Eki2 is upregulated specifically in the testis during mouse sex determination

We have identified a gene with gonad restricted expression throughout mouse development, which is orthologous to human EKI2 (ethanolamine kinase 2). Our studies showed that mouse Eki2 expression became upregulated in the male gonad during the period of sex determination. Expression was restricted to the Sertoli cells of the developing testis. Eki2 has sequence similarity to ethanolamine (73%) and choline kinases (54%).     

The Maestro (Mro) Gene Is Dispensable for Normal Sexual Development and Fertility in Mice

The mammalian gonad arises as a bipotential primordium from which a testis or ovary develops depending on the chromosomal sex of the individual. We have previously used DNA microarrays to screen for novel genes controlling the developmental fate of the indifferent embryonic mouse gonad. Maestro (Mro), which encodes a HEAT-repeat protein, was originally identified as a gene exhibiting sexually dimorphic expression during mouse gonad development. Wholemount in situ hybridisation analysis revealed Mro to be expressed in the embryonic male gonad from approximately 11.5 days post coitum, prior to overt sexual differentiation. No significant expression was detected in female gonads at the same developmental stage. In order to address its physiological function, we have generated mice lacking Maestro using gene targeting. Male and female mice homozygous for a Mro null allele are viable and fertile. We examined gonad development in homozygous male embryos in detail and observed no differences when compared to wild-type controls. Immunohistochemical analysis of homozygous mutant testes of adult mice revealed no overt abnormalities. Expression profiling using DNA microarrays also indicated no significant differences between homozygote embryonic male gonads and controls. We conclude that Maestro is dispensable for normal male sexual development and fertility in laboratory mice; however, the Mro locus itself does have utility as a site for insertion of transgenes for future studies in the fields of sexual development and Sertoli cell function.     

Sox15 is up regulated in the embryonic mouse testis

The mammalian sex determining region on the Y chromosome, SRY, is the founding member of the SOX gene family. SOX genes share a common DNA-binding motif termed the HMG box and have diverse roles in vertebrate embryonic development and tissue differentiation. Sox15 expression was analysed during mouse embryogenesis by whole-mount in situ hybridisation and Real Time RT-PCR. Sox15 was found to be expressed in developing mouse gonads from 11.5 dpc to 13.5 dpc with a peak of expression at 12.5 dpc. Expression was approximately twice as high in the male gonad as in the female gonad.     

Identification of a novel population of adrenal-like cells in the mammalian testis

Steroidogenic cells of the adrenal and gonad are thought to be derived from a common primordium that divides into separate tissues during embryogenesis. In this paper, we show that cells with mixed adrenal and Leydig cell properties are found dispersed in the insterstitium of the embryonic and adult mouse testis. They express the adrenal markers Cyp11b1 and Cyp21 and respond to ACTH. Consistent with these properties, we show that the embryonic testis produces the adrenal steroid corticosterone. These cells also express Cyp17 and respond to hCG stimulation but do not express the Leydig specific marker Insl3 showing that they are a population of steroidogenic cells distinct from Leydig cells. Based on their properties, we refer to these cells as adrenal-like cells of the testis and propose that they are the mouse equivalent of the precursors of human adrenal rests, tumors found primarily in male patients with congenital adrenal hyperplasia. Organ culture studies show that ACTH-responsive cells are present at the gonad/mesonephros border and seem to migrate into the XY but not the XX gonad during development. Consistent with this, using transgenic Cyp11a1 reporter mice, we definitively show that steroidogenic cells can migrate from the mesonephros into the XY gonad. We also show that the region between the mesonephros and the gonad harbors steroidogenic cell precursors that are repressed by the presence of the mesonephros. We propose that this region is the source of the adrenal-like cells that migrate into the testis as it develops and are activated when Leydig cells differentiate. These studies reveal the complex nature of steroidogenic cell differentiation during urogenital development.     

Where does gonadal sex differentiation begin? Gradient of histological sex differentiation in the gonads of pejerrey,Odontesthes bonariensis (Pisces,Atherinidae)

This study investigated the possibility that the histological process of gonadal sex differentiation in pejerrey (Odontesthes bonariensis), a fish with marked temperature-dependent sex determination (TSD), occurs through a predictable gradient of differentiation as opposed to simultaneous or random differentiation throughout the gonad. For this purpose, fish reared at 17 degrees, 24 degrees, and 29 degrees C from hatching were sampled weekly for 11 weeks, fixed, and prepared for histological observation of serial cross-sections of the gonads. The thermal manipulation and sampling procedure ensured the availability of males and females at various degrees of gonadal sex differentiation. The location of the differentiated area(s) was estimated in the right and left gonads of 17 females and 14 males selected among the available specimens so as to represent increasing degrees of differentiation. The analysis revealed that sex differentiation followed a gradient from the anterior to posterior areas of the gonads regardless of sex. Furthermore, plotting of the degree of sex differentiation in the right gonad as a function of the degree of differentiation of the left gonad clearly showed that sex differentiation only begins in the right gonad when 10-30% of the length of the left gonad has already differentiated. The mean rostral edge of the differentiated areas in females was 9% and 10.8% for the left and right gonads, respectively, while for males these values were 7.3% and 7.0%, respectively. Thus, it was established that ovarian and testicular differentiation in pejerrey follow both a cephalocaudal and a left-to-right gradient. Possible explanations for this gradient and its relevance for TSD in pejerrey, that is, as a mechanism to prevent discrepant differentiation of male and female features within the same gonad, are discussed.     

Deciphering the Mammary Epithelial Cell Hierarchy

Clonal assays offer a powerful approach to dissecting the many events involved in the generation and maintenance of complex tissues from an undifferentiated stem cell pool. The application of such quantitative functional methodologies to studies of the hematopoietic system have been key to defining the hierarchy of progenitor subsets that reflect an irreversible stepwise process of lineage restriction. Recent studies now suggest that a similar paradigm applies to the normal mammary gland. The adult mouse mammary gland maintains a population of stem cells that generate biologically distinct and physically separable subpopulations of mammary epithelial progenitors which, in turn, generate terminally differentiated cells. Suggestive parallels between mouse and human mammary cells point to the likelihood that a similarly structured multi-step differentiation program characterizes the mammary gland from both species.     

Expression pattern of anti-Müllerian hormone (amh) in the hybrid fish complex of Squalius alburnoides

In fish of the Squalius alburnoides complex, hybridisation and polyploidy have affected sex ratios, resulting in strong correlations between sex and genotype. The preponderance of females among triploids and the occurrence of an all male lineage among diploids seem to imply that sex ratio deviations should have a strong genetic basis. Until now, no information has been gathered regarding the molecular basis of sex determination in this intricate hybrid system. Thus, putative regulatory elements of the cascade that potentially are involved in sex determination in S. alburnoides have to be investigated. Being reported to have an important role in teleost sex determination, and more particularly in male gonad development, the anti-Müllerian hormone, amh was a good initial candidate. Here we report the isolation, cloning and characterization of the amh ortholog in S. alburnoides and the ancestral species S. pyrenaicus. In adult S. alburnoides and S. pyrenaicus of both sexes, amh shows a gonad specific expression pattern, restricted to the Sertoli cell lineage in testis and to granulosa cells in ovaries. During development, it plays an early role in male gonad differentiation in S. alburnoides. Overall the observed patterns are similar to what has been reported in other teleost species. This suggests a conserved role of amh and implies that its expression dynamics cannot be directly responsible for the sex ratio deviations reported in S. alburnoides. It is possible that a conjunction of other factors could be contributing for sex ratio imbalance. The present results constitute the starting point in the characterization of the S. alburnoides sex determination cascade, a process that we expect to shed some light on the molecular basis of sex distribution, within the context of hybrid system evolution.     

Temperature-dependent sex determination in the American alligator: expression of SF1, WT1 and DAX1 during gonadogenesis

Sex determination in mammals and birds is chromosomal, while in many reptiles sex determination is temperature dependent. Morphological development of the gonads in these systems is conserved, suggesting that many of the genes involved in gonad development are also conserved. The genes SF1, WT1 and DAX1 play various roles in the mammalian testis-determining pathway. SF1 and WT1 are thought to interact to cause male-specific gene expression during testis development, while DAX1 is believed to inhibit this male-specific gene expression. We have cloned SF1 and DAX1 from the American alligator, a species with temperature-dependent sex determination (TSD). SF1, DAX1 and WT1 are expressed in the urogenital system/gonad throughout the period of alligator gonadogenesis which is temperature sensitive. SF1 appears to be expressed at a higher level in females than in males. This SF1 expression pattern is concordant with the observed pattern during chicken gonadogenesis, but opposite to that observed during mouse gonadogenesis. Although the observed sexual dimorphism of gonadal SF1 expression in alligators and chickens is opposite that observed in the mouse, it is probable that SF1 is involved in control of gonadal steroidogenesis in all these vertebrates. DAX1 and WT1 are both expressed during stages 22-25 of both males and females. However, there appear to be no sex differences in the expression patterns of these genes. We conclude that DAX1, WT1 and SF1 may be involved in gonadal development of the alligator. These genes may form part of a gonadal-development pathway which has been conserved through vertebrate evolution.     

Germ cells are not the primary factor for sexual fate determination in goldfish

The presence of germ cells in the early gonad is important for sexual fate determination and gonadal development in vertebrates. Recent studies in zebrafish and medaka have shown that a lack of germ cells in the early gonad induces sex reversal in favor of a male phenotype. However, it is uncertain whether the gonadal somatic cells or the germ cells are predominant in determining gonadal fate in other vertebrate. Here, we investigated the role of germ cells in gonadal differentiation in goldfish, a gonochoristic species that possesses an XX-XY genetic sex determination system. The primordial germ cells (PGCs) of the fish were eliminated during embryogenesis by injection of a morpholino oligonucleotide against the dead end gene. Fish without germ cells showed two types of gonadal morphology: one with an ovarian cavity; the other with seminiferous tubules. Next, we tested whether function could be restored to these empty gonads by transplantation of a single PGC into each embryo, and also determined the gonadal sex of the resulting germline chimeras. Transplantation of a single GFP-labeled PGC successfully produced a germline chimera in 42.7% of the embryos. Some of the adult germline chimeras had a developed gonad on one side that contained donor derived germ cells, while the contralateral gonad lacked any early germ cell stages. Female germline chimeras possessed a normal ovary and a germ-cell free ovary-like structure on the contralateral side; this structure was similar to those seen in female morphants. Male germline chimeras possessed a testis and a contralateral empty testis that contained some sperm in the tubular lumens. Analysis of aromatase, foxl2 and amh expression in gonads of morphants and germline chimeras suggested that somatic transdifferentiation did not occur. The offspring of fertile germline chimeras all had the donor-derived phenotype, indicating that germline replacement had occurred and that the transplanted PGC had rescued both female and male gonadal function. These findings suggest that the absence of germ cells did not affect the pathway for ovary or testis development and that phenotypic sex in goldfish is determined by somatic cells under genetic sex control rather than an interaction between the germ cells and somatic cells.     

Characterization of the Xp21-23 region in the wood lemming, a region involved in XY sex reversal.

The wood lemming (Myopus schisticolor) harbors two types of X chromosome, a normal X and a variant X, designated X*. The X* chromosome contains a mutation that causes XY sex reversal. We have previously demonstrated that the Xp21-23 region is deleted from X* and is associated with XY sex reversal. To further analyze the deleted region, we have constructed and characterized seven X chromosome- and region-specific recombinant DNA libraries. Further, we have screened mouse fetal gonad cDNA libraries with the microdissected Xp21-23 DNA as a probe in an attempt to identify homologous and expressed sequences from the deletion. Fourteen positive clones were isolated, and sequence analyses showed that ten of these contained identical sequences homologous to mouse gamma-satellite sequences. One of the remaining four was perfectly homologous to the mouse gene Ccth (chaperonin containing t-complex polypeptide 1, eta subunit). Southern blot indicated that the Ccth cDNA was located on the X chromosome, not deleted from the X* but closely linked to the deletion region. Although the role of the Ccth containing region in sex determination of the wood lemming requires additional studies, the isolation of the mouse Ccth gene by the deletion Xp21-23 probe could be important since this gene is mainly expressed in testis.     

Molecular cloning and expression in gonad of Rana rugosa WT1 and Fgf9

Sry (sex-determining region on the Y chromosome) is required for testicular differentiation in mammals. In addition to Sry, other genes such as WT1, Fgf9, Dax1, Dmrt1 and Sox9 are widely accepted to be involved in the sex determination in vertebrates. However, the roles of these genes during sex determination still remain unclear in amphibians. This study was undertaken to examine the expression of WT1 and Fgf9 in the developing gonad of amphibians. We first isolated the WT1 cDNA from the frog Rana rugosa. Like WT1 in mice, R. rugosa WT1 showed 2 isoforms; i.e., one had an additional 3 amino acids, KTS, included between the third and fourth zinc fingers. However, 17 amino acids in exon 5 of mammalian WT1 could not be found in R. rugosa WT1, which is also the case in turtle and chicken. The mRNA of both isoforms (+KTS, -KTS) was detected in the lung, kidney and testis, but not in the ovary and muscle of adult frogs. The 2 isoforms were expressed first in the embryos at stage 23. Thereafter, the expressions remained constant in the gonad attached to mesonephros of both sexes during sex determination. We next isolated the R. rugosa Fgf9 cDNA encoding 208 amino acids. The amino acid sequence of Fgf9 had similarity greater than 92% with chicken, mouse and human Fgf9s, suggesting that Fgf9 is highly conserved among vertebrate classes. Fgf9 was expressed in the ovary of an adult frog strongly, but in the lung weakly. In contrast, the Fgf9 mRNA was hardly detected in the kidney, testis and muscle. Moreover, Fgf9 did not show a sexually dimorphic expression pattern during sex determination in R. rugosa. The results, taken together, suggest that both WT1 and Fgf9 are expressed in the indifferent gonad prior to sex determination without any difference in the expression between males and females. Thus, it seems unlikely that they are a key factor to initiate the divergence leading to testicular or ovarian differentiation in R. rugosa.     

Wnt4 expression in the differentiating gonad of the frog Rana rugosa

Wnt4, a member of the Wnt family, is known to influence the sex-determination cascade. In mice having a targeted deletion of Wnt4, masculinization occurs in XX pups. Therefore, in addition to Sry, Wnt4 is also involved in sex determination in mice. In humans, a chromosomal duplication of the WNT4 causes feminization of XY-individuals. Thus, for better understanding of the mechanism of sex determination in vertebrates, it is necessary to examine the expression of Wnt4 at early gonadal development stages in non-mammalians. We first isolated the Wnt4 cDNA from the tetsis of the frog Rana rugosa. R. rugosa Wnt4 had a high similarity (>86%) at the amino acid level with zebra fish, chicken, mouse, and human Wnt4s. We next employed RT-PCR analysis to examine whether Wnt4 was expressed in a sexually dimorphic fashion at early stages of gonadal development in R. rugosa. Wnt4 was transcribed first in the embryos at the late gastrula stage, and its expression was maintained until the indifferent gonad differentiated into a testis or an ovary. Wnt4 expression in the differentiating gonad appeared in a non-sexually dimorphic pattern. The results, taken together, suggest that Wnt4 is highly conserved through evolution, and that its expression in the indifferent gonad takes place with no sexual dimorphism. Thus, Wnt4 is not a key factor to initiate the development of a testis or an ovary from the indifferent gonads in R. rugosa. However, this gene probably forms part of a gonadal-development pathway in this species.