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.

Concerted morphogenesis of genital ridges and nephric ducts in the mouse captured through whole-embryo imaging.

During development of the mouse urogenital complex, the gonads undergo changes in three-dimensional structure, body position and spatial relationship with the mesonephric ducts, kidneys and adrenals. The complexity of genital ridge development obscures potential connections between morphogenesis and gonadal sex determination. To characterize the morphogenic processes implicated in regulating gonad shape and fate, we used whole-embryo tissue clearing and light sheet microscopy to assemble a time course of gonad development in native form and context. Analysis revealed that gonad morphology is determined through anterior-to-posterior patterns as well as increased rates of growth, rotation and separation in the central domain that may contribute to regionalization of the gonad. We report a close alignment of gonad and mesonephric duct movements as well as delayed duct development in a gonad dysgenesis mutant, which together support a mechanical dependency linking gonad and mesonephric duct morphogenesis.

Crustacean endocrinology: Sexual differentiation and potential application for aquaculture.

Crustaceans, which represent a significant subset of arthropods, are classified into three major classes: Ostracoda, Malacostraca, and Branchiopoda. Among them, sex manipulation in decapod species from the Malacostraca class has been extensively researched for aquaculture purposes and to study reproductive physiology and sexual plasticity. Some decapods exhibit sexual dimorphism that influences their biological and economic value. Monosex culture, in which only one sex is cultivated, increases production yields while reducing the risk of invasiveness, as genetic leakage into natural waters is less likely to occur. Differences in yield are also observed when cultivating different sexes, with all-male cultures of Macrobrachium rosenbergii being more profitable than both mixed and all-female cultures. Research on decapod sexual differentiation has led to a better understanding of sex determination and sexual differentiation processes in arthropods. Similar to most mammals and other vertebrate classes, Malacostraca crustaceans, including decapods, exhibit a cell-non-autonomous mode of sexual development. Genetic factors (e.g., sex chromosomes) and endocrine factors (e.g., insulin-like androgenic gland factor and crustacean female sex hormone) play pivotal roles in the development of sexually dimorphic traits. This review synthesizes the existing understanding of sex determination mechanisms and the role of sex hormones in decapod species. Additionally, it provides an overview of the methyl farnesoate, which has been suggested to be involved in male sex differentiation in some crab species, as well as the phenomenon of male-to-female sex reversal in host decapods caused by parasitic crustaceans.

Two phases of gonadal sex differentiation in zebrafish with ZZ/ZW sex determination system.

Zebrafish sex chromosomes have been identified in the wild Nadia (NA) strain, and its sex determination belongs to the female-heterogametic ZZ/ZW system. Here, we investigate the correlation between ZZ/ZW sex chromosomes in the NA strain with sex-related factors, and sort out the complicated process of sex determination in zebrafish. Two phases exist during zebrafish sex differentiation. In the first phase, ZW gonads differentiate into juvenile ovary while ZZ gonads remain indifferent. In the second phase, ZW gonads either continue ovary development or undergo female-to-male transition, while ZZ gonads undergo direct male development. The W chromosome may contribute to the first phase while the abundance of germ cells and other factors may be involved in the second phase of sex differentiation in zebrafish.

The expression profiles of cyp19a1, sf-1, esrs and gths in the brain-pituitary during gonadal sex differentiation in juvenile Japanese eels.

Eels are gonochoristic species whose gonadal differentiation initiates at the yellow eel stage and is influenced by environmental factors. We revealed some sex-related genes were sex dimorphically expressed in gonads during gonadal sex differentiation of Japanese eel (Anguilla japonica); however, the expression of sex-related genes in the brain-pituitary during gonadal sex differentiation in eels is still unclear. This study aimed to investigate the sex-related gene expressions in the brain-pituitary and tried to clarify their roles in the brain and gonads during gonadal sex differentiation. Based on our previous histological study, the control eels developed as males, and estradiol-17ß (E2) was used for feminization. Our results showed that during testicular differentiation, the brain cyp19a1 transcripts and aromatase proteins were increased significantly; moreover, the cyp19a1, sf-1, foxl2s, and esrs (except gperb) transcripts in the midbrain/pituitary also were increased significantly. Forebrain gnrh1 transcripts increased slightly during gonadal differentiation of both sexes, but the gnrhr1b and gnrhr2 transcripts in the midbrain/pituitary were stable during gonadal differentiation. The expression levels of gths and gh in the midbrain/pituitary were significantly increased during testicular differentiation and were much higher in males than in E2-feminized females. These results implied that endogenous estrogens might play essential roles in the brain/pituitary during testicular differentiation, sf-1, foxl2s, and esrs may have roles in cyp19a1 regulation in the midbrain/pituitary of Japanese eels. For the GnRH-GTH axis, gths, especially fshb, may be regulated by esrs and involved in regulating testicular differentiation and development in Japanese eels.

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.

New cells added to the preoptic area during sex change in the common clownfish Amphiprion ocellaris.

Sex differences in cell number in the preoptic area of the hypothalamus (POA) are documented across all major vertebrate lineages and contribute to differential regulation of the hypothalamic-pituitary-gonad axis and reproductive behavior between the sexes. Sex-changing fishes provide a unique opportunity to study mechanisms underlying sexual differentiation of the POA. In anemonefish (clownfish), which change sex from male to female, females have approximately twice the number of medium-sized cells in the anterior POA compared to males. This sex difference transitions from male-like to female-like during sex change. However, it is not known how this sex difference in POA cell number is established. This study tests the hypothesis that new cell addition plays a role. We initiated adult male-to-female sex change in 30 anemonefish (Amphiprion ocellaris) and administered BrdU to label new cells added to the POA at regular intervals throughout sex change. Sex-changing fish added more new cells to the anterior POA than non-changing fish, supporting the hypothesis. The observed effects could be accounted for by differences in POA volume, but they are also consistent with a steady trickle of new cells being gradually accumulated in the anterior POA before vitellogenic oocytes develop in the gonads. These results provide insight into the unique characteristics of protandrous sex change in anemonefish relative to other modes of sex change, and support the potential for future research in sex-changing fishes to provide a richer understanding of the mechanisms for sexual differentiation of the brain.

The development of sex differences in the nervous system and behavior of flies, worms, and rodents.

Understanding how sex differences in innate animal behaviors arise has long fascinated biologists. As a general rule, the potential for sex differences in behavior is built by the developmental actions of sex-specific hormones or regulatory proteins that direct the sexual differentiation of the nervous system. In the last decade, studies in several animal systems have uncovered neural circuit mechanisms underlying discrete sexually dimorphic behaviors. Moreover, how certain hormones and regulatory proteins implement the sexual differentiation of these neural circuits has been illuminated in tremendous detail. Here, we discuss some of these mechanisms with three case-studies-mate recognition in flies, maturation of mating behavior in worms, and play-fighting behavior in young rodents. These studies illustrate general and unique developmental mechanisms to establish sex differences in neuroanatomy and behavior and highlight future challenges for the field.

Neuroimmunology and neuroepigenetics in the establishment of sex differences in the brain.

The study of sex differences in the brain is a topic of neuroscientific study that has broad reaching implications for culture, society and biomedical science. Recent research in rodent models has led to dramatic shifts in our views of the mechanisms underlying the sexual differentiation of the brain. These include the surprising discoveries of a role for immune cells and inflammatory mediators in brain masculinization and a role for epigenetic suppression in brain feminization. How and to what degree these findings will translate to human brain development will be questions of central importance in future research in this field.

Temperature fluctuations and estrone sulfate affect gene expression via different mechanisms to promote female development in a species with temperature-dependent sex determination.

Variation in developmental conditions can affect a variety of embryonic processes and shape a number of phenotypic characteristics that can affect offspring throughout their lives. This is particularly true of oviparous species where development typically occurs outside of the female, and studies have shown that traits such as survival and behavior can be altered by both temperature and exposure to steroid hormones during development. In species with temperature-dependent sex determination (TSD), the fate of gonadal development can be affected by temperature and by maternal estrogens present in the egg at oviposition, and there is evidence that these factors can affect gene expression patterns. Here, we explored how thermal fluctuations and exposure to an estrogen metabolite, estrone sulfate, affect the expression of several genes known to be involved in sexual differentiation: Kdm6b, Dmrt1, Sox9, FoxL2 and Cyp19A1. We found that most of the genes responded to both temperature and estrone sulfate exposure, but that the responses to these factors were not identical, in that estrone sulfate effects occur downstream of temperature effects. Our findings demonstrate that conjugated hormones such as estrone sulfate are capable of influencing temperature-dependent pathways to potentially alter how embryos respond to temperature, and highlight the importance of studying the interaction of maternal hormone and temperature effects.

Igf3 is essential for ovary differentiation in zebrafish†.

Zebrafish gonadal sexual differentiation is an important but poorly understood subject. Previously, we have identified a novel insulin-like growth factor (Igf) named insulin-like growth factor 3 (Igf3) in teleosts. The importance of Igf3 in oocyte maturation and ovulation has been recently demonstrated by us in zebrafish. In this study, we have further found the essential role of Igf3 in gonadal sexual differentiation of zebrafish. A differential expression pattern of igf3 between ovary and testis during sex differentiation (higher level in ovary than in testis) was found in zebrafish. An igf3 knockout zebrafish line was established using TALENs-mediated gene knockout technique. Intriguingly, all igf3 homozygous mutants were males due to the female-to-male sex reversal occurred during sex differentiation. Further analysis showed that Igf3 did not seem to affect the formation of so-called juvenile ovary and oocyte-like germ cells. Oocyte development was arrested at primary growth stage, and the ovary was gradually sex-reversed to testis before 60 day post fertilization (dpf). Such sex reversal was likely due to decreased germ cell proliferation by suppressing PI3K/Akt pathway in early ovaries of igf3 mutants. Estrogen is considered as a master regulator in fish sex differentiation. Here, we found that igf3 expression could be upregulated by estrogen in early stages of ovarian follicles as evidenced in in vitro treatment assays and cyp19a1a mutant zebrafish, and E2 failed to rescue the defects of igf3 mutants in ovarian development, suggesting that Igf3 may serve as a downstream factor of estrogen signaling in sex differentiation. Taken together, we demonstrated that Igf3 is essential for ovary differentiation in zebrafish.

Male fetal sex affects uteroplacental angiogenesis in growth restriction mouse model†.

Abnormally increased angiotensin II activity related to maternal angiotensinogen (AGT) genetic variants, or aberrant receptor activation, is associated with small-for-gestational-age babies and abnormal uterine spiral artery remodeling in humans. Our group studies a murine AGT gene titration transgenic (TG; 3-copies of the AGT gene) model, which has a 20% increase in AGT expression mimicking a common human AGT genetic variant (A[-6]G) associated with intrauterine growth restriction (IUGR) and spiral artery pathology. We hypothesized that aberrant maternal AGT expression impacts pregnancy-induced uterine spiral artery angiogenesis in this mouse model leading to IUGR. We controlled for fetal sex and fetal genotype (e.g., only 2-copy wild-type [WT] progeny from WT and TG dams were included). Uteroplacental samples from WT and TG dams from early (days 6.5 and 8.5), mid (d12.5), and late (d16.5) gestation were studied to assess uterine natural killer (uNK) cell phenotypes, decidual metrial triangle angiogenic factors, placental growth and capillary density, placental transcriptomics, and placental nutrient transport. Spiral artery architecture was evaluated at day 16.5 by contrast-perfused three-dimensional microcomputed tomography (3D microCT). Our results suggest that uteroplacental angiogenesis is significantly reduced in TG dams at day 16.5. Males from TG dams are associated with significantly reduced uteroplacental angiogenesis from early to late gestation compared with their female littermates and WT controls. Angiogenesis was not different between fetal sexes from WT dams. We conclude that male fetal sex compounds the pathologic impact of maternal genotype in this mouse model of growth restriction.

Vasa expression is associated with sex differentiation in the Asian yellow pond turtle, Mauremys mutica.

Vasa, one of the best-studied germ cell markers plays a critical role in germ cell development and differentiation in animals. Vasa deficiency would lead to male-specific sterility in most vertebrates, but female sterility in the fly. However, the role of the vasa gene involved in germ cell differentiation is largely elusive. Here, we first characterized the expression profile of vasa products in the Asian yellow pond turtle by quantitative reverse-transcription polymerase chain reaction and fluorescence immunostaining. The results showed that vasa messenger RNA (mRNA) is initially detected in embryos at stage 16, and then dramatically increased in embryos at stage 19. In particular, like the sex-related genes, vasa mRNA exhibited differential expression in embryos between the male-producing temperature (MPT, 25°C) and the female-producing temperature (FPT, 33°C), whereas there was no difference in methylation levels of vasa promoter detected between FPT and MPT. In contrast, in the adult Asian yellow pond, the level of vasa mRNA was much higher in the testis than ovary. Moreover, the immunostaining on testicular sections and cells showed that Vasa protein was exclusively expressed in germ cells: Weak but detectable in spermatogonia, highest in spermatocytes, moderate and concentrated in chromatid bodies in spermatids and spermatozoa, and bare in somatic cells. The expression profile of Vasa protein is similar in turtle species studied so far but distinct from those in fish species in this study. The findings of this study would provide new insights into our understanding of the conservation and divergence of the vasa gene, even other germ cell genes across phyla.

Sexually anomalous individuals of the black fly Simulium trangense (Diptera: Simuliidae) infected with mermithid parasites (Nematoda: Mermithidae).

Sexually anomalous individuals, typically intersexes or gynandromorphs, bear a mixture of male and female traits. Twelve sexually anomalous individuals of the black fly Simulium (Gomphostilbia) trangense Jitklang, Kuvangkadilok, Baimai, Takaoka & Adler were discovered among 49 adults reared from pupae. All 12 sexually anomalous adults were parasitized by mermithid nematodes, although five additional parasitized adults had no overt external anomalies. Sequence analysis of the 18S rRNA gene revealed that the mermithids, possibly representing a new species, are related to Mesomermis spp., with genetic distances of 5.09-6.87%. All 12 anomalous individuals had female phenotypical traits on the head, thorax, forelegs, midlegs, and claws, but male features on the left and right hind basitarsi. One individual had mixed male and female genitalia. The findings are in accord with the trend that mermithid infections are associated with sexually anomalous adult black flies.

Connection between seminiferous tubules and epididymal duct is originally induced before sex differentiation in a sex-independent manner.

BACKGROUND: Spermatozoa in mammals develop in seminiferous tubules in a testis and are transported through the male reproductive tract. Their developmental origins are, however, different from each other; the seminiferous tubules are testicular (gonadal) structure but the subsequent ducts stem from the mesonephros. Although some mechanisms should function for the connection between these ducts, there are few reports on them. In the present study, basic information such as timing, localization, and cell types involved in the connection was obtained by sequential immunohistochemistry. RESULTS: At the time when the undifferentiated gonad differentiates into the testis or ovary, Adrenal-4 binding protein/steroidogenic factor-1 (Ad4BP/SF-1)-positive gonadal cells were noted in the mesonephric tubules (MT) in both sexes. At an earlier stage, although Ad4BP/SF-1-positive coelomic epithelial cells were adjacent to the MT, a basal membrane around them was not observed. CONCLUSIONS: The connection between the testis cords and MT is suggested to be induced between Ad4BP/SF-1-positive gonadal cells and the MT before sex differentiation in a sex-independent manner.

Microglia and sexual differentiation of the developing brain: A focus on extrinsic factors.

Microglia, the innate immune cells of the brain, have recently been removed from the position of mere sentinels and promoted to the role of active sculptors of developing circuits and cells. Alongside their functions in normal brain development, microglia coordinate sexual differentiation of the brain, a set of processes which vary by region and endpoint like that of microglia function itself. In this review, we highlight the ways microglia are both targets and drivers of brain sexual differentiation. We examine the factors that may drive sex differences in microglia, with a special focus on how changing microenvironments in the developing brain dictate microglia phenotypes and discuss how their diverse functions sculpt lasting sex-specific changes in the brain. Finally, we consider how sex-specific early life environments contribute to epigenetic programming and lasting sex differences in microglia identity.

Microglia and sexual differentiation of the developing brain: A focus on ontogeny and intrinsic factors.

Sexual differentiation of the brain during early development likely underlies the strong sex biases prevalent in many neurological conditions. Mounting evidence indicates that microglia, the innate immune cells of the central nervous system, are intricately involved in these sex-specific processes of differentiation. In this review, we synthesize literature demonstrating sex differences in microglial number, morphology, transcriptional state, and functionality throughout spatiotemporal development as well as highlight current literature regarding ontogeny of microglia. Along with vanRyzin et al. in this issue, we explore the idea that differences in microglia imparted by chromosomal or ontogeny-related programming can influence microglial-driven sexual differentiation of the brain, as well as the idea that extrinsic differences in the male and female brain microenvironment may in turn impart sex differences in microglia.

Sexual differentiation of microglia.

Sex plays a role in the incidence and outcome of neurological illnesses, also influencing the response to treatments. Despite sexual differentiation of the brain has been extensively investigated, the study of sex differences in microglia, the brain's resident immune cells, has been largely neglected until recently. To fulfill this gap, our laboratory developed several tools, including cellular and animal models, which bolstered in-depth studies on sexual differentiation of microglia and its impact on brain physiology, as well as on the onset and progression of neurological disorders. Here, we summarize the current status of knowledge on the sex-dependent function of microglia, and report recent evidence linking these cells to the sexual bias in the susceptibility to neurological brain diseases.

Plasmodium sexual differentiation: how to make a female.

Sexual development is integral to the transmission of Plasmodium parasites between vertebrates and mosquitos. Recent years have seen great advances in understanding the gene expression that underlies commitment of asexual parasites to differentiate into sexual gametocyte stages, then how they mature and form gametes once inside a mosquito. Less well understood is how parasites differentially control development to become males or females. Plasmodium parasites are haploid at the time of sexual differentiation, but a clonal haploid line can produce both male and female gametocytes, so they presumably lack the sex-determining alleles present in some other eukaryotes. Though the molecular switch to initiate male or female development remains hidden, recent studies reveal regulatory proteins needed for the sex-specific maturation of male and female gametocytes. Yuda and collaborators report the characterization of a transcription factor necessary for female gametocyte maturation. With renewed attention on malaria elimination, sex has been an increasing focus because transmission-blocking strategies are likely to be an important component of elimination efforts.

Homozygous mutation of foxh1 arrests oogenesis causing infertility in female Nile tilapia†.

Foxh1, a member of fox gene family, was first characterized as a transcriptional partner in the formation of the Smad protein complex. Recent studies have shown foxh1 is highly expressed in the cytoplasm of oocytes in both tilapia and mouse. However, its function in oogenesis remains unexplored. In the present study, foxh1-/- tilapia was created by CRISPR/Cas9. At 180 dah (days after hatching), the foxh1-/- XX fish showed oogenesis arrest and a significantly lower GSI. The transition of oocytes from phase II to phase III and follicle cells from one to two layers was blocked, resulting in infertility of the mutant. Transcriptomic analysis revealed that expression of genes involved in estrogen synthesis and oocyte growth were altered in the foxh1-/- ovaries. Loss of foxh1 resulted in significantly decreased Cyp19a1a and increased Cyp11b2 expression, consistent with significantly lower concentrations of serum estradiol-17ß (E2) and higher concentrations of 11-ketotestosterone (11-KT). Moreover, administration of E2 rescued the phenotypes of foxh1-/- XX fish, as indicated by the appearance of phase III and IV oocytes and absence of Cyp11b2 expression. Taken together, these results suggest that foxh1 functions in the oocytes to regulate oogenesis by promoting cyp19a1a expression, and therefore estrogen production. Disruption of foxh1 may block the estrogen synthesis and oocyte growth, leading to the arrest of oogenesis and thus infertility in tilapia.