Hugo H. Ortega

Developmental programming: prenatal androgen excess disrupts ovarian steroid receptor balance

Steroid hormones play an important role in reproduction and the receptors through which they signal change in a developmental time, follicle stage, and cell-specific manner. Disruption in steroid receptor expression affects follicle formation and differentiation. In this study, using prenatal testosterone (T) and dihydrotestosterone (DHT)-treated female sheep as model systems, we tested the hypothesis that prenatal androgen excess disrupts the developmental ontogeny of ovarian steroid receptor protein expression. Pregnant Suffolk ewes were injected twice weekly with T propionate or DHT propionate (a non-aromatizable androgen) in cottonseed oil from days 30 to 90 of gestation. Changes in ovarian estrogen receptors (ER; ESR1, ESR2), androgen receptor (AR) and progesterone receptor (PGR) proteins were determined at fetal (days 90 and 140), postpubertal (10 months), and adult (21 months; only prenatal T-treated sheep studied) ages by immunohistochemistry. Prenatal T and DHT treatment induced selective increase in AR but not ER or PGR expression in the stroma and granulosa cells of fetal days 90 and 140 ovaries. An increase in ESR1 and decrease in ESR2 immunostaining coupled with increased AR expression were evident in granulosa cells of antral follicles of 10- and 21-month-old prenatal T but not DHT-treated females (analyzed only at 10 months). These findings provide evidence that an early increase in ovarian AR is the first step in the altered ovarian developmental trajectory of prenatal T-treated females, and manifestations of postnatal ovarian dysfunction are likely facilitated via altered equilibrium of antral follicular granulosa cell ER/AR protein expression.

An imbalance between apoptosis and proliferation contributes to follicular persistence in polycystic ovaries in rats

BackgroundCystic ovarian disease is an important cause of infertility that affects bovine, ovine, caprine and porcine species and even human beings. Alterations in the ovarian micro-environment of females with follicular cysts could alter the normal processes of proliferation and programmed cell death in ovarian cells. Thus, our objective was to evaluate apoptosis and proliferation in ovarian cystic follicles in rats in order to investigate the cause of cystic follicle formation and persistence.MethodsWe compared the number of in situ apoptotic cells by TUNEL assay, expression of active caspase-3 and members of Bcl-2 family by immunohistochemistry; and cell proliferation by the expression of the proliferation markers: PCNA and Ki-67.ResultsThe proliferation index was low in granulosa of tertiary and cystic follicles of light exposed rats when compared with tertiary follicles of control animals, while in theca interna only cystic follicles presented low proliferation index when compared with tertiary follicles (p < 0.05). The granulosa of cysts exhibited a similar cell DNA fragmentation to early atretic follicles. In the granulosa and theca interna, active caspase-3 shown similar immunostaining levels in tertiary and cystic follicles (p < 0.05). The granulosa cells presented high expression of Bcl-2, Bcl-xL and Bcl-w in the tertiary and cystic follicles with diminishing intensity in the atretic follicles, except with Bcl-w where the intensity was maintained in the atretic follicles (p < 0.05). The expression of Bax was weak in the healthy and cystic follicles. In the theca interna, Bcl-2 expression was the same as the pattern found in the granulosa; no differences were found between tertiary and cystic follicles from both groups for Bcl-xL and Bcl-w. The expression of Bax in this layer was higher in the tertiary follicles of the treated animals (p < 0.05) while the values for cystic follicles were similar to those in the tertiary follicles of controls. The theca externa showed low expression of the pro and anti-apoptotic proteins.ConclusionThese results show that the combination of weak proliferation indices and low apoptosis observed in follicular cysts, could explain the cause of the slow growth of cystic follicles and the maintenance of a static condition without degeneration, which leads to their persistence. These alterations may be due to structural and functional modifications that take place in these cells and could be related to hormonal changes in animals with this condition.

Expression of Cytoskeletal Proteins in the Follicular Wall of Induced Ovarian Cysts

Several experimental models have been developed for the study of the polycystic ovarian syndrome in the rat. In the present study, the syndrome was induced by exposure to constant light, and the expression of cytoskeletal proteins in the follicular wall was evaluated by immunohistochemistry. We analyzed the immunohistochemically stained area (IHCSA) by image analysis to evaluate the expression of intermediate filaments (vimentin, desmin, cytokeratins, gliofibrillary acidic protein and neurofilaments) and α-smooth muscle actin (α-SMA) in cystic ovaries in relation to normal ovaries. The granulosa cell layer of cystic follicles had a significantly greater IHCSA for vimentin than the normal antral follicles. This difference was also significant between atretic and antral follicles. Cytokeratins showed a very low expression in the granulosa cells of antral follicles of control ovaries while in granulosa cells of atretic and cystic follicles they showed a significantly higher IHCSA. Immunohistochemical localization of desmin and α-SMA was restricted to the theca externa. Immunoreactivity for gliofibrillary acidic protein and neurofilament was negative. The highest intensity in the staining with vimentin and cytokeratins observed in the granulosa cells of the cystic follicles is probably due to structural and functional changes that occur during the process of cystogenesis and they could be associated with intense changes in the expression of cytoskeletal proteins that may be essential to the proper cellular functioning.

Polycystic ovarian syndrome: temporal characterization of the induction and reversion process in an experimental model

Numerosos modelos experimentais tem sido desenvolvimos para o estudo da sindrome do ovario policistico em ratos. No presente estudo, a sindrome foi inducida por exposicao a luz constante. O processo foi avaliado durante sua inducao e inclusive durante sua reversao. O ciclo estral foi analisado atraves de citologia vaginal; parametros reprodutivos foram avaliados por acasalamento, bem como a morfologia ovariana. Todos animais desenvolveram a sindrome depois de 13 semanas de luz permanente. As caracteristicas histologicas dos ovarios, na semana 15, foram similares aquelas observadas na sindrome do ovario policistico em humanos e outras especies. Apos a regressao da sindrome, nao houve diferenta em nenhum dos parametros reprodutivos avaliados, quando comparados com o grupo controle.

Molecular aspects of bovine cystic ovarian disease pathogenesis

Cystic ovarian disease (COD) is one of the main causes of reproductive failure in cattle and causes severe economic loss to the dairy farm industry because it increases both days open in the post partum period and replacement rates due to infertility. This disease is the consequence of the failure of a mature follicle to ovulate at the time of ovulation in the estrous cycle. This review examines the evidence for the role of altered steroid and gonadotropin signaling systems and the proliferation/apoptosis balance in the ovary with cystic structures. This evidence suggests that changes in the expression of ovarian molecular components associated with these cellular mechanisms could play a fundamental role in the pathogenesis of COD. The evidence also shows that gonadotropin receptor expression in bovine cystic follicles is altered, which suggests that changes in the signaling system of gonadotropins could play a fundamental role in the pathogenesis of conditions characterized by altered ovulation, such as COD. Ovaries from animals with COD exhibit a disrupted steroid receptor pattern with modifications in the expression of coregulatory proteins. These changes in the pathways of endocrine action would trigger the changes in proliferation and apoptosis underlying the aberrant persistence of follicular cysts. Free Spanish abstract: A Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/149/6/R251/suppl/DC1.

Effect of a biological response modifier on cellular death mechanisms at drying off

Agents that increase natural protective mechanisms have been proposed for prevention and treatment of intramammary infections. The objective of this study was to describe the effects of a single intramammary infusion of a lipopolysaccharide (LPS)-based biological response modifier (BRM) on cellular death mechanism in uninfected and Staphylococcus aureus-infected bovine mammary glands during involution. Three groups of 12 cows, each one including 6 Staph. aureus-infected and 6 uninfected, were infused in two mammary quarters with BRM or placebo and slaughtered at 7, 14 and 21 d of involution. In infected quarters, BRM treatment produced a significant increase in percent of stained epithelial cells for the apoptosis-promoting protein Bax at every observation period. In addition, BRM produced a significant increase of immunostained stromal cells for Bax compared with placebo-treated quarters. BRM treatment produced an increase in percentages of epithelial cells staining with active caspase-3 at 7 d and 14 d of involution compared with placebo-treated quarters and a significant decrease in percentages of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive epithelial cells at 7 d and 21 d of involution. In addition, BRM treatment caused an increase in percentage of stromal cells immunostaining for active caspase-3 and TUNEL. An increase of active caspase-3 and TUNEL epithelial and stromal cell immunostaining was observed in Staph. aureus-infected compared with uninfected quarters. Cellular proliferation, determined by Ki-67 immunostaining, was increased in epithelial and stromal cells from Staph. aureus-infected compared with uninfected quarters at every observation period. These results provide new insights into the mechanism of mammary cell death in uninfected and Staph. aureus-infected bovine mammary gland during involution and illustrate the effects of LPS-based BRM on apoptosis and cell proliferation during mammary involution.

Intermediate filament proteins expression and carbohydrate moieties in trophoblast and decidual cells of mature cat placenta

The aim of this study was to characterize cytoskeletal intermediate filament proteins and glycoconjugates of syncytiotrophoblast, cytotrophoblast and decidual cells of feline endotheliochorial placenta. Samples from 12 normal pregnant female cats, after 45 ± 5 days of gestation, were obtained removing the uterine horns by hysterectomy. Sections were processed for routine observation and for immunohistochemistry using anticytokeratin, antivimentin and antidesmin antibodies. In addition, lectin histochemistry was performed using a panel of several biotinylated lectins to characterize glycosides expression profile. Cytotrophoblast and syncytiotrophoblast showed immunoreactivity only with acidic and basic cytokeratins. Decidual cells were only positive to vimentin, consistent with their origin from endometrial fibroblasts. Trophoblast expressed a broad population of glycans, highly exposing terminal N-acetyl glucosamine residues and non-sialylated galactose and N-acetyl galactosamine oligomers. Oligosaccharides bound by Phaseolus vulgaris erythroagglutinin were the only highly branched N-linked residues evidenced in cats, and they were restricted to the syncytium. Unlike results reported on humans, mice and rats on lectin affinity of decidual cells, sialid acids and complex N-linked oligosaccharides were not demonstrated in cats. Glycosylation of proteins determines many of their final properties, thus becoming essential for the embryo-maternal dialogue during implantation and placentation. Changes in glycosylation pattern have been related to pathological pregnancies in other species. Hence, the knowledge about glycosylation profile of the normal cat placenta may lead to a better understanding of both normal and pathological reproductive events.

Role of Glucocorticoids in Cystic Ovarian Disease: Expression of Glucocorticoid Receptor in the Bovine Ovary

The aim of this study was to characterize the expression of glucocorticoid receptor (GR) in the components of normal bovine ovary and in animals with cystic ovarian disease (COD). Changes in the protein and mRNA expression levels were determined in control cows and cows with COD by immunohistochemistry and real-time PCR. GR protein expression in granulosa cells was higher in cysts from animals with spontaneous COD and adrenocorticotropic hormone-induced COD than in tertiary follicles from control animals. In theca interna cells, GR expression was higher in cysts from animals with spontaneous COD than in tertiary follicles from control animals. The increase in GR expression observed in cystic follicles suggests a mechanism of action for cortisol and its receptor through the activation/inactivation of specific transcription factors. These factors could be related to the pathogenesis of COD in cattle.

Characterization of pituitary cell populations in rats with induced polycystic ovaries.

Several hypotheses have been proposed about the pathogenesis of polycystic ovarian syndrome (PCOS), however, the fundamental physiological interactions that initiate the development of follicular cysts have not yet been elucidated. Hence, in this study the proliferation, density and population of gonadotrophs, mammotrophs, somatotrophs and corticotrophs of the pituitary glands of rats with induced follicular cysts have been investigated by 2 experimental models (continuous light exposition and estradiol valerate-treated rats). Specific immunoreactivity associated with follicle-stimulating hormone, luteinizing hormone, prolactin, growth hormone and adrenocorticotropic hormone was evaluated by immunohistochemistry with specific hormone antibodies and proliferation of secretory cells by their colocalization (double-labeling) with proliferating cell nuclear antigen. The results indicate a reduction in the density and proliferation of gonadotrophs in both experimental groups. A reduction in the average density, proliferation and population of lactotrophs and corticotrophs was also observed in estradiol valerate-treated animals. However, no significant differences were found in somatotrophs. The present study contributes to the information about alterations of some cell populations that occur in the pituitary gland of rats with polycystic ovaries, and will enhance our understanding of the pathogenesis of this disease.

Follicular Cysts: A Single Sign and Different Diseases. A View from Comparative Medicine

Ovarian cystic follicles are the sign of important causes of reproductive failure in numerous species. In this review, some morphological, endocrinological and clinical aspects of cystic follicles in women, cows, mares, sows and bitches are discussed. Follicular cysts are the consequence of the failure of a mature follicle to ovulate at the appointed time of ovulation in the estrous cycle. Although the etiology of follicular cysts remains unknown, this review examines the evidence about the role of endocrine signaling systems in the specific disease or syndrome in each of the species mentioned above. This review also describes, the changes in the pathways of endocrine mechanisms that would trigger disturbances in the intraovarian component underlying the aberrant persistence of follicular cysts. The knowledge of the morphological and endocrinological nature of cystic follicles in different species can provide relevant information to better understand specific diseases when it is integrally analyzed from the comparative medicine viewpoint.