Morphometric and gene expression analyses of stromal expansion during development of the bovine fetal ovary

During ovarian development stroma from the mesonephros penetrates and expands into the ovarian primordium and thus appears to be involved, at least physically, in the formation of ovigerous cords, follicles and surface epithelium. Cortical stromal development during gestation in bovine fetal ovaries (n = 27) was characterised by immunohistochemistry and by mRNA analyses. Stroma was identified by immunostaining of stromal matrix collagen type I and proliferating cells were identified by Ki67 expression. The cortical and medullar volume expanded across gestation, with the rate of cortical expansion slowing over time. During gestation, the proportion of stroma in the cortex and total volume in the cortex significantly increased (P  0.05). The expression levels of 12 genes out of 18 examined, including osteoglycin (OGN) and lumican (LUM), were significantly increased later in development (P < 0.05) and the expression of many genes was positively correlated with other genes and with gestational age. Thus, the rate of cortical stromal expansion peaked in early gestation due to cell proliferation, whilst late in development expression of extracellular matrix genes increased.M.D. Hartanti, A K. Hummitzsch, H.F. Irving-Rodgers, W.M. Bonner, K.J. Copping, R.A. Anderson, I.C. McMillen, V.E.A. Perry and R.J. Rodger

Gata4 Is Required for Formation of the Genital Ridge in Mice

In mammals, both testis and ovary arise from a sexually undifferentiated precursor, the genital ridge, which first appears during mid-gestation as a thickening of the coelomic epithelium on the ventromedial surface of the mesonephros. At least four genes (Lhx9, Sf1, Wt1, and Emx2) have been demonstrated to be required for subsequent growth and maintenance of the genital ridge. However, no gene has been shown to be required for the initial thickening of the coelomic epithelium during genital ridge formation. We report that the transcription factor GATA4 is expressed in the coelomic epithelium of the genital ridge, progressing in an anterior-to-posterior (A-P) direction, immediately preceding an A-P wave of epithelial thickening. Mouse embryos conditionally deficient in Gata4 show no signs of gonadal initiation, as their coelomic epithelium remains a morphologically undifferentiated monolayer. The failure of genital ridge formation in Gata4-deficient embryos is corroborated by the absence of the early gonadal markers LHX9 and SF1. Our data indicate that GATA4 is required to initiate formation of the genital ridge in both XX and XY fetuses, prior to its previously reported role in testicular differentiation of the XY gonadHoward Hughes Medical Institut

Morphogenesis and fibronectin in sexual differentiation of rat embryonic gonads.

peer reviewedThe possible role of fibronectin in the organization of the sex-specific gonadal components was studied by immunocytochemistry combined with electron and light microscopy in rat fetuses at the ages of 12-15 days. Fibronectin was evenly distributed in both sexes under the basal lamina of the surface epithelium. Other basal laminae were not seen using light or electron microscopy inside the gonadal ridges at the age of 12 days. As the first sign of sexual differentiation, fibronectin-negative gonadal cords appeared in 13-day-old fetuses. In the males the cords were bigger than those in the females. The cords were clearly separated from the interstitium in 15-day-old fetuses of both sexes. A continuous layer of fibronectin had formed between the testicular surface epithelium and the elongated cords indicating the formation of a tunica albuginea. In females the surface epithelium-cord connection was maintained at all stages. Connections of the gonadal cords to mesonephric tubuli were seen in the rete region of both sexes. The electron optical basal lamina around the gonadal cords became continuous by the age of 15 days. The present results suggest that fibronectin is intimately involved in the sexual differentiation of the gonads, but not under the regulation of H-Y antigen or other testis-organizing factor

Developmental changes in interstitial collagens of fetal rat genital ducts

The distribution of interstitial collagen types I and III was studied by immunocytochemistry in the mesenchyme of progressing and regressing mesonephric and paramesonephric ducts of male and female rat fetuses from the age of 15 days until birth. Immunocytochemistry revealed a collagen-poor mesenchymal area around the genital ducts and in continuation with the coelomic epithelium on the lateral edge of the mesonephric ridge of 15-day-old fetuses. Ultrastructurally, collagen fibrils were accumulated along the continuous lamina densa of the mesonephric ducts, whereas they were absent on the medial side of the male and female paramesonephric ducts. In males, the amount of collagen fibrils increased with the histological maturation of the mesenchyme around the mesonephric duct, whereas around the regressing paramesonephric duct collagens disappeared from the basement membrane region and the surrounding mesenchyme of the 16-day-old male duct. After the completion of the paramesonephric regression, the mesenchyme acquired a uniformly collagen containing interstitial matrix. In females, the collagens increased in the mesenchyme around the progressing paramesonephric duct, and the original site of the regressing mesonephric duct became occupied with a collagen-containing mesenchyme by the age of 19 days. The results suggest a close structural linkage between the mesonephric duct and the established early paramesonephric duct. The differences in the developmental maturation of the periductal mesenchyme and the observed changes in the composition of the interstitial matrix probably reflect the functional differences in the regulatory factors acting on the progression and regression of the male and female genital ducts

Basement membrane in differentiating mesonephric and paramesonephric ducts of male and female rat fetuses.

The differentiation of male and female rat genital ducts and their basement membranes were studied by light- and electron-microscopic localization of type-IV and -V collagen, laminin, and heparan sulfate proteoglycan at the fetal ages of 15-21 days. At 15 days, the basement membrane of the mesonephric duct was continuous in both sexes, whereas on the medial side of the paramesonephric duct, it was incomplete. The male mesonephric duct remained enveloped by a continuous basement membrane. Increasing accumulation of basement-membrane material in the periductal mesenchyme was regarded as incipient epididymal differentiation. Local expansions and slow degradation of the basement membrane were noted in the regressing female mesonephric duct. The female paramesonephric duct had acquired a continuous basement membrane by the age of 16 days. At this age, the incomplete basement membrane in the medial side of the male paramesonephric duct disappeared, and breaks in the lateral portion appeared. The formation of epitheliomesenchymal contacts and basal cytoplasmic blebs in the epithelial cells of the regressing paramesonephric duct coincided with the disappearance of the basement-membrane material in the condensed periductal mesenchyme. The asymmetric regression of the male paramesonephric duct was initiated in the immature medial side. The changes in the periductal matrix are indications of basic differences in the regulation of the development and regression of the genital ducts in different sexes

Epithelio-mesenchymal interface and fibronectin in the differentiation of the rat mesonephric and paramesonephric ducts.

The distribution of fibronectin and the morphological differentiation of the genital ducts was studied in rat fetuses at ages from 15 to 21 days. Fibronectin was localized with the peroxidase-antiperoxidase and avidin-biotin method at the electron- and light-microscope level. In 15-day-old male and female fetuses, fibronectin was localized as a continuous lamella around the mesonephric duct and as a discontinuous lamella around the paramesonephric duct. During the differentiation of the female paramesonephric duct, the fibronectin layer became continuous and remained so after the age of 16 days. The fibronectin layer of the male mesonephric duct remained continuous at all ages. The accumulation of mesenchymal cells on the outer surface of the female mesonephric duct and the concomitant detachment of the fibronectin layer around the duct suggests that mesenchymal regulation plays a role in the regression of the mesonephric duct. In the regressing male paramesonephric duct fibronectin was simultaneously lost in the condensed periductal mesenchyme, the places of epithelio-mesenchymal contact, and the epithelial cytoplasmic protrusions towards the mesenchyme. Ultrastructurally, fibronectin was localized in the basal laminae, on the cell membrane in contact with the extracellular material, and on the surface of the fibrillar and flocculent extracellular material. In addition to auto- and heterophagy, epithelio-mesenchymal interactions seem to play an important role in the regression of the genital ducts, although in different ways in males and females. The present results give additional support to the theory of the possible migration of epithelial cells into the surrounding mesenchyme during the regression of the paramesonephric duct

Conventional and confocal immunocytochemistry of proliferating cell nuclear antigen is a useful tool in evaluating bovine embryo viability after cryopreservation

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