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.

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.

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.