Olga Castro

Control of human luteal steroidogenesis.

The human corpus luteum (CL) undergoes a dynamic cycle of differentiation, steroid hormone production and regression during the course of non-fertile cycles. In humans and other primates, luteal steroidogenesis is absolutely dependent on pituitary-derived LH. However, changes in LH and LH receptor expression do not explain the marked decline in progesterone production at the end of the luteal phase. Changes in the level of the steroidogenic acute regulatory protein (StAR), a gene whose expression is controlled by LH most likely account for the cyclic pattern of progesterone production. During the mid-to-late luteal phase of a fertile cycle, chorionic gonadotropin (hCG) rescues the CL, overcoming the actions of the factors inducing luteolysis. Although the agents causing regression of the CL in a non-fertile cycle are not yet known, intra-luteal growth factors and cytokines that modify the action of LH probably contribute to the reduction of StAR expression and the subsequent fall in progesterone production.

Molecular regulation of progesterone secretion by the human corpus luteum throughout the menstrual cycle

The present study examines the expression of the steroidogenic acute regulatory protein (StAR) within the human corpus luteum (CL) in conjunction with other molecules that regulate the apoptotic process of the CL. Our results indicate that the primary 1.6 kb StAR transcript occurs in greater abundance in early and mid-luteal phase compared with late luteal phase CL. Mature StAR protein (30 kDa) was present in lower amounts within late CL compared with early and mid-luteal phase. The pre-protein (37 kDa), which has been considered the active isoform to favor cholesterol translocation and subsequently steroid hormone synthesis, was also detected in lower amount in late CL. Several molecules, including pro-inflammatory cytokines, reactive oxygen species, steroids and inducible nitric oxide synthase (iNOS), have been linked as pro-apoptotic regulatory agents. Moreover, many of these molecules diminish progesterone synthesis in human cultured luteal cells. Interestingly, these molecules preferentially decrease progesterone biosynthesis in mid and late luteal cells in culture. These data suggest that the inhibitory effect of these molecules, as well as the amount of apoptotic cells in the CL are age dependent. The number of luteal apoptotic cells, as well as luteal cells stained positive for iNOS, increased from early to late CL. To examine the effects of hCG on StAR expression and apoptosis, we used two models-(1) in vivo hCG administration during the late luteal phase; and (2) in vitro incubation of explants of late CL with hCG. hCG increased both the level of StAR expression and the level of anti-apoptotic protein Bcl-2 within the late CL. We conclude that mRNA and protein expression of StAR and bcl-2 are important target elements for hCG during the CL rescue.

Regulation of steroid hormone synthesis by human corpora lutea: failure of follicle-stimulating hormone to support steroidogenesis in vivo and in vitro

Abstract The authors studied the role of follicle-stimulating hormone (FSH) in luteal steroido-genesis by replacing gonadotropin-releasing hormone (GnRH) infusion with pure FSH 48 hours after ovulation in two hypogonadotropic patients. Plasma progesterone (P) and estradiol (E 2 ) decreased after FSH administration. Human luteal cells were cultured for 48 hours in the presence and absence of FSH, human chorionic gonadotropin (hCG), testosterone (T), or dibutyryl cyclic adenosine monophosphate (Bu 2 cAMP). In the presence of T, E 2 synthesis increased significantly, indicating an active aromatase system in these cells. Human chorionic gonadotropin as well as Bu 2 cAMP significantly increased E 2 , T, and P synthesis. Follicle-stimulating hormone did not stimulate luteal E 2 , T, or P synthesis. The authors conclude that FSH does not sustain luteal steroidogenesis. Moreover, the in vitro findings reveal that hCG modulation of luteal E 2 synthesis is mediated principally by an increase in androgen precursors. These in vivo and in vitro results confirm a crucial role for luteinizing hormone (LH) in the maintenance ofluteal steroidogen-esis.

Tumor necrosis factor-alpha activates nuclear factor-kappaB but does not regulate progesterone production in cultured human granulosa luteal cells.

Background. The role of tumor necrosis factor-α (TNF-α) in granulosa luteal cell function and steroidogenesis is still controversial. Our aim was to examine the steroidogenic response, together with the simultaneous expression and activation of nuclear factor-κB (NF-κB), in cultured human granulosa luteal cells (GLCs) following administration of TNF-α. Materials and methods. This prospective controlled study was conducted in the Human Reproduction Division at the Institute of Maternal and Child Research, Faculty of Medicine, University of Chile and the San Borja Arriarán Hospital, National Health Service, Santiago, Chile. GLCs were obtained from aspirates of follicles from women undergoing in vitro fertilization (IVF). Thirty-two women undergoing IVF for tubal-factor and/or male-factor infertility participated in this study. Protein levels of NF-κB, the NF-κB inhibitor IκBα and steroidogenic acute regulatory protein (StAR) were determined by Western blot and localization of NF-κB was studied by indirect immunofluorescence. Progesterone production was determined by radioimmunoassay. Results. TNF-α did not affect the expression of StAR protein or the synthesis of progesterone. NF-κB was expressed in the GLCs and activated by TNF-α, resulting in degradation of IκBα and mobilization of the p65 NF-κB subunit into the nucleus. Conclusions. These results indicate that TNF-α did not modulate steroidogenesis in cultured human GLCs. However, NF-κB was activated by TNF-α. Therefore the activation of NF-κB via the TNF-α pathway is likely associated with other preovulatory granulosa cell processes important for human ovarian function.

Ultrastructural and biochemical evidence for the presence of mature steroidogenic acute regulatory protein (StAR) in the cytoplasm of human luteal cells

The distribution of the steroidogenic acute regulatory protein (StAR) inside thecal and granulosa-lutein cells of human corpus luteum (CL) was assessed by immunoelectron microscopy. We found greater levels of StAR immunolabeling in steroidogenic cells from early- and mid-than in late luteal phase CL and lower levels in cells from women treated with a GnRH antagonist in the mid-luteal phase. Immunoelectron microscopy revealed significant levels of StAR antigen in the mitochondria and in the cytoplasm of luteal cells. The 30 kDa mature StAR protein was present in both mitochondria and cytosol (post-mitochondrial) fractions from homogenates of CL at different ages, whereas cytochrome c and mitochondrial HSP70 were detected only in the mitochondrial fraction. Therefore, we hypothesized that either appreciable processing of StAR 37 kDa pre-protein occurs outside the mitochondria, or mature StAR protein is selectively released into the cytoplasm after mitochondrial processing. The presence of mature StAR in the cytoplasm is consonant with the notion that StAR acts on the outer mitochondrial membrane to effect sterol import, and that StAR may interact with other cytoplasmic proteins involved in cholesterol metabolism, including hormone sensitive lipase.

Effect of the aromatase inhibitor 4-hydroxyandrostene-3,17-dione progesterone synthesis by human luteal cells**Supported in part by Rockefeller Foundation grant Gaps 8523 and Fondecyt 0870.

The authors studied the effects of 4-hydroxyandrostene-3,17-dione (4-OHA) on progesterone (P), 17 beta-estradiol (E2), and 20 alpha-hydroxy-4-pregnen-3-one synthesis and pregnenolone accumulation in cultured human midluteal cells. A dose-dependent inhibition with and without human chorionic gonadotropin (hCG) of E2 and P production was observed. The accumulation of pregnenolone was significantly enhanced three to fourfold by 4-OHA in this culture system, as compared with control value. In addition, a sevenfold increase on pregnenolone accumulation was observed in the presence of 4-OHA plus 10 IU of hCG as compared with control values and 2.2-fold as compared with the 4-OHA treatments. These in vitro findings indicate a direct effect of 4-OHA on luteal steroidogenesis. Nevertheless, the suppressive effect of 4-OHA on P and E2 production is located at different sites of the steroidogenic pathway. In addition, the results demonstrate that hCG in the presence of 4-OHA stimulated pregnenolone accumulation, suggesting that the inhibition of P synthesis is in some steps after the formation of pregnenolone. These data indicate that the actions of 4-OHA on P or E2 formation have different inhibitory mechanisms.