Early Gonad Research Articles

Temporal Differences in Granulosa Cell Specification in the Ovary Reflect Distinct Follicle Fates in Mice1

The embryonic origins of ovarian granulosa cells have been a subject of debate for decades. By tamoxifen-induced lineage tracing of Foxl2-expressing cells, we show that descendants of the bipotential supporting cell precursors in the early gonad contribute granulosa cells to a specific population of follicles in the medulla of the ovary that begin to grow immediately after birth. These precursor cells arise from the proliferative ovarian surface epithelium and enter mitotic arrest prior to upregulating Foxl2. Granulosa cells that populate the cortical primordial follicles activated in adult life derive from the surface epithelium perinatally, and enter mitotic arrest at that stage. Ingression from the surface epithelium dropped to undetectable levels by Postnatal Day 7, when most surviving oocytes were individually encapsulated by granulosa cells. These findings add complexity to the standard model of sex determination in which the Sertoli and granulosa cells of the adult testis and ovary directly stem from the supporting cell precursors of the bipotential gonad.

Disruption of a long distance regulatory region upstream of SOX9 in isolated disorders of sex development

BackgroundThe early gonad is bipotential and can differentiate into either a testis or an ovary. In XY embryos, the SRY gene triggers testicular differentiation and subsequent male development via its...

Expression and Functional Analysis of Dkk1 during Early Gonadal Development

WNT signalling plays a central role in mammalian sex determination by promoting ovarian development and repressing aspects of testis development in the early gonad. Dickkopf homolog 1 (DKK1) is a WNT signalling antagonist that plays critical roles in multiple developmental systems by modulating WNT activity. Here, we examined the role of DKK1 in mouse sex determination and early gonadal development. Dkk1 mRNA was upregulated sex-specifically during testis differentiation, suggesting that DKK1 could repress WNT signalling in the developing testis. However, we observed overtly normal testis development in Dkk1-null XY gonads, and found no significant upregulation of Axin2 or Sp5 that would indicate increased canonical WNT signalling. Nor did we find significant differences in expression of key markers of testis and ovarian development. We propose that DKK1 may play a protective role that is not unmasked by loss-of-function in the absence of other stressors.

Fibroblast Growth Factor-9 in Marsupial Testicular Development

FGF9 is a member of the fibroblast growth factor (FGF) family and is critical for early testicular development and germ cell survival in the mouse. Fgf9 reinforces the testis determinant Sox9 and antagonizes Wnt4, an ovarian factor. To determine whether FGF9 has a conserved role in the mammalian gonad, we examined its expression in the gonads of a marsupial, the tammar wallaby Macropus eugenii, and compared it to WNT4 expression. Marsupial FGF9 is highly conserved with orthologues from eutherian mammals, including humans. FGF9 protein was detected in both the testis and ovary before sexual differentiation, but it subsequently became sexually dimorphic during the period of testicular differentiation. The protein was specifically enriched in the seminiferous cords of the developing testis in the Sertoli and germ cells. FGF9 mRNA expression was upregulated in the tammar testis at the time of seminiferous cord formation and downregulated in the developing ovary in an opposite profile to that of marsupial WNT4. These observations suggest that FGF9 promotes male fate in the early gonad of marsupials through an antagonistic relationship with WNT4 as it does in eutherian mammals.

Ecological basis of heterochrony in early-ontogenesis gonad development in different races of Kura sturgeon (Acipenser guldenstadti persicus Borodin)

Early germ cell development in Persian sturgeon appears to be faster in the marine environment than in aquaculture conditions. Adaptation plasticity differs significantly for gonad formation and sex determination in the sturgeon fry and depends to differing extents on the temperature. The sturgeon gonads are more sensitive to low temperatures on the early stages of sex determination than in the period of gonad formation; the differences between sexes are preconditioned by the earlier start of sex determination in females when compared to males. The rates of the early gonad and germ cell development in sturgeon are conditioned by its reproduction season. In the population of the late spring race, the delay in both early sex determination and germ cell development stays even for the ages of 1+, 2+, and 3+ when compared to the population of the early spring race. The biophysiological quality of parents is transmitted to their offspring.

Sex determination and the control of Sox9 expression in mammals

In mouse sex determination, the presence or absence of Sertoli cells in the developing gonad is essential for the decision to form either a testis or an ovary. The transcription factor SOX9 has emerged as the master regulator of Sertoli cell differentiation during testis development and thus the crucial gene to determine sex. It is the target of two sets of regulatory controls, one positive and one negative, where one set tries to gain dominance over the other in the early gonad and then to establish and maintain the activity or silence of Sox9 throughout life. The data reveal the importance of the positive regulatory loops to reinforce initial decisions, whereas the maintenance of the gonadal phenotype appears to rely on the active repression of the opposite pathway.

Identification of SOX3 as an XX male sex reversal gene in mice and humans

Sex in mammals is genetically determined and is defined at the cellular level by sex chromosome complement (XY males and XX females). The Y chromosome-linked gene sex-determining region Y (SRY) is believed to be the master initiator of male sex determination in almost all eutherian and metatherian mammals, functioning to upregulate expression of its direct target gene Sry-related HMG box-containing gene 9 (SOX9). Data suggest that SRY evolved from SOX3, although there is no direct functional evidence to support this hypothesis. Indeed, loss-of-function mutations in SOX3 do not affect sex determination in mice or humans. To further investigate Sox3 function in vivo, we generated transgenic mice overexpressing Sox3. Here, we report that in one of these transgenic lines, Sox3 was ectopically expressed in the bipotential gonad and that this led to frequent complete XX male sex reversal. Further analysis indicated that Sox3 induced testis differentiation in this particular line of mice by upregulating expression of Sox9 via a similar mechanism to Sry. Importantly, we also identified genomic rearrangements within the SOX3 regulatory region in three patients with XX male sex reversal. Together, these data suggest that SOX3 and SRY are functionally interchangeable in sex determination and support the notion that SRY evolved from SOX3 via a regulatory mutation that led to its de novo expression in the early gonad.

Primordial germ cell proliferation is impaired in Fused Toes mutant embryos

Over the first 4days of their life, primordial germ cells invade the endoderm, migrate into and through the developing hindgut, and traverse to the genital ridge where they cluster and ultimately inhabit the nascent gonad. Specific signal–receptor combinations between primordial germ cells and their immediate environment establish successful migration and colonization. Here we demonstrate that disruption of a cluster of six genes on murine chromosome 8, as exemplified by the Fused Toes (Ft) mutant mouse model, results in severely decreased numbers of primordial germ cells within the early gonad. Primordial germ cell migration appeared normal within Ft mutant embryos; however, germ cell counts progressively decreased during this time. Although no difference in apoptosis was detected, we report a critical decrease in primordial germ cell proliferation by E12.5. The six genes within the Ft locus include the IrxB cluster (Irx3, -5, -6), Fts, Ftm, and Fto, of which only Ftm, Fto, and Fts are expressed in primordial germ cells of the early gonad. From these studies, we have discovered that the Ft locus on mouse chromosome 8 is associated with cell cycle deficits within the primordial germ cell population that initiates just before translocation into the genital ridge.

Protein tyrosine kinase 2 beta (PTK2B), but not focal adhesion kinase (FAK), is expressed in a sexually dimorphic pattern in developing mouse gonads

Sexual reproduction is essential for the propagation and the maintenance of fitness of our species, and is dependent on the correct development of the bipotential genital ridges into testes and ovaries. Although several transcription factors, secreted signaling molecules, and their receptors have been found to be important for testis determination and early gonad development, comparatively little research has been carried out into intracellular signal transduction pathways activated during these processes. Focal adhesion kinase (FAK) and protein tyrosine kinase 2 beta (PTK2B) form one group of cytosolic tyrosine kinases that are known to be important for processes such as cell proliferation, differentiation, and motility. Here, we describe the temporal and spatial expression patterns of Fak and Ptk2b mRNA and protein during sex determination and early gonadogenesis in mouse embryos. Ptk2b mRNA and PTK2B protein were expressed in testes from 11.5 days post coitum onward, predominantly in developing Sertoli cells, in a SOX9-dependent manner. Fak mRNA and FAK protein were expressed in gonads of both sexes at all stages examined. Our data suggest cell type- and stage-specific roles for PTK2B during early testis development.

Gonadal development in a giant threatened reef fish, the humphead wrasse Cheilinus undulatus, and its relationship to international trade

An opportunity arose to obtain humphead wrasse Cheilinus undulatus specimens between 2006 and 2009 from Indonesia, the major source and exporting country of this species, making study on its early gonad development possible for the first time. Protogynous hermaphroditism, previously proposed for this species, was confirmed in this study. Based on histological examination of 178 specimens, mainly <500 mm total length (L(T)) and ranging from 208 to 1290 mm L(T) (119.1 g to 43.0 kg whole body mass), the minimum body sizes for female and male sexual maturation were determined to be 650 and 845 mm L(T), respectively. Primary male development through juvenile sexual differentiation was not detected. A unique blind pouch, with a possible sperm storage function and associated with the testis, was reported for the first time in the Labridae. In Hong Kong retail markets, the global trading centre for this valuable species, live C. undulatus on sale for food were dominated by body sizes <500 mm L(T) between 1995 and 2009, reflecting an international trade largely focused on juveniles. In consideration of these findings, and given the threatened status of this species, management for C. undulatus capture and trade nationally and internationally are discussed with recommendations for ensuring sufficient spawning biomass in exploited populations and for sustainable trade.

Expression of SOX3 in the urogenital ridge is associated with XX male sex reversal in mice

Sox3 is expressed in the progenitor cells of the embryonic CNS and functions as a dosage‐dependent regulator of CNS development in mice and man. To further investigate the role of SOX3, we generated transgenic mice using a Sox3 genomic BAC fragment. Unexpectedly, progeny from one line (termed Sex reversed (Sr)) exhibited a markedly distorted sex ratio in favour of male offspring. Analysis of the reproductive tract in adult sex‐reversed animals indicated that all male‐specific structures were present, indicating complete XX male sex reversal. Transgene expression analysis in Sr embryos demonstrated that Sox3 is ectopically upregulated in the primordium of XX and XY gonads during sex determination due to a position effect. Importantly, morphological, gene expression and co‐transfection studies indicate that SOX3 induces testis differentiation in Sr embryonic gonads by activation of Sox9 in a mechanism analogous to that used by the Y‐linked sex determining gene Sry. Together, these data suggest that SOX3 and SRY proteins are functionally interchangeable in sex determination and provide support for the notion that Sry evolved from Sox3 via a regulatory mutation that led to its de novo expression in the early gonad.

Identification of Suitable Normalizing Genes for Quantitative Real-Time RT-PCR Analysis of Gene Expression in Fetal Mouse Gonads

In biological research, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assays are commonly employed to study mRNA abundance in cells and tissues. This type of assay usually relies on assessing transcript abundance relative to constitutively expressed endogenous reference genes. Therefore, it is important that the reference genes themselves are stably expressed in the cells or tissues analyzed, independent of factors such as age, sex, disease or experimental manipulations. Since no gene is expressed at the same level in all cells at all times, suitable reference genes must be identified for the specific cellular system or tissue being investigated. Here, we sought to identify stably expressed endogenous reference genes during embryonic gonad development in the mouse. We measured the transcript abundance of 10 frequently employed normalizing genes, of which 4 were stably expressed in fetal gonads from 11.5 to 14.5 dpc irrespective of sex. Based on our analysis, we suggest that Rn18s, Rps29, Tbp and Sdha are suitable reference genes for qRT-PCR expression studies during early gonad differentiation in the mouse.

Cloning and expression of candidate sexual development genes in the cane toad (Bufo marinus)

The development of the reproductive system in bufonids (true toads) is unique in several respects: sexual differentiation occurs later than in other anurans, and toads develop a Bidder's organ, a rudimentary ovary that can be manipulated in males to produce mature oocytes. To illuminate the genesis of this unusual reproductive system, we isolated from the cane toad (Bufo marinus) the orthologues of several known vertebrate sex-determining genes, determined their primary structure, and studied their expression by reverse transcriptase-polymerase chain reaction and in situ hybridization of tissue sections. We report here that cane toad Sox9, Dmrt1, and p450aromatase (Cyp19a1) are highly homologous to their counterparts in other vertebrates. They show profiles of expression that generally follow patterns observed in other taxa, but with some novel features. Our data suggest that these genes likely play key roles in sex determination and early gonad development in bufonids.

09-P002 The transcription co-factor Cited2 functions during sex determination and early gonad development

09-P002 The transcription co-factor Cited2 functions during sex determination and early gonad development

The transcription co-factor CITED2 functions during sex determination and early gonad development

The early bi-potential mammalian gonad requires the expression of a Y-linked gene, Sry, during a brief window of time to ensure proper testis development. WT1 and its direct target gene Sf1 function during sex determination as well as in the specified testes and ovaries. We have previously shown that the transcription co-factor CITED2 interacts with WT1 to stimulate the expression of Sf1 in the adrenogonadal primordium to ensure adrenal development. We now show through genetic interactions and expression analyses that Cited2 acts in the gonad with Wt1 and Sf1 to increase the expression of Sry levels to attain a critical threshold to efficiently initiate testis development. Reducing the gene dosage of Wt1 or Sf1 in Cited2 mutant gonads was sufficient to produce partial XY sex reversal while full sex reversal was attained in mutants containing a hypomorphic Sry(POS) allele. A direct correlation was observed between XY sex reversal and reduced expression levels of Sry and Sf1 during sex determination, which indicated that Sry is a downstream target of the CITED2/WT1/SF1 regulatory pathway. Our results provide in vivo evidence for the identification of the first transcription co-factor to function during mammalian sex determination, as part of the WT1/SF1 regulatory mechanism. This highlights the gene dosage sensitivity of the pathway's effect on Sry levels and embryonic gonad development.

Novel markers of early ovarian pre‐granulosa cells are expressed in an Sry‐like pattern

Mammalian gonad differentiation involves sexually dimorphic cell-fate decisions within the bipotential gonadal primordia. Testis differentiation is initiated by a center-to-poles wave of Sry expression that induces supporting cell precursors (SCPs) to become Sertoli rather than granulosa cells. The initiation of ovary differentiation is less well understood. We identified two novel SCP markers, 1700106J16Rik and Sprr2d, whose expression is ovary-biased during early gonad development, and altered in Wnt4, Sf1, Wt1, and Fog2 mutant gonads. In XX and XY gonads, both genes were up-regulated at approximately E11 in a center-to-poles wave, and then rapidly down-regulated in XY gonads in a center-to-poles wave, which is reminiscent of Sry expression in XY gonads. Our data suggest that 1700106J16Rik and Sprr2d may have important roles in early gonad development, and are consistent with the hypothesis that ovarian SCP differentiation occurs in a center-to-poles wave with similar timing to that of testicular SCP differentiation.

The 17β-hydroxysteroid dehydrogenase 4: Gender-specific and seasonal gene expression in the liver of brown trout (Salmo trutta f. fario)

Previously, it was documented that liver peroxisomes display seasonal size changes in the adult Salmo trutta fario, especially in females (and negatively correlated with ovary maturation). It was then hypothesized that decreases in peroxisome size could be paralleled by changes in peroxisomal beta-oxidation and estradiol catabolism actions. The 17beta-hydroxysteroid dehydrogenase 4 has been portrayed as playing an important role in both processes. To elucidate its function in the described peroxisomal pattern, we isolated the cDNA and predicted the protein sequence of the enzyme in that species. The seasonal gene expression pattern in both genders was addressed through quantitative PCR. Fish sampling was in post-spawning period, early and advanced gonad maturation, and pre-spawning. Males did not vary seasonally. As to females, a seasonal pattern was evidenced according to our previous hypothesis. We suggest that the decreased levels observed during vitellogenesis are related to lipid needs for ovary maturation, and, additionally, with the need of modulating estradiol titers.

Seasonal and depth-dependent variations in the size and lipid contents of stage 5 copepodites of Calanus finmarchicus in the waters of the Newfoundland Shelf and the Labrador Sea

We report on the variation in energy reserves of individual C5 copepodites of Calanus finmarchicus from the Newfoundland continental shelf and the Labrador Sea collected from surface and overwintering (or bottom) depths prior to, during and after the expected timing of the onset of diapause. Overall, the trend was for a decreasing average prosome length as the year progressed for all locations although the decline was smallest in the Labrador Sea and greatest in the deep waters of the continental shelf. The size of the oil sac was closely linked to the weight of the copepodite but the form of this relationship showed substantial variations with depth and season. We show a clear increase in the relative oil sac volume for C. finmarchicus between late spring and late summer, by which time some animals had descended to diapause depths. The progressive decrease in oil sac volume of animals sampled at depth in the Labrador Sea between September and December suggests a significant loss of energy reserves during diapause. From the distribution of volumes and body sizes in December we estimate that 23–53% of individuals would not be able to meet the energetic cost of moulting and early gonad development. Overall, some of our observations appear to invalidate earlier hypotheses concerning the governing role of lipids in the life history of C. finmarchicus. However, assessment of the factors that influence entry into dormancy should be based on the relative probabilities of alternative strategies for successful reproduction (e.g. entering dormancy vs. continuing into a second generation).

Gonadal Sex Differentiation in Cultured Juvenile Flounder, Paralichthys orbignyanus (Valenciennes, 1839)

Gonadal Sex Differentiation in Cultured Juvenile Flounder, <i>Paralichthys orbignyanus</i> (Valenciennes, 1839)

Gene discovery from an ovary cDNA library of oriental river prawn Macrobrachium nipponense by ESTs annotation

The oriental river prawn, Macrobrachium nipponense, is an important crustacean species in aquaculture. However, early gonad maturity is a ubiquitous problem which devalues the product quality. While husbandry and nutritional management have achieved little success in tackling this issue, a molecular approach may discover the genes involved in reproduction and development, which will provide the basic knowledge on reproductive control. In this study, a high-quality cDNA library of prawn was constructed from the ovary tissue. A total of 3294 successful sequencing reactions yielded 3256 expressed sequence tags (ESTs) longer than 100 bp. The cluster and assembly analyses yielded 1514 unique sequences including 414 contigs and 1168 singletons. About 719 (47.49%) unique sequences were identified as orthologs of genes from other organisms. By sequence comparability analysis, 28 important genes including cathepsin B, chromobox protein, Cdc2, cyclin B, DEAD box protein and ADF/cofilin protein were expressed. These genes may be involved in reproductive and developmental functions in prawn. Peritrophin consisting of cortical rods was also found in this species. The identification of these EST sequences in M. nipponense would improve our understanding on the genes that regulate reproduction and development in prawn species. This study also lays the groundwork for development of molecular markers related to ovary development in other prawn species.