John J. Peluso

Multiplicity of Progesterone's Actions and Receptors in the Mammalian Ovary

Abstract This minireview summarizes the role that progesterone (P4) plays in regulating granulosa and luteal cell function. These actions include the stimulation of P4 synthesis and the inhibition of estrogen synthesis, mitosis, and apoptosis. P4 also plays a key role in the ovulatory process. Although P4′s actions are well documented, the mechanism or mechanisms that mediate all of these actions have not been defined. In addition to P4-induced gene transcription that is mediated by the nuclear P4 receptors (PGR-A and PGR-B), three other receptor/signal transduction pathways could account for P4′s intraovarian actions. These pathways could be mediated by 1) the PGR localizing at or near the plasma membrane and activating SRC family kinases, 2) a membrane progestin receptor that responds to P4 by lowering intracellular cAMP and increasing MAPK 3/1 activity, and 3) a membrane receptor complex composed of serpine 1 mRNA binding protein (also known as PAIRBP1 or RDA288) and progesterone receptor membrane component 1. Ligand activation of this complex likely leads to an increase in protein kinase G activity, the maintenance of low basal intracellular free calcium, and the inhibition of granulosa and luteal cell mitosis and apoptosis. Given the complexity of P4′s actions within the ovary, it is likely that all of these receptor/signal transduction pathways influence some aspect of ovarian function with the specific P4 response dependent on 1) the expression pattern of these putative P4 receptors, 2) the P4 binding affinity of each receptor system, and 3) the amount of available P4.

Progesterone receptor membrane component 1—Many tasks for a versatile protein

The protein now called Progesterone Receptor Membrane Component 1 (PGRMC1) has been described independently by many groups in different cellular contexts. As a result it has been given an impressive diversity of names. While this protein was initially described on the basis of a singular property, e.g. expression or steroid binding, its possible physiological roles have only recently been reported. Current evidence supports the perception that PGRMC1 may be involved in sterol metabolism or homeostasis and cell survival. Here, we summarize a few sometimes neglected pieces of evidence from the literature along with unpublished findings on the properties and functions of PGRMC1.

Apoptosis as a function of ovarian reserve in women undergoing in vitro fertilization

OBJECTIVE To test the hypothesis that preovulatory follicles from women with diminished ovarian reserve contain fewer numbers of luteinized granulosa cells and have a greater percentage of cells undergoing apoptosis as compared with women with uncompromised ovarian reserve undergoing superovulation for IVF-ET. DESIGN Comparative study. SETTING Academic clinical practice. PATIENTS Luteinized granulosa cell counts were determined from follicular aspirates harvested from 19 women with day 3 serum FSH < or = 6 mIU/mL (conversion factor to SI unit, 1.00) and from 15 women with FSH levels > or = 10 mIU/mL. The second part examined the percent of proliferating and apoptotic cells harvested from nine women with day 3 serum FSH < or = 6 mIU/ mL and from eight women with FSH levels > or = 10 mIU/mL. MAIN OUTCOME MEASURES Cell counts by hemacytometer and percent of proliferating and of apoptotic granulosa cells by flow cytometry. RESULTS The mean number of viable cells isolated per follicle were 2.7 x 10(5) (95% confidence interval [CI] 2.3 to 3.1 x 10(5) from the low FSH group and 1.8 x 10(5) (95% CI 1.4 to 2.3 x 10(5) from the high FSH group. Although the percentage of proliferating cells was the same, the mean percentage of apoptotic cells was 0.5% (95% CI 0.2% to 1.4%) from the low group and 2.0% (95% CI 1.1% to 3.7%) from the high group. CONCLUSIONS Women undergoing IVF with day 3 serum FSH > or = 10 mIU/mL have preovulatory follicles with fewer luteinized granulosa cells and an increase in the percentage of cells undergoing apoptosis as compared with women with day 3 serum FSH < or = 6 mIU/mL.

Alterations in the expression, structure and function of progesterone receptor membrane component-1 (PGRMC1) in premature ovarian failure

Premature ovarian failure (POF) is characterized by hypergonadotropic hypogonadism and amenorrhea before the age of 40. The condition has a heterogeneous background but genetic factors are demonstrated by the occurrence of familial cases. We identified a mother and daughter with POF both of whom carry an X;autosome translocation [t(X;11)(q24;q13)]. RNA expression studies of genes flanking the X-chromosome breakpoint revealed that both patients have reduced expression levels of the gene Progesterone Receptor Membrane Component-1 (PGRMC1). Mutation screening of 67 females with idiopathic POF identified a third patient with a missense mutation (H165R) located in the cytochrome b5 domain of PGRMC1. PGRMC1 mediates the anti-apoptotic action of progesterone in ovarian cells and it acts as a positive regulator of several cytochrome P450 (CYP)-catalyzed reactions. The CYPs are critical for intracellular sterol metabolism, including biosynthesis of steroid hormones. We show that the H165R mutation associated with POF abolishes the binding of cytochrome P450 7A1 (CYP7A1) to PGRMC1. In addition, the missense mutation attenuates PGRMC1s ability to mediate the anti-apoptotic action of progesterone in ovarian cells. These findings suggest that mutant or reduced levels of PGMRC1 may cause POF through impaired activation of the microsomal cytochrome P450 and increased apoptosis of ovarian cells.

Basic Fibroblast Growth Factor Inhibits Apoptosis of Spontaneously Immortalized Granulosa Cells by Regulating Intracellular Free Calcium Levels through a Protein Kinase Cδ-Dependent Pathway1

Previous studies have shown that basic fibroblast growth factor (bFGF) inhibits primary granulosa cells from undergoing apoptosis. The present studies were designed to determine whether spontaneously immortalized granulosa cells (SIGCs) undergo apoptosis when deprived of growth factors and whether bFGF prevents apoptosis. In the absence of serum, the SIGCs lost cell contact and underwent apoptosis as indicated by the presence of annexin V binding, DNA ladders, and nuclear fragmentation. Basic FGF maintained cell contact and reduced the percentage of apoptotic cells. This antiapoptotic action was not observed if bFGF was added 30 min after serum withdrawal. Further, intracellular free calcium ([Ca2+]i) levels gradually increased 3- to 4-fold within 10 min of serum withdrawal. This increase was inhibited by bFGF. The intracellular calcium chelator, BAPTA, completely prevented the SIGCs from undergoing apoptosis in the absence of serum. These observations suggest that bFGF’s ability to regulate [Ca2+]i is an...

Expression and Function of PAIRBP1 Within Gonadotropin-Primed Immature Rat Ovaries: PAIRBP1 Regulation of Granulosa and Luteal Cell Viability

Abstract The protein PAIRBP1, which was initially referred to as RDA288, is involved in mediating the antiapoptotic action of progesterone (P4) in spontaneously immortalized granulosa cells (SIGCs). The present studies were designed to assess the expression and function of PAIRBP1 in the different cell types within the immature rat ovary. Western blot analysis detected PAIRBP1 within whole-cell lysates of immature rat ovaries. Equine gonadotropin (eCG) induced a 3-fold increase in ovarian levels of PAIRBP1. Moreover, human chorionic gonadotropin (hCG), given 48 h after eCG, maintained these elevated levels for up to 4 days. Immunohistochemical analysis confirmed this and further demonstrated that interstitial, thecal, and surface epithelial cells also expressed PAIRBP1. The level of PAIRBP1 in these cells was not influenced by gonadotropin treatment. In contrast, eCG stimulated an increase in PAIRBP1 within the granulosa cells of the developing follicles. Treatment with hCG induced ovulation and ultimately the formation of corpora lutea (CL). High levels of PAIRBP1 expression were also observed within the luteal cells. Immunocytochemical studies on living, nonpermeabilized granulosa and luteal cells revealed that some PAIRBP1 localized to the extracellular surface of these cells. The presence of PAIRBP1 on the extracellular surface was consistent with the observation that an antibody to PAIRBP1 attenuated P4s antiapoptotic action in both granulosa and luteal cells. Although the PAIRBP1 antibody attenuated P4s action, it did not reduce the capacity of cells to specifically bind 3H-P4. Immunoprecipitation with the PAIRBP1 antibody pulled down the membrane P4 binding protein known as progesterone receptor membrane complex-1 (PGRMC1; rat homolog accession number AJ005837). Taken together, these findings suggest that gonadotropins regulate the expression of PAIRBP1 in granulosa and luteal cells and that PAIRBP1 plays an important role in mediating P4s antiapoptotic action in these ovarian cell types. The exact mechanism of PAIRBP1s action remains to be elucidated, but it may involve an interaction with PGRMC1.

Expression of progesterone receptor membrane component 1 and its partner serpine 1 mRNA binding protein in uterine and placental tissues of the mouse and human.

Although activation of the nuclear progesterone (P(4)) receptor (PGR) is required for uterine function, some of the actions of P(4) are mediated through a PGR-independent mechanism. The receptors that account for the PGR-independent actions have not been identified with certainty. The purpose of this study was to assess the expression, localization and hormonal regulation of two novel P(4) receptor candidates, P(4) receptor membrane component (PGRMC) 1 and PGRMC2, as well as the PGRMC1 partner Serpine 1 mRNA binding protein (SERBP1). Unlike Pgrmc1 and Serbp1, which remained unchanged throughout the estrous cycle, Pgrmc2 was highly up-regulated during proestrus and metestrus. Immunohistochemical analyses suggest that PGRMC1 and SERBP1 promote differentiation, since the expression of these proteins increased in endometrial cells undergoing steroid-depended terminal differentiation. Progesterone rather than estrogen appears to be primarily responsible for up-regulating the expression of PGRMCs. PGRMC1 and SERBP1 also showed overlapping patterns of expression in the human placenta and associated membranes with the most abundant expression in smooth muscle of the placental vasculature, villous capillaries and the syncytiotrophoblast. Based on these findings, it is proposed that PGRMC1:SERBP1 protein complex functions in events important to early pregnancy including cellular differentiation, modulation of apoptosis and steroidogenesis. These studies provide a platform from which to build a clearer understanding of P(4) actions in the female reproductive tract and placental tissues that are mediated by non-classical mechanisms.

Progesterone Inhibits Apoptosis in part by PGRMC1-Regulated Gene Expression

Progesterone receptor membrane component-1 (PGRMC1) is present in both the cytoplasm and nucleus of spontaneously immortalized granulosa cells (SIGCs). PGRMC1 is detected as a monomer in the cytoplasm and a DTT-resistant PGRMC1 dimer in the nucleus. Transfected PGRMC1-GFP localizes mainly to the cytoplasm and does not form a DTT-resistant dimer. Moreover, forced expression of PGRMC1-GFP increases the sensitivity of the SIGCs to progesterone (P4)s anti-apoptotic action, indicating that the PGRMC1 monomer is functional. However, when endogenous PGRMC1 is depleted by siRNA treatment and replaced with PGRMC1-GFP, P4 responsiveness is not enhanced, although overall levels of PGRMC1 are increased. P4s anti-apoptotic action is also attenuated by actinomycin D, an inhibitor of RNA synthesis, and P4 activation of PGRMC1 suppresses Bad and increases Bcl2a1d expression. Taken together, the present studies suggest a genomic component to PGRMC1s anti-apoptotic mechanism of action, which requires the presence of the PGRMC1 dimer.

Characterization of a Putative Membrane Receptor for Progesterone in Rat Granulosa Cells

Abstract Progesterone (P4) inhibits granulosa cell apoptosis in a steroid-specific, dose-dependent manner, but these cells do not express the classic nuclear P4 receptor. It has been proposed that P4 mediates its action through a 60-kDa protein that functions as a membrane receptor. The present studies were designed to determine the P4 binding characteristics of this protein. Western blot analysis using an antibody that recognizes the P4 binding site of the nuclear P4 receptor (C-262) confirmed that the 60-kDa protein was localized to the plasma membrane of both granulosa cells and spontaneously immortalized granulosa cells (SIGCs). To determine whether this protein binds P4, proteins were immunoprecipitated with the C-262 antibody, electrophoresed, transferred to nitrocellulose, and probed with a horseradish peroxidase-labeled P4 in the presence or absence of nonlabeled P4. This study demonstrated that the 60-kDa protein specifically binds P4. Scatchard plot analysis revealed that 3H-P4 binds to a single site (i.e., single protein), which is relatively abundant (200 pmol/mg) with a Kd of 360 nM. 3H-P4 binding was not reduced by dexamethasone, mifepristone (RU 486), or onapristone (ZK98299). Further studies with SIGCs showed that P4 inhibited apoptosis and mitogen-activated protein kinase kinase (MEK) activity, and maintained calcium homeostasis. These studies taken together support the concept that the 60-kDa P4 binding protein functions as a low-affinity, high-capacity membrane receptor for P4.

E-Cadherin-Mediated Cell Contact Prevents Apoptosis of Spontaneously Immortalized Granulosa Cells by Regulating Akt Kinase Activity

Abstract The present studies were designed to determine the role that homophilic E-cadherin binding plays in preventing apoptosis of spontaneously immortalized granulosa cells (SIGCs). Although the levels of E-cadherin were similar to serum control levels, the amount of E-cadherin at the plasma membrane was dramatically reduced by 5 h after serum withdrawal. To determine whether disrupting homophilic E-cadherin binding leads to apoptosis, SIGCs were cultured in serum in the presence of either EGTA or an E-cadherin antibody. Treatment with either EGTA, which disrupts all calcium-dependent contacts, or E-cadherin antibody, induced apoptosis. Exposure to EGTA reduced MEK and Akt kinase activity, whereas E-cadherin antibody only attenuated Akt kinase activity. Because Akt kinase controls caspase-3 activity, an important activator of apoptosis, caspase-3 activity was monitored. Caspase-3 activity increased after serum depletion, or EGTA or E-cadherin antibody treatment. Time-series analysis of caspase-3 activity within single cells revealed that during apoptosis cell contact was disrupted then caspase-3 activity was detected. Finally, the caspase inhibitor, Z-VAD-FMK, blocked apoptosis. These data taken together are consistent with the concept that E-cadherin-mediated cell contact, either directly or indirectly, promotes Akt kinase activity, which in turn, inhibits caspase-3 activation and thereby maintains SIGC viability.