Gonad Morphogenesis Research Articles

R-spondin 1/dickkopf-1/beta-catenin machinery is involved in testicular embryonic angiogenesis.

Testicular vasculogenesis is one of the key processes regulating male gonad morphogenesis. The knowledge of the molecular cues underlining this phenomenon is one of today’s most challenging issues and could represent a major contribution toward a better understanding of the onset of testicular morphogenetic disorders. R-spondin 1 has been clearly established as a candidate for mammalian ovary determination. Conversely, very little information is available on the expression and role of R-spondin 1 during testicular morphogenesis. This study aims to clarify the distribution pattern of R-spondin 1 and other partners of its machinery during the entire period of testicular morphogenesis and to indicate the role of this system in testicular development. Our whole mount immunofluorescence results clearly demonstrate that R-spondin 1 is always detectable in the testicular coelomic partition, where testicular vasculature is organized, while Dickkopf-1 is never detectable in this area. Moreover, organ culture experiments of embryonic male UGRs demonstrated that Dickkopf-1 acted as an inhibitor of testis vasculature formation. Consistent with this observation, real-time PCR analyses demonstrated that DKK1 is able to slightly but significantly decrease the expression level of the endothelial marker Pecam1. The latter experiments allowed us to observe that DKK1 administration also perturbs the expression level of the Pdgf-b chain, which is consistent with some authors’ observations relating this factor with prenatal testicular patterning and angiogenesis. Interestingly, the DKK1 induced inhibition of testicular angiogenesis was rescued by the co-administration of R-spondin 1. In addition, R-spondin 1 alone was sufficient to enhance, in culture, testicular angiogenesis.

Novel genes and hormonal regulation for gonadal development during embryogenesis in chickens

Asymmetrical gonadal morphogenesis is well known in female chickens in contrast to males where both gonads develop symmetrically. However, only a few genes have been reported to determine differential morphology between female and male gonads in chicken and their mechanisms of action are unclear. Therefore, we focused on three genes (TOM1L1, TTR, and ZEB1) that are related to cellular proliferation and embryonic development based on previous study indicating up- or down-regulated transcripts in the asymmetric female gonads between embryonic day 6 (E6) and E9 by microarray analyses. To define the validity of the gene expression pattern discovered, q-PCR and in situ hybridization analyses were performed. In the left female gonad between E6 and E9 the expression of TOM1L1, TTR and ZEB1 increased at E9. On the other hand, TOM1L1 and TTR increased significantly in both male gonads between E6 and E9. In addition, recombinant FSH and LH stimulated proliferation of gonadal cells and influenced expression of selected genes in chickens. This suggests that hormonal regulation is involved in growth and development in the embryonic gonad of chickens. Collectively, the results show differential gene expression between the left and right gonads in chicken embryos and that of is regulated by gonadotropin. These results provide novel insights into candidate genes regulating gonad development and differentiation.

Gonad morphogenesis defects drive hybrid male sterility in asymmetric hybrid breakdown of Caenorhabditis nematodes.

Determining the causes and evolution of reproductive barriers to gene flow between populations, speciation, is the key to understanding the origin of diversity in nature. Many species manifest hybrid breakdown when they intercross, characterized by increasingly exacerbated problems in later generations of hybrids. Recently, Caenorhabditis nematodes have emerged as a genetic model for studying speciation, and here we investigate the nature and causes of hybrid breakdown between Caenorhabditis remanei and C. latens. We quantify partial F1 hybrid inviability and extensive F2 hybrid inviability; the ~75% F2 embryonic arrest occurs primarily during gastrulation or embryonic elongation. Moreover, F1 hybrid males exhibit Haldane's rule asymmetrically for both sterility and inviability, being strongest when C. remanei serves as maternal parent. We show that the mechanism by which sterile hybrid males are incapable of transferring sperm or a copulatory plug involves defective gonad morphogenesis, which we hypothesize results from linker cell defects in migration and/or cell death during development. This first documented case of partial hybrid male sterility in Caenorhabditis follows expectations of Darwin's corollary to Haldane's rule for asymmetric male fitness, providing a powerful foundation for molecular dissection of intrinsic reproductive barriers and divergence of genetic pathways controlling organ morphogenesis.

Sex-specific expression of CTNNB1 in the gonadal morphogenesis of the chicken

BackgroundBeta-catenin (CTNNB1), as a key transcriptional regulator in the WNT signal transduction cascade, plays a pivotal role in multiple biological functions such as embryonic development and homeostasis in adults. Although it has been suggested that CTNNB1 is required for gonad development and maintenance of ovarian function in mice, little is known about the expression and functional role of CTNNB1 in gonadal development and differentiation in the chicken reproductive system.MethodsTo examine sex-specific, cell-specific and temporal expression of CTNNB1 mRNA and protein during gonadal development to maturation of reproductive organs, we collected left and right gonads apart from mesonephric kidney of chicken embryos on embryonic day (E) 6, E9, E14, E18, as well as testes, oviduct and ovaries from 12-week-old and adult chickens and performed quantitative PCR, in situ hybridization, and immunohistochemical analyses. In addition, localization of Sertoli cell markers such as anti-Müllerian hormone (AMH), estrogen receptor alpha (ESR1), cyclin D1 (CCND1) and N-cadherin (CDH2) during testicular development was evaluated.ResultsResults of the present study showed that CTNNB1 mRNA and protein are expressed predominantly in the seminiferous cords on E6 to E14 in the male embryonic gonad, and are mainly localized to the medullary region of female embryonic gonads from E6 to E9. In addition, CTNNB1 mRNA and protein are abundant in the Sertoli cells in the testes and expressed predominantly in luminal epithelial cells of the oviduct, but not in the ovaries from 12-week-old and adult chickens. Concomitant with CTNNB1, AMH, ESR1, CCND1 and CDH2 were detected predominantly in the seminiferous cord of the medullary region of male gonads at E9 (after sex determination) and then maintained or decreased until hatching. Interestingly, AMH, ESR1, CCND1 and CDH2 were located in seminiferous tubules of the testes from 12-weeks-old chickens and ESR1, CCND1 and CDH2 were expressed predominantly in the Sertoli cells within seminiferous tubules of adult testes.ConclusionsCollectively, these results revealed that CTNNB1 is present in gonads of both sexes during embryonic development and it may play essential roles in differentiation of Sertoli cells during formation of seminiferous tubules during development of the testes.

The Drosophila actin regulator ENABLED regulates cell shape and orientation during gonad morphogenesis.

Organs develop distinctive morphologies to fulfill their unique functions. We used Drosophila embryonic gonads as a model to study how two different cell lineages, primordial germ cells (PGCs) and somatic gonadal precursors (SGPs), combine to form one organ. We developed a membrane GFP marker to image SGP behaviors live. These studies show that a combination of SGP cell shape changes and inward movement of anterior and posterior SGPs leads to the compaction of the spherical gonad. This process is disrupted in mutants of the actin regulator, enabled (ena). We show that Ena coordinates these cell shape changes and the inward movement of the SGPs, and Ena affects the intracellular localization of DE-cadherin (DE-cad). Mathematical simulation based on these observations suggests that changes in DE-cad localization can generate the forces needed to compact an elongated structure into a sphere. We propose that Ena regulates force balance in the SGPs by sequestering DE-cad, leading to the morphogenetic movement required for gonad compaction.

Mig-38, a novel gene that regulates distal tip cell turning during gonadogenesis in C. elegans hermaphrodites

In Caenorhabditis elegans gonad morphogenesis, the final U-shapes of the two hermaphrodite gonad arms are determined by migration of the distal tip cells (DTCs). These somatic cells migrate in opposite directions on the ventral basement membrane until specific extracellular cues induce turning from ventral to dorsal and then centripetally toward the midbody region on the dorsal basement membrane. To dissect the mechanism of DTC turning, we examined the role of a novel gene, F40F11.2/mig-38, whose depletion by RNAi results in failure of DTC turning so that DTCs continue their migration away from the midbody region. mig-38 is expressed in the gonad primordium, and expression continues throughout DTC migration where it acts cell-autonomously to control DTC turning. RNAi depletion of both mig-38 and ina-1, which encodes an integrin adhesion receptor, enhanced the loss of turning phenotype indicating a genetic interaction between these genes. Furthermore, the integrin-associated protein MIG-15/Nck-interacting kinase (NIK) works with MIG-38 to direct DTC turning as shown by mig-38 RNAi with the mig-15(rh80) hypomorph. These results indicate that MIG-38 enhances the role of MIG-15 in integrin-dependent DTC turning. Knockdown of talin, a protein that is important for integrin activation, causes the DTCs to stop migration prematurely. When both talin and MIG-38 were depleted by RNAi treatment, the premature stop phenotype was suppressed. This suppression effect was reversed upon additional depletion of MIG-15 or its binding partner NCK-1. These results suggest that both talin and the MIG-15/NCK-1 complex promote DTC motility and that MIG-38 may act as a negative regulator of the complex. We propose a model to explain the dual role of MIG-38 in motility and turning.

Raw Functions through JNK signaling and cadherin-based adhesion to regulate Drosophila gonad morphogenesis

To form a gonad, germ cells (GCs) and somatic gonadal precursor cells (SGPs) must migrate to the correct location in the developing embryo and establish the cell–cell interactions necessary to create proper gonad architecture. During gonad morphogenesis, SGPs send out cellular extensions to ensheath the individual GCs and promote their development. We have identified mutations in the raw gene that result in a failure of the SGPs to ensheath the GCs, leading to defects in GC development. Using genetic analysis and gene expression studies, we find that Raw negatively regulates JNK signaling during gonad morphogenesis, and increased JNK signaling is sufficient to cause ensheathment defects. In particular, Raw functions upstream of the Drosophila Jun-related transcription factor to regulate its subcellular localization. Since JNK signaling regulates cell adhesion during the morphogenesis of many tissues, we examined the relationship between raw and the genes encoding Drosophila E-cadherin and β-catenin, which function together in cell adhesion. We find that loss of DE-cadherin strongly enhances the raw mutant gonad phenotype, while increasing DE-cadherin function rescues this phenotype. Further, loss of raw results in mislocalization of β-catenin away from the cell surface. Therefore, cadherin-based cell adhesion, likely at the level of β-catenin, is a primary mechanism by which Raw regulates germline-soma interaction.

Gonad morphogenesis and distal tip cell migration in the Caenorhabditis elegans hermaphrodite.

Cell migration and morphogenesis are key events in tissue development and organogenesis. In Caenorhabditis elegans, the migratory path of the distal tip cells determines the morphology of the hermaphroditic gonad. The distal tip cells undergo a series of migratory phases interspersed with turns to form the gonad. A wide variety of genes have been identified as crucial to this process, from genes that encode components and modifiers of the extracellular matrix to signaling proteins and transcriptional regulators. The connections between extracellular and transmembrane protein functions and intracellular pathways are essential for distal tip cell migration, and the integration of this information governs gonad morphogenesis and determines gonad size and shape.

Identification of a novel ADAMTS9/GON-1 function for protein transport from the ER to the Golgi

A disintegrin-like and metalloprotease with thrombospondin type I motif (ADAMTS9) is a member of the secreted metalloprotease family that is believed to digest extracellular matrix (ECM) proteins outside of cells. Its Caenorhabditis elegans orthologue, GON-1, is involved in ECM degradation and is required for gonad morphogenesis. ADAMTS9 and GON-1 have similar domain structures, and both have a unique C-terminal domain called the "GON domain," whose function remains unknown. Here we show that down-regulation of human ADAMTS9 and C. elegans GON-1 results in the inhibition of protein transport from the endoplasmic reticulum (ER) to the Golgi. This phenotype was rescued by the expression of the GON domain localizing in the ER in human cells and C. elegans. We propose a novel function of ADAMTS9 and GON-1 in the ER that promotes protein transport from the ER to the Golgi. This function is GON-domain dependent but protease activity independent.

Multiple functions of PBRM-1/Polybromo- and LET-526/Osa-containing chromatin remodeling complexes in C. elegans development

The SWI/SNF-like chromatin remodeling complexes consist of two evolutionarily conserved subclasses, which are characterized by specific accessory components, the OSA/BAF250 and Polybromo proteins. These complexes regulate the expressions of distinct sets of target genes, with some overlap, and the regulatory components are thought to determine the target specificity for each complex. Here we isolated C. elegans mutants of the genes for the OSA/BAF250 homolog, LET-526, and the Polybromo homolog, PBRM-1, in a screen for the abnormal asymmetric cell division phenotype. In the asymmetric division of the T cell, both LET-526 and PBRM-1 regulated the asymmetric expression of psa-3/Meis between the T cell daughters, suggesting that the two subclasses share the same target. In the gonad, PBRM-1 regulated gonad primordium formation during embryogenesis, whereas LET-526 was required post-embryonically for distal tip cell (DTC) production from the gonad primordium, suggesting that these proteins have distinct targets for DTC development. Thus, the same cellular process is regulated by LET-526 and PBRM-1 in the asymmetric division of the T cell, but they regulate distinct cellular processes in the gonad morphogenesis. Although disruption of the core component PSA-1 or PSA-4 caused similar defects in the gonad and T cell, it also caused early embryonic arrest, which was not observed in the let-526, pbrm-1, or let-526 pbrm-1 double mutants, suggesting that some targets of SWI/SNF-like complexes do not require LET-526 or PBRM-1 for their transcription. Our results show that the target selection by SWI/SNF-like complexes during C. elegans development is intricately regulated by accessory components.

Somatic gonadal cells: The supporting cast for the germline

Cell-cell signaling and adhesion are critical for establishing tissue architecture during development and for maintaining tissue architecture and function in the adult. Defects in adhesion and signaling can result in mislocalization of cells, uncontrolled proliferation and improper differentiation, leading to tissue overgrowth, tumor formation, and cancer metastasis. An important example is found in the germline. Germ cells that are not incorporated into the gonad exhibit a greater propensity for forming germ cell tumors, and defects in germline development can reduce fertility. While much attention is given to germ cells, their development into functional gametes depends upon somatic gonadal cells. The study of model organisms has provided great insights into how somatic gonadal cells are specified, the molecular mechanisms that regulate gonad morphogenesis, and the role of germline-soma communication in the establishment and maintenance of the germline stem cell niche. This work will be discussed in the context of Drosophila melanogaster.

The ribosomal protein RACK1 is required for microRNA function in both C. elegans and humans.

Despite the importance of microRNAs (miRNAs) in gene regulation, it is unclear how the miRNA-Argonaute complex--or miRNA-induced silencing complex (miRISC)--can regulate the translation of their targets in such diverse ways. We demonstrate here a direct interaction between the miRISC and the ribosome by showing that a constituent of the eukaryotic 40S subunit, receptor for activated C-kinase (RACK1), is important for miRNA-mediated gene regulation in animals. In vivo studies demonstrate that RACK1 interacts with components of the miRISC in nematodes and mammals. In both systems, the alteration of RACK1 expression alters miRNA function and impairs the association of the miRNA complex with the translating ribosomes. Our data indicate that RACK1 can contribute to the recruitment of miRISC to the site of translation, and support a post-initiation mode of miRNA-mediated gene repression.

Brca2 in zebrafish ovarian development, spermatogenesis, and tumorigenesis

Humans with inherited mutations in BRCA2 are at increased risk for developing breast and ovarian cancer; however, the relationship between BRCA2 mutation and these cancers is not understood. Studies of Brca2 mutation by gene targeting in mice are limited, given that homozygous Brca2 mutation typically leads to early embryonic lethality. We established a zebrafish line with a nonsense mutation in brca2 exon 11 (brca2(Q658X)), a mutation similar in location and type to BRCA2 mutations found in humans with hereditary breast and ovarian cancer. brca2(Q658X) homozygous zebrafish are viable and survive to adulthood; however, juvenile homozygotes fail to develop ovaries during sexual differentiation. Instead, brca2(Q658X) homozygotes develop as infertile males with meiotic arrest in spermatocytes. Germ cell migration to the embryonic gonadal ridge is unimpaired in brca2(Q658X) homozygotes; thus, failure of ovarian development is not due to defects in early establishment of the embryonic gonad. Homozygous tp53 mutation rescues ovarian development in brca2(Q658X) homozygous zebrafish, reflecting the importance of germ cell apoptosis in gonad morphogenesis. Adult brca2(Q658X) homozygous zebrafish are predisposed to testicular neoplasias. In addition, tumorigenesis in multiple tissues is significantly accelerated in combination with homozygous tp53 mutation in both brca2(Q658X) homozygous and brca2(Q658X) heterozygous zebrafish. These studies reveal critical roles for brca2 in ovarian development and tumorigenesis in reproductive tissues.

KDR-LacZ-expressing cells are involved in ovarian and testis-specific vascular development, suggesting a role for VEGFA in the regulation of this vasculature

Our objectives were to evaluate kinase insert domain protein receptor (KDR)-β-galactosidase (LacZ) expression as a marker for vascular development during gonadal morphogenesis and to determine whether any novel non-angiogenic KDR-LacZ expression was present in mouse testes or ovaries. Gonads were collected from mice expressing LacZ driven by the Kdr promoter (KDR-LacZ) from embryonic day 11 (E11) through postnatal day 60 (P60). At E11.5, mesonephric cells expressing KDR-LacZ seemed to migrate into the developing testis and surrounded developing seminiferous cords. Cells expressing KDR-LacZ appeared in the ovary with no apparent migration from the adjacent mesonephros, suggesting a different origin of endothelial cells. Testis organ cultures from E11 mice were treated with 8μM VEGFR-TKI, a vascular endothelial growth factorA signal transduction inhibitor; subsequently, the amount of KDR-LacZ staining was reduced by 66%-99% (P<0.002), and the ability of KDR-expressing cells to form a densely organized vascular network was inhibited. Novel non-angiogenic KDR-LacZ staining was detected in the testis on specific subsets of germ cells at E16, E17, P4, P20, P30, and P60. In ovaries, staining was present on oocytes within oocyte cysts at E17 and within late secondary follicles of postnatal mice. Thus, KDR is an excellent marker for analyzing vascular development in the gonads. Inhibition of VEGFA signal transduction prevents the development of testis-specific vasculature. Furthermore, non-vascular KDR-LacZ staining suggests that KDR directly affects both spermatogenesis and somatic-oocyte interactions during gametogenesis.

Gonadal morphogenesis and sex differentiation in cultured chub mackerel, Scomber japonicus

The timing of primordial germ-cell (PGC) migration with regard to the gonadal anlagen, gonad formation and sex differentiation was examined histologically in the chub mackerel (Scomber japonicus) at 5–190 days post hatching (dph). At 5 dph, PGCs appeared on the peritoneal epithelium surface or in the mesentery, on the dorsal side of the abdominal cavity. By 10 dph, stromal cells around the PGCs proliferated. The gonadal primordium was formed by 15 dph. The gonadosomatic index was 0.01% at 30 dph and increased thereafter (0.32% in females and 0.04% in males at 160 dph). Ovarian differentiation occurred at 30–40 dph, indicated by ovarian cavity formation (elongation and fusion of the upper and lower ovarian edges). Meiosis was subsequently initiated. A few meiotic oocytes surrounded the cavity at 50 dph; most were in the perinucleolus stage at 60 dph and attained a diameter of 60–70 μm at 190 dph. Testicular differentiation occurred at 30 dph, indicated by the formation of the sperm duct primordium. Spermatogonia gradually proliferated, developing into spermatocytes at the chromatin–nucleolus stage (after 90 dph) and subsequently into spermatids and spermatozoa (160 dph). These data could aid the development of seeding and cell-engineering technologies for scombrid fish.

The actin regulator Enabled is essential for proper gonad morphogenesis in Drosophila

The actin regulator Enabled is essential for proper gonad morphogenesis in Drosophila

Gonadal Morphogenesis and Gene Expression in Reptiles with Temperature-Dependent Sex Determination

In reptiles with temperature-dependent sexual determination, the thermosensitive period (TSP) is the interval in which the sex is defined during gonadal morphogenesis. One-shift experiments in a group of eggs define the onset and the end of the TSP as all and none responses, respectively. Timing for sex-undetermined (UG) and -determined gonads (DG) differs at male- (MPT) or female-producing temperatures (FPT). During the TSP a decreasing number of embryos respond to temperature shifts indicating that in this period embryos with both UG and DG exist. Although most UG correspond to undifferentiated gonads, some embryos extend UG after the onset of histological differentiation. Thus, temperature affects gonadal cells during the process of morphogenesis, but timing of commitment depends on individual embryos. A correlation between gonadal morphogenesis, TSP, and gene expression suggests that determination of the molecular pathways modulated by temperature in epithelial cells (surface epithelium and medullary cords) holds the key for a unifying hypothesis on temperature-dependent sex determination.

Cell-surface Processing of the Metalloprotease Pro-ADAMTS9 Is Influenced by the Chaperone GRP94/gp96

A disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs 9 (ADAMTS9) is a highly conserved metalloprotease that has been identified as a tumor suppressor gene and is required for normal mouse development. The secreted ADAMTS9 zymogen undergoes proteolytic excision of its N-terminal propeptide by the proprotein convertase furin. However, in contrast to other metalloproteases, propeptide excision occurs at the cell surface and leads to decreased activity of the zymogen. Here, we investigated the potential cellular mechanisms regulating ADAMTS9 biosynthesis and cell-surface processing by analysis of molecular complexes formed by a construct containing the propeptide and catalytic domain of pro-ADAMTS9 (Pro-Cat) in HEK293F cells. Cross-linking of cellular proteins bound to Pro-Cat followed by mass spectrometric analysis identified UDP-glucose:glycoprotein glucosyltransferase I, heat shock protein gp96 (GRP94), BiP (GRP78), and ERdj3 (Hsp40 homolog) as associated proteins. gp96 and BiP were present at the cell surface in an immunoprecipitable complex with pro-ADAMTS9 and furin. Treatment with geldanamycin, an inhibitor of the HSP90alpha family (including gp96), led to decreased furin processing of pro-ADAMTS9 and accumulation of the unprocessed pro-ADAMTS9 at the cell surface. gp96 siRNA down-regulated the levels of cell-surface pro-ADAMTS9 and furin, whereas the levels of cell-surface pro-ADAMTS9, but not of cell-surface furin, were decreased upon treatment with BiP siRNA. These data identify for the first time the cellular chaperones associated with secretion of an ADAMTS protease and suggest a role for gp96 in modulating pro-ADAMTS9 processing.

OVEX1, a novel chicken endogenous retrovirus with sex-specific and left-right asymmetrical expression in gonads.

BackgroundIn chickens, as in most birds, female gonad morphogenesis is asymmetrical. Gonads appear first rather similarly, but only the left one undergoes full differentiation and gives rise to a functional ovary. The right gonad, in which the cortex does not develop, remains restricted to the medulla and finally regresses. Opportunity was taken of this left-right asymmetry to perform a suppression subtractive hybridization screening to select for transcripts preferentially expressed in the developing left ovary as compared to the right one, and thus identify genes that are potentially involved in the process of ovarian differentiation.ResultsOne of these transcripts, named Ovex1 according to its expression profile, corresponds to an endogenous retrovirus that has not been previously characterized. It is transcribed as full-length and singly spliced mRNAs and contains three uninterrupted open reading frames coding potentially for proteins with homology to Gag and Pro-Pol retroviral polyproteins and a third protein showing only a weak similarity with Env glycoproteins. Ovex1 is severely degenerated; it is devoid of typical long terminal repeats and displays some evidence of recombination. An orthologous Ovex1 locus was identified in the genome of zebra finch, a member of a different bird order, and similar sequences were detected in turkey, guinea fowl, and duck DNA. The relationship between these sequences follows the bird phylogeny, suggesting vertical transmission of the endogenous retrovirus for more than 100 million years.Ovex1 is transcribed in chicken gonads with a sex-dependent and left-right asymmetrical pattern. It is first expressed in the cortex of the left indifferent gonads of both sexes. Expression is transient in the left testis and absent in the right one. In developing ovaries, Ovex1 transcription increases sharply in the left cortex and is weakly detected in the medulla. After folliculogenesis, Ovex1-expressing cells constitute the follicular granulosa cell layer. Ovex1 expression highlights a striking desquamation process that leads to profound cortical remodeling associated with follicle morphogenesis.ConclusionEvidence for a selection pressure at the protein level suggests that this endogenous retrovirus, expressed in the ovarian supporting cell lineage, might play an active role in bird ovarian physiology.

Expression Profiles of SRY and SOX9 in Rabbit Gonads: The Classical Model of Mammalian Sex Differentiation

Sex determination in mammalian gonads depends on the concerted action of SRY and SOX at the genital ridge. Although most research on the mechanisms involved in sex determination has been done in mice, the study of non-model organisms may indicate the extent of the generalizations currently based on model systems. The present study investigated the correlation between SRY/SOX9 expression patterns and the process of morphogenesis in the developing gonad of the rabbit. In males, the onset of SRY/SOX9 expression closely followed the establishment of undifferentiated genital ridges at 13 to 14 dpc. In contrast to mouse, real-time PCR in the rabbit revealed that levels of SRY/ SOX9 peak after the onset of seminiferous cord formation, while in mouse this occurs before. Furthermore, rabbit gonads maintain low levels of SRY and SOX9 expression in male and female gonads, respectively. In situ hybridization suggests that cells of the mesonephric Bowman capsules, which do not express SRY, may become SOX9-expressing pre-Sertoli cells during the long period of seminiferous cord formation in the rabbit. In contrast to mouse, current results indicate that the patterns of SRY/SOX9 expression associated with the process of gonadal morphogenesis in rabbit appear similar to those of other mammals, including humans.