed throughout their lifetime. However, approximately 500 species (~ 2%) of extant teleost fishes change sex during their lifetime. Although phylogenetic and evolutionary ecological studies have recently revealed that the extant sequential hermaphroditism in teleost fish is derived from gonochorism, the evolution of this transsexual ability remains unclear. We revealed in a previous study that the tunica of the ovaries of several protogynous hermaphrodite groupers contain functional androgen-producing cells, which were previously unknown structures in the ovaries of gonochoristic fishes. Additionally, we demonstrated that these androgen-producing cells play critical roles in initiating female-to-male sex change in several grouper species. In the present study, we widened the investigation to include 7 genera and 18 species of groupers and revealed that representatives from most major clades of extant groupers commonly contain these androgen-producing cells, termed testicular-inducing steroidogenic (TIS) cells. Our findings suggest that groupers acquired TIS cells in the tunica of the gonads for successful sex change during their evolution. Thus, TIS cells trigger the evolution of sex change in groupers.

Sex identification in embryos and adults of Darwin's finches.

Darwin's finches are an iconic example of adaptive radiation and evolution under natural selection. Comparative genetic studies using embryos of Darwin's finches have shed light on the possible evolutionary processes underlying the speciation of this clade. Molecular identification of the sex of embryonic samples is important for such studies, where this information often cannot be inferred otherwise. We tested a fast and simple chicken embryo protocol to extract DNA from Darwin's finch embryos. In addition, we applied minor modifications to two of the previously reported PCR primer sets for CHD1, a gene used for sexing adult passerine birds. The sex of all 29 tested embryos of six species of Darwin's finches was determined successfully by PCR, using both primer sets. Next to embryos, hatchlings and fledglings are also impossible to distinguish visually. This extends to juveniles of sexually dimorphic species which are yet to moult in adult-like plumage and beak colouration. Furthermore, four species of Darwin's finches are monomorphic, males and females looking alike. Therefore, sex assessment in the field can be a source of error, especially with respect to juveniles and mature monomorphic birds outside of the mating season. We caught 567 juveniles and adults belonging to six species of Darwin's finches and only 44% had unambiguous sex-specific morphology. We sexed 363 birds by PCR: individuals sexed based on marginal sex specific morphological traits; and birds which were impossible to classify in the field. PCR revealed that for birds with marginal sex specific traits, sexing in the field produced a 13% error rate. This demonstrates that PCR based sexing can improve field studies on Darwin's finches, especially when individuals with unclear sex-related morphology are involved. The protocols used here provide an easy and reliable way to sex Darwin's finches throughout ontogeny, from embryos to adults.

Interplay between sex determination cascade and major signaling pathways during Drosophila eye development: Perspectives for future research.

Understanding molecular mechanisms of sexually dimorphic organ growth is a fundamental problem of developmental biology. Recent quantitative studies showed that the Drosophila compound eye is a convenient model to study the determination of the final organ size. In Drosophila, females have larger eyes than males and this is evident even after correction for the larger body size. Moreover, female eyes include more ommatidia (photosensitive units) than male eyes and this difference is specified at the third larval instar in the eye primordia called eye imaginal discs. This may result in different visual capabilities between the two sexes and have behavioral consequences. Despite growing evidence on the genetic bases of eye size variation between different Drosophila species and strains, mechanisms responsible for within-species sexual dimorphism still remain elusive. Here, we discuss a presumptive crosstalk between the sex determination cascade and major signaling pathways during dimorphic eye development. Male- and female-specific isoforms of Doublesex (Dsx) protein are known to control sex-specific differentiation in the somatic tissues. However, no data on Dsx function during eye disc growth and patterning are currently available. Remarkably, Sex lethal (Sxl), the sex determination switch protein, was shown to directly affect Hedgehog (Hh) and Notch (N) signaling in the Drosophila wing disc. The similarity of signaling pathways involved in the wing and eye disc growth suggests that Sxl might be integrated into regulation of eye development. Dsx role in the eye disc requires further investigation. We discuss currently available data on sex-biased gene expression in the Drosophila eye and highlight perspectives for future studies.

Differential expression analysis and identification of sex-related genes by gonad transcriptome sequencing in estradiol-treated and non-treated Ussuri catfish Pseudobagrus ussuriensis.

The Ussuri catfish (Pseudobagrus ussuriensis) has an XX/XY sex determination system but its sex determination gene(s) remain unknown. To better understand the molecular sex determination mechanism, transcriptome analysis was conducted to obtain sex-related gene expression profiles. Transcriptome analyses were made of male and female developing/differentiating gonads by high-throughput RNA sequencing, including gonads from fish given an estradiol-induced sex reversal treatment. A total of 81,569 unigenes were assembled and 39,904 were significantly matched to known unique proteins by comparison with public databases. Twenty specifically expressed and 142 differentially expressed sex-related genes were extracted from annotated data by comparing the treatment groups. These genes are involved in spermatogenesis (e.g., Dnali1, nectin3, klhl10, mybl1, Katnal1, Eno4, Mns1, Spag6, Tsga10, Septin7), oogenesis (e.g., Lagr5, Fmn2, Npm2, zar1, Fbxo5, Fbxo43, Prdx4, Nrip1, Lfng, Atrip), gonadal development/differentiation (e.g., Cxcr4b, Hmgb2, Cftr, Ch25h, brip1, Prdm9, Tdrd1, Star, dmrt1, Tut4, Hsd17b12a, gdf9, dnd, arf1, Spata22), and estradiol response (e.g., Mmp14, Lhcgr, vtg1, vtg2, esr2b, Piwil1, Aifm1, Hsf1, gdf9). Dmrt1 and gdf9 may play an essential role in sex determination in P. ussuriensis. The expression patterns of six random genes were validated by quantitative real-time PCR, which confirmed the reliability and accuracy of the RNA-seq results. These data provide a valuable resource for future studies of gene expression and for understanding the molecular mechanism of sex determination/differentiation and gonadal development/differentiation (including hormone-induced sexual reversal) in Ussuri catfish. This has the potential to assist in producing monosex Ussuri catfish to increase aquacultural productivity.

The rise and fall of the ancient northern pike master sex-determining gene.

The understanding of the evolution of variable sex determination mechanisms across taxa requires comparative studies among closely related species. Following the fate of a known master sex-determining gene, we traced the evolution of sex determination in an entire teleost order (Esociformes). We discovered that the northern pike (Esox lucius) master sex-determining gene originated from a 65 to 90 million-year-old gene duplication event and that it remained sex linked on undifferentiated sex chromosomes for at least 56 million years in multiple species. We identified several independent species- or population-specific sex determination transitions, including a recent loss of a Y chromosome. These findings highlight the diversity of evolutionary fates of master sex-determining genes and the importance of population demographic history in sex determination studies. We hypothesize that occasional sex reversals and genetic bottlenecks provide a non-adaptive explanation for sex determination transitions.

Characteristics and Cryopreservation of Semen of Sex-Reversed Females of Salmonid Fish.

Sex reversal has been used as a breeding strategy by salmonid fish to produce genetically and phenotypically single sex populations. Production of all-female fish has great importance for the creation of monosex female triploids of salmonid fish, which are valued for their sterility, lack of female maturation, and larger commercial size. Among salmonids, the majority of rainbow trout (Oncorhynchus mykiss) production is based on all-female production with a high proportion of all-female triploid production in Europe. The main aim of this review is to present the recent knowledge regarding sex-reversed females (SRFs) of salmonid fish. We discuss the methods of sex reversal as well as their effects on the morphology and histology of the reproductive tract. We focus on the characteristics of SRF semen as well as the factors determining semen quality. The lower quality of SRF sperm compared to that of normal males has resulted in the need for the artificial maturation of semen. Most importantly, methods of semen storage-both short-term and long-term (cryopreservation)-that can improve hatchery operations are presented with the special emphasis on recent progress in development of efficient cryopreservation procedures and use of cryopreserved semen in hatchery practice. Moreover, we also address the emerging knowledge concerning the proteomic investigations of salmonid sperm, focusing primarily on the proteomic comparison of normal male and SRF testicular semen and presenting changes in SRF rainbow trout sperm proteome after in vitro incubation in artificial seminal plasma.

Physical interactions facilitate sex change in the protogynous orange-spotted grouper, Epinephelus coioides.

Sex change in teleost fishes is commonly regulated by social factors. In species that exhibit protogynous sex change, such as the orange-spotted grouper Epinephelus coioides, when the dominant males are removed from the social group, the most dominant female initiates sex change. The aim of this study was to determine the regulatory mechanisms of socially controlled sex change in E. coioides. We investigated the seasonal variation in social behaviours and sex change throughout the reproductive cycle of E. coioides, and defined the behaviour pattern of this fish during the establishment of a dominance hierarchy. The social behaviours and sex change in this fish were affected by season, and only occurred during the prebreeding season and breeding season. Therefore, a series of sensory isolation experiments was conducted during the breeding season to determine the role of physical, visual and olfactory cues in mediating socially controlled sex change. The results demonstrated that physical interactions between individuals in the social groups were crucial for the initiation and completion of sex change, whereas visual and olfactory cues alone were insufficient in stimulating sex change in dominant females. In addition, we propose that the steroid hormones 11-ketotestosterone and cortisol are involved in regulating the initiation of socially controlled sex change.

Sex determination, gonadal sex differentiation, and plasticity in vertebrate species.

A diverse array of sex determination (SD) mechanisms, encompassing environmental to genetic, have been found to exist among vertebrates, covering a spectrum from fixed SD mechanisms (mammals) to functional sex change in fishes (sequential hermaphroditic fishes). A major landmark in vertebrate SD was the discovery of the SRY gene in 1990. Since that time, many attempts to clone an SRY ortholog from nonmammalian vertebrates remained unsuccessful, until 2002, when DMY/dmrt1by was discovered as the SD gene of a small fish, medaka. Surprisingly, however, DMY/dmrt1by was found in only 2 species among more than 20 species of medaka, suggesting a large diversity of SD genes among vertebrates. Considerable progress has been made over the last 3 decades, such that it is now possible to formulate reasonable paradigms of how SD and gonadal sex differentiation may work in some model vertebrate species. This review outlines our current understanding of vertebrate SD and gonadal sex differentiation, with a focus on the molecular and cellular mechanisms involved. An impressive number of genes and factors have been discovered that play important roles in testicular and ovarian differentiation. An antagonism between the male and female pathway genes exists in gonads during both sex differentiation and, surprisingly, even as adults, suggesting that, in addition to sex-changing fishes, gonochoristic vertebrates including mice maintain some degree of gonadal sexual plasticity into adulthood. Importantly, a review of various SD mechanisms among vertebrates suggests that this is the ideal biological event that can make us understand the evolutionary conundrums underlying speciation and species diversity.

Exogenous Oestrogen Impacts Cell Fate Decision in the Developing Gonads: A Potential Cause of Declining Human Reproductive Health.

The increasing incidence of testicular dysgenesis syndrome-related conditions and overall decline in human fertility has been linked to the prevalence of oestrogenic endocrine disrupting chemicals (EDCs) in the environment. Ectopic activation of oestrogen signalling by EDCs in the gonad can impact testis and ovary function and development. Oestrogen is the critical driver of ovarian differentiation in non-mammalian vertebrates, and in its absence a testis will form. In contrast, oestrogen is not required for mammalian ovarian differentiation, but it is essential for its maintenance, illustrating it is necessary for reinforcing ovarian fate. Interestingly, exposure of the bi-potential gonad to exogenous oestrogen can cause XY sex reversal in marsupials and this is mediated by the cytoplasmic retention of the testis-determining factor SOX9 (sex-determining region Y box transcription factor 9). Oestrogen can similarly suppress SOX9 and activate ovarian genes in both humans and mice, demonstrating it plays an essential role in all mammals in mediating gonad somatic cell fate. Here, we review the molecular control of gonad differentiation and explore the mechanisms through which exogenous oestrogen can influence somatic cell fate to disrupt gonad development and function. Understanding these mechanisms is essential for defining the effects of oestrogenic EDCs on the developing gonads and ultimately their impacts on human reproductive health.

FGF9 is a downstream target of SRY and sufficient to determine male sex fate in ex vivo XX gonad culture.

Fibroblast growth factor 9 (FGF9) is an autocrine/paracrine growth factor that plays critical roles in embryonic and organ developments and is involved in diverse physiological events. Loss of function of FGF9 exhibits male-to-female sex reversal in the transgenic mouse model and gain of FGF9 copy number was found in human 46, XX sex reversal patient with disorders of sex development. These results suggested that FGF9 plays a vital role in male sex development. Nevertheless, how FGF9/Fgf9 expression is regulated during testis determination remains unclear. In this study, we demonstrated that human and mouse SRY bind to -833 to -821 of human FGF9 and -1010 to -998 of mouse Fgf9, respectively, and control FGF9/Fgf9 mRNA expression. Interestingly, we showed that mouse SRY cooperates with SF1 to regulate Fgf9 expression, whereas human SRY-mediated FGF9 expression is SF1 independent. Furthermore, using an ex vivo gonadal culture system, we showed that FGF9 expression is sufficient to switch cell fate from female to male sex development in 12-16 tail somite XX mouse gonads. Taken together, our findings provide evidence to support the SRY-dependent, fate-determining role of FGF9 in male sex development.

Loss of dmrt1 restores zebrafish female fates in the absence of cyp19a1a but not rbpms2a/b.

Sex determination and differentiation is a complex process regulated by multiple factors, including factors from the germline or surrounding somatic tissue. In zebrafish, sex-determination involves establishment of a bipotential ovary that undergoes sex-specific differentiation and maintenance to form the functional adult gonad. However, the relationships among these factors are not fully understood. Here, we identify potential Rbpms2 targets and apply genetic epistasis experiments to decipher the genetic hierarchy of regulators of sex-specific differentiation. We provide genetic evidence that the crucial female factor rbpms2 is epistatic to the male factor dmrt1 in terms of adult sex. Moreover, the role of Rbpms2 in promoting female fates extends beyond repression of Dmrt1, as Rbpms2 is essential for female differentiation even in the absence of Dmrt1. In contrast, female fates can be restored in mutants lacking both cyp19a1a and dmrt1, and prolonged in bmp15 mutants in the absence of dmrt1. Taken together, this work indicates that cyp19a1a-mediated suppression of dmrt1 establishes a bipotential ovary and initiates female fate acquisition. Then, after female fate specification, Cyp19a1a regulates subsequent oocyte maturation and sustains female fates independently of Dmrt1 repression.