Leptin gene expression in subcutaneous adipose tissue in girls before and during puberty.

OBJECTIVES: Leptin functions as a neuroendocrine hormone and it is related to the onset of puberty in animal models. Its role in normal human sexual maturation is still incompletely defined. The aim of the study was to assess the relationships between leptin mRNA (gene) expression, thickness of subcutaneous fat tissue and the serum concentration of leptin in girls before and during puberty. STUDY DESIGN: Twenty-nine lean girls were studied (mean age 10.8+/-1.9 years). The subjects were divided into two groups according to pubertal status. The first group consisted of 14 prepubertal girls and second group of 15 girls who were in puberty. Body height, weight, arm circumference, skin fold thickness at abdominal, triceps and subscapular sites were measured. Serum leptin was assessed by RIA method. Leptin mRNA was measured in subcutaneous abdominal adipose tissue by semi-quantitative assays based on reverse transcription (RT) of the mRNA and polymerase chain reaction (PCR) amplification of the cDNA. RESULTS: Girls in pubertal stages had higher serum leptin concentration than prepubertal girls. The mean values of leptin mRNA level in subcutaneous abdominal adipose tissue were not statistically different between groups. There was also no difference between the thickness of skin folds in investigated girls. A positive correlation between leptin mRNA expression and skin fold thickness, BMI and arm circumference as well as between the leptin concentration and skin fold thickness, BMI and arm circumference were observed. CONCLUSIONS: The level of leptin gene expression and serum leptin concentrations depend on the amount of fat tissue. We can propose that initiation of pubertal events does not result from increased of leptin mRNA expression in subcutaneous abdominal fat cells or from its increased concentration in blood.

Selective alterations in insulin receptor substrates-1, -2 and -4 in theca but not granulosa cells from polycystic ovaries.

The elevated insulin concentrations that occur in many women with polycystic ovary syndrome (PCOS) can contribute significantly to ovarian hyperandrogenism. The objective of the present study was to compare the content of proximal insulin signalling molecules in theca and granulosa cells between polycystic ovaries and regular cycling controls. Individual follicles (3-7 mm) were obtained from 11 women with PCOS and 10 regularly cycling control women. The theca and granulosa cells were microdissected from each follicle. Total protein was extracted and signalling proteins were measured by western blot analysis. There was no difference in insulin receptor content between PCOS and controls in either theca or granulosa cells. Insulin receptor substrate (IRS)-1 and -2 were increased (P<0.05), but IRS-4 was decreased (P<0.03) in PCOS theca cells. There were no changes in IRS-1, -2 or -4 in granulosa cells. IRS-3 was undetectable in all samples. There were no changes in phosphatidyl inositol-3 kinase catalytic subunits p110alpha or p110beta in either theca or granulosa cells. These data demonstrate cell-specific alterations in IRS protein concentrations in theca cells from polycystic ovaries that are consistent with an exaggerated amplification of the insulin signal and which may play an important role in ovarian hyperandrogenism and thecal hyperplasia.

Leptin messenger ribonucleic acid (mRNA) content in the human placenta at term: relationship to levels of leptin in cord blood and placental weight.

During fetal life, placental tissue represents an additional source of leptin for the mother and conceptus. It has been suggested that feto-placental production of leptin may be involved in placental and fetal growth regulation. The aim of this study was to examine the correlation between leptin mRNA expression in the placenta and the concentrations of leptin in cord blood. A total of 30 healthy, pregnant women who gave birth to healthy neonates were included in the study. Maternal blood (obtained from the cubital vein) and umbilical cord blood were drawn immediately after birth. Serum leptin concentration was determined by enzyme-linked immunosorbent assay and serum insulin concentration was measured by radioimmunoassay. Leptin mRNA was measured in placental tissue by a reverse transcriptase-polymerase chain reaction. The estimated mean leptin mRNA expression in placenta was 4.65 +/- 1.83 pg mRNA/microg DNA. Leptin mRNA correlated with cord serum leptin concentrations (r = 0.3691, p = 0.045). Placental weight correlated with placental leptin mRNA (r = 0.3686, p = 0.045). The mean leptin concentration in cord serum at birth was slightly lower (3.1 +/- 1.9 ng/ml) than that found in maternal serum (3.9 +/- 1.2 ng/ml). A positive correlation was observed between cord and maternal serum leptin levels (r = 0.58, p = 0.001). The mean insulin concentration in maternal serum was not significantly higher than that in umbilical serum: 22.2 +/- 17.8 microIU/ml vs. 6.9 +/- 3.6 microIU/ml; r = 0.069, p = 0.71). Neither maternal nor umbilical insulin concentrations correlated with leptin concentration in cord or maternal peripheral serum.

Effects of leptin administration and feed restriction on thecal leucocytes in the preovulatory rat ovary and the effects of leptin on meiotic maturation, granulosa cell proliferation, steroid hormone and PGE2 release in cultured rat ovarian follicles.

Leptin is expressed by adipocytes and is thought to play a role in regulating food intake and in reproduction. It has been demonstrated that acute leptin administration to immature gonadotrophin-primed rats in vivo inhibits ovulation and causes a decline in food intake. However, feed restriction alone does not inhibit ovulation. Two experiments were designed to investigate the mechanism of leptin-induced inhibition of ovulation. In the first experiment, which was prompted by the importance of ovarian leucocytes in ovulation, the role of leucocytes in leptin-induced inhibition of ovulation was investigated. The second experiment investigated whether high leptin concentrations could inhibit other factors important to ovulation, such as meiotic competence of oocytes, granulosa cell proliferation, steroid or PGE(2) release, and interleukin 1beta production, in vitro. In the first experiment, the populations of neutrophils and monocytes-macrophages in the preovulatory follicles of gonadotrophin-primed, leptin-treated and -untreated rats were examined. A decrease in food intake, as a result of either leptin treatment or feed restriction, specifically reduced the numbers of neutrophils and monocytes-macrophages infiltrating the theca interna of preovulatory follicles without affecting the numbers found in the stroma. The findings show that reduced infiltration of thecal neutrophils and macrophages into preovulatory follicles is a response to reduced food intake. Furthermore, this reduction is not the direct cause of the leptin-induced inhibition of ovulation. In the second experiment, ovarian follicles were cultured for 4 or 12 h in the presence or absence of the following hormones: FSH (500 miu), insulin-like growth factor I (IGF-I) (50 ng ml(-1)), LH (100 ng ml(-1)) and leptin (300 ng ml(-1)). The results demonstrated that high concentrations of leptin in follicle culture do not affect meiotic maturation or steroid release, but tend to inhibit release of PGE 2 (although this result was not significant). DNA synthesis in granulosa cells was not inhibited by leptin in FSH- and IGF-I-supplemented culture media. These results are in agreement with previous studies that have shown that leptin inhibits the stimulatory effects of IGF-I on FSH-stimulated oestradiol production in rat granulosa cells without affecting progesterone production. In summary, leptin does not appear to have an adverse effect on the components of ovulation tested in this study, and therefore must impact on the ovulatory cascade in a way that remains to be defined.

Expression of the long (OB-RB) and short (OB-RA) forms of the leptin receptor throughout the oestrous cycle in the mature rat ovary.

Leptin is secreted by adipocytes and exerts its effects by interacting with the long form of the leptin receptor, OB-RB. The leptin protein and leptin receptors have been localized in the ovary, and acute leptin treatment directly inhibits ovulation in the rat ovary. It was hypothesized that expression of the leptin receptor gene varies throughout the oestrous cycle to modulate the sensitivity of the ovary to leptin. In this study, expression of genes for the long and short isoforms of the leptin receptor in the adult ovary was investigated at different stages of the rat oestrous cycle. Vaginal cytology was used to determine the stage of the oestrous cycle. Ovaries were collected and RNA was extracted for real-time RT-PCR analysis of leptin receptor gene expression. OB-RB gene expression was low in pro-oestrus (3.13 +/- 0.18 fg RNA per microg total DNA) and dioestrus II (2.52 +/- 0.19 fg RNA per microg total DNA) of the oestrous cycle, whereas expression was high in oestrus (5.9 +/- 0.27 fg RNA per microg total DNA) and dioestrus I (4.6 +/- 0.24 fg RNA per microg total DNA) (P < 0.001). Expression of the gene for the short form of the leptin receptor (OB-RA) was at a maximum in dioestrus I (65.5 +/- 0.8 fg RNA per ng total DNA), high in oestrus (39.0 +/- 0.8 fg RNA per ng total DNA) and low at pro-oestrus (5.0 +/- 0.2 fg RNA per ng total DNA) and dioestrus II (1.1 +/- 0.09 fg RNA per ng total DNA) (P < 0.001). Plasma oestradiol concentrations (pg ml-1) were highest at pro-oestrus (19.38 +/- 1.3), and similar at the remaining three stages studied (oestrus: 13.7 +/- 1.9; dioestrus I: 12.4 +/- 1.0; dioestrus II: 10.3 +/- 0.9) (P < 0.05). Plasma progesterone concentrations (ng ml-1) were higher in the luteal phases of the oestrous cycle (dioestrus I: 18.6 +/- 2.3; dioestrus II: 14.7 +/- 2.5) than during pro-oestrus (5.12 +/- 0.6) and oestrus (5.9 +/- 0.8) (P < 0.05). Plasma leptin concentrations were detectable only in pro-oestrus (0.35 +/- 0.05 ng ml(-1)) and were below the detection limit of the assay at other stages of the oestrous cycle. In summary, mRNA content for the long and short isoforms of the leptin receptor is lower in pro-oestrus and dioestrus II than in oestrus and dioestrus I of the rat oestrous cycle. The fluctuations in leptin receptor mRNA content may be a response to the concentrations of circulating steroid hormones and leptin. This research supports the initial hypothesis and shows that ovarian leptin receptor concentrations vary throughout the oestrous cycle in response to the changing environment of the ovary.

Luteinizing hormone receptor, steroidogenesis acute regulatory protein, and steroidogenic enzyme messenger ribonucleic acids are overexpressed in thecal and granulosa cells from polycystic ovaries.

Recent data suggest that steroidogenic enzyme messenger ribonucleic acids (mRNAs) may be overexpressed in thecal cells, and LH receptors may be prematurely expressed in granulosa cells in women with polycystic ovaries. The purpose of this study was to determine whether there is abnormal gene expression in thecal and granulosa cells from polycystic ovaries. Ovarian tissue specimens were obtained from 12 women with PCOS and 24 regularly cycling control women. The granulosa cells and the theca interna were microdissected from individual follicles. LH receptor, steroidogenesis acute regulatory protein (StAR), cholesterol side-chain cleavage cytochrome P450 (CYP11A), and 17alpha-hydroxylase/C(17-20) lyase cytochrome P450 (CYP17) mRNAs were measured by RT-PCR. There was no difference between 3- to 7-mm control follicles and dominant follicles with respect to LH receptor mRNA expression in either thecal or granulosa cells. CYP11A and CYP17 mRNAs were higher in thecal cells from 3- to 7-mm follicles than in dominant follicles, but StAR expression was not different. In granulosa cells, StAR and CYP11A mRNA expression was higher in dominant follicles than in 3- to 7-mm follicles. The mean levels of LH receptor, StAR, CYP11A, and CYP17 mRNA expression were higher in thecal cells from PCOS follicles than in size-matched control follicles. In granulosa cells, the mean levels of LH receptor and CYP11A, but not StAR, mRNA expression were higher in PCOS than in control follicles. These data demonstrate that regulatory protein and steroidogenic enzyme mRNAs are overexpressed in thecal and granulosa cells from polycystic ovaries and support the conclusions that the thecal cells are hyperstimulated and the granulosa cells may be prematurely luteinizing.

Stem cell factor and insulin-like growth factor-I stimulate luteinizing hormone-independent differentiation of rat ovarian theca cells.

The signal initiating ovarian theca cell (TC) differentiation is gonadotropin independent because theca precursor cells do not contain LH receptors. Previously we demonstrated that preantral follicles produce paracrine TC differentiating factors that promote androgen production by an LH-independent mechanism. This study tested the effects of two granulosa cell-produced peptides, insulin-like growth factor-I (IGF-I) and stem cell factor (SCF), on TC differentiation and androgen production. Neutralizing antibodies to either IGF-I or SCF blocked the stimulatory effects of follicle-conditioned medium on TC precursor differentiation more than 90%. The TC isolated from the ovaries of hypophysectomized immature rats by percoll gradient centrifugation were cultured (48 h) with and without SCF (0-100 ng/ml) and IGF-I (0-100 ng/ml) to test their effects on TC differentiation. Androsterone in the medium was measured by RIA. Luteinizing hormone receptor, steroidogenesis acute regulatory protein (StAR), CYP11A, CYP17, and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) mRNAs were measured by specific reverse transcriptase polymerase chain reaction assays. Stem cell factor or IGF-I alone did not stimulate androsterone production but in combination caused a concentration-dependent increase in androsterone levels. Maximum androsterone levels were less than those stimulated by LH (0.1 ng/ml) alone. Although IGF-I synergistically augmented LH stimulation of androsterone production, SCF did not alter LH-stimulated androsterone production in the presence or absence of IGF-I. Stem cell factor alone had no effect on LH receptor, StAR, CYP11A, and 3beta-HSD mRNA expression but decreased CYP17 mRNA levels. Insulin-like growth factor-I alone had no effect on StAR or CYP17 mRNA expression but increased LH receptor, CYP11A, and 3beta-HSD mRNA levels. In combination, SCF plus IGF-I increased the expression of all five mRNAs. These data support the conclusion that IGF-I and SCF are important regulators of TC differentiation.

The in vivo and in vitro effects of exogenous leptin on ovulation in the rat.

Leptin, a hormonal product of the Lep gene, is expressed by adipocytes and is thought to play a role in regulating food intake and reproduction. The leptin protein has been localized in many reproductive tissues, including the ovary. Several publications indicate that the ovary is directly affected by leptin and that leptin may be a factor linking obesity and reproductive dysfunction. In this study, the effect of systemic leptin administration on ovulation in the rat ovary, both in vivo and in vitro, was investigated. Ip administration of leptin (30 microg at 3 hourly intervals for 15 h) to immature gonadotropin-primed rats caused a decline in ovulation in vivo, from 15.9+/-2.0 oocytes in the control animals to 5.3+/-1.6 oocytes in the leptin-treated animals (P < 0.001). Plasma progesterone and estradiol levels were analyzed immediately before ovulation, and neither was altered significantly in animals receiving the leptin treatment. Food consumption and body weight decreased following leptin treatment; however, a loss in body weight alone (pair-fed controls) was insufficient to explain the decrease in ovulation observed in the leptin-treated animals. In vitro perfusion of FSH-primed whole ovaries showed that treatment with leptin in combination with LH significantly decreased ovulations from 5.7+/-1.6 per ovary perfused with LH alone to 1.3+/-0.6 in those with LH and 1 microg/ml leptin (P < 0.05). Progesterone and estradiol levels in the samples taken during the perfusion period were unaffected by leptin treatment. In summary, leptin administration resulted in fewer ovulations, both in vivo and in vitro, but did not influence steroid levels. Systemic leptin administration at these doses can therefore inhibit ovulation, a process that occurs through a direct effect on the ovary.

Leptin impairs the synergistic stimulation by transforming growth factor-beta of follicle-stimulating hormone-dependent aromatase activity and messenger ribonucleic acid expression in rat ovarian granulosa cells.

Leptin blocks the insulin-like growth factor-I-induced increase in FSH-dependent estradiol-17beta (E(2)) production by rat ovarian granulosa cells (GC) in vitro. To determine whether the leptin effect extended to another positive modulator of FSH-dependent E(2) production, the direct ovarian effects of leptin on transforming growth factor beta (TGF-beta) were investigated. Reverse transcription-polymerase chain reaction demonstrated that theca-interstitial cells (TIC) from hypophysectomized rats expressed only a nonsignal-transducing isoform (OB-Ra) of leptin receptor mRNA. Leptin had no effect on TIC androgen production. In contrast, mRNAs for OB-Ra and the signal-transducing (OB-Rb) leptin receptor isoforms were expressed in GC. When GC obtained from 26-day-old rats were cultured (48 h) with FSH and androstenedione, both estrone (E(1)) and E(2) levels increased over those in untreated controls. In the presence of FSH (0.1 IU/ml), TGF-beta (10 ng/ml) potentiated E(2) and E(1) accumulation by 2.7- and 1.45-fold, respectively. Leptin did not alter basal or FSH-stimulated E(2) and E(1) levels. However, leptin suppressed the effect of TGF-beta on FSH-dependent E(2) and E(1) production by 39% and 29%, respectively. Aromatase cytochrome P450 (P450(arom)) mRNA expression and P450(arom) activity were increased by FSH and further augmented by the addition of TGF-beta. Leptin abolished the TGF-beta effect on P450(arom) mRNA expression, and it decreased P450(arom) activity by approximately 27%. These data support the hypothesis that leptin antagonizes the stimulatory effects of TGF-beta on FSH-dependent estrogen production by a mechanism involving the leptin-induced attenuation of P450(arom) activity and mRNA expression in GC.

5alpha-reductase activity in women with polycystic ovary syndrome.

The recent demonstration of high concentrations of 5alpha-androstane-3,17-dione in the follicular fluid of polycystic ovaries suggests a potential role for 5alpha-reduced androgens in the etiology of polycystic ovary syndrome (PCOS). The purpose of the present study was to determine whether there is increased 5alpha-reductase activity or messenger ribonucleic acid (mRNA) expression in polycystic ovaries. 5alpha-Reductase 1 and 5alpha-reductase 2 mRNAs were measured in thecal (TC) and granulosa (GC) cells from individual follicles of 18 women with PCOS and 26 regularly cycling control women. Both 5alpha-reductase 1 and 2 mRNA expression was higher in GC than in TC, and 5alpha-reductase 2 mRNA levels were approximately 3-fold higher than 5alpha-reductase 1 mRNA. 5alpha-Reductase 1 and 2 mRNA expression were similar in GC from PCOS and control women, but 5alpha-reductase mRNA was decreased in TC from PCOS follicles. In control women, 5alpha-reductase 2 mRNA was highest in GC from 3- to 5-mm follicles and decreased to undetectable levels in GC from 7-mm follicles. A similar pattern of expression was present in GC from PCOS follicles, but detectable levels of 5alpha-reductase 2 mRNA were present in GC from 7-mm follicles. 5alpha-Reductase activity was measured in whole follicles by measuring the conversion of radiolabeled testosterone to dihydrotestosterone. Kinetic analysis of total 5alpha-reductase activity at physiological pH revealed a Km of 1.46 micromol/L and a maximal velocity of 0.31 nmol/min x mg protein, indicating predominantly type 1 activity. The total 5alpha-reductase activity was approximately 4-fold higher in PCOS follicles than in control follicles. These data demonstrate elevated 5alpha-reductase activity in polycystic ovaries and support the hypothesis that 5alpha-reduced androgens may play a role in the pathogenesis of PCOS.

Leptin regulation of aromatase activity in adipose stromal cells from regularly cycling women.

OBJECTIVES AND DESIGN: Leptin, a product of adipocytes, is a cytokine with multiple effects on the reproductive axis. Leptin causes the activation of STAT proteins within target cells. The aromatase gene promoter in adipose stromal cells contains a functional STAT binding region, leading to the hypothesis that leptin may regulate aromatase activity in fat tissue. To test this hypothesis, adipose stromal cells were isolated from subcutaneous abdominal fat or breast fat then placed into tissue culture. MATERIALS AND METHODS: The cells were treated for three days with increasing concentrations of recombinant human leptin. Aromatase activity in the stromal cells was measured by the release of 3H2O from radiolabeled androstenedione precursor. RESULTS: Basal aromatase activity varied markedly between, but there were no differences between abdominal fat and breast fat. Leptin concentrations in the physiological range of normal weight or thin women (10 ng/ml) had no effect on aromatase activity. In 2 of 8 abdominal fat cultures and 1 of 2 breast fat cultures, a high obese concentration of leptin (100 ng/ml) stimulated a significant increase in aromatase activity. In the remaining subjects there was no effect of leptin, even at high concentrations. CONCLUSIONS: These data demonstrate that in approximately 30 percent of our subject population leptin was able to stimulate aromatase activity in adipose stromal cells at high concentrations. The elevated levels of aromatase activity may contribute to increase circulating estrogen levels in certain obese women and suggest that elevated leptin concentrations in obese women may cause locally elevated estrogen concentrations in the breast and thereby promote tumor formation.

Leptin antagonizes the insulin-like growth factor-I augmentation of steroidogenesis in granulosa and theca cells of the human ovary.

There is increasing evidence that leptin is a physiological link between obesity and infertility. Although leptin receptors have been demonstrated in human ovaries, there is no information regarding the effects of leptin on cells from developing ovarian follicles. To test the direct effects of leptin on human ovarian cells, granulosa cells (GC) and theca cells were isolated from the ovaries of regularly cycling women. Serum was obtained at the time of surgery, and follicular fluid was aspirated from the follicles before isolation of the ovarian cells. Leptin concentrations were similar in follicular fluid and serum. RT-PCR analysis demonstrated that the long, signaling form of the leptin receptor was expressed in both theca and GC. In cultured GC, leptin had no effect on estradiol production, alone or in the presence of FSH, but caused a concentration-related inhibition of the insulin-like growth factor I (IGF-I) augmentation of FSH-stimulated estradiol production. The effect of leptin was specific, because there was no effect on progesterone production. In cultured theca cells, leptin did not alter androstenedione production, alone or in the presence of LH. Leptin caused a concentration-related inhibition of the IGF-I augmentation of LH-stimulated androstenedione production. These data demonstrate that leptin can directly inhibit IGF-I action in ovarian theca and GC at concentrations commonly present in obese women.

Intact leptin receptor is selectively expressed in human fetal pituitary and pituitary adenomas and signals human fetal pituitary growth hormone secretion.

Leptin, a circulating hormone secreted by adipocytes, communicates peripheral nutritional status to hypothalamic centers affecting satiety, energy expenditure, and body weight. The intact leptin receptor (OB-R), a single membrane-spanning peptide containing an approximately 300-amino acid intracellular domain, is highly expressed in the hypothalamus, whereas shorter OB-R isoforms with truncated cytoplasmic regions resulting from alternative splicing have also been identified. We studied expression of OB-R isoforms in human fetal pituitaries, adult anterior pituitaries, and human pituitary adenomas. Using RT-PCR, messenger ribonucleic acid expression of the OB-R intact isoform was detected in fetal anterior pituitary tissues, but not in adult anterior pituitary glands, whereas both fetal and adult tissues expressed the short forms. Messenger ribonucleic acid of both intact and short OB-R isoforms were expressed in 4 of 5 GH-secreting, all 9 PRL-secreting, and 26 of 29 nonfunctioning pituitary adenomas. Recombinant human leptin (3-6 nmol/L) specifically stimulated GH secretion from primary human fetal pituitary cultures by 40-90% (P < 0.05) without altering fetal ACTH, PRL, or gonadotropin secretion. Thus, the intact OB-R is selectively expressed in human fetal and adult pituitary tumor tissues, but not in normal adult pituitary. Leptin specifically stimulates GH release from normal fetal somatotrophs, substantiating the functionality of its intact receptor in the fetal pituitary. Thus, pituitary adenomas appear to revert to a fetal phenotype of leptin receptor expression.

Inhibin A, inhibin B and activin A concentrations in follicular fluid from women with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is characterized by arrested follicle development at the early antral stage. Alterations in inhibin production by developing follicles could be involved in PCOS by suppressing follicle stimulating hormone concentrations during the follicular phase of the menstrual cycle as well as by increasing thecal androgen production. Inhibin B appears to be more important than inhibin A during the follicular phase; however, there are no data regarding the follicular fluid concentrations of inhibin B in PCOS. The purpose of this study was to compare inhibin A, inhibin B and activin A concentrations in the follicular fluid from regularly cycling women and women with PCOS. Inhibin A, inhibin B and activin A were measured in the follicular fluid of 4-7 mm follicles from PCOS ovaries and size-matched follicles from normally cycling women by specific and sensitive two-site enzyme-linked immunosorbent assays. In both control and polycystic ovaries, inhibin B was approximately 10-fold higher than activin A and more than 100-fold higher than inhibin A. There was no difference in activin A concentrations between PCOS and control follicles. In control ovaries, the inhibin B and inhibin A concentrations in dominant follicles were significantly higher than in cohort follicles. While inhibin A concentrations were lower in PCOS follicles than in normal cohort follicles, there was no difference in inhibin B concentrations between PCOS follicles and normal cohort follicles. These data are consistent with the concept that inhibin B is the physiologically most important form of inhibin during the follicular phase of the menstrual cycle and indicate that PCOS is not associated with increased inhibin B concentrations in follicular fluid.

Aromatase mRNA expression in individual follicles from polycystic ovaries.

Polycystic ovary syndrome (PCOS) is a common reproductive disorder characterized by arrested follicular development prior to selection of a dominant follicle. Dominant follicles produce large amounts of oestradiol but PCOS follicles do not. With several potential aromatase (P450AROM) inhibitors in follicular fluid, the question arises whether P450AROM is expressed in PCOS granulosa cells, but the activity is inhibited, or whether P450AROM is not expressed in PCOS. The purpose of the present study was to determine whether P450AROM mRNA expression is altered in PCOS and to correlate P450AROM mRNA expression in individual follicles with aromatase stimulatory bioactivity and oestradiol in the follicular microenvironments. P450AROM mRNA was measured in individual follicles from 16 PCOS and 48 regularly cycling control women by quantitative polymerase chain reaction (PCR) and correlated with follicular fluid oestradiol concentrations and aromatase stimulating bioactivity measured by the rat granulosa cells aromatase bioassay. Follicular fluid oestradiol was low in all control follicles <7 mm in diameter. Some follicles > or = 7 mm contained elevated oestradiol values (P < 0.01) and all had an androstenedione:oestradiol ratio of <4. Only in granulosa cells from follicles > or = 7 mm with an androstenedione:oestradiol ratio of <4 were P450AROM mRNA levels increased (P < 0.05). These same follicles also contained increased levels of aromatase stimulating bioactivity whereas follicles <7 mm or with androstenedione:oestradiol ratio of >4 contained little or no bioactivity. All PCOS follicles contained low levels of oestradiol, P450AROM mRNA and aromatase stimulating bioactivity similar to size-matched control follicles. These data indicate that P450AROM mRNA expression and oestradiol production begin in developing follicles when they reach approximately 7 mm in diameter. Oestradiol production is low in PCOS follicles because there is insufficient aromatase stimulating bioactivity to increase P450AROM mRNA expression.

Inhibin A, inhibin B and activin A in the follicular fluid of regularly cycling women.

Recent measurements of circulating inhibin A and inhibin B concentrations indicate that inhibin B may play an important role in the selection of dominant follicles. The concentrations of inhibin A, inhibin B and activin A were measured in the follicular fluids of 61 individual follicles (4.8-20 mm in diameter) from 47 regularly cycling women using specific two-site enzyme-linked immunosorbent assays. The microenvironment of each follicle was characterized by measuring follicular fluid androstenedione and oestradiol concentrations. The mean activin A concentrations were < 8 ng/ml for follicles of all sizes (4-17 mm). Inhibin A concentrations were < 1 ng/ml in follicles < 6 mm, and progressively increased to concentrations > 50 ng/ml in follicles > or = 13 mm. Follicles with androstenedione/oestradiol ratios < or = 4 had higher concentrations of inhibin A than follicles with androstenedione/oestradiol ratios > 4. Inhibin B concentrations were higher than inhibin A concentrations in all follicles, increasing from 19.2 +/- 8.3 ng/ml in 4 mm follicles to 409 +/- 9.6 ng/ml in 13 mm follicles and then declining to 275 +/- 47 ng/ml in 17 mm follicles. These results support the hypothesis that inhibin B may play a more important paracrine role in developing follicles and a greater regulatory role with respect to follicle stimulating hormone (FSH) secretion than inhibin A.

Ontogeny of steroidogenic enzyme gene expression in ovarian theca-interstitial cells in the rat: regulation by a paracrine theca-differentiating factor prior to achieving luteinizing hormone responsiveness.

The theca cells (TC) first become identifiable in preantral follicles after the granulosa cells (GC) begin to divide. It remains unknown when the TC first respond to LH and acquire the capacity to produce androgens. The signal initiating TC differentiation is also unknown since pre-theca cells do not contain LH receptors. Since the first wave of follicle development in the rat occurs postnatally, we correlated the function of dispersed ovarian cells from 4-, 5-, 6-, 7-, and 10-day-old rats with the morphological differentiation of TC. The largest follicles in ovaries from 4-day-old rats were primary follicles without associated TC. These cells were unable to produce cAMP or steroids in vitro in response to hCG. At 5 days, the first theca were associated with follicles containing 2-3 layers of GC. These cells were responsive to hCG, producing cAMP, progesterone, androstenedione, and androsterone. Responses to hCG increased progressively through 10 days of age. Cholesterol side-chain cleavage (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and 17 alpha-hydroxylase/C17-20 lyase (P450(17 alpha)) enzymes were localized exclusively to the theca interna. Messenger RNAs for LH receptor, P450scc, 3 beta-HSD, and P450(17 alpha) were expressed prior to the time the TC become responsive to LH or morphologically differentiated. To determine the source of the signal regulating TC differentiation, dispersed cells from 4-day-old rat ovaries that were unresponsive to LH were treated with preantral follicle-conditioned medium containing thecal differentiating factor (TDF) activity. The TDF activity stimulated androgen production and expression of LH receptor, P450scc, 3 beta-HSD, and P450(17 alpha) mRNAs. These data demonstrate that a paracrine signal from the preantral follicle can initiate TC differentiation prior to expression of LH receptors. TC become responsive to LH and capable of producing androgens coincident with morphological differentiation.

Hepatocyte growth factor regulates ovarian theca-interstitial cell differentiation and androgen production.

During ovarian follicle growth, precise regulation of the onset of androgen production by ovarian theca-interstitial cells (TIC) is necessary for maintaining follicle viability. Thus, temporary suppression of TIC androgen production in preantral follicles is the key to promoting follicle development. Evidence indicates that this process is coordinated via intraovarian growth factors. Hepatocyte growth factor (HGF) can induce granulosa cell (GC) proliferation and suppress follicular atresia, indicating a role for HGF in promoting follicle growth and viability. To determine whether HGF could reversibly suppress androgen production, this study investigated the effect of HGF on TIC differentiation and steroid production. Twenty-six-day-old rats were used in all studies. HGF messenger RNA (mRNA) expression in TIC and GC was determined by reverse transcription-PCR. Agarose gel electrophoresis of the PCR products yielded a single band corresponding to the 290-bp HGF product for both TIC and GC. HGF expression in cultured TIC and GC was not blocked by gonadotropins or HGF. To investigate the effects of HGF on TIC steroidogenesis, TIC were isolated from the ovaries of hypophysectomized rats. TIC (3.0 x 10(4) cells/well) were cultured with LH (0-3 ng/ml) and/or HGF (0-100 ng/ml) for 48 h, and androsterone levels were measured by RIA. HGF did not alter androsterone levels in the absence of LH; however, HGF reversibly impaired LH-dependent androsterone production by as much as 57% (IC50 = 1.5 +/- 0.01 ng/ml). LH (0.3 ng/ml) stimulated progesterone (P4) synthesis by TIC (1201 +/- 190 pg/ml) compared to that by control cells (210 +/- 30 pg/ml). HGF stimulated basal P4 production, and LH-dependent P4 synthesis was augmented 2.6-fold by HGF (ED50 = 0.3 +/- 0.01 ng/ml). The DNA content and cell viability in TIC cultures were not affected by HGF. The effect of HGF on steroidogenic enzyme gene expression in TIC was also investigated via PCR. HGF did not alter the level of basal or LH-induced P450 side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase mRNAs; however, LH-dependent P45017 alpha hydroxylase/C17,20 lyase mRNA content was reduced 4.5 fold in the presence of HGF. Thus, HGF is expressed in both TIC and GC obtained from the immature rat ovary, suggesting its presence in growing follicles. In TIC, HGF stimulated P4 synthesis, but impaired androgen production, concurrent with a down-regulatory effect on P45017 alpha hydroxylase/C17,20 lyase gene expression. Collectively, these results indicate that HGF reversibly impairs LH-stimulated androgen production in TIC. Such effects may help promote folliculogenesis.

Direct intraovarian effects of leptin: impairment of the synergistic action of insulin-like growth factor-I on follicle-stimulating hormone-dependent estradiol-17 beta production by rat ovarian granulosa cells.

The Ob gene product, leptin, is secreted by adipocytes and is required for fertility in the mouse. Leptin-deficient mice are obese and infertile, symptoms reminiscent of polycystic ovary syndrome (PCOS). Prior studies have shown that serum leptin levels are elevated in a significant sub-population of anovulatory women with PCOS, suggesting that elevated leptin levels may adversely affect ovarian function. Since leptin receptor mRNA has been detected in the ovary, this study was designed to test the hypothesis that leptin may impair granulosa cell (GC) estradiol-17 beta (E2) production by a direct mechanism. GC were isolated from the ovaries of 26-day-old Sprague-Dawley rats, and were cultured (60,000 GC/well) in 96-well plates in the presence and absence of ovine FSH (0.001-100 ng/ml) and androstenedione (0.1 microM), with and without recombinant murine leptin (0.1-100 ng/ml) for 48 h. Leptin alone had no effect on E2 production. FSH caused a dose-related increase in E2 production by GC (ED50 = 1.9 +/- 0.4 ng/ml). Addition of leptin did not alter FSH-stimulated B2 levels. Concomitant treatment with FSH and IGF-I (30 ng/ml) augmented maximal FSH-dependent E2 production five-fold. Leptin caused a dose-dependent (IC50 = 2.7 +/- 0.6 ng/ml) inhibition (30-50%) of the IGF-I increase in FSH-stimulated E2 production. The inhibitory effect of leptin was specific for E2 production since there was no effect on basal, FSH-, or FSH+ IGF-I-dependent progesterone levels. The results of this study demonstrate that leptin can directly impair the IGF-I-mediated augmentation of FSH-stimulated E2 synthesis by GC. These data raise the possibility that high leptin levels may contribute to infertility in some women with PCOS by counteracting the sensitizing effects of IGF-I in dominant follicles.

Serum immunoreactive leptin concentrations in women with polycystic ovary syndrome.

Recent data in the mouse demonstrate that leptin, a protein hormone produced by fat cells, is required for fertility. In the absence of leptin the mice become obese, diabetic and infertile. Polycystic ovary syndrome (PCOS), a common cause of infertility in women, is associated with obesity and insulin resistance. Because of the increased frequency of PCOS in obese women we tested the hypothesis that alterations in serum leptin concentrations might be associated with PCOS. Immunoreactive leptin concentrations were measured in 58 women with PCOS and 70 regularly menstruating (control) women. As has previously been shown there was a positive correlation between leptin levels and body mass index (BMI). Although the leptin levels in the majority of women with PCOS fell within the control range, 29% of PCOS women had leptin levels above the 99% prediction interval for their BMI and none had low leptin levels. There were also positive correlations of leptin levels with free testosterone and insulin sensitivity in control women. In women with PCOS, 13% and 9.5% exhibited higher than expected leptin concentrations with respect to free testosterone and insulin sensitivity, respectively. Insulin resistant PCOS women had higher leptin levels than controls. The data demonstrate that a substantial proportion of women with PCOS have leptin levels that are higher than expected for their BMI, free testosterone and insulin sensitivity. These results suggest that abnormalities in leptin signaling to the reproductive system may be involved in certain cases of PCOS.