Analysis of different cell death processes of prepubertal rat oocytes in vitro.

The processes of cell death were studied in vitro in populations of oocytes isolated from prepubertal rats. In order to identify apoptosis, the externalized phosphatidylserine was recognized with Annexin-V coupled to FITC and the fragmentation of DNA was demonstrated by means of electrophoresis. Oocytes were tested for autophagy by means of the incorporation of monodansylcadaverine and monitoring Lc3-I/Lc3-II by western blot. The expression of mRNA marker genes of autophagy and of apoptosis was studied by means of RT-PCR in pure populations of oocytes. Some oocytes expressed at least one of the following markers: caspase-3, lamp1 and Lc3. Some oocytes were positive to Annexin-V or to monodansylcadaverine. However, most of them were simultaneously positive to both markers. The relative frequency of oocytes simultaneously positive to markers of apoptosis and autophagy did not change in the different ages studied. The transformation of Lc3-I in Lc3-II was present in all populations of oocytes studied. The mRNAs for caspase-3, lamp1 and Lc3 were present in all populations of oocytes analyzed. Our results demonstrate that oocytes of rats from new born to prepubertal age are eliminated by means of three different cell death processes: apoptosis, autophagy and a mixed event in which both routes to cell death participate in the same cell.

Follicle-stimulating hormone treatment reverses the effect of hypophysectomy on cell proliferation in the chicken embryo ovary.

The present study evaluated the effect of FSH treatment on ovarian cell proliferation in the hypophysectomized chicken embryo. Hypophysectomy (Hx) was performed by the partial decapitation technique. Two series of experiments were performed: (a) Hx embryos were treated at 8 days of development with recombinant human FSH (rhFSH) and evaluated at 9 days by measuring BrdU incorporation; (b) Hx embryos were injected with rhFSH and rhCG at 9 days of development and the proliferation rate was measured at 13 days. The presence of mRNA for FSHR and LHR in the ovary of control and Hx embryos was demonstrated by RT-PCR analysis. There was a decrease in the percentage of BrdU labeled cells in the absence of hypophysis at 9 and 13 days of incubation. The decrease was reversed with rhFSH treatment. This effect was observed in the ovarian surface epithelium and the somatic cells of the cortex and the medulla in the 9-day-old embryo. Moreover, the number of somatic, steroidogenic, and germ cells was reduced at 13 days of incubation in the Hx embryo; when treated with rhFSH the number of cells increased to the level of controls. In another experiment, ovaries of 9-day-old chicken embryos were organ cultured for 48 h in a serum-free medium with rhFSH and rhCG separately. The proliferation index was incremented by rhFSH compared to control and rhCG-treated embryos. Therefore, FSH stimulates somatic cell proliferation in the chicken embryo ovary as early as 9 days of development.

Heterogeneity of growth hormone immunoreactivity in lymphoid tissues and changes during ontogeny in domestic fowl.

Growth hormone (GH) expression is not confined to the pituitary and occurs in many extrapituitary tissues. Here, we describe the presence of GH-like moieties in chicken lymphoid tissues and particularly in the bursa of Fabricius. GH-immunoreactivity (GH-IR), determined by ELISA, was found in thymus, spleen, and in bursa of young chickens, but at concentrations <1% of those in the pituitary gland. Although the GH concentration in the spleen and bursa was approximately 0.82 and 0.23% of that in the pituitary at 9-weeks of age, because of their greater mass, the total GH content in the spleen, bursa, and in thymus were 236, 5.18, and 31.5%, respectively, of that in the pituitary gland. This GH-IR was associated with several proteins of different molecular size, as in the pituitary gland, when analyzed by SDS-PAGE under reducing conditions. While most of the GH-IR in the pituitary was associated with the 26 kDa monomer (40%), the putatively glycosylated 29 kDa variant (16%), the 52 kDa dimer (14%) and the 15 kDa submonomeric isoform (16%), GH-IR in the lymphoid tissues was primarily associated (27-36%) with a 17 kDa moiety, although bands of 14, 26, 29, 32, 37, 40, and 52 kDa were also identified in these tissues. The heterogeneity pattern and relative abundance of bursal GH-IR bands were determined during development between embryonic day 13 (ED13) and 9-weeks of age. The relative proportion of the 17 kDa GH-like band was higher (45-58%) in posthatched birds than in the 15 and 18-day old embryos (21 and 19%, respectively). The 26 kDa isoform was minimally present in embryos (<4% of total GH-IR) but in posthatched chicks it increased to 12-20%. Conversely, while GH-IR of 37, 40, and 45 kDa were abundantly present in embryonic bursa ( approximately 30% at ED13 and approximately 52-55% at ED15 and ED18, respectively), in neonatal chicks and juveniles they accounted for less than 5%. These ontogenic changes were comparable to those previously reported for similar GH-IR proteins in the chicken testis during development. In summary, these results demonstrate age-related and tissue-specific changes in the content and composition of GH in immune tissues of the chicken, in which GH is likely to be an autocrine or paracrine regulator.

Follicle-stimulating hormone increases cell proliferation in the ovary and the testis of the chick embryo.

Previous studies have demonstrated that FSH stimulates cell proliferation in the ovary and the testis of the chick embryo. This study analyzed the presence of FSH receptor and the cell subpopulations that proliferate in response to FSH in chick embryo gonads. FSH receptor mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) in the male and female gonads of the 6 to 14-day-old chick embryo. Somatic cells of the ovary expressed the FSH receptor in the 14-day-old chick embryo. Ovarian surface epithelium of the 14-day-old chick embryo increased the mitotic index 15-18 h after FSH treatment. Similarly, the mitotic index in oogonia was increased 24 h after receiving a pulse of FSH; this result was confirmed by an increase in the number of germ cells that incorporated bromodeoxyuridine (BrdU). Somatic cells of the medullary cords in the ovary displayed an increase in the mitotic index 15-21 h after the FSH injection. In the chick embryo testis, at the same stage of development, the treatment with FSH increased the mitotic index in cells of the seminiferous tubules and to a lesser extent in cells at a peritubular and interstitial location. Present results demonstrate that in the chick embryo, FSH stimulates the proliferation of ovarian surface epithelium, oogonia in the cortex, and somatic cells of the medullary cords of the chick embryo ovary. In the chick embryo testis, FSH stimulates cell proliferation in seminiferous tubules and peritubular cells.

Follicle-stimulating hormone regulates steroidogenic enzymes in cultured cells of the chick embryo ovary.

This investigation addresses the potential regulation of enzymes involved in the biosynthesis of steroid hormones during early stages of gonadal development by follicle-stimulating hormone (FSH). Gonadal cells of 10-day-old chick embryo and cells of the left ovary of 18-day-old chick embryo were cultured for 60 h in a defined medium with or without the addition of FSH (2.0 IU/ml). At the end of the culture, cells were recovered and evaluated by biotransformation of tritiated steroid precursors and mRNA levels were evaluated by RT-PCR. The production of estrone from androstenedione was increased in the FSH-treated cells, both human FSH (hFSH) and recombinant human FSH (rhFSH), indicating a stimulatory effect on aromatase (P450arom). Similarly, the intensity of the band corresponding to P450arom mRNA was higher in hFSH and rhFSH than in control and chorionic gonadotropin (hCG) groups. The P450arom stimulation was observed in the ovary of 10- and 18-day-old chick embryo. The transformation of dehydroepiandrosterone to androstenedione was taken as evidence of 3beta-hydroxysteroid dehydrogenase function. This enzyme was stimulated in the cultured ovarian cells of 18-day-old chick embryos treated with hFSH and rhFSH compared with controls. The production of pregnenolone in the mitocondrial fraction of 18-day-old chick embryo ovary was increased when cultured with hFSH and rhFSH. This observation together with the increase in the band intensity corresponding to mRNA of P450 cholesterol side-chain cleavage indicates stimulation by FSH treatment; hCG produced a similar effect. Somatic cells of the medullary cords are proposed to be FSH target cells in the ovary of the chick embryo.

The P450 aromatase (P450 arom) gene is asymmetrically expressed in a critical period for gonadal sexual differentiation in the chick.

Steroid hormones appear to play an important role in gonadal sex differentiation of birds. Here we studied the steady-state level of the P450 arom mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) in the left and the right presumptive ovary and testis of developing chicken embryos. The gonads were evaluated every hour during the undifferentiated period, at 144-156 h of incubation (h/i), and every 24 h after sexual differentiation at 168 and 192 h/i. Activity of P450 arom was determined by estrone production from [3H]androstenedione at 144-192 h/i. Moreover, morphological development of the gonad was also examined by light microscopy. Results show that onset of P450 arom mRNA and its protein activity were simultaneously detected in the left and the right ovaries at 147 h/i. Asymmetric function of P450 arom gene expression was observed at 156 h/i when morphological gonadal differentiation is first recognized. Biotransformation of [3H]androstenedione to estrone was also asymmetrically detected between the left and right gonad at 156 h/i and asymmetry was maintained throughout the analyzed stages. It is proposed that there is a gene in birds that is asymmetrically expressed in the undifferentiated stage of the female and the male gonad. In the female this gene could promote P450 arom gene expression, increasing estrogen production, which in turn could induce ovarian cortex proliferation and expression of other structural estrogen-regulated genes involved in ovarian sexual determination.

Evidence for estrogen receptor expression in germ cell and somatic cell subpopulations in the ovary of the newly hatched chicken.

Estrogens are involved in the gonadal morphogenesis of vertebrates, and almost all hormonal effects of 17beta-estradiol are mediated through specific receptors. At the time of sexual differentiation in the chicken, or even before, there is evidence of the presence of estrogen receptors and the secretion of 17beta-estradiol. However, no information is available regarding the cellular types that express the estrogen receptor in the immature chick ovary. The present study analyzes estrogen receptor expression in germ and somatic cells of the ovary in the newly hatched chicken. Highly purified cell subpopulations of germ and somatic cells were evaluated for specific 17beta-estradiol nuclear binding. In addition, the estrogen receptor was localized at the ultrastructural level by the immunogold technique. Finally, reverse transcription and polymerase chain reaction procedures detected a steady-state level of mRNA for the estrogen receptor. Somatic cells including typical steroidogenic cells showed specific 17beta-estradiol nuclear binding, displayed the estrogen receptor, and possessed estrogen receptor transcripts. The same result was observed in primary oocytes, together with the ultrastructural localization of estrogen receptor in extended chromatin filaments. Our experimental data support the hypothesis that estrogens are involved in the function of somatic and germ cells subpopulations in the immature chicken ovary.

Proliferative and steroidogenic effects of follicle-stimulating hormone during chick embryo gonadal development.

The aim of this study was to evaluate the in vitro effect of human follicle-stimulating hormone (hFSH) on cellular proliferation and steroid hormone secretion in the left ovary, the right ovary, and the testis of the chick embryo. Gonads from 8- to 18-day-old chick embryo were cultured in a defined medium during 60 h under basal and hFSH-stimulated conditions (0.5 IU/ml of culture medium). At the end of the culture, the incorporation of ¿(3)Hthymidine and the total number of cells were measured to estimate gonadal cell proliferation. The secretion of 17beta-estradiol and testosterone in the culture medium was radioimmunologically assayed in order to evaluate the steroidogenic function of the cultured gonadal cells. The response to hFSH stimulation was observed in the left ovary, the right ovary, and the testis from the 8-day-old chick embryo. In the left ovary, cellular proliferation was not augmented by hFSH in the 8-, 10-, and 13-day-old chick embryo; meanwhile, the proliferative stimulus of hFSH was observed in the 15- and 18-day-old embryos. In the same ovary, 17beta-estradiol and testosterone secretion were stimulated after hFSH treatment at all evaluated stages (8-18 days of chick embryo development). In the right ovary, an increment in proliferation and steroidogenesis was induced by hFSH in the 8-, 10-, and 13-day-old chick embryo. Afterward, the right gonad did not respond to hFSH. Testis cells displayed hFSH response as an increment in cell proliferation at all embryonic ages (8-18 days of chick embryo development). There was a transient lack of response to hFSH in testosterone secretion at 10 and 13 days of development. The in vitro effect of hFSH on cell proliferation and steroid hormone secretion changed in the ovary and the testis according to the age of the embryo. These changes could be attributed to the growth of the left ovary and the testis and the regression of the right ovary. Probably, paracrine factors modulated the gonadotropin effect on the target cells during embryonic development of chick embryo gonads.

[Physiopathologic bases for preeclampsia: a hypothesis]. / Bases fisiopatológicas de la preeclampsia: una hipótesis.

Preeclampsia represents the main medical complication of pregnancy and one of the most important causes of maternal mortality around the world. At this time, the etiology of the illness is unidentified and there is not sure predictors for early identification. The objective of this article was to provide an integrative hypotheses that suggests the possibility of to relate an increment of placental lactogen, insulin resistance, hyperinsulinemia and risk of preeclampsia.

Steroid metabolism in granulosa and theca interna cells from preovulatory follicles of domestic hen (Gallus domesticus).

The capability of granulosa and theca interna cells, from preovulatory follicles of the domestic hen, to metabolize steroid precursors was evaluated. Granulosa and theca interna cells were isolated from ovarian preovulatory follicles at three different developmental stages: F1, F3 and F5. Tritiated pregnenolone (P5), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4) and testosterone (T) were employed as precursors and their metabolic products were evaluated. The major metabolite of P5 by granulosa cells was P4, but we also observed low amounts of 5beta-pregnandione. DHEA metabolism by granulosa cells yielded mainly A4, and minute quantities of 5beta-androstan-3,17-dione (5beta-dione) were detected. The only significant metabolite obtained in granulosa cells from A4 was 5beta-dione, whereas T was only transformed into A4. On the other hand, P5 metabolism by theca interna cells yielded A4 as the main product, also P4, 17alpha-OHP4, 17alpha-OHP5, 5beta-pregnandione, and DHEA, were found. When DHEA was the precursor A4 was produced in higher amounts than 5beta-dione. A4 was mainly transformed into 5beta-dione. In similar conditions, T was transformed into A4. These results show that granulosa cells have enzymatic activities of 3beta-hydroxysteroid dehydrogenase/5-4 isomerase (3beta-HSD from P5 and DHEA), 17beta-hydroxysteroid dehydrogenase (17beta-HSD from T) and 5beta-reductase (from P5, DHEA and A4). Whereas theca interna cells have enzymatic activities of cytochrome P450c17 (from P5 and P4), 3beta-HSD (from P5 and DHEA), 17beta-HSD (from T) and 5beta-reductase (from P4, DHEA and A4). These data support the concept that theca interna cells have the ability to synthesize androgens from progestins produced in granulosa cells. In addition, since theca interna cells did not show the capacity to aromatize androgens suggests that interaction between theca interna and theca externa cells occurs in vivo, thus confirming the three cell model for estrogen production. Furthermore, the fact that other metabolites were produced both in granulosa and theca interna cells, but in a different extent, suggests that complex mechanisms are participating in the regulation of steroid synthesis in avian ovary follicles.

Follicle stimulating hormone increases somatic and germ cell number in the ovary during chick embryo development.

The aim of the present study was to evaluate the effect of human follicle stimulating hormone (hFSH) on cellular proliferation in the chick embryo ovary. Chick embryos (Babcock B300) were injected on chorioallantoic membrane with a single dose of hFSH (2.0 IU/ embryo) at Days 7, 9, or 13 of incubation or with hCG (2.0 IU/embryo) at Day 13 of incubation. At 17 days of incubation and within 24 h after hatching, left ovaries were dissected and completely dissociated. Cells from the whole ovary were classified into germ cells (primary oocytes), typical steroidogenic cells, and poorly differentiated somatic cells and counted with the aid of a hemocytometer. Aliquots of the cell suspension from the whole left ovary were analyzed by flow cytometry, in order to determine the percentage of cells at each phase of the cell cycle. In addition, samples of the suspension (1.0 x 10(6 )cells) were incubated for 2 h in basal and stimulated conditions measuring 17beta-estradiol secretion in the medium. The ovarian cell number at 17 days of incubation showed that hFSH treatment at Day 7 did not modify the cell number in any of the subpopulations evaluated; treatment at Day 9 resulted in an increase in poorly differentiated somatic cell number, without changes in steroidogenic and germ cells, whereas hFSH treatment at Day 13 augmented the number of poorly differentiated, steroidogenic, and germ cells. The percentage of cells in S-phase was increased 12 and 15 h after hFSH treatment (Day 13). Secretion of 17beta-estradiol was increased in the hFSH-treated group (Day 13) measured at 17 days of incubation. The increase in cell number of the three subpopulations was still observed in the left ovary of the newly hatched chicken. Treatment with hCG at Day 13 of incubation did not change the number of poorly differentiated, steroidogenic, and germ cells in the left ovary, neither in the 17-day-old chick embryo nor in the newly hatched chicken. The 17beta-estradiol secretion in hCG-treated embryos was similar to controls. The present study is the first evidence of an effect of FSH on somatic and germ cell number, together with an increase in 17beta-estradiol production during chick embryo ovary development.

Proliferative effect in vitro of follicle-stimulating hormone on the left ovary of the chick embryo.

The purpose of the present study was to evaluate the effect of human follicle-stimulating hormone (hFSH) on cellular proliferation in the chick embryo ovary. Left ovaries from 18-day-old chick embryos (Babcock B300) were dissociated by trypsin (0.25%) treatment. In some experiments, dissociated ovarian cells were further separated by a continuous metrizamide gradient (0-20%). Four cellular subpopulations were recovered from the density gradient: (a) typical steroidogenic cells (F1, density 1.026 g/ml), (b) primary oocytes (F2, 1.048 g/ml), (c) pregranulosa cells, and (d) poorly differentiated epithelial cells (both cellular types were found in F3, 1.059 g/ml, and F4, 1.071 g/ml). Samples (5 x 10(5) cells) of dissociated cells of the whole ovary and of the four cellular subpopulations obtained from the density gradient were cultured on polycarbonate membranes in a defined medium with 0.1 microCi of [3H]thymidine for 60 hr. When necessary, 17beta-estradiol, hFSH, recombinant human FSH, and hCG were added to the medium at the begining of the culture. The total number of cells and the incorporation of [3H]thymidine to the ovarian cell aggregate at the end of the culture increased when hFSH and recombinant hFSH (0.5 IU/ml) were added. No changes were produced with hCG (2.0 IU/ml) and 17beta-estradiol (200 ng/ml) treatments. The secretion of 17beta-estradiol by cultured ovarian cells was stimulated in the hFSH-, recombinant hFSH-, and hCG-treated groups. The dose-response curve to hFSH resulted in an ED50 of 0.03 IU/ml. In the temporal curve, the stimulatory effect of hFSH on the total number of cells and the [3H]thymidine incorporation were observed at 36 hr of culture and maintained up to 60 hr. The proliferative effect of hFSH measured as [3H]thymidine incorporation was observed only in the F4 fraction of the density gradient, which includes poorly differentiated epithelial cells and pregranulosa cells from the ovarian medulla and cortex, respectively. Present results demonstrate a dose-dependent stimulation of DNA synthesis by hFSH in the inmature ovary of the 18-day-old chick embryo. Either pregranulosa cells or poorly differentiated epithelial cells or both subpopulations would be the target cells for the proliferative effect of the gonadotropin.

Steroid metabolism in theca externa cells from preovulatory follicles of domestic hen (Gallus domesticus).

The purpose of this study was to evaluate the ability of theca externa cells from preovulatory follicles of the domestic hen to metabolize tritiated steroid precursors. Theca externa cells were isolated from ovarian preovulatory follicles at three different developmental stages; F1 (35 mm), F3 (26 mm), and F5 (13 mm). Tritiated pregnenolone (P5), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4), and testosterone (T) were employed as precursors and their metabolic products were evaluated after separation by thin-layer chromatography. The major metabolite of P5 by theca externa cells was 5-pregnen-3 beta, 20 beta-diol (F5, 47%; F3, 39%; and F1, 24%), but minimal quantities of P4 were detected. Progesterone metabolism yielded mainly 4-pregnen-20 beta-ol-3-one (F5, 52%; F3, 34%; and F1, 49%). When DHEA was used as precursor, A4 was produced in higher amounts (F5, 29%; F3, 23%; and F1, 11%) than estrone (E1) (F5, 1.5%; F3, 0.9%; and F1, 0.4%). Androstenedione was mainly transformed into E1 (F5, 11.9%; F3, 12.2%; and F1, 0.2%) but lower quantities of T and 17 beta-estradiol (E2) were found. Testosterone was actively transformed into A4 (F5, 50%; F3, 50%; and F1, 30%), but a low transformation to E2 (F5, 1.9%; F3, 1.7%; and F1, 1.4%) and E1 (F5, 2%; F3, 1%; and F1, 0.5%) was found. These results show that theca externa cells from preovulatory follicles of hen have enzymatic activities of 20 beta-reductase (from P5 and P4), 3 beta-hydroxysteroid dehydrogenase/5-4 isomerase (from P5 and DHEA), 17 beta-hydroxysteroid dehydrogenase (from A4 and T), and aromatase (from A4 and T). Furthermore, the enzyme activities decrease with follicular maturation, except for 20 beta-reductase which is constant. These data support the concept that theca externa cells have the ability to synthesize different steroids than reported in theca externa cells. In addition, since theca externa cells did not show the capacity to produce androgens but these steroids were aromatized to estrogens by these cells, it was suggested that the interaction between theca interna cells and theca externa cells occurs in vivo, thus supporting the multicellular theory for estrogen production.

Androstenedione metabolism in the indifferent stage of bovine gonad development.

Suspensions of cells obtained from genital ridges and gonads of bovine embryos of 1.0-3.4 cm in crown-rump (C-R) length were used for studies of the metabolism of radiolabeled exogenous steroid precursor. Tritiated androstenedione was employed as precursor and the final products of their metabolism were evaluated after separation by thin-layer chromatography. Genetic sex was determined by the karyotype of hepatocytes from the same embryos. The extent of conversion of tritiated androstenedione was higher in cells from male embryos than cells from female embryos. Furthermore, androstenedione was metabolized mainly to testosterone in male embryos. By contrast, cells obtained from female embryos transformed androstenedione to estrone and 17 beta-estradiol. The onset of this activity was observed at 1.8 cm in C-R length in males and at 2.2 cm in C-R length in females. In all cases, after the onset, the metabolic activity increased in relation to the age of the embryos. These data show that in the bovine embryo gonad, the activity of aromatase and 17 beta-hydroxysteroid dehydrogenase are present when the morphological differentiation of the gonadal sex has not yet been established.

Newly hatched chick ovarian cell subpopulations metabolize distinctively progestin and androgen precursors.

The aim of this study was to characterize the ability of four different cell fractions (F1, F2, F3, and F4), isolated from the ovary of newly hatched chick by means of subsequent metrizamide gradients (0-15%), to metabolize progestins and androgens. The results showed the presence of 17 alpha-hydroxylase/C17-20 lyase and 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) activities in the typical steroidogenic cells isolated in F1 (d = 1.012 g/ml). Primary oocytes present in F2 (d = 1.037 g/ml) did not show relevant steroid metabolism in the various assays that were carried out. Fractions 3 (d = 1.055 g/ml) and 4 (d = 1.071 g/ml), which contained a mixture of prefollicular and poorly differentiated epithelial cells, presented both 5 beta-reductase and aromatase activities, whereas 17 beta-HSD activity was mainly located in the cells of fraction 3. It is highly possible that poorly differentiated epithelial cells of fractions 3 and 4 are responsible for the steroidogenic activity. We conclude that in newly hatched chick ovary, typical steroidogenic cells metabolize progestins to androgens, and poorly differentiated epithelial cells further aromatize androgens to estrogens. In addition, we suggest the existence of at least two metabolically distinct poorly differentiated epithelial cell subpopulations, one presenting 5 beta-reductase and aromatase activities and another exhibiting 17 beta-HSD activity.

Morphological changes in the thecal layer during the maturation of the preovulatory ovarian follicle of the domestic fowl (Gallus domesticus).

The aim of the present study was to evaluate changes in the steroidogenic tissue, the blood vessels and the undifferentiated cell islets observed in the theca of developing follicles in the hen ovary. Samples of preovulatory follicles (F1 to F5 in order of decreasing size) and small yellow follicles (SYF) were obtained from White Leghorn hens for light microscopy studies. Cell suspensions of the whole theca obtained from follicles at the same stage of development were analyzed for 17 beta-estradiol and testosterone secretion. Typical steroidogenic cells with abundant cytoplasmic lipid droplets were identified in the theca interna beside blood vessels and nerve fibers. Islets of undifferentiated cells were observed in the theca externa. The steroidogenic tissue of the theca interna was evident in the small yellow follicle. However, considering the growth of the developing follicle, the total volume of steroidogenic cells increased from the SYF up to F1 follicle. The blood vessels reached the maximum development in the largest follicle (F1). On the other hand, undifferentiated cell islets were markedly reduced in the F1 follicle. The structural changes herein described could be correlated to modifications in the steroid hormone secretion during the maturation of the ovarian follicle.

Steroid metabolism in the cortex and the medulla of the early fetal bovine ovary.

Suspensions of cells obtained from the cortex and the medulla of the ovary of early bovine fetuses of 4-10 cm in crown-rump (C-R) length were used for studies of the metabolism of radiolabeled exogenous steroid precursors. Tritiated dehydroepiandrosterone, androstenedione, and testosterone were employed as precursors and their metabolic products were evaluated after separation by thin-layer chromatography. The extent of conversion of tritiated dehydroepiandrosterone to androstenedione was higher in cells from medulla than in those from the cortex. Androstenedione was metabolized mainly to estrone, and the percent of transformation was similar in the cortex and the medulla, but the rate of formation of 17 beta-estradiol was higher in the medulla than in the cortex. Furthermore, cells obtained from the cortex and the medulla aromatized testosterone to 17 beta-estradiol at a similar rate. In all cases, the percent of transformation was higher in ovaries from fetuses of 4.0 cm in C-R length than those from fetuses of 9.0-10.0 cm in C-R length. These data suggest that, in the bovine fetal ovary, the activity of 3 beta-hydroxysteroid dehydrogenase/isomerase and 17 beta-hydroxysteroid dehydrogenase predominates in the medulla, whereas aromatase activity is similar in the cortex and the medulla of the early fetal bovine ovary.

Effect of 17 beta-estradiol on somatic and germ cell subpopulations in the ovary of the newly hatched chick.

The involvement of estrogens in avian ovary differentiation and morphogenesis has been previously demonstrated by several authors. The aim of the present study is to provide evidence regarding which cell subpopulations of the ovary are target cells for 17 beta-estradiol during chick development. White Leghorn chick embryos received 200 ng of the hormone or the vehicle on the chorioallantoic membrane at 11, 13, and 15 days of incubation. Within 24 hr after hatching, the left ovary was processed for total cell count, study of the meiotic prophase of oocytes, and the morphometric evaluation of the cortical and the medullary regions of the ovary. Germ cell number was not modified by the treatment. Similarly, no changes were observed in the stage of the prophase and the cellular area of oocytes. The total number of somatic cells was increased in the newly hatched chick ovary treated with 17 beta-estradiol. The number of undifferentiated cells in the juxtacortical medulla was higher in the treated ovary than in the control group. Moreover, the pregranulosa cells of the cortical region were hypertrophied in the 17 beta-estradiol treated ovary. However, no changes were observed in the typical steroidogenic cells after the estrogen treatment. In conclusion, two subpopulations of somatic cells are estrogen target cells in the prefollicular chicken ovary: the pregranulosa cells of the ovarian cortex and the undifferentiated cells in the juxtacortical medulla.

Steroidogenic cell subpopulations obtained from the theca of preovulatory follicles in the ovary of the domestic fowl.

The purpose of the present study is to provide further evidence in support of the theory which suggests that two cells are involved in the secretion of estrogens and androgens in the theca of the preovulatory ovarian follicles in the domestic fowl. The third (F3), the fourth (F4), and the fifth (F5), in order of decreasing size follicles, were obtained from white Leghorn hens. Theca cells were dispersed by trypsin treatment, and isolated cells were centrifuged in a continuous Percoll gradient (0-100%). Testosterone and 17 beta-estradiol secretion from cell samples in each density gradient fraction were radioimmunologically assayed. The highest secretion of 17 beta-estradiol was registered in cells isolated in density fractions ranging from 1.059 to 1.070 g/ml, while the maximal testosterone production was found in fractions from 1.037 to 1.048 g/ml. The different results were obtained from the three types of follicles studied (F3-F5). Morphological study of the F4 follicle showed typical steroidogenic cells with abundant lipid droplets in the cytoplasm in the theca interna, whereas clusters of poorly differentiated epithelial cells were visualized between fibroblast layers of the theca externa. The secretion of aromatizable androgens by the typical steroidogenic cells of the theca interna and estrogen production in the epithelial cells of the theca externa are postulated.

Ultrastructure of the ovarian medulla in the newly hatched chick treated with human chorionic gonadotropin.

The ultrastructural characteristics of the ovarian medulla of the newly hatched white leghorn chick were studied in control animals and compared with chicks that were treated with human chorionic gonadotropin during embryonic development. The ovarian medulla was mainly occupied by epithelial cells which formed cords or islets surrounded by a basal lamina. Within this epithelial compartment, steroidogenic cells, poorly differentiated cells and a lacunary system could be recognized. When chicks were treated with human chorionic gonadotropin, steroidogenic cells became discernible; there was an increment in the amount of cytoplasm and the area of mitochondria. Poorly differentiated cells exhibited signs of stimulation, and transitional images suggested the transformation of these cells into steroidogenic cells. The epithelial cells of the lacunar system also displayed stimulated cytoplasmic organelles. Evidence was supplied suggesting that relatively undifferentiated cells persist in the ovarian medulla until hatching and can develop into steroidogenic cells under gonadotropic stimulation.