Differential expression of functionality markers in mid-secretory endometrium of infertile women under treatment with ovulation-inducing agents.

OBJECTIVES: To analyze the expression of protein markers related to cell proliferation and death, as well as oestrogen and progesterone receptors in the endometrium of infertile women with hypothalamic-pituitary dysfunction treated with clomiphene citrate (CC) or recombinant follicle-stimulating hormone (rFSH), and compare them with ovulatory women. STUDY DESIGN: The study included 12 control ovulatory women and 29 anovulatory women, 19 of whom underwent ovulation induction with CC (n = 12) or rFSH (n = 5). Endometrial biopsies were obtained by Pipelle during the mid-secretory phase. Samples were stained with haematoxylin and eosin. Immunohistochemistry of proteins related to cell proliferation and cell death, as well as steroid receptors, was undertaken, and apoptosis was determined using TUNEL analysis. RESULTS: Immunohistochemical analysis of Ki67 expression showed significantly higher expression in the glandular epithelium of ovulatory women compared with the other groups. Glandular oestrogen receptor α expression was significantly lower in rFSH-treated women compared with ovulatory women. The number of apoptotic cells, Bax expression and progesterone receptor expression were similar in all groups. In contrast, Bcl-2 expression was significantly lower in the glandular epithelium of rFSH-treated women. CONCLUSIONS: In infertile women with hypothalamic-pituitary dysfunction, treatment with ovulation-inducing agents modifies the expression of proteins involved in cell proliferation and death, as well as the expression of steroid hormone receptors in the endometrium. These differences may help to explain, at the molecular level, the functionality of the endometrium during the implantation window, and may help to optimize pregnancy rates obtained with these treatments.

Protective effect of testosterone on early apoptotic damage induced by streptozotocin in rat pancreas.

Beta-cell apoptosis is responsible for the development of insulin-dependent diabetes mellitus in the streptozotocin (STZ) rat model. It has been demonstrated that steroid hormones possess antioxidant and protective antiapoptotic effects in many tissues. The aim of the present study was to investigate the early apoptotic damage induced by STZ in rat pancreas, and the effect of testosterone in preventing apoptosis of pancreatic beta cells. Intact and castrated adult male Wistar rats were subjected to a unique injection of STZ 60 mg/kg (body weight) in citrate buffer, and the kinetics of apoptosis in beta cells was assessed. Insulin and glucose were measured by RIA and a glucometer respectively, and in pancreatic tissue by immunohistochemistry. At 6 h after STZ injection, a marked increase in apoptotic beta cells was detected; however, glucose and insulin serum levels were not significantly different from the controls. The castrated animals presented higher percentages of apoptotic beta cells (65.75 +/- 5.42%) than intact males (20.6 +/- 4.38%) and castrated, testosterone-substituted males (30.66 +/- 1.38%). The decrease in apoptotic beta cells induced by testosterone was reversed by the antiandrogen flutamide (67.69 +/- 3.45%). The overall results indicate that early apoptotic damage produced by STZ in castrated animals was reversed by testosterone, suggesting that this hormone exerts a natural protective effect in rat pancreas. This effect could help to explain some sexual differences in diabetes mellitus incidence in man, reinforcing the idea that new approaches in steroid hormone therapies should be considered for treatment of this disease.

[Tumor suppressor gene p53: mechanisms of action in cell proliferation and death]. / El gen supresor de tumores p53: mecanismos de acción en la proliferación y muerte celular.

Normal development is a balance process, which includes proliferation and cell death. Indeed both proliferation and apoptotic cell death are very complex process that involves the participation of many genes. In both events, the tumor suppressor p53 is one of the most important and studied genes. This transcription factor activates several genes, which results in the arrest of the cellular cycle and cellular repair or apoptosis. Many are the signals that activate p53 function including: DNA damage by gamma or ultraviolet radiation and chemical agents and hypoxia, among others. When p53 is activated it can either induces the expression of p21 (Waf1, Cip-1), which participates in the cellular arrest between G1-S transition, or the expression of bax, PIGs, IGF-BP3, Fas, FasL and DR5. The former genes participate in the cascade of events that induce apoptosis. Cellular arrest or apoptosis depends of the degree of cellular damage. The final outcome of the different mechanisms of action of p53 is to maintain the genomic stability of the cell. Thus, the absence of this protein contributes to genomic instability, the accumulation of mutations and increased tumorigenesis. It has been demonstrated that p53 present mutations in 50-55% of all types of reported human cancer. These mutations are primary located in DNA binding domain of the protein, which results in the loss of its biological activity. Frequently, tumors that present wild type p53 have a better response towards therapy than those that present p53 mutations. This review is focused on the knowledge of the normal p53 cellular pathways and their alterations in cancer. It is clear that the understanding of p53 function in the development of this pathology may give new insights in future therapeutic strategies including gene therapy for cancer.

c-Fos gene expression pattern in the hypothalamus and the preoptic area of defeminized rats.

The object of the present study was to determine the c-fos gene expression pattern in the hypothalamus (HYP) and the preoptic area (POA) after estradiol and testosterone priming during the critical period of sexual differentiation of the rat brain. Three-day-old female rats were injected s.c. with a single dose of 17beta-estradiol (200 microg), testosterone enantate (200 microg) or vehicle (corn oil). HYP and POA were dissected 2 h, 24 h and 14 days after treatments and on the day of vaginal opening (VO). Other animals, previously treated as above, were acutely injected with 17beta-estradiol (5 microg) on the day of VO; HYP and POA were obtained 3 h later. Total RNA was extracted and processed for semiquantitative RT-PCR. We observed that c-fos gene expression was markedly increased in POA of the animals treated with estradiol or testosterone 2 h after treatments, while a non-significant increase in c-fos gene expression was observed in the HYP of these animals. We found a significant increase in c-fos expression in HYP and POA on the day of VO in both estradiol and testosterone defeminized rats. Interestingly, the acute estradiol administration on the day of VO did not induce c-fos gene expression in either HYP or POA of defeminized animals, instead a diminution in its expression was observed in animals treated with testosterone in POA. The overall results suggest that estradiol and testosterone imprinting during critical postnatal period of sexual differentiation of the brain permanently modifies the regulation of c-fos gene expression.

Variations of progesterone receptor and c-fos gene expression in the rat uterus after treatment with norethisterone and its A-ring reduced metabolites.

It has been suggested that some contraceptive derivatives of 19-nor-testosterone possess estrogenic activity that may facilitate the development of breast cancer. The aim of this work was to investigate the estrogenic properties of norethisterone (NET) and its A-ring-reduced derivatives by determining progesterone receptor (PR) and c-fos mRNA content of two estrogen-regulated genes in the uterus of ovariectomized rats. mRNA content was evaluated by Northern blot 1-6 h after 17 beta-estradiol administration. The highest PR and c-fos mRNA content was observed 3 h and 2 h after 17 beta-estradiol administration, respectively. NET did not modify either PR or c-fos mRNA content. In contrast, 5 alpha- and 3 beta, 5 alpha-NET significantly increased mRNA content of both genes. The increase in c-fos mRNA content induced by these reduced compounds was lower than that found with estradiol treatment. The overall results indicate that NET administration can indirectly induce estrogenic effects through the action of its 5 alpha-dihydro and 3 beta, 5 alpha-tetrahydro derivatives.

Differential expression of the estrogen-regulated proto-oncogenes c-fos, c-jun, and bcl-2 and of the tumor-suppressor p53 gene in the male mouse chronically infected with Taenia crassiceps cysticerci.

Chronic infection with Taenia crassiceps cysticerci produces a 200-fold increase in serum estradiol levels in male mice. The aim of this study was to investigate the expression pattern of c-fos and c-jun, two estradiol-regulated genes, as well as that of p53 and bcl2 in the testes, spleen, and thymus of male mice infected with T. crassiceps cysticerci. In parasitized animals the c-fos mRNA content was significantly increased in all tissues studied, whereas the c-jun mRNA content was increased only in the thymus. The p53 mRNA content was markedly reduced in all tissues of the parasitized animals analyzed, whereas bcl-2 gene expression was abolished in the thymus. On the other hand, thymic cell analysis performed by flow cytometry showed a diminution in the content of CD3+, CD4+, and CD8+ subpopulations in the parasitized mice. Our results suggest that the increase in estradiol levels of the host should change the expression pattern of several genes that participate in apoptosis regulation in the thymus of male mice during chronic infection with T. crassiceps cysticerci.

C-fos expression pattern in the hypothalamus and the preoptic area of the rat during proestrus.

c-fos gene expression in the hypothalamus, the preoptic area, the hippocampus and the frontal cerebral cortex of the rat was determined every two hours from 09:00 h to 19:00 h on the day of proestrus by using reverse transcription coupled to polymerase chain reaction. Rats under a 14:10 h light-dark cycle, with lights on at 06:00 h were used. We found a marked increase in c-fos gene expression in the studied regions but the hippocampus at 13:00 h, followed by a significant diminution in the subsequent hours of proestrus day. The high expression of c-fos gene at 13:00 could be associated to the increase in estradiol seric levels observed both at 11:00 h and at 13:00 h. Our results correlate with the increase in the number of FOS immunoreactive cells in some forebrain areas in proestrus afternoon related to gonadotropin releasing hormone secretion.

Detection and expression of corticosteroid binding protein gene in human pathogenic fungi.

Participation of steroid hormones in the growth of several fungal species has been widely reported. The aim of the present study was to detect the presence and expression of the corticosteroid binding protein (CBP) gene in different pathogenic fungal isolates from human clinical specimens. Genomic DNA and total RNA were obtained from six different pathogenic fungal species and submitted to Southern blot and Reverse Transcription-Polymerase Chain Reaction respectively. The results indicated that all the fungi studied presented and expressed CBP gene. The sequence of a PCR product of CBP gene fragment corresponding to the carboxyl terminal region in Trichophyton mentagrophytes, which presented the highest CBP expression, showed an identity of 98% as compared to the previously reported gene sequence from Candida albicans. The overall results indicate that CBP is a highly conserved gene in fungi and suggest that steroid hormones should play an important physiological role in these eukaryotic organisms.