Integrin alpha5 is expressed on human luteinizing granulosa cells during corpus luteum formation, and its expression is enhanced by human chorionic gonadotrophin in vitro

Beta-adrenergic sensitivity and counterregulatory hormone and symptomatic responses to hypoglycaemia were studied in a 22-year-old man before and 3 and 34 weeks after removal of an insulinoma. The beta-adrenergic sensitivity was measured by the effect of an isoprenaline infusion on the heart rate, and the dose needed to increase the heart rate by 25 beats min(-1) (I25) calculated from regression lines. The glucose thresholds for the hormonal responses and symptoms were studied during a gradual fall in plasma glucose using a hypoglycaemic clamp technique. As compared with preoperative values, beta-adrenergic sensitivity was unchanged 3 weeks after surgery, but showed a marked improvement after 34 weeks, the I25 (in microg isoprenaline) being 0.96, 0.86, and 0.56, respectively. The hormone responses to hypoglycaemia were earlier, but with no improvement in symptom generation at 3 weeks. After 34 weeks, the thresholds for both hormone release and symptom generation occurred at a plasma glucose approximately 1 mmol l(-1) higher than before surgery. Thus, in our patient, there was a marked improvement in beta-adrenergic sensitivity, an earlier release of counterregulatory hormones, and an earlier recognition of hypoglycaemic symptoms after surgery. However, the restoration of these responses took more than 3 weeks.     

The effect of luteal "rescue" on the expression and localization of matrix metalloproteinases and their tissue inhibitors in the human corpus luteum

Luteolysis is associated with tissue remodeling probably involving the matrix metalloproteinases (MMPs) and their specific tissue inhibitors (TIMPs). This study investigated the expression and localization of the major MMPs and TIMPs in the human corpus luteum throughout the luteal phase and after luteal rescue with hCG. Corpora lutea (n = 9) were collected at hysterectomy and were dated by serial urinary LH estimation. In addition, corpora lutea (n = 3) were collected from women who had received daily doubling doses of hCG to mimic the hormonal changes of early pregnancy. MMP-1, MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 were investigated by zymography, reverse zymography, Northern blotting, and in situ hybridization. There was no change in the expression of MMP-1, TIMP-1, and TIMP-2 throughout the luteal phase or after luteal rescue. Little TIMP-3 could be detected in the corpus luteum. MMP-9 activity peaked in the early and late luteal phase. The expression and activity of MMP-2 were maximal in the late luteal phase. Exposure to hCG during luteal rescue in vivo was associated with a reduction (P < 0.05) in the expression and activity of MMP-2. Messenger ribonucleic acids (mRNAs) for MMP-1, MMP-2, and TIMP-2 were localized to the connective tissue stroma and the thecal-lutein cells of the corpus luteum. In contrast, TIMP-1 mRNA was localized to the granulosa-lutein cells, and MMP-9 mRNA was expressed in scattered cells within the steroidogenic and nonsteroidogenic cell layers. In conclusion, during maternal recognition of pregnancy, hCG prevents the normal increase in MMP-2 in the late luteal phase. MMPs can function in an environment containing large amounts of TIMP-1, as they have a different cellular localization.     

Effect of adenosine analogues on the expression of matrix metalloproteinases and their inhibitors from human dermal fibroblasts

The effect of the cytostatic and antiviral adenosine analogues 3-deazaadenosine (c3Ado) and 3-deaza-(+/-)-aristeromycin (c3Ari) on human skin fibroblasts was studied. Variables examined were cell morphology, viability, DNA fragmentation, expression of matrix metalloproteinases (MMPs) and matrix metalloproteinase inhibitors (TIMPs). None of these variables were changed when cells were exposed to c3Ari concentrations ranging from 10(-5) to 10(-3) M or 10(-5) M c3Ado. However, large changes in cell morphology, viability and expression of MMPs and MMP inhibitors occurred when fibroblasts were treated with 10(-4) or 10(-3) M c3Ado. Cells rounded up, shrank in volume, some detached and viability was lost without any detectable fragmentation of DNA. These changes in morphology and viability were associated with a differentiated expression of MMPs and MMP inhibitors. A large increase in collagenase activity occurred, and depending on the concentration of the adenosine analogue and the length of treatment, this change in activity could be shown to be due to one or a combination of the following factors: an increased synthesis of the collagenase protein, a decreased production of TIMP-1 or an increased activity of the collagenase superactivator, stromelysin. In contrast to this, treatment with c3Ado resulted in a decreased gelatinase activity, which in part could be attributed to an increased production of an inhibitor that seemed to affect gelatinase but not collagenase. The cellular changes induced by c3Ado seemed to reflect some of the alteration in the metabolic machinery that appears during a drug-induced or programmed/controlled death of a dermal cell. The different effects exerted by these two adenosine analogues on dermal fibroblasts can at least in part explain why c3Ado have previously been shown to be more toxic than c3Ari in animal models.     

Programmed cell death in human ovary is a function of follicle and corpus luteum status

Although extensive investigation on follicular apoptosis (programmed cell death) has been conducted in the infraprimate ovary, there is little information regarding apoptosis and its relationship to follicular status in the human. In this study, apoptosis was investigated in 116 human ovarian follicles (primordial to dominant) and 5 corpora lutea from a total of 27 premenopausal women. Follicles and corpora lutea were evaluated for the presence of DNA fragmentation, characteristic of apoptosis, by two methods: in situ hybridization using 3' end-labeling of DNA with digoxigenin-labeled nucleotides and subsequent digoxigenin antibody and peroxidase staining, and/or biochemical analysis of low molecular weight DNA laddering. Follicle functional status was evaluated by determining follicle sizes and follicular fluid androgen/estrogen (A/E) ratios. No apoptosis was observed in 67 primordial, primary, or secondary follicles. Positive staining for DNA fragmentation was found in a few granulosa cells in 0.1- to 2-mm follicles, whereas abundant staining in granulosa was detected in 2.1- to 9.9-mm follicles. In contrast, no DNA fragmentation was detected in dominant follicles (10-16 mm). The frequency of apoptosis in follicles was calculated to be 37% in 0.1- to 2-mm follicles, 50% in 2.1- to 5-mm follicles, and 27% in 5.1- to 9.9-mm follicles. Abundant low molecular weight DNA laddering was only found in androgen-dominant follicles and not in estrogen-dominant follicles. Positive staining for DNA fragmentation and low molecular weight DNA laddering were observed in degenerating but not healthy-appearing corpora lutea. In the former, DNA fragmentation was found primarily in large luteal cells. These data suggest that follicular atresia in human ovary results from normal programmed cell death and primarily occurs in the granulosa cell layers of the early antral and < 10-mm antral follicles primarily. Furthermore, because apoptosis occurs as early as the 200-mm stage, follicle selection may begin as early as the initial formation of the antrum. The results also suggest that degeneration of the corpus luteum occurs by apoptotic mechanisms.     

Endothelial cell proliferation follows the mid-cycle luteinizing hormone surge, but not human chorionic gonadotrophin rescue, in the human corpus luteum

The corpus luteum is essential for the maintenance of early pregnancy in women. Angiogenesis may be one factor involved in luteal rescue. The aim of this study was to determine the changes in endothelial cell proliferation throughout the luteal phase and in human chorionic gonadotrophin (HCG)-simulated early pregnancy. Human corpora lutea obtained throughout the luteal phase and in simulated early pregnancy were immunostained with antibodies for endothelial and proliferating cells. Number and distribution of endothelial and proliferating cells were examined. Endothelial cells were least abundant in the early luteal phase, increasing in the mid-luteal phase (P < 0.03). Endothelial numbers did not differ significantly between the late and the rescued corpora lutea. Endothelial cell proliferation was greatest in the early luteal phase and continued at a lower level during later stages. Simulated early pregnancy resulted in no change in endothelial cell proliferation. These results showed that a high degree of endothelial cell proliferation is associated with formation of the human corpus luteum. Unchanging levels of proliferation following HCG treatment (for 5-8 days from day 12 to day 16 post-ovulation, at 125 IU to 16,000 IU, following a daily doubling of dose) suggest that alternative processes are involved during luteal rescue.     

Human microvascular endothelial cells differ from macrovascular endothelial cells in their expression of matrix metalloproteinases

Matrix metalloproteinase (MMP) secretion by microvascular endothelial cells is an essential first step in the formation of new blood vessels (angiogenesis). Since angiogenesis does not occur in large blood vessels, we investigated whether the secretion of MMPs and tissue inhibitor of MMP (TIMP1) differs between micro- and macro-vascular endothelial cells. We compared the secretion of MMPs and TIMP1 by human endothelial cells derived from neonatal foreskin (FSE) and umbilical vein (HUVE) sources. The cells were incubated for 24 hr in the presence or absence of the angiogenic agents, phorbol myristate acetate (PMA, 100 ng/ml) or tumour necrosis factor-alpha (TNF, 100 ng/ml). The cell supernatants were removed and assayed for MMPs and TIMP1 using a spectrophotometric assay for MMP1, zymography, Western blotting and Northern analysis. When endothelial cells were incubated in basal medium for 24 hr they secreted MMP1, MMP2 and TIMP1 but not MMP9. HUVE secreted substantially higher levels of these proteins compared to FSE. In addition, HUVE secreted two low molecular mass bands representing activated forms of MMP2. These activated forms were not present in supernatants derived from FSE. In response to PMA, both FSE and HUVE increased secretion of MMP1 and TIMP1. However, there was a dramatic difference in level of response by the two cell types with FSE secreting substantially more TIMP1 and MMP9 compared to HUVE. These data clearly show that cultured endothelial cells derived from microvascular vs macrovascular tissues exhibit different MMP and TIMP secretory profiles.     

Macrophages, cell proliferation, and cell death in the human menstrual corpus luteum

We studied the presence and numbers of macrophages in the different compartments of the human menstrual corpus luteum (CL) in relation to the proliferative activity and apoptosis in luteal cells. Macrophages were recognized by immunohistochemical demonstration of the lysosome-associated glycoprotein CD68, and proliferating cells by immunohistochemical detection of the cell cycle-related protein Ki67 and by counting mitotic cells. In general, changes in the number of macrophages were parallel to the functional activity of the CL. Macrophage numbers increased up to the end of the early luteal phase, remained relatively unchanged during the midluteal phase, and decreased at the late luteal phase. Furthermore, macrophages showed prominent morphological changes during the cycle. They showed round or elongated cytoplasm during the early and late luteal phases, and dendritic features in the midluteal phase. Proliferating cells were very abundant on Days 15-16 and showed a significant decrease thereafter. Most proliferating cells corresponded to stromal (mainly vascular) cells. However, about 5% of granulosa-lutein cells and about 15% of theca-lutein cells were proliferating during the early and midluteal phases. Regression of the CL at the late luteal phase was associated with both a decrease in the number of proliferating cells and an increase in the number of apoptotic cells, which were highly increased on Days 25-27 of the cycle. The number of macrophages was not related to cell proliferation nor to cell death during the luteal phase. The observed changes in both macrophage number and morphology suggest the existence of a bidirectional communication between macrophages and steroidogenic cells in the human CL, or regulation of both cell populations by similar mechanisms.     

Increased expression of matrix metalloproteinases in vivo in scleritis tissue and in vitro in cultured human scleral fibroblasts

Scleritis is a sight-threatening inflammatory disorder of the eye characterized by the degradation of scleral matrix. Matrix metalloproteinases (MMPs) are ubiquitous proteolytic enzymes important in physiological and pathological processes, the activity of which is stringently controlled by the action of a family of natural antagonists, the tissue inhibitors of matrix metalloproteinases (TIMPs). We hypothesized that enhanced expression of MMPs, without the negative regulatory influence of TIMPs, may be a key feature of tissue destruction in inflammatory eye diseases, such as scleritis. The aim of this study was to localize and characterize cells expressing MMPs and TIMPs in sclera affected by necrotizing scleritis and, in a parallel study, to establish whether cytokines modulate MMP expression in cultured human scleral fibroblasts. In situ hybridization and immunohistochemical analyses indicated that resident scleral fibroblasts as well as inflammatory cells such as macrophages and T lymphocytes express stromelysin, gelatinase B, and TIMP-1 in necrotizing scleritis tissue. In addition, cytoplasmic immunoreactivity for tumor necrosis factor-alpha, an inducer of MMPs, was detected in infiltrating inflammatory cells. Cultured scleral fibroblasts stimulated with the combination of interleukin-1 alpha plus tumor necrosis factor-alpha increased TIMP-1 mRNA twofold above constitutive levels. By contrast, these cytokines induced a sevenfold increase in the steady-state levels of stromelysin mRNA. Using Western blotting, stromelysin and TIMP-1 protein production paralleled mRNA induction in cytokine-stimulated human scleral fibroblasts. Culture supernatants harvested from cytokine-stimulated human scleral fibroblasts were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis gelatin substrate zymography. Our results revealed a prominent 92-kd gelatinolytic band corresponding to gelatinase B, which was inducible with interleukin-1 alpha. These data provide evidence for our hypothesis, that an imbalance between enzyme/inhibitor ratios may be the underlying mechanism of the tissue destruction characteristic of scleritis. Our results demonstrate the potential involvement of MMPs and their modulation by cytokines produced by infiltrating inflammatory cells in destructive ocular inflammation.     

Effect of an inhibitor of matrix metalloproteinases on spontaneous osteoarthritis in guinea pigs

A 67-year-old patient with large granular lymphocyte (LGL) leukemia is described. At fluorescence-activated cell sorting (FACS) analysis of the peripheral blood, the lymphocytes were positive for CD3, CD4, CD5, CD29, CD45RA, CD57, and TCR alpha/beta and negative for CD7, CD8, CD16, CD56, CD19, CD22, and TCR gamma/delta. Bone marrow histology and immunohistochemistry did not reveal any lymphocyte infiltration. Cytogenetic examination of peripheral blood cultures showed a clone with the karyotype 46,XY,t(3;5)(p26;q13). Molecular analysis revealed rearrangement of the gamma-T-cell-receptor chain. The region 3p25-3p26 which harbors the von Hippel-Lindau tumor suppressor gene and the RAF1 oncogene has been rearranged in a few cases of T-cell leukemia. The translocation in this case has not yet been described and may reflect an alternative mechanism in the pathogenesis of these disorders.     

Influence of nicotine on progesterone and estradiol production of cultured human granulosa cells

A case of peripheral ameloblastoma in a 57-years-old woman is presented, along with a discussion of the clinical and histological characteristics of the lesion. After clinical and radiographic examinations, and with a differential diagnosis of pyogenic granuloma, an excisional biopsy was performed and the material collected was sent for histological examination. On the basis of the histopathological diagnosis, a second operation was performed with a wide safety margin, including bone tissue, which did not show any involvement with the odontogenic neoplasm.     

Effect of matrix metalloproteinases activity on outflow in perfused human organ culture

PURPOSE: To test the hypothesis that extracellular matrix turnover, mediated by the matrix metalloproteinases, modulates aqueous humor outflow facility in a human outflow model. METHODS: Matrix metalloproteinase activity was manipulated and outflow facility evaluated using perfused human anterior segment organ culture. Purified matrix metalloproteinases, tissue inhibitors of metalloproteinases (TIMPs), and several families of synthetic inhibitors of matrix metalloproteinases were added to the perfusion medium. Matrix metalloproteinase expression was increased by adding recombinant interleukin (IL)-1alpha. Kinetic inhibition analysis was conducted for stromelysin, gelatinase A, and gelatinase B with the various inhibitors. Live-dead staining was used to evaluate culture viability. RESULTS: Increasing metalloproteinase activity, by adding purified metalloproteinases or by inducing their expression by IL-1alpha treatment, increased outflow facility. Inhibition of endogenous trabecular metalloproteinase activity using TIMP or several families of synthetic metalloproteinase inhibitors reduced outflow rates. The elevation and the reduction of outflow rates were reversible, with changes requiring 1 to 3 days. Kinetic enzyme inhibition analysis produced 50% inhibitory concentration values for these inhibitors that were compatible with the concentration ranges for outflow inhibition. CONCLUSIONS. The ability of several specific matrix metalloproteinase inhibitors to reduce outflow facility implies that endogenous extracellular matrix turnover by these enzymes was required for the maintenance of trabecular outflow resistance, at least in this human culture model. These observations provide support for the hypothesis that controlled extracellular matrix turnover is important in the regulation of aqueous humor outflow facility.     

Immunohistochemical study of matrix metalloproteinases and their tissue inhibitors in pulmonary Langerhans' cell granulomatosis

OBJECTIVE: To evaluate the role of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in the pathogenesis of the lesions of pulmonary Langerhans' cell granulomatosis. DESIGN: Immunohistochemical and confocal microscopic studies were made of lung biopsy specimens from five patients with pulmonary Langerhans' cell granulomatosis. RESULTS: The reactivity of Langerhans' cells was moderate to intense for MMP-2, weaker for MMP-9, and faint for TIMP-1 and TIMP-2. Type IV collagen colocalized with MMP-2 in areas of damage to epithelial basement membranes, a finding that emphasizes the potential importance of this enzyme in the pathogenesis of the destructive lesions of pulmonary Langerhans' cell granulomatosis. In the more advanced fibrotic lesions, TIMP-2 colocalized with basement membranes and with fibrillar collagen, suggesting that it contributes to the permanence of the fibrosis. CONCLUSION: These results indicate an important role for MMPs and TIMPs in pulmonary Langerhans' cell granulomatosis.     

Effect of lipoproteins on cultured human mesangial cells

It was recently reported that low-density lipoprotein (LDL) promotes mesangial cell proliferation, and oxidized LDL is cytotoxic for mesangial cells. However, there have been few studies about the effects of other lipoproteins on mesangial cells. Accordingly, we investigated the effect of various lipoproteins on cultured human mesangial cells using 3H-thymidine (3H-TdR) incorporation and cell counting assays. We also investigated the levels of several cytokines in mesangial cell culture supernatants after stimulation by the lipoproteins. Addition of very-low-density lipoprotein (VLDL) at concentrations up to 100 micrograms/mL, intermediate-density lipoprotein (IDL) at up to 50 micrograms/mL, and LDL at up to 50 micrograms/mL induced the proliferation of cultured human mesangial cells, whereas cell growth was inhibited at higher concentrations. Oxidized LDL caused a concentration-dependent decrease of 3H-TdR incorporation. High-density lipoprotein (HDL) had no proliferative effective effect at any concentration. Exposure to VLDL, IDL, LDL, or a high concentration of HDL enhanced the secretion of interleukin-6, platelet-derived growth factor, and transforming growth factor-beta by mesangial cells, whereas tumor necrosis factor-alpha secretion was stimulated by oxidized LDL. These finding indicate that triglyceride (TG)-rich lipoproteins (VLDL and IDL) promote mesangial cell proliferation as well as LDL, whereas oxidized LDL has the reverse effect. These effects of lipoproteins may be related to modulation of various cytokines. Accordingly, TG-rich lipoproteins, LDL, and oxidized LDL may be involved in mesangial cell proliferation and injury in patients with mesangial proliferative glomerulonephritis.     

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in reactive and neoplastic lymphoid cells

We have studied the expression of gelatinase A, gelatinase B, interstitial collagenase, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in reactive lymphoid cells, as well as in a series of cell lines derived from neoplasms of B- and T-cell lineage. Using both Northern blot analysis and zymography, gelatinase B activity was detected by zymography in two Burkitt cell lines and in a tonsillar cell suspension, while gelatinase A and interstitial collagenase activities were not detected by either method. TIMP-1 expression was demonstrated by Northern blot analysis in the multipotential neoplastic K-562 cell line, the high grade Burkitt's B-cell lymphoma lines, isolated tonsillar B cells and at low levels in peripheral blood T cells, but was not expressed in any of the neoplastic T-cell lines or isolated peripheral blood B cells. In contrast, TIMP-2 expression was restricted to tissues containing cells of T-cell lineage with high levels being observed in the neoplastic T-cell lines and lower levels in normal peripheral blood T cells and hyperplastic tonsil. Expression of TIMP-1 and TIMP-2 was confirmed at the protein level by reverse zymography and immunofluorescence assays using antihuman TIMP polyclonal antibodies. Expression of gelatinase B by the high grade B-cell Burkitt's lymphoma cell lines is consistent with previous findings in large cell immunoblastic lymphomas and indicates that this enzyme may play an important role in high grade non-Hodgkin's lymphomas. TIMP expression correlated with cell lineage in that TIMP-1 was primarily observed in B cells and TIMP-2 was restricted to T cells.     

Coordinated expression of tissue-type plasminogen activator and plasminogen activator inhibitor type 1 during corpus luteum formation and luteolysis in the adult pseudopregnant rat

Proteolytic activity generated by the plasminogen activator (PA) system is associated with many biological processes. Using an adult pseudopregnant rat model, we have studied how two components of the PA system, tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI-1), are expressed temporally and spatially during different developmental stages of the corpus luteum (CL). Northern blot analysis, in situ hybridization, in situ zymography, and fibrin overlay were used to analyze the expression and distribution of tPA and PAI-1 messenger RNA (mRNA) as well as PA activity in CL of different ages. We demonstrated that during the luteinization period (approximately days 1-2), tPA mRNA was highly and evenly expressed in newly formed CL, whereas PAI-1 mRNA was mainly detected in the central part of the same CL. In accordance with these findings, proteolytic activity generated by tPA was detected in the outer region of newly formed CL by in situ zymography. During the luteotropic period (approximately days 3-10), tPA mRNA expression was very low. PAI-1 mRNA expression was also low, but increased on day 10. As expected, proteolytic activity was very low during this period. During functional luteolysis (days 13-14) and subsequent structural luteolysis, tPA mRNA was elevated. PAI-1 mRNA was also expressed during this period. Moreover, the net PA activity, as determined by fibrin overlay, was relatively high during this period. Our studies indicate that tPA and PAI-1 are coordinately expressed in the CL, resulting in increased proteolytic activities during the luteinization and luteolytic periods. PA-mediated proteolysis may, therefore, play a role in both CL formation and luteolysis in rats.     

Proteolysis of extracellular matrix by invadopodia facilitates human breast cancer cell invasion and is mediated by matrix metalloproteinases

Breast cancer cell lines vary in invasive behavior and one highly invasive cell line (MDA-MB-231) proteolytically degrades extracellular matrix with invadopodia (Thompson et al. 1992, J Cell Physiol, 150, 534-44; Chen et al 1994, Breast Cancer Res Treat, 31, 217-26). Invadopodial proteolysis of extracellular matrix is thought to be necessary for invasion; however, this has not been demonstrated directly. To obtain such evidence, normal (HBL-100) and malignant (MCF-7, MDA-MB-231) breast cells were evaluated for invadopodial proteolysis of extracellular matrix and invasive behavior. We report that invadopodial proteolysis of immobilized fibronectin is positively correlated with invasion of cells into type I collagen gels. Moreover, reducing the proteolytic activity of invadopodia with the metalloproteinase inhibitor, batimastat (BB-94), also decreases invasion indicating that breast cancer cell invasion is dependent upon proteolytically active invadopodia.     

The effects of insulin on 3 beta-hydroxysteroid dehydrogenase expression in human luteinized granulosa cells

OBJECTIVE: We determined the relative effects of insulin and FSH on progesterone accumulation as well as activity, protein content, and mRNA expression of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in human luteinized granulosa cells. METHODS: Luteinized granulosa cells obtained from women undergoing in vitro fertilization were plated and grown to near confluence and treated with FSH, insulin, or a combination of insulin and FSH. Progesterone production as well as enzyme activity, protein content, and mRNA expression for 3 beta HSD were evaluated. RESULTS: Progesterone production was not affected by insulin alone but increased threefold in the presence of FSH (50 ng/microL) alone. The presence of FSH plus insulin (100 nmol/L) caused a significant increase in progesterone accumulation greater than that of FSH alone. The already high basal levels of 3 beta HSD activity were unaffected by insulin alone but increased 1.7-fold in the presence of FSH. The combination of FSH (50 ng/mL) and insulin (100 nmol/L) increased activity 1.3-fold over FSH alone (P < .02). Insulin (greater than 100 nmol/L) alone increased 3 beta HSD protein content as measured by Western analysis 1.8-2-fold over basal levels, whereas FSH alone increased protein content 2.8-fold, and was further augmented by the addition of insulin in a dose-related fashion up to 3.5-fold over basal levels. Insulin increased 3 beta HSD mRNA twofold over basal levels; FSH alone increased mRNA expression of 3 beta HSD 3.2-fold. In the presence of insulin plus FSH, 3 beta HSD mRNA expression increased 7.6-fold over basal levels. For comparison, insulin also stimulated cytochrome P450 aromatase activity, P450 aromatase protein, and mRNA but to a greater degree than that seen for 3 beta HSD. CONCLUSION: Insulin is a regulator of both 3 beta HSD and aromatase expression in human granulosa cells. Elevated insulin levels could therefore affect steroid production in human granulosa cells and presumably alter the menstrual cycle and fertility.