A soluble class II cytokine receptor, IL-22RA2, is a naturally occurring IL-22 antagonist.

IL-22 is an IL-10 homologue that binds to and signals through the class II cytokine receptor heterodimer IL-22RA1/CRF2-4. IL-22 is produced by T cells and induces the production of acute-phase reactants in vitro and in vivo, suggesting its involvement in inflammation. Here we report the identification of a class II cytokine receptor designated IL-22RA2 (IL-22 receptor-alpha 2) that appears to be a naturally expressed soluble receptor. IL-22RA2 shares amino acid sequence homology with IL-22RA1 (also known as IL-22R, zcytor11, and CRF2-9) and is physically adjacent to IL-20Ralpha and IFN-gammaR1 on chromosome 6q23.3-24.2. We demonstrate that IL-22RA2 binds specifically to IL-22 and neutralizes IL-22-induced proliferation of BaF3 cells expressing IL-22 receptor subunits. IL-22RA2 mRNA is highly expressed in placenta and spleen by Northern blotting. PCR analysis using RNA from various tissues and cell lines showed that IL-22RA2 was expressed in a range of tissues, including those in the digestive, female reproductive, and immune systems. In situ hybridization revealed the dominant cell types expressing IL-22RA2 were mononuclear cells and epithelium. Because IL-22 induces the expression of acute phase reactants, IL-22RA2 may play an important role as an IL-22 antagonist in the regulation of inflammatory responses.

A potential role for insulin-like growth factor binding protein-4 proteolysis in the establishment of ovarian follicular dominance in cattle.

A critical transition in ovarian follicular development is the selection of a dominant follicle, capable of ovulating, from a cohort of synchronously growing antral follicles. However, little is known about mechanisms and factors that regulate the selection and growth of dominant ovarian follicles. We have investigated whether a component of the insulin-like growth factor (IGF) system, namely IGFBP-4 protease, is associated with the establishment of follicular dominance in cattle. IGFBP proteases degrade IGFBPs, freeing IGFs to interact with their receptors. In experiment 1, follicular fluid from preovulatory follicles (n = 4) degraded about 80% of the added recombinant human (rh) IGFBP-4 within 18 h of incubation. The IGFBP-4 protease exhibited optimal activity at neutral/basic pH and its sensitivity to various protease inhibitors suggested a metalloprotease. The decline in the intensity of the band corresponding to intact rhIGFBP-4 was accompanied by the appearance of immunoreactive fragments of molecular weights approximately 18 and 14 kDa, which were not detectable by ligand blot analysis. In experiment 2, follicular fluid samples were collected from dominant and subordinate follicles on Day 2 or 3 of the first follicular wave, after ovariectomy (experiment 2a, n = 3/day) or by ultrasound-guided follicular aspiration (experiment 2b, n = 4-5/day). Estradiol concentrations in follicular fluid from dominant vs. subordinate follicles confirmed their identities and indicated that the dominant follicle had been selected by Day 2 of the follicular wave. In both experiments 2a and 2b, IGFBP-4 proteolytic activity was 2- to 3.5-fold (P < 0.05) and 5-fold (P < 0.01) higher in follicular fluid from dominant than subordinate follicles on Days 2 and 3 of the follicular wave, respectively. The finding that IGFBP-4 proteolytic activity is higher in dominant, estrogen-active follicles than in subordinate follicles of the same cohort, as early as Day 2 of the follicular wave, strongly suggests a role for IGFBP-4 protease in the establishment of ovarian follicular dominance.

Pregnancy-associated plasma protein-a is the insulin-like growth factor binding protein-4 protease secreted by human ovarian granulosa cells and is a marker of dominant follicle selection and the corpus luteum.

Insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), and IGFBP proteases are important in ovarian function. IGFs stimulate granulosa steroidogenesis, an effect that is inhibited by IGFBP-4 and augmented by IGFBP-4 proteolysis. We have recently identified the IGFBP-4 protease in human ovarian follicular fluid (FF) as pregnancy-associated plasma protein-A (PAPP-A). In the current study, we identify the IGFBP-4 protease secreted by cultured human ovarian granulosa cells as PAPP-A, based on specific immunoinhibition and immunodepletion of the IGFBP-4 protease activity with PAPP-A polyclonal antibodies and immunorecognition by PAPP-A monoclonal antibodies in ELISA. PAPP-A was barely detectable in conditioned media (CM) from granulosa derived from /=9 mm, coincident with dominant follicle selection, and by luteinizing granulosa. PAPP-A levels in CM from the latter did not change in response to IGF-II or hCG (100 ng/mL). A naturally occurring inhibitor of PAPP-A, proform of eosinophil major basic protein (proMBP), was detected by ELISA in estrogen-dominant follicular fluid FF, but not in CM from granulosa or luteinizing granulosa cells treated with IGF-II (0-200 ng/mL), FSH (0-100 ng/mL) or hCG (0-100 ng/mL), suggesting an alternative source (other than granulosa) for proMBP, compared to PAPP-A. The data demonstrate granulosa cells as a source of PAPP-A in human ovary and suggest that PAPP-A is a marker of ovarian follicle selection and corpus luteum formation. In addition the data suggest complex regulation of this system in human ovary.

Interleukin 20: discovery, receptor identification, and role in epidermal function.

A structural, profile-based algorithm was used to identify interleukin 20 (IL-20), a novel IL-10 homolog. Chromosomal localization of IL-20 led to the discovery of an IL-10 family cytokine cluster. Overexpression of IL-20 in transgenic (TG) mice causes neonatal lethality with skin abnormalities including aberrant epidermal differentiation. Recombinant IL-20 protein stimulates a signal transduction pathway through STAT3 in a keratinocyte cell line, demonstrating a direct action of this ligand. An IL-20 receptor was identified as a heterodimer of two orphan class II cytokine receptor subunits. Both receptor subunits are expressed in skin and are dramatically upregulated in psoriatic skin. Taken together, these results demonstrate a role in epidermal function and psoriasis for IL-20, a novel cytokine identified solely by bioinformatics analysis.

Growth factor regulation of insulin-like growth factor binding protein secretion by cultured human granulosa-luteal cells.

OBJECTIVE: To examine the effects of epidermal growth factor (EGF), transforming growth factor-beta (TGF-beta), and fibroblast growth factor (FGF) on insulin-like growth factor binding protein (IGFBP) secretion by cultured human granulosa-luteal cells. DESIGN: Granulosa-luteal cells obtained at the time of oocyte harvest for IVF were cultured in serum-free medium in the presence or absence of EGF, TGF-beta, or FGF. Conditioned medium then was analyzed by Western ligand blot and immunoradiometric assays. SETTING: An academic medical center. PATIENT(S): Women undergoing IVF. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): IGFBP-1 secretion. RESULT(S): By Western ligand blot analysis, IGFBP-1 levels were 1.4-fold to 7.4-fold higher in conditioned medium from cells cultured in the presence of EGF than in control medium. By immunoradiometric assay, IGFBP-1 levels increased from 1.6 to 9.9 times over control. The TGF-beta had no apparent effect, and FGF did not consistently stimulate IGFBP-1 secretion. CONCLUSION(S): The EGF may decrease intrafollicular bioavailable IGF levels by increasing inhibitory IGFBPs, thereby leading to arrest of follicular development. Interactions between the EGF and IGF systems may be involved in the processes governing human ovarian follicle maturation and atresia.

Human granulosa cells use high density lipoprotein cholesterol for steroidogenesis.

This study examines the ability of human high density lipoproteins (HDL3) to deliver cholesteryl esters to human granulosa cells and describes the selective cholesterol pathway by which this occurs. Luteinized cells obtained from subjects undergoing in vitro fertilization-embryo transfer procedures were incubated with native HDL3 (or radiolabeled or fluorescently labeled HDL cholesteryl esters) to determine whether cells from humans (in which HDL is not the primary circulating lipoprotein species) can nevertheless interiorize and appropriately process cholesteryl esters for steroidogenesis. The results indicate that hormone-stimulated granulosa cells actively and efficiently use human HDL-derived cholesterol for progesterone production. More than 95% of the mass of HDL cholesteryl esters entering cells does so through the nonlysosomal (selective) pathway, i.e. cholesteryl esters released from HDL are taken up directly by the cells without internalization of apoproteins. Once internalized, the cholesteryl esters are either hydrolyzed and directly used for steroidogenesis or stored in the cells as cholesteryl esters until needed. The utilization of the internalized cholesteryl esters is a hormone-regulated event; i.e. luteinized human granulosa cells internalize and store large quantities of HDL-donated cholesteryl esters when available, but further processing of the cholesteryl esters (hydrolysis, re-esterification, or use in steroidogenesis) does not occur unless the cells are further stimulated to increase progesterone secretion.

Insulin-like growth factors and insulin-like growth factor binding proteins in androgen-dominant ovarian follicles from testosterone-treated female-to-male trans-sexuals.

OBJECTIVE: To determine insulin-like growth factor (IGF)-I and IGF-II levels, IGF binding protein (IGFBP) profile, and IGFBP-4 protease activity in androgen-dominant follicular fluid (FF) from female-to-male trans-sexuals and to compare with those in follicles from normocycling women. DESIGN: Follicular fluid samples were obtained from four female-to-male trans-sexuals and 15 women with normo-ovulatory cycles at the Dijkzigt Academic Hospital. Western ligand blot analysis and protease assays were used to determine IGFBP profile, and immunoradiometric assays were used to detect IGF levels. SETTING: The study was performed in two academic medical centers. PATIENT(S): Female-to-male trans-sexuals and women with normo-ovulatory cycles. INTERVENTIONS: None. MAIN OUTCOME MEASURE(S): Determination of IGF levels and IGFBP profile. RESULT(S): Insulin-like growth factor I levels in FF from female-to-male trans-sexuals were not significantly different compared with levels in androgen-dominant FF and estrogen-dominant FF. Significantly lower levels of IGF-II were observed in FF from female-to-male trans-sexuals than in estrogen-dominant FF, whereas IGF-II levels in FF from female-to-male trans-sexuals were not significantly different than those in androgen-dominant FF. Similar IGFBP profiles from FF from female-to-male trans-sexuals and androgen-dominant FF were noted, with markedly elevated levels of the 31- and 24-kd IGFBPs and a 28-kd IGFBP, compared with estrogen-dominant FF. An IGFBP-4-specific metalloserine protease that is active in estrogen-dominant FF likewise was undetected in FF from female-to-male trans-sexuals. CONCLUSION(S): Follicles developing under the influence of exogenous androgens in ovaries in female-to-male trans-sexuals appear to be similar to androgen-dominant follicles in normo-ovulatory women with regard to IGF-I and IGF-II levels, IGFBP profile, and the absence of IGFBP-4 protease activity.

Serum and follicular fluid levels of insulin-like growth factor I (IGF-I), IGF-II, and IGF-binding protein-1 and -3 during the normal menstrual cycle.

Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) have important regulatory functions in ovarian follicular development. Although most studies have investigated the IGF system in ovarian cells in vitro, investigation of the IGF system in the peripheral circulation and in follicles of varying sizes throughout the menstrual cycle in large numbers of subjects has been lacking. In the current study we performed daily IGF-I, IGF-II, IGFBP-1, and IGFBP-3 measurements in 9 healthy regularly cycling volunteers throughout the menstrual cycle. In addition, we investigated IGF-I, IGF-II, IGFBP-1, and IGFBP-3 levels in 13 samples of androgen-dominant follicular fluid [FFa androstenedione to estradiol (AD:E2) ratio, > 4] and 19 samples of estrogen-dominant follicular fluid (FFe; AD:E2 ratio, 4) obtained from 21 regularly cycling subjects and in 18 samples of fluid from luteinizing follicles obtained from patients undergoing in vitro fertilization (IVF) treatment (FFivf). IGF-I, IGF-II, IGFBP-1, and IGFBP-3 were measured using two-site immunoradiometric assays. No significant day to day differences were observed in IGF-I, IGF-II, IGFBP-1, and IGFBP-3 levels across the menstrual cycle. Median IGF-II levels in FFe (630 ng/mL; range, 212-1000) were significantly higher compared to those in FFa (474 ng/mL; range, 272-603; P = 0.002). Median IGFBP-3 levels in FFe (2955 ng/mL; range, 388-3448) were also significantly higher than those in FFa (2352 ng/mL; range, 756-2604; P = 0.003). Median IGF-I (192 ng/mL; range, 29-256) and IGFBP-1 (12 ng/mL; range, 2-281) levels in FFe were not significantly different from those in FFa [149 (range, 22-232) and 21 (range, 5-32) ng/mL, respectively). In contrast, significantly lower IGFBP-1 levels were found in FFe compared to FFivf (79 ng/mL; range, 57-234; P = 0.002), whereas there was no significant difference between FFe and FFivfe IGF-I, IGF-II, or IGFBP-3 levels, respectively. IGF-II levels were correlated with follicle diameter (r = 0.52; P = 0.002), cycle day (r = 0.47; P = 0.0065), E2 levels (r = 0.53; P = 0.003), AD:E2 ratio (r = -0.58; P = 0.001), and P concentrations (r = 0.60; P = 0.001) in all follicles, whereas no such correlations were found with IGF-I. In conclusion, as circulating levels of IGF-I, IGF-II, IGFBP-1, and IGFBP-3 are not menstrual cycle dependent, it is unlikely that these growth factors and these binding proteins play an endocrine role in cyclic ovarian follicle development, although both cycle-dependent delivery to the ovary and modification of their actions locally within the ovary cannot be excluded. With regard to FF1 the findings that IGF-II levels in FF1 are elevated compared to those in FFa and correlate with follicular functional status support a role for IGF-II during development of the dominant follicle. In addition, as IGFBP-3 in estrogen-dominant follicles mirrors the rise of IGF-II, this IGFBP may be a primary regulator of IGF-II action within the estrogen-dominant follicle. Finally, the finding of elevated levels of IGFBP-1 in luteinizing (IVF) follicles suggests an important role for this peptide in corpus luteum regulation.

Insulin receptor mediates inhibitory effect of insulin, but not of insulin-like growth factor (IGF)-I, on IGF binding protein 1 (IGFBP-1) production in human granulosa cells.

Insulin-like growth factor binding proteins (IGFBPs) may participate in regulating ovarian function by modifying effects of insulin-like growth factors (IGFs) or by directly affecting ovarian steroidogenesis in both normal and pathological circumstances. The latter include hyperinsulinemic insulin resistant states, such as polycystic ovary syndrome. We examined regulation of IGFBP-1 production in human granulosa cells by insulin and IGF-I. The cells were obtained during in vitro fertilization, plated in McCoy-5A tissue culture medium supplemented with 10% fetal calf serum (10(5) cells/0.5 mL), and incubated at 37 C, 90% humidity, 5% CO2 for 48 h. After additional 24 h incubation without fetal calf serum, 1, 10, or 100 ng/mL of insulin or IGF-I were added with or without 2 h preincubation with 10 micrograms/mL monoclonal anti insulin receptor antibody IR-47-9. After 48 h incubation with insulin or IGF-I, the medium was collected and IGFBP-1 and progesterone concentrations were measured, using kits from Diagnostic Systems Laboratories, Webster, TX. Progesterone concentration ranged between 50-100 ng/mL/10(5) cells, without consistent stimulatory effect of either insulin or IGF-I. Control cells produced 7.0 +/- 1.7 ng/mL of IGFBP-1. Incubation with 1 or 10 ng/mL of insulin resulted in culture medium IGFBP-1 concentrations of 7.1 +/- 1.3 ng/mL and 5.4 +/- 0.7 ng/mL, respectively (P = NS). Incubation with 100 ng/mL of insulin reduced IGFBP-1 culture medium concentration to 1.6 +/- 0.3 ng/mL (P < 0.01, compared with controls). 1, 10, and 100 ng/mL of IGF-I inhibited IGFBP-1 concentrations in the conditioned culture medium to 1.3 +/- 0.3 ng/mL, 0.4 +/- 0.1 ng/mL and 0.3 +/- 0.1 ng/mL, respectively (P < 0.01, compared with controls). Preincubation with antiinsulin receptor antibody IR-47-9 alleviated inhibitory effect of insulin, but not of IGF-I on IGFBP-1 production. After preincubation with IR-47-9, IGFBP-1 culture medium concentrations were 5.9 +/- 0.8 ng/mL, 4.9 +/- 1.2 ng/mL, and 4.8 +/- 1.3 ng/mL for 1, 10, and 100 ng/mL of insulin, respectively. The latter number was significantly higher than IGFBP-1 concentration in the medium collected from cells incubated with 100 ng/mL of insulin without IR-47-9 (1.6 +/- 0.3 ng/mL, P < 0.01) and not significantly different from the control cells. For cells preincubated with IR-47-9 and then incubated with 1, 10, or 100 ng/mL of IGF-I, the IGFBP-1 conditioned culture medium concentrations were 1.7 +/- 0.1 ng/mL, 0.5 +/- 0.2 ng/mL, and 0.3 +/- 0.1 ng/mL, respectively. None of these were significantly different from the IGFBP-1 concentrations in the medium collected from cells incubated with the respective concentrations of IGF-I without preincubation with IR-47-9. We conclude that 1) both insulin and IGF-I inhibit IGFBP-1 production by cultured human granulosa cells; 2) IGF-I is a more potent inhibitor of IGFBP-1 production than insulin; 3) in the range of hormone concentrations tested, insulin exerts its inhibitory effect on IGFBP-1 production via insulin receptor, while IGF-I appears to exert its effect via another receptor.

Estrogen- but not androgen-dominant human ovarian follicular fluid contains an insulin-like growth factor binding protein-4 protease.

Estrogen-dominant follicular fluid (FFe) and granulosa-luteal cell conditioned media, in contrast to androgen-dominant FF (FFa), contain barely detectable levels of insulin-like growth factor binding protein-4 (IGFBP-4) by ligand binding techniques. The current study was designed to evaluate the possibility of an IGFBP-4 protease in FFe, which may alter the affinity of IGFBP-4 for insulin-like growth factors (IGFs), rendering IGFBP-4 undetectable by ligand binding techniques. FFe and FFa were obtained from regularly menstruating women, and FFe was also obtained during in vitro fertilization procedures. Mixing experiments were performed by using human recombinant IGFBP-4 or IGFBP-4 in nonpregnancy serum (NPS) as substrate and FF as the source of the putative protease. Incubation of NPS at 37C for 5 h in the presence of FFe resulted in the reduction of IGFBP-4 to barely detectable levels when analyzed by Western ligand blotting, with no change occurring in the levels of the other binding proteins present in NPS. In contrast, incubation of FFa with NPS under similar conditions had no effect on the levels of IGFBP-4. The disappearance of IGFBP-4 when NPS was mixed with FFe exhibited optimal pH-dependence at pH 7-9. Inhibition of the putative protease by aprotinin, ethylenediaminetetraacetic acid, and 1,10-phenanthroline supports its identification as a metalloserine protease. Western immunoblot analysis detected the presence of a proteolytic fragment of approximately 17-18 kDa after incubation of recombinant IGFBP-4 in the presence of FFe but not in the presence of FFa. Similar incubation of other recombinant human IGFBPs did not reveal their degradation, further suggesting that the protease in FFe is specific for IGFBP-4. These data demonstrate the presence of an IGFBP-4-specific metalloserine protease in FFe but not in FFa, and they suggest that proteolytic cleavage may be responsible for effectively decreasing levels of inhibitory IGFBP-4 and thus increasing bioavailability of IGF peptides within estrogen-dominant follicles. The importance of this mechanism may lie in providing the dominant follicle with more available IGFs to synergize with gonadotropins in stimulating estradiol production and in inhibiting this synergy in androgen-dominant and atretic follicles.

Circulating and ovarian IGF binding proteins: potential roles in normo-ovulatory cycles and in polycystic ovarian syndrome.

IGFs function as co-gonadotropins in the ovary, facilitating steroidogenesis and follicle growth. IGFBP-1 to -5 are expressed in human ovary and mostly inhibit IGF action in in vitro ovarian cell culture systems. In the clinical disorder of polycystic ovarian syndrome (PCOS), which is characterized by hyperandrogenemia, polycystic ovaries and anovulation, follicles have a higher androgen: estradiol (A : E2) content and growth is arrested at the small antral stage. In the PCOS follicle, follicle stimulating hormone (FSH) and IGF levels are in the physiologic range, and even in the face of abundant androstenedione (AD) substrate, aromatase activity and E2 production are low. When PCOS granulosa are removed from their ovarian environment, they respond normally or hyperrespond to FSH. It has been postulated that an inhibitor of IGF's synergistic actions with FSH on aromatase activity may be one (or more) of the IGFBPs, which contributes to the arrested state of follicular development commonly observed in this disorder. High levels of IGFBP-2 and IGFBP-4 are present in follicular fluid (FF) from androgen-dominant follicles (FFa) from normally cycling women and in women with PCOS. This is in marked contrast to the near absence of these IGFBPs in estrogen-dominant FF (FFe), determined by Western ligand blotting. Regulation of granulosa-derived IGFBPs is effected by gonadotropins and insulin-like peptides. In addition, an IGFBP-4 metallo-serine protease is present in FFe, but not in FFa in ovaries from normally cycling women and those with PCOS, although the IGFBP-4 protease is present in PCOS follicles hyperstimulated for in vitro fertilization. Recent studies demonstrate that IGF-II in FFe is higher than in FFa' whereas IGF-I, IGFBP-3 and IGFBP-1 levels do not differ, underscoring the importance of local IGF-II production by the granulosa and the importance of IGFBP-4 and IGFBP-2 in regulation of IGF-II action within the follicle during its developmental pathway as an E2- or A-dominant follicle. In the androgen-treated female-to-male transsexual (TSX) model for PCOS, IGF-I, IGF-II, IGFBP-3 and IGFBP-1 levels do not differ.

Initiation of periovulatory events in primate follicles using recombinant and native human luteinizing hormone to mimic the midcycle gonadotropin surge.

The amplitude and duration of the midcycle LH surge required for periovulatory changes in the primate follicle are incompletely defined. We reported that short (4- to 14-h) LH surges were insufficient to induce periovulatory events after multiple follicular development in macaques. In contrast, an 18- to 24-h LH surge induced oocyte maturation plus granulosa cell luteinization, but did not support corpus luteum function. In this study, the periovulatory changes following LH surges of 48 h elicited using pituitary (pit) or recombinant (r) human (h) LH were compared to those after 24-h LH surge durations or after urinary hCG (u-hCG) treatment. Beginning at menses, rhesus monkeys were treated with human gonadotropins for 9 days to stimulate follicular growth. On day 10, animals (n = 3-5/group) received 1) a single injection of u-hCG [79 +/- 3 micrograms RP-1 equivalents (equiv), im], 2) two injections of pit-hLH (91 +/- 4 micrograms RP-1 equiv, im), 3) one injection of r-hLH (21 +/- 1 micrograms RP-1 equiv, im), or 4) two injections of r-hLH (21 +/- 1 micrograms RP-1 equiv). Oocytes and granulosa cells were obtained via follicle aspiration 27 h after the initial LH or hCG injection. In all groups, serum estradiol rose to similar peak levels by day 10. Circulating LH-like bioactivity was elevated for more than 48 h after u-hCG. Peak serum LH bioactivities were proportional to the administered LH doses, as determined in the in vitro bioassay. Two injections of either r-hLH or pit-hLH elicited surge levels (> 100 ng/mL) of bioactive LH for 36-48 h, whereas one injection sustained surge levels for only 18-24 h. The proportions of oocytes resuming meiosis (68-76%) were similar in all groups. Immunocytochemical staining for progesterone receptor and in vitro progesterone production by granulosa cells in all LH-treated groups were comparable to those of cells form the hCG-treated group. Peak levels of progesterone in the luteal phase were comparable in monkeys treated with two doses of pit-hLH and r-hLH (18.5 +/- 10.4 vs. 8.1 +/- 1.5 ng/mL) and approached that in u-hCG treated monkeys (39.5 +/- 18.0 ng/mL). However, progesterone levels in animals treated once with r-hLH (3.4 +/- 1.5 ng/mL) were less (P < 0.05) than those in u-hCG-treated monkeys.(ABSTRACT TRUNCATED AT 400 WORDS)

Progesterone receptor, but not estradiol receptor, messenger ribonucleic acid is expressed in luteinizing granulosa cells and the corpus luteum in rhesus monkeys.

Estrogens (i.e. estradiol) and progestins (i.e. progesterone) may act as local regulators of ovarian function in various species. This study tested the hypothesis that if progesterone and estradiol act via receptor-mediated pathways in the primate ovary, then receptor messenger RNAs (mRNAs) should be detectable in ovarian cells. The reverse transcription-polymerase chain reaction (RT-PCR) was employed to detect progesterone and estradiol receptor (PR and ER, respectively) mRNAs in the rhesus monkey ovary. Total RNA was isolated from macaque uterine myometrium (positive control), spleen (negative control), whole ovary, germinal (surface) epithelium-enriched cortical and medullary compartments of the ovary, granulosa cells in preovulatory follicles before and after an ovulatory stimulus, and corpora lutea from early (days 3-5), mid (days 7 and 8)-, and late (days 14 and 15) luteal phase of the menstrual cycle. Using primers to the hormone-binding region encoded by the receptor mRNAs, RT-PCR products of the expected sizes were detected for PR and ER from 1 microgram myometrial RNA, whereas products were not obtained from spleen. PR mRNA product was detected in all ovaries, germinal epithelium-enriched cortical and medullary compartments, and corpora lutea from all three stages of the luteal phase (n = 3/stage). PR mRNA product was detected as a strong band in one of three preparations obtained from granulosa cells before an ovulatory stimulus. In contrast, PR mRNA was detected in granulosa cells from all animals after an ovulatory dose of hCG. ER mRNA was detected in whole ovary and in germinal epithelium-enriched cortical compartments, with a barely visible product occasionally observed in medullary compartments of the ovary. In contrast to PR mRNA, ER mRNA was not detected in any corpora lutea throughout the luteal phase or in granulosa cells obtained before or after an ovulatory stimulus. To confirm the specificity of the RT-PCR products, restriction enzymes cleaved the PR product from myometrium, germinal epithelium-enriched cortical compartment, and corpus luteum into the predicted size fragments. Similarly, the ER product from the myometrium and the germinal epithelium-enriched compartment was cleaved into the expected size fragments. Sequence analysis of the PR and ER RT-PCR products revealed 99% homology to the complementary DNA for the hormone-binding region of human PR and ER, respectively. Thus, PR mRNA detection supports the hypothesis of progesterone action via classical receptor-mediated pathways in the luteinizing follicle and corpus luteum of the primate ovary.(ABSTRACT TRUNCATED AT 400 WORDS)

Gonadotrophic and local control of the developing corpus luteum in rhesus monkeys.

The actions of the mid-cycle gonadotrophin surge to convert the pre-ovulatory follicle into the corpus luteum are not well understood in primate species. In experiments titrating the surge duration required in macaque monkeys during artificial in-vitro fertilization (IVF)-related cycles, short (< or = 14 h) LH surges similar to those in rodents and domestic animals failed to initiate peri-ovulatory events. Attenuated (24 h) surges, one-half the duration of spontaneous surges in primates, reinitiated oocyte meiosis and initial luteinization of granulosa cells. However, only surges of > or = 48 h sustained luteal development and function to produce luteal phases of approximately 13 days. Three approaches-immunocytochemistry of steroid receptors, binding of radiolabelled steroid, and reverse transcription-polymerase chain reaction of mRNA-indicate that progesterone receptor (PR), but not oestrogen receptor, is expressed in the macaque corpus luteum. Studies on cells collected before and after the gonadotrophin surge identified a novel action of LH to induce PR expression in luteinizing granulosa cells. Thus, the LH surge requirements vary between non-primate and primate species, as well as between peri-ovulatory events, and the LH surge may promote cellular recognition of paracrine and autocrine factors (e.g. progesterone) that become predominant in the developing corpus luteum.

The potential roles of intraovarian peptides in normal and abnormal mechanisms of reproductive physiology.

Normal and abnormal follicular growth and steroidogenesis depend on gonadotropins as well as intraovarian peptide and polypeptide growth factors, which may mediate or potentiate gonadotropin action. Epidermal growth factor is mitogenic to ovarian granulosa and is a potent inhibitor of granulosa aromatase. It may be involved in the apparent arrest of follicular development commonly seen in women with polycystic ovarian syndrome as well as in the blunted response to gonadotropins seen in this syndrome. Insulin-like growth factors are also mitogenic to ovarian granulosa, but in contrast to epidermal growth factor, insulin-like growth factor-I, both alone and in synergy with gonadotropins, is a potent stimulus of aromatase and granulosa estradiol production. Insulin-like growth factor binding proteins-2 and -4, known inhibitors of insulin-like growth factor action, are higher in follicular fluid from atretic and polycystic ovarian syndrome follicles compared with estrogenic follicles and may be inhibitors of gonadotropin action in follicle selection and in polycystic ovarian syndrome. Cytokines including interleukins, tumor necrosis factor-alpha and interferon-gamma also appear to play a role in modulating ovarian steroidogenesis. Activins, inhibins, and follistatin (activin-binding protein) also affect follicular development and steroidogenesis and may play a role in dominant follicle selection and follicular atresia.

Administration of human luteinizing hormone (hLH) to macaques after follicular development: further titration of LH surge requirements for ovulatory changes in primate follicles.

After stimulation of multiple follicular development, endogenous LH surges elicited by GnRH or GnRH agonist were of insufficient duration (4-14 h) to evoke oocyte maturation and luteinization in this species. In this study, periovulatory LH surge requirements were further titrated using hLH as the ovulatory stimulus. Beginning at menses, rhesus monkeys were treated with human gonadotropins for 9 days to stimulate follicular growth. To induce ovulatory maturation on day 10, animals received: 1) hCG (1000 IU, im; n = 8); 2) highly-purified, urinary hLH (2542 IU, im; n = 4); or 3) hLH (2542 IU, im) followed by three injections of hLH (200 IU, im) at 8-h intervals (0800, 1600, 2400 h) daily during the luteal phase until menses (n = 3). Oocytes and luteinizing granulosa cells were obtained via follicle aspiration 27 h after the initial hLH or hCG injection. Estradiol and progesterone levels were measured in daily serum samples by RIA. Bioactive LH levels were determined at selected intervals within 36 h of the hLH ovulatory stimulus. Nuclear maturity of oocytes was evaluated as an indicator for reinitiation of meiosis. Luteinizing granulosa cells were processed for indirect immunocytochemistry using a monoclonal antibody to human progesterone receptor. In vitro progesterone production by luteinizing granulosa cells over 24 h was also assessed in the absence and presence of hCG. In all groups, serum estradiol rose to similar peak levels on day 10. After hLH, bioactive LH levels peaked (1262 +/- 79 ng/mL; mean +/- SEM) by 2-6 h, declined thereafter but remained above surge levels (100 ng/mL) for 18-24 h. Within 24 h of hLH injection, serum progesterone increased to 13 +/- 3 nmol/L, but returned to baseline in 1-6 days. In contrast, higher levels of progesterone were observed after hCG (114 +/- 51 nmol/L) and during luteal phase treatment with hLH (137 +/- 25 nmol/L) and the luteal phase was longer (11.5 +/- 0.4 and 14.3 +/- 0.7 days, respectively). Of the total cohort of oocytes aspirated, the proportion of oocytes resuming meiotic maturation (metaphase I plus metaphase II) was similar after hCG (76%) and hLH (74%). However, the proportion of oocytes maturing to metaphase II tended to be less (P = 0.08) after hLH (13%) than hCG (22%). Fertilization rates were similar between the two groups. Progesterone receptor was detected in nuclei of luteinizing granulosa cells from all animals receiving hCG, but only in some given hLH.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibin production by macaque granulosa cells from pre- and periovulatory follicles: regulation by gonadotropins and prostaglandin E2.

Although inhibin (IN) is secreted by granulosa cells (GC) of preovulatory follicles, the major source of immunoreactive IN circulating during the primate ovarian cycle is the corpus luteum. The aims of this study were (1) to investigate culture conditions for optimal IN production by luteinized GC (LGC) from rhesus monkeys and (2) to compare IN and progesterone (P) production by nonluteinized GC (NGC) and LGC in response to putative agonists. Animals were treated for up to 9 days with human menopausal gonadotropins to promote the development of multiple preovulatory follicles. GC were obtained from large follicles before (NGC) or 27 h after (LGC) an ovulatory injection of hCG. For Aim 1, cells were cultured in Hams F-10 medium +/- hCG (100 ng/ml) with or without the addition of insulin/transferrin/selenium, 10% fetal bovine serum, or 10% Serum-Plus (JRH Biosciences, Lenexa, KS). Medium was changed on Days 1, 2, 4, 6, and 8, and IN and P concentrations were determined by RIA. Basal (unstimulated) IN production by LGC was enhanced and maintained for 6-8 days in the presence of serum, but rapidly declined in the absence of serum. In contrast, basal P secretion declined regardless of exposure to serum. Human CG consistently increased (p less than 0.05) IN production only in the presence of serum but stimulated (p less than 0.05) P production under all conditions. For Aim 2, cells were cultured for 4 days in Ham's F-10 medium + 10% macaque serum +/- hCG (100 ng/ml), hFSH (100 ng/ml), prostaglandin E2(PGE2; 14 microns), or dibutyryl(db)-cAMP (5 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Titrating luteinizing hormone surge requirements for ovulatory changes in primate follicles. I. Oocyte maturation and corpus luteum function.

The amplitude and duration of the midcycle LH surge required for ovulatory maturation of the follicle and its enclosed oocyte in primates are unknown. To titrate periovulatory LH requirements, female rhesus monkeys received human gonadotropins (FSH with/without LH) for 9 days beginning at menses to promote the development of multiple preovulatory follicles. The next day, animals (n = 4-6/group) received: 1) no ovulatory stimulus; 2) 1000 IU hCG, im; 3) one injection of 100 micrograms GnRH, sc (GnRH-1); 4) three injections of GnRH (GnRH-3) at 3-h intervals (0800, 1100, and 1400 h); or 5) two injections of 50 micrograms GnRH agonist (GnRHa), sc, 8 h apart (0800 and 1700 h) to induce ovulatory maturation. Follicles were aspirated 27 h after the hCG or initial GnRH/GnRHa injection or on days 8 and 10 in animals receiving no ovulatory stimulus. Nuclear maturity of oocytes was evaluated as a marker for reinitiation of meiosis. Estradiol and progesterone levels were determined in daily serum samples by RIA. Levels of LH(-like) bioactivity were measured at selected intervals after hCG injection and within 24 h of GnRH/GnRHa treatment. In all groups, estradiol continuously rose to similar peak levels on day 10. The hCG treatment markedly elevated circulating LH-like bioactivity for up to 3 days. In GnRH-1, bioactive LH increased to 433.1 +/- 170.2 ng/mL (mean +/- SEM; n = 3) within 1-2 h, but then decreased to baseline (4.9 +/- 1.5 ng/mL) within 6 h. GnRH-3 and GnRHa treatment extended the interval of elevated bioactive LH to 8 and 14 h, respectively. There was no difference in the peak levels of LH(-like) bioactivity reached after hCG, GnRH, or GnRHa injection. Functional luteal phases were absent in monkeys receiving no ovulatory stimulus, whereas hCG treatment increased progesterone levels to 101 +/- 9 nmol/L (n = 6) and elicited functional luteal phases of 11.8 +/- 0.4 days. In contrast, only one animal in the GnRH/GnRHa groups (i.e. one GnRH-3 monkey) displayed elevated progesterone levels in the luteal phase. Of the total cohort of oocytes aspirated from follicles, a greater (P less than 0.05) proportion were classified as being in metaphase I or II of meiosis after hCG treatment (86%) compared to no ovulatory stimulus (13%), GnRH-1 (0%), GnRH-3 (43%), and GnRHa (12%). Thus, GnRH elicits a transient LH surge that can be extended by GnRH-3 or GnRHa in stimulated cycles of monkeys.(ABSTRACT TRUNCATED AT 400 WORDS)

Titrating luteinizing hormone surge requirements for ovulatory changes in primate follicles. II. Progesterone receptor expression in luteinizing granulosa cells.

The events in granulosa cells that are initiated by the midcycle LH surge during luteinization of the primate follicle are poorly defined. This study was designed 1) to determine whether an ovulatory dose of hCG can induce progesterone receptors (PR) in macaque granulosa cells, and if so, 2) to begin titrating gonadotropin requirements for PR expression and progesterone production by luteinizing granulosa cells. Rhesus monkeys were treated with human FSH and LH for up to 9 days to stimulate the growth of multiple follicles. The next day, animals (n = 4-5/group) received: 1) no ovulatory stimulus; 2) 1000 IU hCG, im; 3) one injection of 100 micrograms GnRH, sc (GnRH-1); 4) three injections of GnRH (GnRH-3) at 3-h intervals (0800, 1100, and 1400 h); or 5) two injections of 50 micrograms GnRH agonist (GnRHa), sc, 8 h apart (0800 and 1700 h). Granulosa cells obtained by follicle aspiration 27 h after the hCG or initial GnRH/GnRHa injection or on days 8 or 10 from animals receiving no ovulatory stimulus were processed for indirect immunocytochemistry using a monoclonal antibody to human PR (JZB39). Specific staining for PR, determined by comparing cells incubated with PR antibody vs. a nonspecific antibody, was undetectable in granulosa cells from monkeys without an ovulatory stimulus. In contrast, the majority (64 +/- 5%) of cells from hCG-treated animals stained intensely for PR. In the GnRH/GnRHa groups, granulosa cells from only one animal (i.e. one GnRH-3 monkey) showed positive staining for PR. During 24-h culture in Ham's F-10 medium containing 10% monkey serum, basal progesterone production by cells from the hCG-treated group (2163 nmol/L.8 x 10(4) cells) was higher than that by cells from the no ovulatory stimulus/GnRH-1/GnRH-3/GnRHa groups (60, 111, 194, and 332 nmol/L, respectively). However, granulosa cells from the hCG-treated group were less responsive to hCG in vitro in terms of enhanced progesterone production (2 times control levels) than cells from the other four groups (up to 30 times control levels). This study provides direct evidence that an ovulatory dose of hCG induces PR expression in granulosa cells of luteinizing follicles during stimulated cycles in rhesus monkeys. However, repeated injections of GnRH/GnRHa that produced surge levels (greater than 100 ng/mL) of endogenous LH for up to 14 h failed to induce PR expression or progesterone production by granulosa cells. Thus, an extended LH surge more typical of that in the normal menstrual cycle (48-50 h) may be necessary for PR expression and luteinization of granulosa cells in primate follicles.