Cortisol/glucocorticoid receptor: a critical mediator of the ovulatory process and luteinization in human periovulatory follicles.

STUDY QUESTION: Do cortisol/glucocorticoid receptors play an active role in the human ovary during ovulation and early luteinization? SUMMARY ANSWER: The ovulatory hCG stimulation-induced glucocorticoid receptor signaling plays a crucial role in regulating steroidogenesis and ovulatory cascade in human periovulatory follicles. WHAT IS KNOWN ALREADY: Previous studies reported an increase in cortisol levels in the human follicular fluid after the LH surge or ovulatory hCG administration. However, little is known about the role of cortisol/glucocorticoid receptors in the ovulatory process and luteinization in humans. STUDY DESIGN, SIZE, DURATION: This study was an experimental prospective clinical and laboratory-based study. An in vivo experimental study was accomplished utilizing the dominant ovarian follicles from 38 premenopausal women undergoing laparoscopic sterilization. An in vitro experimental study was completed using the primary human granulosa/lutein cells (hGLC) from 26 premenopausal women undergoing IVF. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study was conducted in a private fertility clinic and academic medical centers. Dominant ovarian follicles were collected before the LH surge and at defined times after hCG administration from women undergoing laparoscopic sterilization. Primary hGLC were collected from women undergoing IVF. hGLC were treated without or with hCG in the absence or presence of RU486 (20 µM; dual antagonist for progesterone receptor and glucocorticoid receptor) or CORT125281 (50 µM; selective glucocorticoid receptor antagonist) for 12 or 36 h. The expression of genes involved in glucocorticoid receptor signaling, steroidogenesis, and ovulatory cascade was studied with RT-quantitative PCR and western blotting. The production of cortisol, corticosterone, and progesterone was assessed by hormone assay kits. MAIN RESULTS AND THE ROLE OF CHANCE: hCG administration upregulated the expression of hydroxysteroid 11-beta dehydrogenase 1 (HSD11B1), nuclear receptor subfamily 3 group C member 1 (NR3C1), FKBP prolyl isomerase 5 (FKBP5), and FKBP prolyl isomerase 4 (FKBP4) in human ovulatory follicles and in hGLC (P < 0.05). RU486 and CORT125281 reduced hCG-induced increases in progesterone and cortisol production in hGLC. The expression of genes involved in glucocorticoid receptor signaling, steroidogenesis, and the key ovulatory process was reduced by RU486 and/or CORT125281 in hGLC. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The role of cortisol/glucocorticoid receptors demonstrated using the hGLC model may not fully reflect their physiological roles in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Successful ovulation and luteinization are essential for female fertility. Women with dysregulated cortisol levels often suffer from anovulatory infertility. Deciphering the functional role of glucocorticoid receptor signaling in human periovulatory follicles enhances our knowledge of basic ovarian physiology and may provide therapeutic insights into treating infertility in women. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by P01HD71875 (to M.J., T.E.C., and M.B.) and R01HD096077 (to M.J.) from the Foundation for the National Institutes of Health and the BTPSRF of the University of Kentucky Markey Cancer Center (P30CA177558). The authors report no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Molecular Influences on the Quantification of Lewis Acidity with Phosphine Oxide Probes.

Gutmann-Beckett-type measurements with phosphine oxide probes can be used to estimate effective Lewis acidity with 31P nuclear magnetic resonance spectroscopy, but the influence of the molecular structure of a given probe on the quantification of Lewis acidity remains poorly documented in experimental work. Here, a quantitative comparison of triethyl (E), trioctyl (O), and triphenyl (P) phosphine oxides as molecular probes of Lewis acidity has been carried out via titration studies in MeCN with a test set of six mono- and divalent metal triflate salts. In comparison to E, the bulkier O displays a similar range of chemical shift values and binding affinities for the various test metal ions. Spectral linewidths and speciation properties vary for individual cation-to-probe ratios, however, confirming probe-specific properties that can impact the data quality. Importantly, P displays a consistently narrower dynamic range than both E and O, illustrating how electronic changes at phosphorus can influence the NMR response. Comparative parametrizations of the effective Lewis acidities of a broader range of metal ions, including the trivalent rare earth ions Y3+, Lu3+, and Sc3+ as well as the uranyl ion (UO22+), can be understood in light of these results, providing insight into the fundamental chemical processes underlying the useful approach of single-point measurements for quantification of effective Lewis acidity. Together with a study of counteranion effects reported here, these data clarify the diverse ensemble of factors that can influence the measurement of Lewis acid/base interactions.

In vivo phenotypic vascular dysfunction extends beyond the aorta in a mouse model for fibrillin-1 (Fbn1) mutation.

In individuals with Marfan Syndrome (MFS), fibrillin-1 gene (FBN1) mutations can lead to vascular wall weakening and dysfunction. The experimental mouse model of MFS (Fbn1C1041G/+) has been advantageous in investigating MFS-associated life-threatening aortic aneurysms. It is well established that the MFS mouse model exhibits an accelerated-aging phenotype in elastic organs like the aorta, lung, and skin. However, the impact of Fbn1 mutations on the in vivo function and structure of various artery types with the consideration of sex and age, has not been adequately explored in real-time and a clinically relevant context. In this study, we investigate if Fbn1 mutation contributes to sex-dependent alterations in central and cerebral vascular function similar to phenotypic changes associated with normal aging in healthy control mice. In vivo ultrasound imaging of central and cerebral vasculature was performed in 6-month-old male and female MFS and C57BL/6 mice and sex-matched 12-month-old (middle-aged) healthy control mice. Our findings confirm aortic enlargement (aneurysm) and wall stiffness in MFS mice, but with exacerbation in male diameters. Coronary artery blood flow velocity (BFV) in diastole was not different but left pulmonary artery BFV was decreased in MFS and 12-month-old control mice regardless of sex. At 6 months of age, MFS male mice show decreased posterior cerebral artery BFV as compared to age-matched control males, with no difference observed between female cohorts. Reduced mitral valve early-filling velocities were indicated in MFS mice regardless of sex. Male MFS mice also demonstrated left ventricular hypertrophy. Overall, these results underscore the significance of biological sex in vascular function and structure in MFS mice, while highlighting a trend of pre-mature vascular aging phenotype in MFS mice that is comparable to phenotypes observed in older healthy controls. Furthermore, this research is a vital step in understanding MFS's broader implications and sets the stage for more in-depth future analyses, while providing data-driven preclinical justification for re-evaluating diagnostic approaches and therapeutic efficacy.

In Vivo Phenotypic Vascular Dysfunction Extends Beyond the Aorta in a Mouse Model for Fibrillin-1 ( FBN1 ) Mutation.

In individuals with Marfan Syndrome (MFS), fibrillin-1 gene ( FBN1 ) mutations can lead to vascular wall weakening and dysfunction. The experimental mouse model of MFS ( FBN1 C1041G/+ ) has been advantageous in investigating MFS-associated life-threatening aortic aneurysms. Although the MFS mouse model presents an accelerated-aging phenotype in elastic organs (e.g., lung, skin), the impact of FBN1 mutations on other central and peripheral arteries function and structure with the consideration of the impact of sex remains underexplored. In this study, we investigate if FBN1 mutation contributes to sex-dependent alterations in central and cerebral vascular function similar to phenotypic changes associated with normal aging in healthy control mice. In vivo ultrasound imaging of central and cerebral vasculature was performed in 6-month-old male and female MFS and C57BL/6 mice and sex-matched 12-month-old (middle-aged) healthy control mice. Our findings confirm aortic enlargement (aneurysm) and wall stiffness in MFS mice, but with exacerbation in male diameters. Coronary artery blood flow velocity (BFV) in diastole was not different but left pulmonary artery BFV was decreased in MFS and 12-month-old control mice regardless of sex. At 6 months of age, MFS male mice show decreased posterior cerebral artery BFV as compared to age-matched control males, with no difference observed between female cohorts. Reduced mitral valve early-filling velocities were indicated in MFS mice regardless of sex. Male MFS mice also demonstrated left ventricular hypertrophy. Overall, these results underscore the significance of biological sex in vascular function and structure in MFS mice, while highlighting a trend of pre-mature vascular aging phenotype in MFS mice that is comparable to phenotypes observed in older healthy controls.

Influence of endogenous angiotensin II on control of sympathetic nerve activity in human dehydration.

Arterial blood pressure can often fall too low during dehydration, leading to an increased incidence of orthostatic hypotension and syncope. Systemic sympathoexcitation and increases in volume regulatory hormones such as angiotensin II (AngII) may help to maintain arterial pressure in the face of decreased plasma volume. Our goals in the present study were to quantify muscle sympathetic nerve activity (MSNA) during dehydration (DEH), and to test the hypothesis that endogenous increases in AngII in DEH have a mechanistic role in DEH-associated sympathoexcitation. We studied 17 subjects on two separate study days: DEH induced by 24 h fluid restriction and a euhydrated (EUH) control day. MSNA was measured by microneurography at the peroneal nerve, and arterial blood pressure, electrocardiogram, and central venous pressure were also recorded continuously. Sequential nitroprusside and phenylephrine (modified Oxford test) were used to evaluate baroreflex control of MSNA. Losartan (angiotensin type 1 receptor (AT1) antagonist) was then administered and measurements were repeated. MSNA was elevated during DEH (42 +/- 5 vs. EUH: 32 +/- 4 bursts per 100 heartbeats, P = 0.02). Blockade of AT1 receptors partially reversed this change in MSNA during DEH while having no effect in the control EUH condition. The sensitivity of baroreflex control of MSNA was unchanged during DEH compared to EUH. We conclude that endogenous increases in AngII during DEH contribute to DEH-associated sympathoexcitation.

Negative pressure oxygen breathing and head-down tilt increase nitrogen elimination.

Negative pressure breathing (NPB) increases the rate of nitrogen elimination, which is thought to be due to an increase in cardiac output due to augmented venous return to the heart. Hyperoxia, however, decreases the rate of nitrogen elimination. The effect of hyperoxia on the increase in nitrogen elimination during NPB is not known. We hypothesized that NPB as and head down tilt (HDT), which is also thought to increase cardiac output, would counteract the detrimental effects of hyperoxia on nitrogen elimination. Nitrogen elimination was measured in 12 subjects while they lay supine breathing 100% O2 supplied at atmospheric pressure (control), -10 cm H2O (NPOB(-10)), and -15 cm H2O (NPOB(-15)). Nitrogen elimination was also measured in the subjects while they breathed 100% O2 supplied at atmospheric pressure in the supine position with a 6 degrees HDT. Over a two-hour washout period, NPOB significantly increased nitrogen elimination by more than 14%, although there was no significant difference between NPOB(-10) and NPOB(-15). HDT also significantly increased nitrogen elimination by almost 8%. Neither NPOB nor HDT significantly affected cardiac output but calf blood flow was significantly lower during NOPB(-15). Combining NPB or HDT with 100% oxygen breathing appear to be useful means of increasing nitrogen elimination and should be considered in situations where this effect may be beneficial, such as with oxygen prebreathing prior to decompression.

Molecular changes in the equine follicle in relation to variations in antral follicle count and anti-Müllerian hormone concentrations.

REASONS FOR PERFORMING STUDY: The wide variation in circulating anti-Müllerian hormone (AMH) concentrations between mares is attributed to differences in antral follicle count (AFC) which may reflect follicular function. There are few data regarding variations in AFC and associated regulatory factors for AMH in the equine follicle during follicular development. OBJECTIVES: To examine molecular and hormonal differences in the equine follicle in relation to variations in AFC and circulating AMH concentrations during follicular development and to identify genes co-expressed with AMH in the equine follicle. STUDY DESIGN: Observational study. METHODS: Plasma AMH concentrations and AFC were determined in 30 cyclic mares. Granulosa cells, theca cells and follicular fluid were recovered from growing (n = 17) or dominant follicles (n = 13). The expression of several genes, known to be involved in folliculogenesis and steroidogenesis, was examined using real-time reverse transcriptase polymerase chain reaction and immunohistochemistry. Intrafollicular oestradiol and AMH concentrations were determined by immunoassay. RESULTS: Within growing follicles, the expression of AMH, AMHR2, ESR2 and INHA in granulosa cells was positively correlated with AFC and plasma AMH concentrations. In addition, the expression of ESR1 and FSHR was positively associated with plasma AMH concentrations. No significant associations were detected in dominant follicles. Furthermore, there was no association between AMH or oestradiol concentrations in follicular fluid and variations in AFC. Finally, the expression of AMH and genes co-expressed with AMH (AMHR2, ESR2 and FSHR) in granulosa cells as well as intrafollicular AMH concentrations decreased during follicular development while intrafollicular oestradiol concentrations increased and were inversely related to intrafollicular AMH concentrations. CONCLUSIONS: This study indicates that variations in AFC and circulating AMH concentrations are associated with molecular changes in the growing equine follicle.

Cellular localization of ovarian proopiomelanocortin messenger RNA during follicular and luteal development in the rat.

Opioid peptides are expressed in the reproductive system and have been reported to regulate reproductive function. The present study used in situ hybridization to selectively localize ovarian cells containing high levels of proopiomelanocortin (POMC) mRNA, an opioid precursor, during different stages of ovarian development. Prepubertal rats were primed with PMSG to stimulate follicular development, followed by hCG to induce ovulation. Treatment groups consisted of control (no treatment), PMSG (2 days post-PMSG), 1 day corpus luteum (CL; 1 day post-hCG), and 8 day CL (8 days post-hCG). POMC mRNA-containing cells were present in antral follicles, CL, and the interstitial compartment. With gonadotropin treatment, the percentage of follicles containing heavily labeled cells increased in the PMSG and 1 day CL groups. The number of POMC mRNA-containing cells per follicle also increased in the 1 day CL group. In the CL, no difference was observed in the percentage of CL exhibiting labeled cells between the 1 day CL and 8 day CL groups; however, more labeled luteal cells per CL were present in the 1 day CL group. A marked increase in POMC mRNA-containing cells was observed in the interstitial compartment of the 1 day CL group. These results indicate that the number of POMC mRNA-containing cells increases with follicular development and CL formation; however, the ovarian distribution suggests that the labeled cells could be nonendocrine cells, possibly white blood cells. The in situ hybridization findings are indicative of low total concentrations of ovarian POMC mRNA, suggesting mainly an autocrine or paracrine role for POMC or POMC-derived peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of prodynorphin gene expression in the ovary: distal DNA regulatory elements confer gonadotropin regulation of promoter activity.

Examination of the regulation of prodynorphin (pro-DYN) promoter activity is limited by the absence of a good cell model. The discovery of pro-DYN mRNA and derived peptides in the reproductive tract led us to examine the cellular localization and hormonal regulation of ovarian pro-DYN expression and to evaluate normal granulosa cells as a model for studying pro-DYN gene regulation. Ovarian pro-DYN mRNA levels were significantly elevated in PMSG-primed immature rats 12-24 h after receiving an ovulatory dose of hCG. Levels peaked 2 days after hCG, remained elevated throughout the ensuing pseudopregnancy, and rose again at the end of pseudopregnancy. In situ hybridization localized pro-DYN expression predominantly to granulosa and luteal cells. Transfection of primary cultures of granulosa cells revealed that the activity of the rat pro-DYN promoter [-1858 to 133 base pairs (bp)] was increased 18- to 19-fold by hCG and human FSH treatments and 7-fold by cAMP analog treatment. Deletion analysis identified a 358 bp fragment as the primary hormone-responsive sequence (-1858 to -1500 bp; containing three potential cAMP-responsive elements); its deletion resulted in severely reduced FSH responsiveness, and its ligation to hormone-unresponsive basal promoter sequences completely restored FSH responsiveness. This is an unusually distal position for cAMP-responsive elements compared to other cAMP-regulated genes. These data demonstrate specific expression of pro-DYN in granulosa and luteal cells, which is under sensitive gonadotropin regulation, and identify a distal hormone-responsive sequence within the promoter.

Divergent effects of interleukin-1 beta on steroidogenesis and matrix metalloproteinase inhibitor expression and activity in cultured rat granulosa cells.

The periovulatory increase in ovarian matrix metalloproteinase inhibitor (MMPI) expression is regulated both in vitro and in vivo by LH, but the intermediary steps in this process are uncertain. The purpose of this experiment was to determine whether interleukin-1 beta (IL-1 beta), a known modulator of MMPI expression in other systems and one that is induced by LH in the ovary, is capable of regulating granulosa matrix metalloproteinase inhibitor expression and activity. Using an in vitro rat granulosa cell model, these parameters were assessed in response to IL-1 beta or LH administration alone or in combination. Granulosa cells were obtained from 24-day-old immature female rats primed with 20 IU PMSG at 22 days of age. Cells were cultured under serum-free conditions for 24 h at 37 C in the presence of medium (control), LH (100 ng/ml), IL-1 beta (10 ng/ml), or LH plus IL-1 beta. MMPI activity in the conditioned medium was assessed using a colorimetric assay (n = 6), whereas progesterone and estrogen concentrations in the conditioned medium were determined by RIA (n = 6). RNA was isolated from the granulosa cells and assessed for Northern analysis of tissue inhibitor of metalloproteinase-1 (TIMP-1; n = 4), TIMP-2 (n = 3), and TIMP-3 (n = 3) expression. When added to granulosa cells, IL-1 beta and LH each significantly (P < 0.05) increased MMPI activity in granulosa cell-conditioned medium above control values (40.9 +/- 3.0% inhibition for IL-1 beta and 67.1 +/- 5.6% inhibition for LH vs. 31.4 +/- 2.4% inhibition for controls). When added in combination, IL-1 beta had no effect on LH-stimulated inhibitor activity (67.1 +/- 5.6% inhibition vs. 69.9 +/- 5.1% inhibition for LH and LH plus IL-1 beta, respectively). Methylamine hydrochloride treatment revealed that the majority of inhibitor activity in all treatment groups was derived from TIMPs. The patterns of TIMP-1, TIMP-2, and TIMP-3 messenger RNA expression among the treatment groups paralleled the TIMP-derived inhibitor activity, in that both IL-1 beta and LH alone stimulated transcript expression of all three TIMPs. In addition, an increase in progesterone production was associated with IL-1 beta-stimulated (1.22-fold over control values; P = 0.0006) and LH-stimulated (9.6-fold over control values; P = 0.007) MMPI expression and activity. Lastly, IL-1 beta and LH significantly (P < 0.05) decreased estrogen production by approximately 33% compared to that in cultures with LH only. It is concluded from the current study that IL-1 beta is a mediator of MMPI expression as well as granulosa cell steroidogenesis, and that this cytokine has divergent actions in the presence and absence of LH.

Expression and localization of PPARs in the rat ovary during follicular development and the periovulatory period.

PPARs are a family of nuclear hormone receptors involved in various processes that could influence ovarian function. We investigated the cellular localization and expression of PPARs during follicular development in ovarian tissue collected from rats 0, 6, 12, 24, and 48 h post-PMSG. A second group of animals received human CG (hCG) 48 h post-PMSG. Their ovaries were removed 0, 4, 8, 12, and 24 h post-hCG to study the periovulatory period. mRNAs corresponding to the PPAR isotypes (alpha, delta, and gamma) were localized by in situ hybridization. Changes in the levels of mRNA for the PPARs were determined by ribonuclease protection assays. PPAR gamma mRNA was localized primarily to granulosa cells, and levels of expression did not change during follicular development. Four hours post-hCG, levels of mRNA for PPAR gamma decreased (P < 0.05) but not uniformly in all follicles. At 24 h post-hCG, levels of PPAR gamma mRNA were reduced 64%, but some follicles maintained high expression. In contrast, mRNAs for PPAR alpha and delta were located primarily in theca and stroma, and their levels did not change during the intervals studied. To investigate the physiologic significance of PPAR gamma in the ovary, granulosa cells from PMSG-primed rats were cultured for 48 h with prostaglandin J(2) (PGJ(2)) and ciglitazone, PPAR gamma activators. Both compounds increased progesterone and E2 secretion (P < 0.05). These data suggest that PPAR gamma is involved in follicular development, has a negative influence on the luteinization of granulosa cells, and/or regulates the periovulatory shift in steroid production. The more general and steady expression of PPARs alpha and delta indicate that they may play a role in basal ovarian function.

Spatiotemporal messenger ribonucleic acid expression of ovarian tissue inhibitors of metalloproteinases throughout the rat estrous cycle.

The tissue inhibitors of metalloproteinases (TIMPs) within the ovary closely regulate the matrix metalloproteinases, enzymes capable of degrading components of the extracellular matrix. The purpose of this study was to examine the spatial and temporal messenger RNA (mRNA) expression of the TIMPs in the ovaries of normally cycling rats. Ovaries were collected at 1100 h on each day of the 4-day estrous cycle, and TIMP mRNA expression was examined by Northern blot, RT-PCR, or in situ hybridization. TIMP-1 mRNA levels were significantly higher on estrus than on any other day. Although the 1.0-kb TIMP-2 transcript did not change across the cycle, the 3.5-kb transcript decreased significantly between metestrus and diestrus. Expression of TIMP-3 mRNA decreased significantly between proestrus and estrus. TIMP-1, TIMP-2, and TIMP-3 mRNAs were primarily localized to the theca, stroma, and corpora lutea (CL) on all days of the cycle, but with distinct cyclic changes. Thecal expression of TIMP-1 and TIMP-2 mRNAs was especially high immediately before and after ovulation. TIMP-1 and TIMP-3 mRNAs, which were low to undetectable in the granulosa cells of preovulatory follicles, were greatly increased in the luteinizing cells of newly forming CL on estrus. Although the presence of TIMP-1 mRNA in the granulosa cells of preovulatory follicles by in situ hybridization was near background levels, it was specifically identified in granulosa cells of follicles on all days of the cycle using laser capture microdissection and RT-PCR. Both TIMP-2 and TIMP-3 transcripts were up-regulated in luteinized follicles on proestrus and were present throughout the cycle in regressing CL. In summary, the unique and dynamic expression patterns of the TIMPs suggest that they have important, yet distinct, functions in the ovary. The high levels of TIMP-1 mRNA in the CL on estrus indicate a likely role for this inhibitor in luteal formation. The presence of TIMP-2 mRNA in regressing CL suggests an involvement in luteal demise, whereas TIMP-3 may play a role in the health of the follicle as well as in CL regression.

Hormonal regulation of matrix metalloproteinase inhibitors in rat granulosa cells and ovaries.

Metalloproteinases, such as collagenase or gelatinase, and their associated inhibitors appear to control connective tissue remodeling during follicular rupture. We examined the regulation of metalloproteinase inhibitor activity by various treatments in cultured rat granulosa cells. Granulosa cells were harvested from immature PMSG-primed rats and cultured with LH, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), cAMP, or forskolin. Inhibitor activity was measured in the medium. Increasing concentrations of either LH (0.1-1000 ng/ml) or TPA (2.5-100 nM) resulted in a dose-dependent increase in metalloproteinase inhibitor activity (2.9- and 2.4-fold increases above control, respectively). There was also a time-dependent induction of inhibitor activity in cells incubated in the presence of LH (100 ng/ml) for 6, 12, 18, or 24 h. Forskolin (0.1 mM) or cAMP (1 mM) treatment increased inhibitor activity 2.8- and 1.6-fold above that in control cultures. LH and TPA treatment in combination resulted in an additive increase in inhibitor activity compared to LH or TPA treatment alone. This finding suggested that the granulosa cell inhibitor activity might be induced through separate intracellular pathways. The inhibitor present in conditioned medium was isolated by chromatographic separation on a Sepharose 6B mol wt exclusion column. The inhibitor present was approximately 28,000 mol wt, which is consistent with the size of tissue inhibitor of metalloproteinase (TIMP). In addition to the granulosa cell experiments, changes in ovarian mRNA levels for TIMP were determined. There was a preovulatory increase in TIMP mRNA from whole rat ovaries, with the highest levels detected 12 h after hCG administration. The present study establishes that metalloproteinase inhibitor activity from rat granulosa cells is induced through separate pathways: a LH-cAMP-dependent protein kinase-A pathway and a cAMP-independent protein kinase-C pathway. Furthermore, a TIMP-like protein is observed in granulosa cell-conditioned medium, while TIMP mRNA is present in rat ovaries and increases before ovulation, suggesting that the granulosa cell metalloproteinase inhibitor is TIMP. We propose that TIMP acts in part to control the site and extent of follicular connective tissue remodeling associated with ovulation.

The preovulatory increase in ovarian collagenase activity in the rat is independent of prostaglandin production.

In the present study, we have examined the role of gonadotropins and prostaglandins in the preovulatory increase of ovarian collagenase activity in the rat. Whole ovaries of immature PMSG-primed rats (20 IU) were removed before and 8 h after the rats were treated with human (h) CG, Nembutal, and/or indomethacin. The ovaries were homogenized in a solution containing Triton X-100 (0.25%) and centrifuged. Collagenase was extracted by resuspending the pellets in buffer containing 100 mM CaCl2, heating to 60 C for 6 min, and centrifuging. The supernatants were treated with dithiothreitol (2 mM) and iodoacetamide (5 mM) to inactivate collagenase inhibitors. Collagenase activity was measured as the percent digestion of 3H-type I collagen/100 microliters aliquot of ovarian sample. At zero time (52 h after PMSG), ovarian collagenase activity was 4.2 +/- 1.2% digestion (mean +/- SEM, n = 3). In ovaries collected 8 h after the endogenous LH surge or 8 h after the administration of 10 IU hCG at time zero, collagenase activity rose to 19.6 +/- 2.1 (n = 6) and 22.5 +/- 1.7% digestion (n = 11), respectively. Indomethacin (1.5 mg/100 g BW) administered 30 min after hCG, produced no change in collagenase activity (24.8 +/- 2.5% digestion, n = 7) although the expected increase in ovarian prostaglandin E after hCG treatment was blocked. When the endogenous LH surge was blocked with Nembutal (3 mg/100 g BW), collagenase activity in 8-h ovaries was 6.8 +/- 1.1% digestion (n = 10). The Nembutal block of the preovulatory collagenase increase was overcome by administration of hCG (8-h ovarian enzyme activity = 22.7 +/- 3.2% digestion, n = 8). These observations demonstrate that hCG stimulates ovarian collagenase activity and that this stimulation is not dependent on prostaglandin synthesis.

Alpha 2-macroglobulin and tissue inhibitor of metalloproteinases: collagenase inhibitors in human preovulatory ovaries.

Extensive remodeling of the follicular extracellular matrix occurs during the process of ovulation. This remodeling involves the breakdown of collagen, which is regulated, in part, by the action of the metalloproteinase collagenase and its associated inhibitors. In the present study, follicular metalloproteinase inhibitors were characterized to determine whether they were serum-borne or of ovarian origin, possibly a tissue-derived inhibitor known as tissue inhibitor of metalloproteinase (TIMP). Human follicular fluid and granulosa cells were obtained from preovulatory follicles of patients in an in vitro fertilization program. Chromatographic separation of follicular fluid on Sepharose 6B resulted in two peaks of inhibitory activity. The large molecular radius (Mr) inhibitor was similar in size to the serum-borne metalloproteinase inhibitor alpha 2-macroglobulin (i.e. Mr 700,000) whereas the small Mr inhibitor approximated the size of TIMP (i.e. Mr 29,000). Incubation of aliquots from either of the two peaks of inhibitor activity or an alpha 2-macroglobulin standard with an antibody to alpha 2-macroglobulin decreased the inhibitory activity in both the large Mr peak and the alpha 2-macroglobulin standard by 86.6 +/- 1.7% and 71.5 +/- 7.7% (n = 4, P less than 0.005), respectively, implying cross-reactivity with the alpha 2-macroglobulin antibody. The inhibitory activity in the small Mr peak, however, was unchanged. Northern analysis of total granulosa cell RNA demonstrated TIMP messenger RNA (mRNA) in all eight granulosa cell samples examined whereas alpha 2-macroglobulin mRNA was virtually undetectable. A positive correlation (r = 0.85, P less than 0.01) was observed between the levels of TIMP mRNA and the ratio of the follicular estradiol-progesterone concentration. However, inhibitor activity in the follicular fluid was not correlated with the levels of TIMP mRNA (r = 0.05). These findings confirm the presence of alpha 2-macroglobulin in follicular fluid and demonstrate that human preovulatory granulosa cells contain mRNA for TIMP, an inhibitor that regulates metalloproteinases such as collagenase, gelatinase, and proteoglycanase. Additionally, the expression of TIMP mRNA is steroid related and may be hormonally regulated. It is proposed that TIMP produced in the granulosa cell compartment in conjunction with alpha 2-macroglobulin from the serum may act to control the site and extent of ovarian connective tissue remodeling.

Galanin: a novel intraovarian regulatory peptide.

Galanin is a 29-amino acid peptide that acts as a neuropeptide in many tissues. To date, galanin action and the hormonal regulation of galanin gene expression have not been described in the ovary of any species. To study possible ovarian expression and regulation of galanin, immature gonadotropin-primed rats were given hCG (10 IU), and their ovaries were collected 0, 4, 8, 12, and 20 h after hCG treatment for determination of galanin messenger RNA (mRNA) concentration by solution hybridization. Galanin mRNA levels progressively increased after hCG administration, peaking at 12 h (2.4-fold increase vs. 0 h), with a subsequent return to 0 h levels at 20 h. To determine a possible ovarian role for galanin, rats were killed 48 h after gonadotropin administration, their ovaries were removed, and granulosa cells were harvested. These cells and the ovarian tissue remaining after granulosa cell collection (i.e. "shells") were each cultured for 24 h with increasing concentrations of galanin (0, 10, 100, and 1000 nM) in the presence or absence of LH. The medium was examined for steroid production and metalloproteinase inhibitor activity. In granulosa cell cultures, galanin increased the levels of estradiol by 26% and had no effect on progesterone, but decreased metalloproteinase inhibitor activity by 61% in the conditioned medium. In the shell cultures, galanin increased estradiol, progesterone, and androstenedione in the medium, suggesting that galanin acts on cells other than granulosa cells or that galanin action requires a paracrine interaction between granulosa and thecal cells. Our data demonstrate that galanin message is increased by hCG, and that galanin acts to amplify ovarian steroidogenesis while decreasing metalloproteinase inhibitor activity. These findings establish that ovarian galanin mRNA is hormonally stimulated and that galanin acts as an intraovarian regulatory peptide.

Identification and characterization of metalloproteinase inhibitor activity in human ovarian follicular fluid.

Metalloproteinase inhibitors regulate collagenase activity in the extracellular matrix. To assess the role of metalloproteinase inhibitors in the ovulatory process, inhibitor activity was examined in human follicular fluid collected 2-4 h before ovulation. The relationship between inhibitor activity and steroid content was determined, and the inhibitors were partially purified and characterized. Inhibitory activity in follicular fluid (n = 25) correlated with both follicular estradiol (P less than 0.001) and progesterone (P less than 0.02) concentrations per follicle. Chromatographic separation of the follicular fluid on Sepharose 6B isolated two peaks of inhibitory activity. The inhibitor from the small mol wt (Mr) peak shared many of the properties of tissue inhibitors of metalloproteinase. It was stable in response to heat (60 C) and methylamine (200 mM), and was destroyed by reduction and alkylation, a procedure reported to destroy previously characterized inhibitors. Partial purification by affinity and ion exchange chromatography demonstrated the inhibitor to be a glycoprotein with an approximate Mr of 28-29K. The large Mr inhibitor had an approximate size of 700K and exhibited many of the characteristics of alpha 2-macroglobulin, a serum-derived metalloproteinase inhibitor. It was sensitive to heat, methylamine, and reduction and alkylation. Thus, follicular fluid contains metalloproteinase inhibitor activity that is steroid related and may be hormonally regulated. Ovarian metalloproteinase inhibitors may act to regulate connective tissue remodeling during follicular rupture.

Plasminogen activator and plasminogen activator inhibitor in human preovulatory follicular fluid.

To evaluate the roles of plasminogen activator (PA) and PA inhibitor (PAI) in human ovulation, we obtained follicular fluid and granulosa cells from individual preovulatory follicles of patients undergoing gamete intrafallopian tube transfer. The follicular fluid samples (n = 25) were analyzed for total tissue-type PA antigen, PA enzyme activity by fibrin autography, PAI activity, PAI type 1 (PAI-1) antigen, and PAI-1 mRNA. The follicular fluid of preovulatory follicles contained low levels of total tissue-type PA antigen (less than 1 ng/mL). Fibrin autography experiments indicated little or no detectable PA activity associated with free or unbound PA. The results of the PAI activity assay and PAI-1 antigen determination support the concept of a relative abundance of PAI compared with PA. Hybridization analysis was used to measure the relative amounts of granulosa cell PAI-1 mRNA. The levels of PAI-1 mRNA correlate with follicular fluid PAI concentrations in individual follicles. Taken together, these results support the idea that there is very little free, or active, PA in follicular fluid of human preovulatory follicles, but there is an abundance of PAI. Furthermore, PAI-1 produced by the granulosa cells may represent a major form of PAI in follicular fluid.

Expression of plasminogen activator (PA) and a PA inhibitor in human granulosa cells from preovulatory follicles.

Studies with rat ovarian cells indicate that proteolytic enzymes, such as plasminogen activator (PA), play a role in the tissue remodeling that occurs before ovulation. In the rat, gonadotropins appear to increase granulosa cell tissue-type plasminogen activator (t-PA) content by increasing the cellular concentration of t-PA mRNA as well as by modulating the activity of a specific PA inhibitor (PAI). We obtained granulosa cells from preovulatory follicles of women undergoing either in vitro fertilization or gamete intra-fallopian tube transfer in order to evaluate the roles of PA and PAI in human ovulation. Samples of granulosa cell total RNA were hybridized with probes for t-PA, urokinase-type PA, PAI type 1 (PAI-1), or inhibin A-chain (as a control). Northern analyses revealed that the RNA of granulosa cells from preovulatory follicles contained little or no detectable PA mRNA. In contrast, two species of PAI mRNA were detected in relative abundance. The signal intensity produced by the PAI-1 probe varied by about 8-fold among patient samples, suggesting that PAI-1 may be useful as a marker of follicular maturation and differentiation. These results demonstrate that human granulosa cells collected immediately before ovulation contain PA inhibitor mRNA, yet have little or no PA mRNA.

Identification of a null allele of CYP2C9 in an African-American exhibiting toxicity to phenytoin.

Cytochrome P450 (CYP) 2C9 is the principal enzyme responsible for the metabolism of numerous clinically important drugs. Two polymorphic alleles CYP2C9*2 and CYP2C9*3 have been documented which affect the metabolism and clinical toxicity of drugs such as phenytoin, warfarin, glipizide, and tolbutamide. The present study reports the first example of a null polymorphism in CYP2C9. This mutation dramatically affects the half-life and clinical toxicity of phenytoin. The study subject was a female African-American presented to the emergency department with phenytoin toxicity evidenced by mental confusion, slurred speech, memory loss and the inability to stand. She exhibited extremely poor clearance of phenytoin with an elimination half-life of approximately 13 days. Genotyping studies demonstrated that the patient did not possess any known variant CYP2C9 alleles. Phenytoin is metabolized to a minor extent by the polymorphic CYP2C19, but this individual did not possess any variant CYP2C19 alleles. Sequencing studies revealed that the individual was homozygous for a new CYP2C9 allele (CYP2C9*6) with the deletion of an adenine at base pair 818 of the cDNA. The clearance of phenytoin in this individual is estimated to be approximately 17% of that observed in normal patients. The frequency of this allele was 0.6% (95% confidence limits of 0.1 to 3.5%) in 79 African-Americans and 0% (95% confidence limits of 0 to 1.1%) in 172 Caucasians. The study also demonstrates the severe clinical consequences to patients with a null mutation in CYP2C9 after treatment with normal doses of phenytoin.