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.

Sex Lethal Gene Manipulates Gonadal Development of Medaka, Oryzias latipes, through Estrogenic Interventions.

Germ cells are pivotal for gonadal sexuality maintenance and reproduction. Sex lethal (sxl), the somatic sex determining gene of Drosophila, is the known regulator and initiator of germ cell femininity in invertebrates. However, the role of the Sxl homologue has rarely been investigated in vertebrates. So, we used medaka to clarify the role of sxl in vertebrate gonadogenesis and sexuality and identified two Sxl homologues, i.e., Sxl1a and Sxl1b. We found that sxl1a specifically expresses in the primordial germ cells (PGC), ovary, (early gonia and oocytes), while sxl1b distributions are ubiquitous. An mRNA overexpression of sxl1a accelerated germ cell numbers in 10 DAH XY fish, and sxl1a knockdown (KD), on the other hand, induced PGC mis-migration, aberrant PGC structuring and ultimately caused significant germ cell reduction in XX fish. Using an in vitro promoter analysis and in vivo steroid treatment, we found a strong link between sxl1a and estrogenic germ cell-population maintenance. Further, using sxl1a-KD and erß2-knockout fish, we determined that sxl1 acts through erß2 and controls PGC sexuality. Cumulatively, our study highlights the novel role of sxl1a in germ cell maintenance and sexual identity assignment and thus might become a steppingstone to understanding the commonalities of animal sexual development.

A Tandem Duplicate of Anti-Müllerian Hormone with a Missense SNP on the Y Chromosome Is Essential for Male Sex Determination in Nile Tilapia, Oreochromis niloticus.

Variation in the TGF-ß signaling pathway is emerging as an important mechanism by which gonadal sex determination is controlled in teleosts. Here we show that amhy, a Y-specific duplicate of the anti-Müllerian hormone (amh) gene, induces male sex determination in Nile tilapia. amhy is a tandem duplicate located immediately downstream of amhΔ-y on the Y chromosome. The coding sequence of amhy was identical to the X-linked amh (amh) except a missense SNP (C/T) which changes an amino acid (Ser/Leu92) in the N-terminal region. amhy lacks 5608 bp of promoter sequence that is found in the X-linked amh homolog. The amhΔ-y contains several insertions and deletions in the promoter region, and even a 5 bp insertion in exonVI that results in a premature stop codon and thus a truncated protein product lacking the TGF-ß binding domain. Both amhy and amhΔ-y expression is restricted to XY gonads from 5 days after hatching (dah) onwards. CRISPR/Cas9 knockout of amhy in XY fish resulted in male to female sex reversal, while mutation of amhΔ-y alone could not. In contrast, overexpression of Amhy in XX fish, using a fosmid transgene that carries the amhy/amhΔ-y haplotype or a vector containing amhy ORF under the control of CMV promoter, resulted in female to male sex reversal, while overexpression of AmhΔ-y alone in XX fish could not. Knockout of the anti-Müllerian hormone receptor type II (amhrII) in XY fish also resulted in 100% complete male to female sex reversal. Taken together, these results strongly suggest that the duplicated amhy with a missense SNP is the candidate sex determining gene and amhy/amhrII signal is essential for male sex determination in Nile tilapia. These findings highlight the conserved roles of TGF-ß signaling pathway in fish sex determination.

A new relaxin-like gonad-stimulating peptide identified in the starfish Asterias amurensis.

Relaxin-like gonad-stimulating peptide (RGP) of starfish Asterina pectinifera was the first invertebrate gonadotropin to have its chemical structure identified. However, it is unclear whether gonadotropic hormones in other species starfish are relaxin-like peptides. Thus, this study tried to identify the molecular structure of gonadotropic hormone in Asterias amurensis. As a result, we identified A. amurensis gonadotropic hormone as the RGP (AamRGP). The DNA sequence encoding AamRGP consisted of 330 base pairs with an open reading frame encoding a peptide of 109 amino acids (aa), including a signal peptide (26 aa), B-chain (20 aa), C-peptide (38 aa) and A-chain (25 aa). Comparing with A. pectinifera RGP (ApeRGP), the amino acid identity levels between AmaRGP and ApeRGP were 58% for the A-chain and 73% for the B-chain. Furthermore, chemical synthetic AamRGP induced gamete spawning and oocyte maturation in ovarian fragments of A. amurensis. In contrast, the ovary of A. pectinifera failed to respond to the AamRGP. This suggested that AamRGP is a new relaxin-like peptide.

Starvation beneficially influences the liver physiology and nutrient metabolism in Edwardsiella tarda infected red sea bream (Pagrus major).

Dietary compromises, especially food restrictions, possess species-specific effects on the health status and infection control in several organisms, including fish. To understand the starvation-mediated physiological responses in Edwardsiella tarda infected red sea bream, especially in the liver, we performed a 20-day starvation experiment using 4 treatment (2 fed and 2 starved) groups, namely, fed-placebo, starved-placebo, fed-infected, and starved-infected, wherein bacterial exposure was done on the 11th day. In the present study, the starved groups showed reduced hepatosomatic index and drastic depletion in glycogen storage and vacuole formation. The fed-infected fish showed significant (P<0.05) increase in catalase and superoxide dismutase activity in relation to its starved equivalent. Significant (P<0.05) alteration in glucose and energy metabolism, as evident from hexokinase and glucose-6-phosphate dehydrogenase activity, was recorded in the starved groups. Interestingly, coinciding with the liver histology, PPAR (peroxisome proliferator activated receptors) α transcription followed a time-dependent activation in starved groups while PPARγ exhibited an opposite pattern. The transcription of hepcidin 1 and transferrin, initially increased in 0dai (days after infection) starved fish but reduced significantly (P<0.05) at later stages. Two-color immunohistochemistry and subsequent cell counting showed significant increase in P63-positive cells at 0dai and 5dai but later reduced slightly at 10dai. Similar results were also obtained in the lysosomal (cathepsin D) and non-lysosomal (ubiquitin) gene transcription level. All together, our data suggest that starvation exerts multidirectional responses, which allows for better physiological adaptations during any infectious period, in red sea bream.

Sexually dimorphic expression of the sex chromosome-linked genes cntfa and pdlim3a in the medaka brain.

In vertebrates, sex differences in the brain have been attributed to differences in gonadal hormone secretion; however, recent evidence in mammals and birds shows that sex chromosome-linked genes, independent of gonadal hormones, also mediate sex differences in the brain. In this study, we searched for genes that were differentially expressed between the sexes in the brain of a teleost fish, medaka (Oryzias latipes), and identified two sex chromosome genes with male-biased expression, cntfa (encoding ciliary neurotrophic factor a) and pdlim3a (encoding PDZ and LIM domain 3 a). These genes were found to be located 3-4 Mb from and on opposite sides of the Y chromosome-specific region containing the sex-determining gene (the medaka X and Y chromosomes are genetically identical, differing only in this region). The male-biased expression of both genes was evident prior to the onset of sexual maturity. Sex-reversed XY females, as well as wild-type XY males, had more pronounced expression of these genes than XX males and XX females, indicating that the Y allele confers higher expression than the X allele for both genes. In addition, their expression was affected to some extent by sex steroid hormones, thereby possibly serving as focal points of the crosstalk between the genetic and hormonal pathways underlying brain sex differences. Given that sex chromosomes of lower vertebrates, including teleost fish, have evolved independently in different genera or species, sex chromosome genes with sexually dimorphic expression in the brain may contribute to genus- or species-specific sex differences in a variety of traits.

Identification and expression profiles of prdm1 in medaka Oryzias latipes.

Mouse Prdm1, also known as Blimp1, plays important roles in maturation and survival of lymphoid cells, as well as in organogenesis of muscle, limb, sensor organs and primordial germ cells. The homologues of mouse prdm1 have been identified in a diverse of animals including zebrafish and fugu. Here, we report the identification and expression profiles of two homologues of prdm1, namely prdm1a and prdm1b in medaka, Oryzias latipes. The transcripts of prdm1a and prdm1b were detectable in all the tissues including immune organs such as gill, spleen, kidney, liver and intestine that we have checked on. The transcripts of prdm1a could be detected in the embryonic shield at mid-gastrula stage and later in the somite, eye, otic vesicle, branchial arches, fin, intestine and cloaca during embryogenesis using in situ hybridization. Moreover, the expression of prdm1a in the liver of both medaka and zebrafish could be up-regulated by the immune stimuli including lipopolysaccharide, polyI:C and the grass carp reovirus, similarly to the up-regulation of IL1B. These results indicate that Prdm1a may play important roles in embryogenesis and also in immune response in fish.

Characterization of gonadal soma-derived factor expression during sex change in the protogynous wrasse, Halichoeres trimaculatus.

BACKGROUND: Sex change in fishes provides a good experimental model for understanding the mechanisms and plasticity of sex determination and differentiation. The three-spot wrasse, Halichoeres trimaculatus is a protogynous hermaphrodite. During sex change from female to male, the ovary is replaced by the testis through the degeneration of oocytes and subsequent spermatogenesis. In the present study, we cloned a cDNA-encoding gonadal soma-derived factor (GSDF) from protogynous wrasse and examined its expression pattern in the sexually mature gonads and the sex-changing gonad induced experimentally by aromatase inhibition. RESULTS: Expression of gsdf was predominantly observed in the testis, and it was mainly localized to the supporting cells surrounding the spermatogonia. In the ovary, only slight expression of gsdf was observed in morphologically undifferentiated supporting cells in contact with oogonia. During sex change, strong expression of gsdf appeared first in the supporting cells surrounding the gonial germ cells before the onset of spermatogenesis. Thereafter, the expression of gsdf continually increased in the supporting cells surrounding the proliferating spermatogonia throughout the sex change. CONCLUSIONS: These results suggest that gsdf is involved in the proliferation of spermatogonia and subsequent spermatogenesis in both the testis and the gonad in the early stages of sex change.

An oocyte-specific astacin family protease, alveolin, is released from cortical granules to trigger egg envelope hardening during fertilization in medaka (Oryzias latipes).

It has long been hypothesized that in fishes the contents of cortical granules are involved in the hardening of egg envelope following fertilization. We previously purified the egg envelope hardening initiation factor from the exudates released from activated medaka (Oryzias latipes) eggs and tentatively termed this protein alveolin. Alveolin is a member of the astacin metalloprotease family and was proposed to be a protease which hydrolyzes ZPB at one restricted position to allow starting cross-linking with ZPC. Here, we investigated the complete pathway from biosynthesis and accumulation to secretion of alveolin. A single alveolin transcript was detected only in ovarian preparations, confirming the specific expression of alveolin in the ovary. In situ hybridization indicated that the alveolin mRNA is already expressed in the very early previtellogenic oocytes. However, immunocytochemical studies revealed that the appearance of alveolin protein was delayed until the beginning of the vitellogenic stage. The cortical granules isolated from unfertilized eggs contained a high molecular weight form of glycosylated alveolin with a 50kDa relative molecular mass. Hypotonic treatment burst isolated granules in vitro and transformed alveolin to a 21.5kDa form, which is the same size as that of natural alveolin released from eggs upon fertilization. This transformation was inhibited in the presence of leupeptin and 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), suggesting that a serine protease is involved in alveolin activation upon fertilization. Furthermore, the phylogenetic relationship of alveolin with other vertebrate astacin family members was analyzed. The result shows that alveolin and its teleostean homologs make a new group which is separate from either the hatching enzyme, meprin and BMP1/tolloid groups.

Ol4E-T, a eukaryotic translation initiation factor 4E-binding protein of medaka fish (Oryzias latipes), can interact with nanos3 and vasa in vitro.

Maternal factors have essential roles in the specification and development of germ cells in metazoans. In Drosophila, a number of genes such as oskar, vasa, nanos, and tudor are required for specific steps in pole cell formation and further germline development. Drosophila cup, another maternal factor, is confirmed as a main factor in normal oogenesis, maintenance, and survival of female germ-line stem cells by interaction with Nanos. Through searching for the homolog of Drosophila cup in the medaka, the homolog of eukaryotic translation initiation factor 4E (eIF4E)-transporter, named Ol4E-T, was identified. Reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization revealed that Ol4E-T is maternally deposited in the embryo and Ol4E-T expression is maintained throughout embryogenesis. Ol4E-T is predominantly expressed in the adult gonads. In the testes, Ol4E-T is expressed in the same regions where medaka vasa, named olvas is expressed. In the ovary, expression of Ol4E-T conforms to that of nanos3 and olvas. Ol4E-T harbors a well-conserved eIF4E-binding motif, YTKEELL, by which Ol4E-T interacts with eIF4E in medaka. Additionally, Ol4E-T can interact with medaka Nanos3 and Olvas, as shown by yeast two hybridization. The spatial expression and interactions between Ol4E-T with germ cell markers Olvas and Nanos3 suggest a role for Ol4E-T in germ-line development in medaka.

Dmrt1 mutation causes a male-to-female sex reversal after the sex determination by Dmy in the medaka.

DMRT1, which is found in many vertebrates, exhibits testis-specific expression during the sexual differentiation period, suggesting a conserved function of DMRT1 in the testicular development of vertebrate gonads. However, functional analyses have been reported only in mammals. The current study focused on the Dmrt1 function in the teleost medaka, Oryzias latipes, which has an XX-XY sex determination system. Although medaka sex is determined by the presence or absence of the Y chromosome-specific gene Dmy, we demonstrated that in one Dmrt1 mutant line, which was found by screening a gene-driven mutagenesis library, XY mutants developed into normal females and laid eggs. Histological analyses of this mutant revealed that the XY mutant gonads first developed into the normal testis type. However, the gonads transdifferentiated into the ovary type. The mutant phenotype could be rescued by transgenesis of the Dmrt1 genomic region. These results show that Dmrt1 is essential to maintain testis differentiation after Dmy-triggered male differentiation pathway.

Developmental tracing of luteinizing hormone ß-subunit gene expression using green fluorescent protein transgenic medaka (Oryzias latipes) reveals a putative novel developmental function.

BACKGROUND: Luteinizing hormone (LH) and follicle stimulating hormone (FSH), produced in gonadotrope cells in the adenohypophysis are key regulators of vertebrate reproduction. The differential regulation of these hormones, however, is poorly understood and little is known about gonadotrope embryonic development. We developed a stable transgenic line of medaka with the LH beta subunit gene (lhb) promotor driving green fluorescent protein (gfp) expression to characterize development of LH-producing gonadotropes in whole larvae and histological sections. Additionally, developmental and tissue-specific gene expression was examined. RESULTS: The lhb gene is maternally expressed during early embryogenesis. Transcript levels increase by stage 21 (36 hours post fertilization [hpf]) and then decrease during continued larval development. Examination of the expression of pituitary marker genes show that LH-producing cells are initially localized outside the primordial pituitary, and they were localized to the developing gut tube by 32 hpf. At hatching, lhb-GFP is clearly detected in the gut epithelium and in the anterior digestive tract. lhb-GFP expression later consolidate in the developing pituitary by 2 weeks postfertilization. CONCLUSIONS: During embryonic development, lhb is primarily expressed outside the central nervous system and pituitary. The novel expression of lhb in the embryonic gut suggests that LH has a hitherto unidentified developmental function.

R-spondins are involved in the ovarian differentiation in a teleost, medaka (Oryzias latipes).

BACKGROUND: In mammals, R-spondin (Rspo), an activator of the Wnt/ß-catenin signaling pathway, has been shown to be involved in ovarian differentiation. However, the role of the Rspo/Wnt/ß-catenin signaling pathway in fish gonads is still unknown. RESULTS: In the present study, full-length cDNAs of Rspo1, 2 and 3 were cloned from the gonads of medaka (Oryzias latipes). The deduced amino acid sequences of mRspo1-3 were shown to have a similar structural organization. Phylogenetic analysis showed that Rspo1, 2 and 3 were specifically clustered into three distinct clads. Tissue distribution revealed that three Rspo genes were abundantly expressed in the brain and ovary. Real-time PCR analysis around hatching (S33-5dah) demonstrated that three Rspo genes were specifically enhanced in female gonads from S38. In situ hybridization (ISH) analysis demonstrated that three Rspo genes were expressed in the germ cell in ovary, but not in testis. Fluorescence multi-color ISH showed that Rspo1 was expressed in both somatic cells and germ cells at 10dah. Exposure to ethinylestradiol (EE2) in XY individuals for one week dramatically enhanced the expression of three Rspo genes both at 0dah and in adulthood. CONCLUSIONS: These results suggest that the Rspo-activating signaling pathway is involved in the ovarian differentiation and maintenance in medaka.

Specific expression of Olpiwi1 and Olpiwi2 in medaka (Oryzias latipes) germ cells.

Piwi is necessary for germ stem cell survival in Drosophila and homologues have been identified in a diverse range of organisms. Here, we identify and characterize two homologous genes of piwi, Olpiwi1 and Olpiwi2, in the model fish medaka (Oryzias latipes). Olpiwi1 is similar to Ziwi in zebrafish or Miwi in the mouse, and Olpiwi2 is similar to Zili in zebrafish or Mili in the mouse. Moreover, Olpiwi2 mRNA is produced from two different chromosomes. RT-PCR showed expression of Olpiwi1 and Olpiwi2 predominantly in the gonads. In situ hybridization revealed germ cell-specific expression of Olpiwi1 and Olpiwi2 throughout the development of oocytes from oogonia to mature oocytes in the ovary, and from spermatogonia to spermatocytes in the testes of adults. RT-PCR and whole mount in situ hybridization showed that both Olpiwi1 and Olpiwi2 were maternally deposited in the embryo. Olpiwi1 and Olpiwi2 were detected in primordial germ cells during embryonic development. These results suggest that both Olpiwi1 and Olpiwi2 are germ cell specific, and may play important roles in germ cell development and gametogenesis in this model species.

Female-specific target sites for both oestrogen and androgen in the teleost brain.

To dissect the molecular and cellular basis of sexual differentiation of the teleost brain, which maintains marked sexual plasticity throughout life, we examined sex differences in neural expression of all subtypes of nuclear oestrogen and androgen receptors (ER and AR) in medaka. All receptors were differentially expressed between the sexes in specific nuclei in the forebrain. The most pronounced sex differences were found in several nuclei in the ventral telencephalic and preoptic areas, where ER and AR expression were prominent in females but almost completely absent in males, indicating that these nuclei represent female-specific target sites for both oestrogen and androgen in the brain. Subsequent analyses revealed that the female-specific expression of ER and AR is not under the direct control of sex-linked genes but is instead regulated positively by oestrogen and negatively by androgen in a transient and reversible manner. Taken together, the present study demonstrates that sex-specific target sites for both oestrogen and androgen occur in the brain as a result of the activational effects of gonadal steroids. The consequent sex-specific but reversible steroid sensitivity of the adult brain probably contributes substantially to the process of sexual differentiation and the persistent sexual plasticity of the teleost brain.

Expression patterns of gonadotropin hormones and their receptors during early sexual differentiation in Nile tilapia Oreochromis niloticus.

In Nile tilapia, sex-specific expression of foxl2 and cyp19a1a in XX gonads and dmrt1 in XY gonads at 5-6 days after hatching (dah) is critical for differentiation of the gonads into either ovaries or testes. The factors triggering sexually dimorphic expression of these genes are unknown, and whether the gonadotropin hormones are involved in early gonadal sex differentiation of the Nile tilapia has been unclear. In the present study, we determined the precise timing of expression of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the pituitary and that of their receptors (fshra and lhcgrbb) in the undifferentiated gonad in both XX and XY tilapia fry by quantitative RT-PCR and immunohistochemical analysis. Expression of fshb mRNA and Fsh protein in the pituitary was detected from the first sampling day (3 dah) to 25 dah in both XX and XY tilapia larvae without sexual dimorphism and increased gradually after 25 dah in the pituitary. fshra mRNA was expressed beginning 5 dah and was present at significantly higher levels in XX gonads than in the XY gonads at 6-25 dah. These results indicate that the level of Fsh protein in the pituitary was not critical for differentiation of gonads into ovaries or testes, but the expression level of its receptor, fshra, in undifferentiated gonads appeared to be involved in determining gonadal sexual differentiation. Based on these observations, it is likely that in XX gonads, up-regulation of fshra may be necessary to induce cyp19a1a expression, which stimulates estradiol-17beta (E(2)) production and subsequent ovarian differentiation. On the other hand, lhb mRNA was not detected until 25 dah in the pituitaries of both sexes, and sexual dimorphism in lhcgrbb mRNA levels appeared later (10-25 dah) than that of fshra in the gonads, indicating the limited role of LH and lhcgrbb in gonadal differentiation of the Nile tilapia.

Histological observation of the urogenital papillae in the bi-directional sex-changing gobiid fish, Trimma okinawae.

The gobiid fish Trimma okinawae changes its sex bi-directionally according to its social status. Morphological changes in the urinogenital papillae (UGP) of this fish have been reported during sex change. However, there have been no detailed observations of such changes. Here, we histologically examined the UGP structure of male- and female-phase fish. UGPs of fish in female and male phase contained both oviducts and sperm ducts. Both ducts were coalesced into one duct within the posterior region of the UGP. Female-phase fish had many longitudinal folds in the hypertrophied tunica mucosa of the oviduct, which was found to be responsible for the transport of eggs and the removal of follicular cells from the oocyte. In contrast, male-phase fish had an immature oviduct and a mature sperm duct in the UGP. In the male-phase fish, the co-existence of spermatozoa and fibrillar secretions was observed in the sperm duct during spermiation.

Participation of Gs-proteins in the action of relaxin-like gonad-stimulating substance (GSS) for 1-methyladenine production in starfish ovarian follicle cells.

Gonad-stimulating substance (GSS) in starfish is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to gonadotropins in vertebrates. Recently, we purified GSS from radial nerves in the starfish Asterina pectinifera and identified the chemical structure as a heterodimer composed of two different peptides (A- and B-chain) with disulfide cross-linkages. This study examined the hormonal action of GSS on ovarian follicle cells obtained from ovaries in growing (stage IV) and fully grown (stage V) stages, and particularly the mode of signal transduction. The action of GSS on 1-MeAde production by follicle cells in stage V was mediated through the production of cAMP. In contrast, GSS failed to induce 1-MeAde and cAMP production by follicle cells in stage IV. According to competitive experiments using radioiodinated and radioinert GSS, highly specific binding was observed in follicle cells, though their affinities and numbers in stage IV were inferior to those in stage V. Interestingly, Gsα was not detected immunologically in follicle cell membranes of stage IV. Gß was also faint in stage IV. Although adenylyl cyclase activity in stage V was dose-dependently activated by GSS in the presence of GTP, neither GSS in the presence of GTP nor nonhydrolyzable GTP analogs were effective on the activity in stage IV. These findings strongly suggest that the failure of GSS to produce 1-MeAde is because of a lack of Gs-proteins in follicle cells at stage IV.

Expression of 3ß-hydroxysteroid dehydrogenase (hsd3b), star and ad4bp/sf-1 during gonadal development in medaka (Oryzias latipes).

In most vertebrates, sex steroids play a critical role in gonadal development, maturation of germ cells, and development of secondary sexual characteristics. Sex steroids are synthesized in steroid-producing cells (SPCs) in the testis known as Leydig cells, as well as in thecal and granulosa cells in the ovary. In SPCs, cholesterol is sequentially catalyzed by a set of steroidogenic factors and enzymes in order to produce sex steroids. Therefore, integrated expression of the genes involved in steroidogenesis is critical for the proper production of sex steroids. In the present study, regulatory mechanisms of steroidogenic factors and enzymes were examined. We focused on hsd3b, star and ad4bp/sf-1 as well as the description of temporal and spatial expression of these genes during gonadal development in medaka (Oryzias latipes). During testicular development, hsd3b, star and ad4bp/sf-1 were co-expressed in the interstitial somatic cells subsequent to the formation of the seminiferous tubule precursor, suggesting that ad4bp/sf-1 regulated the transcription of both hsd3b and star. During ovarian development, the expression pattern of hsd3b coincided with that of cyp11a1, but not with that of aromatase. Although ad4bp/sf-1 was mainly expressed in presumptive follicular cells, it was also detected in hsd3b positive interstitial cells in the developing ovary. Contrary to our expectations, the onset of star expression occurred during a later stage of ovarian development than the expression of other steroidogenic enzymes. Thus, the regulation mechanism of star transcription appears to differ from that of the other steroidogenic enzymes in the developing ovary, but not in the developing testis.

GATA4 is involved in the gonadal development and maturation of the teleost fish tilapia, Oreochromis niloticus.

GATA4, a member of the GATA family, is a well-known transcription factor implicated in the regulation of sex determination and sexual differentiation in mammals. However, little is known about the possible role of GATA4 in fish reproduction. In the present study, a full-length GATA4 cDNA from the tilapia was cloned and characterized. The tilapia GATA4 gene contained an open reading frame (ORF) of 1179 nucleotides encoding a protein of 392 amino acids. Sequence alignment revealed that the tilapia GATA4 protein shared higher homology (ranging from 63.1 to 74.6%) with other vertebrates. RT-PCR analysis indicated that the GATA4 gene is expressed in the ovary, testis, liver, intestine and heart in adult tilapia. In situ hybridization was performed to examine the temporal and spatial expression patterns of GATA4 during tilapia gonadal differentiation and development. In the undifferentiated gonad, GATA4 was expressed in the somatic cells of both sexes. Subsequently, GATA4 expression persisted in the differentiated, juvenile and adult ovary and testis in tilapia. Our data indicate for the first time that GATA4 is not only necessary for the onset of gonadal differentiation, but also important for gonadal development and maturation.