Desmoplakin (Dsp) conditional knockout in NR5A1+ somatic cells affects germ cell survival in developing mouse gonads.

Cell to cell interactions are crucial for morphogenesis and tissue formation. Desmoplakin (encoded by the Dsp gene) is a component of desmosomes and anchors the transmembrane adhesion proteins to the cytoskeleton. Its role in gonad development remains vague. To study the role of desmoplakin in gonad development, we used a tissue-specific knockout of the Dsp gene in the NR5A1+ somatic cells of the gonads. We show here that desmoplakin is necessary for the survival of germ cells in fetal testes and ovaries. The Dspknockout in NR5A1+ somatic cells in testes decreased the number of germ cells, and thus the size of the testes, but did not affect the Sertoli cells or the structure of testis cords and interstitium. The Dspknockout in NR5A1+ somatic cells in ovaries decreased the number of female germ cells and drastically reduced the formation of ovarian follicles. Dsp knockout in NR5A1+ somatic cells did not affect the sex determination and sexual differentiation of the gonads, as judged from an unchanged expression of genes essential for these processes. We conclude that mediation by desmoplakin cell adhesion between the gonadal cells is necessary for germ cell survival.

Matrix metalloproteinase-dependent regulation of extracellular matrix shapes the structure of sexually differentiating mouse gonads.

The extracellular matrix (ECM) proteins play an important role in the establishment of the sex-dependent structure of developing gonads. The matrix metalloproteinases (MMPs) are the major players in the regulation of ECM. Our hypothesis was that the MMPs-dependent regulation of EMC is crucial for the establishment of the correct, either testis or ovary, structure of developing gonad. We cultured developing mouse gonads in vitro in the presence of the MMPs inhibitors (α-2-macroglobulin, leupeptin, phosphoramidon) or the MMPs activator, APMA (4-aminophenylmercuric acetate). These inhibitors and activator inhibit/activate, to a different degree, matrix metalloproteinases, but the exact mechanism of inhibition/activation remains unknown. We found that the MMP inhibitors increased accumulation of ECM in the developing gonads. The α-2-macroglobulin had the weakest, and the phosphoramidon the strongest effect on the ECM and the structure of the gonads. The α-2-macroglobulin caused a slight increase of ECM and did not disrupt the gonad structure. Leupeptin led to the strong accumulation of ECM, resulted in the formation of the structures resembling testis cords in both testes and ovaries, and caused increase of apoptosis and complete loss of germ cells. Phosphoramidon caused the strongest accumulation of ECM, which separated individual cells and completely prevented intercellular adhesion both in the testes and in the ovaries. As a result of aberrant morphology, the sex of the phosphoramidon-treated gonads was morphologically unrecognizable. The APMA - the activator of MMP caused ECM loss, which led to the loss of cell adhesion, cell dispersion and an aberrant morphology of the gonads. These results indicate that the ECM accumulation is MMPs-dependent and that the correct amount and distribution of ECM during gonad development plays a key role in the formation of the gonad structure.

The Central Role of Cadherins in Gonad Development, Reproduction, and Fertility.

Cadherins are a group of membrane proteins responsible for cell adhesion. They are crucial for cell sorting and recognition during the morphogenesis, but they also play many other roles such as assuring tissue integrity and resistance to stretching, mechanotransduction, cell signaling, regulation of cell proliferation, apoptosis, survival, carcinogenesis, etc. Within the cadherin superfamily, E- and N-cadherin have been especially well studied. They are involved in many aspects of sexual development and reproduction, such as germline development and gametogenesis, gonad development and functioning, and fertilization. E-cadherin is expressed in the primordial germ cells (PGCs) and also participates in PGC migration to the developing gonads where they become enclosed by the N-cadherin-expressing somatic cells. The differential expression of cadherins is also responsible for the establishment of the testis or ovary structure. In the adult testes, N-cadherin is responsible for the integrity of the seminiferous epithelium, regulation of sperm production, and the establishment of the blood-testis barrier. Sex hormones regulate the expression and turnover of N-cadherin influencing the course of spermatogenesis. In the adult ovaries, E- and N-cadherin assure the integrity of ovarian follicles and the formation of corpora lutea. Cadherins are expressed in the mature gametes and facilitate the capacitation of sperm in the female reproductive tract and gamete contact during fertilization. The germ cells and accompanying somatic cells express a series of different cadherins; however, their role in gonads and reproduction is still unknown. In this review, we show what is known and unknown about the role of cadherins in the germline and gonad development, and we suggest topics for future research.

Transcriptome profiling reveals male- and female-specific gene expression pattern and novel gene candidates for the control of sex determination and gonad development in Xenopus laevis.

Xenopus laevis is an amphibian (frog) species widely used in developmental biology and genetics. To unravel the molecular machinery regulating sex differentiation of Xenopus gonads, we analyzed for the first time the transcriptome of developing amphibian gonads covering sex determination period. We applied microarray at four developmental stages: (i) NF50 (undifferentiated gonad during sex determination), (ii) NF53 (the onset of sexual differentiation of the gonads), (iii) NF56 (sexual differentiation of the gonads), and (iv) NF62 (developmental progression of differentiated gonads). Our analysis showed that during the NF50, the genetic female (ZW) gonads expressed more sex-specific genes than genetic male (ZZ) gonads, which suggests that a robust genetic program is realized during female sex determination in Xenopus. However, a contrasting expression pattern was observed at later stages (NF56 and NF62), when the ZW gonads expressed less sex-specific genes than ZZ gonads, i.e., more genes may be involved in further development of the male gonads (ZZ). We identified sexual dimorphism in the expression of several functional groups of genes, including signaling factors, proteases, protease inhibitors, transcription factors, extracellular matrix components, extracellular matrix enzymes, cell adhesion molecules, and epithelium-specific intermediate filaments. In addition, our analysis detected a sexually dimorphic expression of many uncharacterized genes of unknown function, which should be studied further to reveal their identity and if/how they regulate gonad development, sex determination, and sexual differentiation. Comparison between genes sex-specifically expressed in developing gonads of Xenopus and available transcriptome data from zebrafish, two reptile species, chicken, and mouse revealed significant differences in the genetic control of sex determination and gonad development. This shows that the genetic control of gonad development is evolutionarily malleable.

Tissue-specific knockout of E-cadherin (Cdh1) in developing mouse gonads causes germ cells loss.

The normal course of gonad development is critical for the sexual development and reproductive capacity of the individual. During development, an incipient bipotential gonad which consists of unorganized aggregate of cells, must differentiate into highly structured testis or ovary. Cell adhesion molecules (CAMs) are a group of proteins crucial for segregation and aggregation of different cell types to form different tissues. E-cadherin (Cdh1) is one of the CAMs expressed in the developing gonads. We used tissue-specific knockout of Cdh1 gene in OCT4+ germ cells and, separately, in SF1+ somatic cells of developing gonads. The knockout of E-cadherin in somatic cells caused decrease in the number of germ cells, while the knockout in the germ cells caused their almost complete loss. Thus, the presence of E-cadherin in both the germ and somatic cells is necessary for the survival of germ cells. Although the lack of E-cadherin did not impair cell proliferation, it enhanced apoptosis, which was a possible cause of germ cell loss. However, the somatic cells of the gonad differentiated normally into Sertoli cells in the testis cords, and into follicular cells in the ovaries. The testis and ovigerous cords maintained their integrity; they were covered by continuous basement membranes. The testicular interstitium with steroidogenic fetal Leydig cells did not show any noticeable changes. However, in the female gonads, because of the lack of germ cells, the ovarian follicles were absent. The sex determination and sexual differentiation of the gonad were not impaired. These results underscore an important role of E-cadherin in germ cell survival and gonad development.

Transcriptome analysis identifies genes involved in sex determination and development of Xenopus laevis gonads.

Development of the gonads is a complex process, which starts with a period of undifferentiated, bipotential gonads. During this period the expression of sex-determining genes is initiated. Sex determination is a process triggering differentiation of the gonads into the testis or ovary. Sex determination period is followed by sexual differentiation, i.e. appearance of the first testis- and ovary-specific features. In Xenopus laevis W-linked DM-domain gene (DM-W) had been described as a master determinant of the gonadal female sex. However, the data on the expression and function of other genes participating in gonad development in X. laevis, and in anurans, in general, are very limited. We applied microarray technique to analyze the expression pattern of a subset of X. laevis genes previously identified to be involved in gonad development in several vertebrate species. We also analyzed the localization and the expression level of proteins encoded by these genes in developing X. laevis gonads. These analyses pointed to the set of genes differentially expressed in developing testes and ovaries. Gata4, Sox9, Dmrt1, Amh, Fgf9, Ptgds, Pdgf, Fshr, and Cyp17a1 expression was upregulated in developing testes, while DM-W, Fst, Foxl2, and Cyp19a1 were upregulated in developing ovaries. We discuss the possible roles of these genes in development of X. laevis gonads.

Development of Xenopus laevis bipotential gonads into testis or ovary is driven by sex-specific cell-cell interactions, proliferation rate, cell migration and deposition of extracellular matrix.

Information on the mechanisms orchestrating sexual differentiation of the bipotential gonads into testes or ovaries in amphibians is limited. The aim of this study was to investigate the development of Xenopus laevis gonad, to identify the earliest signs of sexual differentiation, and to describe mechanisms driving these processes. We used light and electron microscopy, immunofluorescence and cell tracing. In order to identify the earliest signs of sexual differentiation the sex of each tadpole was determined using genotyping with the sex markers. Our analysis revealed a series of events participating in the gonadal development, including cell proliferation, migration, cell adhesion, stroma penetration, and basal lamina formation. We found that during the period of sexual differentiation the sites of intensive cell proliferation and migration differ between male and female gonads. In the differentiating ovaries the germ cells remain associated with the gonadal surface epithelium (cortex) and a sterile medulla forms in the ovarian center, whereas in the differentiating testes the germ cells detach from the surface epithelium, disperse, and the cortex and medulla fuse. Cell junctions that are more abundant in the ovarian cortex possibly can favor the persistence of germ cells in the cortex. Also the stroma penetrates the female and male gonads differently. These finding indicate that the crosstalk between the stroma and the coelomic epithelium-derived cells is crucial for development of male and female gonad.

Histological Analysis of Gonadal Ridge Development and Sex Differentiation of Gonads in Three Gecko Species.

Reptiles constitute a highly diverse group of vertebrates, with their evolutionary lineages having diverged relatively early. The types of sex determination exemplify the diversity of reptiles; however, there are limited data regarding the gonadal development in squamate reptiles. Geckos constitute a group that is increasingly used in research and that serves as a potential reptilian model organism. The aim of this study was to trace the changes in the structure of developing gonads in the embryos of three gecko species: the crested gecko, leopard gecko, and mourning gecko. These species represent different families of the Gekkota infraorder and exhibit different types of sex determination. Gonadal development was examined from the formation of the earliest gonadal ridges through the development of undifferentiated gonadal structures, sex differentiation of gonads, and the formation of testicular and ovarian structures. The study showed that the gonadal primordia of these three gecko species formed on the most dorsally located surface of the dorsal mesentery, and both the coelomic epithelium and the nephric mesenchyme contributed to their development. As in other reptile species, primordial germ cells settled in the gonadal ridges, and the undifferentiated gonad was composed of a cortex and a medulla. Ovarian differentiation started with the thickening of the gonadal cortex and proliferation of germ cells in this region. A characteristic feature of the developing gecko ovaries was the thickened crescent-shaped cortex on the medial and ventral surfaces of the ovaries. The ovarian medulla also grew and exhibited diverse tendencies to form cords. In the leopard gecko, advanced cord-like structures with lumens were observed in the ovaries, which were not seen in the crested gecko. Testicular differentiation was characterized by cortical thinning and the disappearance of germ cells in this region. In the medulla, the development of distinct cords with early lumen formation was noted. A characteristic feature of embryonic gonads was their growth in a horizontal plane. In this study, gonadal development was characterized by several features that are shared by geckos and other reptiles, along with features that are specific only to geckos.

Histological analysis of early gonadal development in three bird species reveals gonad asymmetry from the beginning of gonadal ridge formation and a similar course of sex differentiation.

The developing gonads constitute a valuable model for studying developmental mechanisms because the testes and ovaries, while originating from the same primordia, undergo two different patterns of development. So far, gonadal development among birds has been described in detail in chickens, but literature on the earliest stages of gonadogenesis is scarce. This study presents changes in the structure of the gonads in three species of breeding birds (chicken, duck, and pigeon), starting from the first signs of gonadal ridge formation, that is, the thickenings of the coelomic epithelium. It appears that both gonads show asymmetry from the very beginning of gonadal ridge formation in both genetic sexes. The left gonadal ridge is thicker than the right one, and it is invaded by a higher number of primordial germ cells. Undifferentiated gonads, both left and right, consist of the primitive cortex and the medulla. The primitive cortex develops from the thickened coelomic epithelium, while the primitive medulla - by the aggregation of mesenchymal cells. This study also describes the process of sex differentiation of the testes and ovaries, which is initiated at the same embryonic stage in all three studied species. The first sign of gonadal sex differentiation is the decrease in the number of cortical germ cells and a reduction in cortical thickness in the differentiating testes. This is followed by an increase in the number of germ cells in the medulla. The cortical asymmetry and difference in size between the left and right testes diminishes during later development. However, the differentiating left ovary shows an increase in the number of cortical germ cells and cortical thickness. No regression is seen in the right ovary, although its development is slower. The right ovarian cortex undergoes testis-specific reduction, while the medulla undergoes ovary-specific development. The process of gonadogenesis is similar in the three studied species, with only slight differences in gonadal structure.

Differential effects of testosterone and 17ß-estradiol on gonadal development in five anuran species.

Sex hormones are essential for sexual differentiation and play a key role in the development of gonads in amphibians. The goal of this study was to evaluate the influence of exogenous sex steroids, testosterone, and 17ß-estradiol (E(2)) on development of gonads in five anuran species differing in their evolutionary positions, sex determination, and mode of gonadogenesis. We found that in two closely related species of fire-bellied toad, Bombina bombina and Bombina variegata, testosterone and E(2) exposure results in sex reversal as well as intersex and undifferentiated gonads. Similarly, sex reversal was observed in Hyla arborea after exposure to male or female sex steroids. Xenopus laevis was sensitive to E(2) but only moderately to testosterone. In Bufo viridis, treatment with either sex hormone provoked a developmental delay in gonads and Bidder's organs. Therefore, susceptibility to hormonal sex reversal appeared species dependent but unrelated to genetic sex determination and the type of gonadogenesis. We also found that the onset of sex steroid exposure influences gonad differentiation and the meiotic status of the germ cells depends on their location within the gonad. Our findings reveal differential sensitivity of amphibians to testosterone and E(2), establishing a hierarchy of sensitivity to these hormones among different anuran species.

Giant Multinucleated Cells in Aging and Senescence-An Abridgement.

This review introduces the subject of senescence, aging, and the formation of senescent multinucleated giant cells. We define senescence and aging and describe how molecular and cellular senescence leads to organismal senescence. We review the latest information on senescent cells' cellular and molecular phenotypes. We describe molecular and cellular features of aging and senescence and the role of multinucleated giant cells in aging-related conditions and cancer. We explain how multinucleated giant cells form and their role in aging arteries and gonads. We also describe how multinucleated giant cells and the reversibility of senescence initiate cancer and lead to cancer progression and metastasis. We also describe molecules and pathways regulating aging and senescence in model systems and their applicability to clinical therapies in age-related diseases.

The Role of Vitamin D in COVID-19 and the Impact of Pandemic Restrictions on Vitamin D Blood Content.

Vitamin D is a hormone regulating the immune system and playing a pivotal role in responses to microbial infections. It regulates inflammatory processes by influencing the transcription of immune-response genes in macrophages, T cells, and dendritic cells. The proven role of vitamin D in many infectious diseases of the respiratory tract indicated that vitamin D should also play a role in SARS-CoV-2 infection. Vitamin D inhibits cytokine storm by switching the pro-inflammatory Th1 and Th17 to the anti-inflammatory Th2 and Treg response. Vitamin D is therefore expected to play a role in preventing, relieving symptoms, or treating SARS-CoV-2 infection symptoms, including severe pneumonia. There are several possible mechanisms by which vitamin D may reduce the risk of COVID-19 infection, such as induction of the transcription of cathelicidin and defensin. Also a nongenomic antiviral action of vitamin D and lumisterol, the molecule closely related to vitamin D, was reported. Despite this enormous progress, currently, there is still insufficient scientific evidence to support the claim that vitamin D supplementation may help treat COVID-19 infection. The pandemic restrictions were also shown to impact vitamin D uptake by limiting exposure to sunlight.

Diversity of Balbiani body formation in internally and externally fertilizing representatives of Osteoglossiformes (Teleostei: Osteoglossomorpha).

During the early stages of oogenesis, the Balbiani body is formed in the primary oocytes. It consists of the Golgi apparatus, endoplasmic reticulum (ER), and numerous mitochondria aggregated with germ plasm, but its form may differ among animals. Hypothetically, during oogenesis oocytes become adapted to future development in two different environments depending on internal or external fertilization. We aimed to investigate, using light and transmission electron microscopy, the development of the Balbiani body during oogenesis in representatives of Osteoglossiformes, one of the most basal Teleostei groups. We analyzed the structure of oogonia and primary oocytes in the internally fertilizing butterflyfish Pantodon buchholzi and the externally fertilizing Osteoglossum bicirrhosum and Arapaima gigas to compare formation of the Balbiani body in relation to modes of fertilization. We demonstrated that the presence of the germ plasm as well as the fusion and fission of mitochondria are the conserved features of the Bb. However, each species exhibited also some peculiar features, including the presence of three types of ooplasm with different electron density and mitochondria-associated membranes in P. buchholzi; annulate lamellae, complexes of the Golgi apparatus, ER network, and lysosome-like bodies in O. bicirrhosum; as well as karmellae and whorls formed by the lamellae of the ER in A. gigas. Moreover, the form of the germ plasm observed in close contact with mitochondria differed between osteoglossiforms, with a "net-like" structure in P. buchholzi, the presence of numerous strings in O. bicirrhosum, and irregular accumulations in A. gigas. These unique features indicate that the extreme diversity of gamete structure observed so far only in the spermatozoa of osteoglossiforms is also characteristic for oocyte development in these basal teleosts. Possible reason of this variability is a period of about 150 million years of independent evolution of the lineages.

Differentiation and development of gonads in the yellow-bellied toad, Bombina variegata L., 1758 (Amphibia: Anura: Bombinatoridae).

The aim of this study was to investigate consecutive stages of gonadal development of the yellow-bellied toad (Bombina variegata) with particular emphasis on the origin of somatic and germ cell lineages as well as the timing of gonial cell migration. Changes in gonadal basal lamina distribution helped to explain the exceptional mode of gonadal differentiation in this species. Atypical and rapid differentiation of the male gonad in B. variegata is the result of the ability of gonial cells to migrate into the center of the gonad relatively early. Thus, the testis medulla contains germ cells from the onset of gonadal differentiation into cortex and medulla, whereas in other anurans a sterile medulla is characteristic of both future testes and ovaries; germ cells translocate into the medulla during the subsequent stage of testis development. This atypical testiculogenesis is probably the result of an acceleration of the sex determination period, indicating a contribution of sex determination heterochrony to the course of gonadogenesis. The results also suggest that medullar cells are derived from proliferating coelomic epithelial cells. Moreover, Sertoli cells constitute an integral part of the germinal epithelium in B. variegata, as in other vertebrates. Spermatids do not contact Sertoli cells just before spermiation and do not form bundles.

Molecular mechanisms underlying female sex determination--antagonism between female and male pathway.

Molecular interactions in a developing gonad are crucial for an individual since they determine its phenotypic sex. The process of sex determination is complicated because of the antagonistic interactions between the male and female pathway. Factors responsible for the determination of femaleness make the female pathway. This pathway has to inhibit a complex network of male-determining factors and also has to induce the expression of genes that drive differentiation of the ovary. Morphological description of the ovary development suggests that this process is simple, however, the analysis of the robust gene expression indicates that genetic control of the ovary differentiation is active and complicated at the molecular level. A plethora of genes is expresed in developing gonads. Nevertheless, there are only a couple of genes the role in ovary development of which has been described till now. RSPO1 seems the main gene participating in the establishment of the ovary fate. The loss of functional R-spondin1 causes the complete female-to-male sex reversal in human. The second important factor is WNT4 which plays an opposite role to R-spondin1 in the gonad but also is decisive for the ovarian fate. WNT4 and RSPO1 drive the disposition of beta-catenin in cells and thus these factors regulate gene transcription and cell-cell adhesion. Foxl2 is another gene contributing to the development of the ovary. In females also germ cells seem to play important role in sex determination.

Expression of primary cilia-related genes in developing mouse gonads.

Mechanisms governing differentiation of the bipotential gonad into the testes or ovaries are complex and still vague. The primary cilium is an organelle involved in cell signaling, which controls the development of many organs, but the role of primary cilium in the sex determination and sexual differentiation of gonads is com-pletely unknown. Here we studied the expression of genes involved in primary cilium formation and function-ing in fetal mouse gonads, before, during and after sexual differentiation. We studied the expression of 175 primary cilia-related genes using microarray technique. 144 of these genes were ubiquitously expressed in all studied cell types with no significant differences in expression level. Such a high level of expression of primary cilia-related genes in developing mouse gonads suggests that the primary cilia and/or primary cilia-related genes are important for the development of both somatic and germline component of the gonads. Only 31 genes showed a difference in expression between different cell types, which suggests that they have different functions in the somatic and germ cells. These results justify further studies on the role of primary cilia and the primary cilia-related genes in gonad development.

N-Cadherin Is Critical for the Survival of Germ Cells, the Formation of Steroidogenic Cells, and the Architecture of Developing Mouse Gonads.

Normal gonad development assures the fertility of the individual. The properly functioning gonads must contain a sufficient number of the viable germ cells, possess a correct architecture and tissue structure, and assure the proper hormonal regulation. This is achieved by the interplay between the germ cells and different types of somatic cells. N-cadherin coded by the Cdh2 gene plays a critical role in this interplay. To gain an insight into the role of N-cadherin in the development of mouse gonads, we used the Cre-loxP system to knock out N-cadherin separately in two cell lines: the SF1+ somatic cells and the OCT4+ germ cells. We observed that N-cadherin plays a key role in the survival of both female and male germ cells. However, the N-cadherin is not necessary for the differentiation of the Sertoli cells or the initiation of the formation of testis cords or ovigerous cords. In the later stages of gonad development, N-cadherin is important for the maintenance of testis cord structure and is required for the formation of steroidogenic cells. In the ovaries, N-cadherin is necessary for the formation of the ovarian follicles. These results indicate that N-cadherin plays a major role in gonad differentiation, structuralization, and function.

Transcriptional profiling validates involvement of extracellular matrix and proteinases genes in mouse gonad development.

Extracellular matrix (ECM) plays an important scaffolding role in the establishment of organs structure during development. A great number of ECM components and enzymes (proteinases) regulating formation/degradation of ECM during organ remodeling have been identified. In order to study the role of ECM in the mouse gonad development, especially during sexual differentiation of the gonads when the structure of the testis and ovary becomes established, we performed a global analysis of transcriptome in three main cell types of developing gonad (supporting, interstitial/stromal and germ cells) using transgenic mice, cell sorting and microarray. The genes coding for ECM components were mostly expressed in two gonadal cell lines: supporting and interstitial/stromal cells. These two cell lines differed in the expression pattern of ECM components, which suggests that ECM components might be crucial for differentiation of gonad compartments (for example testis cords vs. interstitium in XY gonads). Collagens and proteoglycans coding genes were mainly expressed in the interstitium/stromal cells, while non-collagen glycoproteins and matricellular coding genes were expressed in both cell lines. We also analyzed the expression of genes encoding ECM enzymes that are secreted to the ECM where they remodel the scaffolding of developing organs. We found that the ECM enzyme genes were also mostly expressed in supporting and interstitial/stromal cells. In contrast to the somatic cells, the germ cells expressed only limited number of ECM components and enzymes. This suggests that the germ line cells do not participate, or play only a minor role, in the sculpting of the gonad structure via ECM synthesis and remodeling. Importantly, the supporting cells showed the sex-specific pattern of expression of ECM components. However, the pattern of expression of most ECM enzymes in the somatic and germ cells is independent on the sex of the gonad. Further studies are required to elucidate the exact roles of identified genes in sexual differentiation of the gonads.

Cell adhesion molecules expression pattern indicates that somatic cells arbitrate gonadal sex of differentiating bipotential fetal mouse gonad.

Unlike other organ anlagens, the primordial gonad is sexually bipotential in all animals. In mouse, the bipotential gonad differentiates into testis or ovary depending on the genetic sex (XY or XX) of the fetus. During gonad development cells segregate, depending on genetic sex, into distinct compartments: testis cords and interstitium form in XY gonad, and germ cell cysts and stroma in XX gonad. However, our knowledge of mechanisms governing gonadal sex differentiation remains very vague. Because it is known that adhesion molecules (CAMs) play a key role in organogenesis, we suspected that diversified expression of CAMs should also play a crucial role in gonad development. Using microarray analysis we identified 129 CAMs and factors regulating cell adhesion during sexual differentiation of mouse gonad. To identify genes expressed differentially in three cell lines in XY and XX gonads: i) supporting (Sertoli or follicular cells), ii) interstitial or stromal cells, and iii) germ cells, we used transgenic mice expressing EGFP reporter gene and FACS cell sorting. Although a large number of CAMs expressed ubiquitously, expression of certain genes was cell line- and genetic sex-specific. The sets of CAMs differentially expressed in supporting versus interstitial/stromal cells may be responsible for segregation of these two cell lines during gonadal development. There was also a significant difference in CAMs expression pattern between XY supporting (Sertoli) and XX supporting (follicular) cells but not between XY and XX germ cells. This indicates that differential CAMs expression pattern in the somatic cells but not in the germ line arbitrates structural organization of gonadal anlagen into testis or ovary.

Methods for the Study of Gonadal Development.

Current knowledge on gonadal development and sex determination is the product of many decades of research involving a variety of scientific methods from different biological disciplines such as histology, genetics, biochemistry, and molecular biology. The earliest embryological investigations, followed by the invention of microscopy and staining methods, were based on histological examinations. The most robust development of histological staining techniques occurred in the second half of the nineteenth century and resulted in structural descriptions of gonadogenesis. These first studies on gonadal development were conducted on domesticated animals; however, currently the mouse is the most extensively studied species. The next key point in the study of gonadogenesis was the advancement of methods allowing for the in vitro culture of fetal gonads. For instance, this led to the description of the origin of cell lines forming the gonads. Protein detection using antibodies and immunolabeling methods and the use of reporter genes were also invaluable for developmental studies, enabling the visualization of the formation of gonadal structure. Recently, genetic and molecular biology techniques, especially gene expression analysis, have revolutionized studies on gonadogenesis and have provided insight into the molecular mechanisms that govern this process. The successive invention of new methods is reflected in the progress of research on gonadal development.