Development of the ovary and ontongeny of mRNA and protein for P450 aromatase (arom) and estrogen receptors (ER) alpha and beta during early fetal life in cattle.

Estradiol-17beta is the predominant steroid produced during early stages of ovarian development in ruminants and steroid hormones have been hypothesized to regulate ovigerous cord formation, germ cell meiosis and ovarian vascular development. Therefore, the objective was to determine the presence and localization of mRNA and protein encoding cytochrome P450 aromatase (P450arom), and estrogen receptors alpha (ERalpha) and beta (ERbeta) during ovarian development in fetuses of cattle on days 35, 45, 60, 75, 90 and 105 after breeding (n=4/age) using in situ hybridization and immunohistochemistry. No ovarian tissue was found in the day 35 fetuses, but was found in all later ages studied. There appeared to be little organization of specific structures in ovaries on days 45 and 60, although germ cells could be identified. Evidence of the beginning of ovigerous cord formation was found on day 60. By day 75 of gestation, the ovigerous cords were more extensive and mesonephric-derived cell streams were detectable. By day 90 (and still present at day 105), both ovigerous cords and cell streams/rete tubules were definitive structures of the developing ovaries. Ovaries appeared to develop in "lobular" segments around the periphery of the ovary. Some lobes appeared to be at slightly different developmental stages, as assessed by the extent or definition of ovigerous cord formation. The localization of mRNAs for P450arom, ERalpha and ERbeta were closely associated with protein content. At days 45 and 60, mRNA and protein of P450arom and ERbeta were located throughout ovaries with signal in medulla being denser than in the cortex. P450arom mRNA or protein was punctate, but not evident in germ cells. From day 75, P450arom was increasingly becoming localized to cell streams or clusters of cells (rete tubules) in the medulla, and by days 90 and 105 of gestation, was more definitively localized to cell streams and/or rete tubules. Similar to P450arom, ERbeta mRNA and protein were observed in cells in the medulla, and also in germ cells, pre-granulosa cells and some surface epithelial cells. ERalpha mRNA and protein were predominately in the surface epithelium in ovaries of all ages with fainter signal for ERalpha protein also being observed in pre-granulosa and stromal cells including the cell streams/rete tubules. ERalpha protein was also detected in a few germ cells at days 90 and 105 of gestation. Thus, in cattle, estradiol-17beta has the potential to regulate, in an autocrine/paracrine manner, a number of different cell types during ovarian development.

Morphological development and characterization of aromatase and estrogen receptors alpha and beta in fetal ovaries of cattle from days 110 to 250.

To better understand the role of estradiol-17beta in fetal ovarian development, presence and localization of cytochrome P450 aromatase (P450arom) and estrogen receptors alpha (ERalpha) and beta (ERbeta) proteins were characterized in fetal ovaries of cattle using immunohistochemistry. Fetal cattle ovaries were collected from an abattoir and sorted into fetal age groups (days 110, 130, 150, 170, 190, 210, 230, 250+) based on crown-rump length. In addition to immunohistochemistry, morphological analysis of ovarian and follicular formation was made. Ovaries appeared lobular at day 110, but by the end of gestation (day 250+) ovaries were oval-shaped similar to those found in adult animals. Ovarian structures within different lobes appeared to be at different developmental stages. At day 110, oocytes and pre-granulosa cells were observed in ovigerous cords that were still open to the surface epithelium. Most ovigerous cords appeared to be closed to the surface epithelium on day 130, all closed by day 150 and were no longer present at day 210. Ovarian follicles were classified as follows: Type 1(primordial): single layer of flattened granulosa cells, Type 1a (transitory): single layer of mixed flattened and cuboidal granulosa cells, Type 2 (primary): at least one but less than two layers of cuboidal granulosa cells, Type 3 (small preantral): two to three layers of granulosa cells, Type 4 (large preantral): four to six layers of granulosa, and the theca layer is forming around the follicle, Type 5 (antral): contain greater than six layers of granulosa cells, several layers of theca cells and the antrum has formed. Type 1 follicles were observed in day 110 ovaries. Follicle Types 1a and 2 were first observed on day 130. Type 3 follicles were first observed on day 150 and Types 4 and 5 were first observed on day 170. P450arom protein was localized in granulosa cells of follicle Types 2-5 and cells of rete tubules throughout the experimental period. There was punctate expression within stroma and rete masses. There was ERalpha protein localization in pre-granulosa cells and germ cells of ovigerous cords and all surface epithelial cells. There was also localization in granulosa cells and oocytes of all follicle types and cells of rete tubules. There was punctate ERalpha protein expression in stroma and rete masses. ERbeta protein was localized in pre-granulosa cells and germ cells of ovigerous cords. Expression was also localized to granulosa cells of all follicle types and cells of rete tubules. ERbeta protein was punctate in oocytes of follicles, surface epithelial cells, stroma and rete masses. Thus, the fetal ovary of cattle has the steroidogenic enzyme (P450arom) to convert androgens to estradiol-17beta, and estrogen receptors alpha and beta to facilitate an estrogen response within the fetal ovary.