Aging-related premature luteinization of granulosa cells is avoided by early oocyte retrieval.

Why IVF pregnancy rates decline sharply after age 43 is unknown. In this study, we compared granulosa cell (GC) function in young oocyte donors (n=31, ages 21-29), middle-aged (n=64, ages 30-37) and older infertile patients (n=41, ages 43-47). Gene expressions related to gonadotropin activity, steroidogenesis, apoptosis and luteinization were examined by real-time PCR and western blot in GCs collected from follicular fluid. FSH receptor (FSHR), aromatase (CYP19A1) and 17ß-hydroxysteroid dehydrogenase (HSD17B) expression were found down regulated with advancing age, while LH receptor (LHCGR), P450scc (CYP11A1) and progesterone receptor (PGR) were up regulated. Upon in vitro culture, GCs were found to exhibit lower proliferation and increased apoptosis with aging. While FSH supplementation stimulated GCs growth and prevented luteinization in vitro. These observations demonstrate age-related functional declines in GCs, consistent with premature luteinization. To avoid premature luteinization in women above age 43, we advanced oocyte retrieval by administering human chorionic gonadotropin at maximal leading follicle size of 16  mm (routine 19-21  mm). Compared to normal cycles in women of similar age, earlier retrieved patients demonstrated only a marginal increase in oocyte prematurity, yet exhibited improved embryo numbers as well as quality and respectable clinical pregnancy rates. Premature follicular luteinization appears to contribute to rapidly declining IVF pregnancy chances after age 43, and can be avoided by earlier oocyte retrieval.

Testosterone, not 5α-dihydrotestosterone, stimulates LRH-1 leading to FSH-independent expression of Cyp19 and P450scc in granulosa cells.

Androgens are crucial for normal folliculogenesis and female fertility as evidenced in androgen receptor-null and granulosa cell conditional knockout mice. It is thought, however, that the multiple effects of androgens in the ovary are mainly complementary to the actions of gonadotropins. Using primary rat granulosa cells, we demonstrated that in the absence of gonadotropins, testosterone (T) increases aromatase (Cyp19) and P450 side-change cleavage expression, two enzymes crucial for normal ovarian function. T can be converted into estradiol, a classical estrogen, by Cyp19 and into 5α-dihydrotestosterone, a pure androgen, by 5α-reductase. However, inhibition of Cyp19 and/or 5α-reductase did not prevent the stimulatory effects of T. In contrast, the effect of this steroid was potentiated by blocking 5α-reductase. Additionally, T, not 5α-dihydrotestosterone, stimulates liver receptor homolog-1 (LRH-1) expression, whereas the expression of steroidogenic factor-1 (SF-1) was not affected by either steroid. LRH-1 and SF-1 are transcription factors known to be involved in the regulation of Cyp19. Accordingly, small interference RNA against LRH-1 prevented Cyp19 and P450 side-change cleavage up-regulation whereas anti-SF-1 small interference RNA had no effects. Chromatin immunoprecipitation demonstrated that T stimulation of LRH-1 leads to the recruitment of LRH-1 to the native Cyp19 promoter, which was not affected by cotreatment with 5α-reductase and Cyp19 inhibitors. Finally, gel shift and supershift analysis demonstrated that the androgen receptor binds to an androgen response element located within the LRH-1 promoter. These results provide novel evidence that T has a direct effect on the expression of genes involved in granulosa cell differentiation.

Mouse cumulus-denuded oocytes restore developmental capacity completely when matured with optimal supplementation of cysteamine, cystine, and cumulus cells.

Our objectives were to study how cysteamine, cystine, and cumulus cells (CCs), as well as oocytes interact to increase oocyte intracellular glutathione (GSH) and thereby to establish an efficient in vitro maturation system for cumulus-denuded oocytes (DOs). Using M16 that contained no thiol as maturation medium, we showed that when supplemented alone, neither cystine nor cysteamine promoted GSH synthesis of mouse DOs, but they did when used together. Although goat CCs required either cysteamine or cystine to promote GSH synthesis, mouse CCs required both. In the presence of cystine, goat CCs produced cysteine but mouse CCs did not. Cysteamine reduced cystine to cysteine in cell-free M16. When TCM-199 that contained 83 microM cystine was used as maturation medium, supplementation with cysteamine alone had no effect, but supplementation with 100 microM cysteamine and 200 microM cystine increased blastulation of DOs matured with CC coculture to a level as high as achieved in cumulus-surrounded oocytes (COCs). Similar numbers of young were produced after two-cell embryos from mouse COCs or CC-cocultured DOs matured with optimal thiol supplementation were transferred to pseudopregnant recipients. It is concluded that 1) mouse CCs can use neither cysteamine nor cystine to promote GSH synthesis, but goat CCs can use either one; 2) goat CCs promote mouse oocyte GSH synthesis by reducing cystine to cysteine, but how they use cysteamine requires further investigation; and 3) mouse DOs can use neither cystine nor cysteamine for GSH synthesis, but they restore developmental capacity completely when matured in the presence of optimum supplementation of cysteamine, cystine, and CCs.

The interactions between cysteamine, cystine and cumulus cells increase the intracellular glutathione level and developmental capacity of goat cumulus-denuded oocytes.

To improve in vitro maturation (IVM) of denuded oocytes (DOs), we observed the interactive effects of cysteamine, cystine and cumulus cells on the glutathione (L-gamma-glutamyl-L-cysteinyl-glycine; GSH) level and developmental capacity of goat IVM oocytes. Cysteamine supplementation increased the GSH level and blastocyst rates of both cumulus-oocyte complexes (COCs) and DOs, while the addition of cystine increased the GSH level and blastulation only in the presence of cumulus cells (COCs or DOs co-cultured on a cumulus cell monolayer). Simultaneous supplementation of cysteamine and cystine increased the GSH content and blastulation of co-cultured DOs to a level similar to that of COCs matured without thiol supplementation. Co-culture without thiol supplementation improved DOs' GSH synthesis but not blastulation. The results suggest that DOs cannot utilize cystine for GSH synthesis unless exogenous cysteamine is supplied by either cumulus cells or supplementation. Thus, while the addition of cystine alone is enough to improve IVM of COCs, improvement of DOs requires supplementation of both cystine and cysteamine. Synergic actions between cysteamine, cystine and cumulus cells restore the GSH level and developmental capacity of goat DOs.