Culture of bovine ovarian follicle wall sections maintained the highly estrogenic profile under basal and chemically defined conditions

Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17β-estradiol (E2) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P4) and E2 concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P4 throughout the culture period; however, P4 concentration was significantly higher in NDM. In both media, E2 concentration was increased at 24 h, followed by a decrease at 48 h. The E2:P4 ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E2:P4 ratio in FWS cultures.

Polimorfismos en los genes CYP11α y CYP17 y etiología del hiperandrogenismo en pacientes con poliquistosis ovárica / Polymorphism in CYP11alpha and CYP17 genes and the etiology of hyperandrogenism in patients with polycystic ovary syndrome

El síndrome de poliquistosis ovárica (PCOS) es un desorden endocrino-metabólico de naturaleza multifactorial, con una marcada predisposición genética, que afecta al 6% de las mujeres en edad reproductiva. Se caracteriza por la presencia de hiperandrogenismo, oligo-anovulación y ovarios poliquísticos. Entre los genes candidatos se encuentran aquellos que codifican para enzimas que actúan en la síntesis de andrógenos. Dos de los genes candidatos son el CYP17 y el CYP11alfa que codifican para la 17alfa hidroxilasa (P45017alfa) y para el P450scc (colesterol side chain cleavage) respectivamente. Los polimorfismos en estos genes están asociados al desarrollo del fenotipo hiperandrogénico. Nuestro objetivo fue analizar las frecuencias alélicas de los polimorfismos de los dos genes mencionados en población con PCOS, compararla con población normal y analizar la relación de cada variante alélica con el fenotipo hiperandrogénico correspondiente. Se analizaron 65 pacientes y 58 controles sanos en los que se determinaron niveles de testosterona y frecuencia de polimorfismos en los genes mencionados. Se observó una diferencia estadísticamente significativa cuando se asoció el grupo de mayor nivel de androgenemia con la presencia del genotipo A2/A2 del gen CYP17, y se hallaron mayores niveles de andrógenos circulantes en las pacientes con PCOS portadoras del alelo 216- del gen CYP11alfa. Nuestros resultados sugieren que ambos alelos juegan un rol menor en el desarrollo de PCOS y podrían ser considerados como potenciales marcadores de riesgo genético para el desarrollo del fenotipo hiperandrogénico.

The polycystic ovary syndrome (PCOS) is a heterogeneous multifactorial endocrine metabolic disorder with genetic predisposition affecting 6% of women in the reproductive age. This syndrome is characterized by the presence of oligo-anovulation, hyperandrogenism and polycystic ovaries. Several genes have been postulated as responsible for the etiology of this disorder. Among these genes are those encoding the enzymes involved in the ovarian androgen biosynthesis. Two of the candidate genes are the CYP17 and the CYP11alpha, encoding the 17-alpha-hydroxylase (P45017alpha) and the cholesterol side chain cleavage (P450scc) respectively. The polymorphisms of these genes are linked to the development of an hyperandrogenic phenotype. The aim of this work was to analyze the allelic frequencies of such polymorphisms in a cohort of women with PCOS and to compare them with those of healthy women. Furthermore, the correlation between each allelic variant and the corresponding hyperandrogenic phenotype was also assessed. Therefore, 65 patients and 58 age matched healthy controls were analyzed. The serum levels of testosterone and the frequency of each polymorphism were determined. When the PCOS population was analyzed, a significant statistical difference was found when relating the group with the highest androgenemia level with the presence of A2/A2 genotype of CYP 17 gene, and a higher level of circulating androgen was found in PCO women carrying the 216- allele of CYP11alpha gene (that did not reach statistical significance). Our results suggest that both alleles play a minor role in the development of PCOS and could be a genetic risk marker of the hyperandrogenic phenotype.

Role of the pentanucleotide (tttta)n polymorphisms of Cyp11alpha gene in the pathogenesis of hyperandrogenism in Chinese women with polycystic ovary syndrome / 华中科技大学学报（医学）（英德文版）

To determine the (tttta)n repeat polymorphisms at the promoter region of CYP11alpha gene, and study its linkage to hyperandrogenism of polycystic ovary syndrome (PCOS) in Chinese women, a case-control study was conducted in the Reproductive Medical Center of the Second Affiliated Hospital of Zhengzhou University (Zhengzhou, China). 96 PCOS patients and 78 healthy control women were included. CYP11alpha (tttta)n repeat-polymorphism genotyping analysis was performed by using polymerase chain reaction (PCR). Serum pituitary hormone and total testosterone levels were measured by ELISA. 4 different CYP11alpha (tttta)n allelles were identified, corresponding to 4-, 6-, 8-, and 9-repeat-unit alleles. The frequency and distribution of these alleles are 0.16, 0.33, 0.38, and 0.13 respectively in PCOS patients, as compared with 0.20, 0.34, 0.35, and 0.11 respectively in healthy controls. There were no significant differences between these two groups. Moreover, no correlation between the polymorphism of CYP11alpha gene and serum testosterone level of patients with PCOS and controls was observed. It is concluded that microsatellite polymorphism (tttta)n of gene CYP11alpha exists in Chinese women and the polymorphism of CYP11alpha gene does not play an important role in the pathogenesis of Chinese patients with PCOS, especially in patients with hyperandrogenism.