Probing single biomolecules with atomic force microscopy

An ovary implanted into the spleen of an ovariectomized rat develops into a luteinized tumor, growing in response to gonadotrophins. Previously, it was shown that in vivo Buserelin, a gonadotrophin-releasing hormone (GnRH) analog, inhibited tumor growth. To determine if GnRH had a direct effect on tumor cells, the presence of GnRH receptors as well as the endocrine effects of buserelin were studied on tumoral tissue. GnRH receptors were present in luteoma in similar concentrations and dissociation constant (Kd) to control estrous ovaries. In vivo treatment with buserelin did not modify luteoma GnRH receptors. In organ incubations, luteoma secreted significantly higher estradiol and lower progesterone than estrous ovaries; addition of buserelin did not modify steroid secretion. The same difference in basal steroid secretion between luteoma cells and luteal cells superovulated prepubertal ovaries was observed in cell cultures. Although luteinizing-hormone (LH)-stimulated progesterone in both kinds of cells, buserelin significantly inhibited LH-stimulated progesterone only in luteoma cells. These results describe clear differences in basal steroid secretion between tumoral and normal tissue. Furthermore, they show that luteoma possess GnRH receptors similar to those in normal ovarian tissue, and that GnRH analogs have endocrine effects on these cells. Therefore, a direct effect of buserelin on luteoma cells can be postulated.     

The onset of the ovarian cycle following calving

The onset of ovarian activity post partum was investigated by measuring progesterone concentrations in milk samples, in two dairy herds consisting of 118 cows with an average milk yield of 8340 kg FCM. Samples were taken three times a week till 50 days post partum. In 17 cows (14.4%) anoestrus occurred. The daily milk yield in this group was 2.65 kg FCM higher than the average yield in the group returning to oestrus before day 50 post partum. In cows returning to oestrus within 50 days post partum the first rise in progesterone was detected on average 27.6 days after calving. In first calvers (31.4 +/- 10.2) and in multiparous cows in the winter period (26.9 +/- 9.4) the onset of ovarian activity was delayed compared to start of ovarian activity in the summer period. In the first cycle only 28% of the cows had a normal luteal phase (12-17 days), 36% of the cows had a shortened luteal phase (less than 6 days), and 24% of the cows had a short luteal phase (6-11 days). In 12% of the cows the luteal phase was longer than 17 days. In the second cycle 56% of the cows had a normal luteal phase while 17% had a shortened luteal phase, and 17% had a short luteal phase. Pregnancy rates after first insemination in cows with a short dioestrus (10-25 days) were higher than in cows with a prolonged dioestrus (26-50 days). On the basis of these result it might be expected that postponing the first insemination until the second or even the third cycle in high-yielding cows will have only a marginal effect on the number of open days and a large effect on the number of inseminations per pregnancy.     

Interleukin (IL)-1beta increases glucose uptake and induces glycolysis in aerobically cultured rat ovarian cells: evidence that IL-1beta may mediate the gonadotropin-induced midcycle metabolic shift

This communication explores the possibility that interleukin (IL)-1beta, a putative intermediary in the ovulatory process, may take part in the gonadotropin-driven midcycle diversion of ovarian carbohydrate metabolism toward glycolysis. We examined the effect of treatment with IL-1beta on glucose metabolism in aerobically cultured whole ovarian dispersates from immature rats. Treatment with IL-1beta increased cellular glucose consumption/uptake, stimulated extracellular lactate accumulation and media acidification, and decreased extracellular pyruvate accumulation in a receptor-mediated, time-, dose- and cell density-dependent manner. Endogenous IL-1beta-like bioactivity was shown to mediate the ability of gonadotropins to exert these same metabolic effects. The IL-1beta effect was also (1) apparent over a broad range of glucose concentrations, inclusive of the putative physiological window; (2) relatively specific, because tumor necrosis factor-alpha and insulin were inactive; (3) contingent upon cell-cell cooperation (4) and reliant on de novo protein synthesis. Comparison of the molar ratios of lactate accumulation to glucose consumption in IL-1beta-replete vs. IL-1beta-deplete cultures suggests that IL-beta promotes the conversion of all available glucose to lactate but that other substrates for lactate production may also exist. However, no lactate was generated by cells grown under glucose-free conditions. Taken together, our data suggest that IL-1beta may act as a metabolic hormone in the ovary. Subject to the limitations of the in vitro paradigm, our data also suggest that IL-1beta may mediate the gonadotropin-associated midcycle shift in ovarian carbohydrate metabolism. By converting the somatic ovarian cells into a glucose-consuming glycolytic machinery, IL-1beta may establish glycolysis as the main energy source of the relatively hypoxic preovulatory follicle and the resultant cumulus-oocyte complex. The consequent oxygen sparing may conserve the limited supply of oxygen needed for vital biosynthetic processes such as steroidogenesis. This adaptational response may also provide the glycolytically incompetent oocyte with the obligatory tricarboxylic cycle precursors it depends on to meet the increased energy demands imposed upon it by the resumption of meiosis.     

Cellular studies of mammary tissue from cows hormonally induced into lactation: lactose and fatty acid synthesis

Temporal changes in ability of mammary gland to synthesize lactose and fatty acids were identified during the treatment of cows hormonally induced to lactate, and animal differences were compared to subsequent milk production. Hormonal treatment involved 17 beta-estradiol + progesterone on days 1 to 7 and dexamethasone on days 17 to 19. Mammary tissue obtained by biopsy on days 0, 8, 16, and 26 of treatment was examined for biosynthetic capacity by tissue slice incubations. In terms of peak daily milk yield, one cow was very successful (greater than 30 kg), two were intermediate (9 to 10 kg), and one cow was unsuccessful (less than 3 kg). Differences between cows in the capability to synthesize lactose and fatty acids were evident as early as day 8 and were further magnified by day 16. In particular, the tissue from the successful cow was undergoing lactogenesis by day 8 while this was not evident until the day 16 biopsy sample in the less successful cows. In contrast to the other cows, tissues from the unsuccessful animal regressed in its ability to synthesize lactose and fatty acids between day 16 and 26. Relative differences between animals in measurements of metabolic capacity were consistent with subsequent milk production.     

Cellular studies of mammary tissue from cows hormonally induced into lactation: histology and ultrastructure

Cytological changes were investigated in mammary parenchyma of cows hormonally induced to lactate. Four cows were induced with injections of 17 beta-estradiol plus progesterone (days 1 to 7) and dexamethasone (days 17 to 19). Tissue biopsies were obtained on days 0, 8, 16, and 26; and milking commenced on day 18 or 21 of the treatment period. Mammary tissue of cow A, the most successful animal, showed marked cellular proliferation and differentiation by day 8. By day 16, the parenchyma of cow A had undergone lactogenesis and exhibited histological and ultrastructural characteristics associated with actively secreting mammary tissue. Mammary epithelia of cow D, the least successful animal, showed minimal development by day 8 and ultrastructurally resembled nondifferentiated mammary cells. Mammary parenchyma of cow D had not undergone differentiation and was involuting by day 16. Mammary tissues of cow B and C showed intermediate responses with only slight development by day 16 and only limited areas of secretory activity by day 26. Results suggest that animals successfully induced into lactation undergo critical periods of cellular proliferation and differentiation which unsuccessful animals do not experience. Cytological differences were first evident by day 8, further magnified by day 16 of the treatment period, and subsequently expressed in terms of milk production (peak milk yield equaled 30.5, 10.0, 9.2, and 2.4 kg/day for cows A, B, C, and D).     

Efficacy of recombinant bovine somatotrophin in the treatment of fat cow syndrome

Recombinant bovine somatotrophin was used in addition to conventional therapy to treat a sample of post-calving cows in a herd suffering a high incidence of fat cow syndrome. Somatotrophin was also given to cows before they calved. There were no significant differences between the treated and untreated groups in the plasma concentrations of glucose, beta-hydroxybutyrate, albumin, total protein or calcium. Significant decreases in mean plasma urea concentration were observed in the pre-calving cows seven and 10 days after treatment with somatotrophin, and a significant decrease in mean plasma urea concentration was also recorded in post-calving cows with the fat cow syndrome. There was no evidence that somatotrophin, with its many potent actions in cows with acetonemia, exacerbated clinical ketosis. The authors' subjective clinical impression was that somatotrophin was helpful in the treatment of cows with the fat cow syndrome.     

Ovarian activity in the scimitar-horned oryx (Oryx dammah) determined by faecal steroid analysis

Ultrasonography and radioimmunoassay (RIA) of serum oestradiol-17beta, luteinizing hormone (LH) and progesterone, and faecal oestrogen and progestin was used to assess ovarian activity in the scimitar-horned oryx (Oryx dammah). Ovarian examination using ultrasonography revealed maximal follicle and corpus luteum (CL) diameters of 15 and 32 mm, respectively. Steroid hormone metabolite distribution among individual faecal pellets within the same defaecation was relatively homogeneous with coefficients of variation averaging 10.2+/-1.8% and 16.2+/-4.6% for oestrogens and progestins, respectively. Elevated faecal oestrogen concentrations were associated with large (> 10 mm) antral follicles detected by ultrasonography. Periovulatory peaks in faecal oestrogen excretion, coincident with nadirs in progestin excretion, were detected in three females. Faecal progestin excretion exhibited a similar temporal pattern to serum progesterone concentrations, with a time lag of approximately 16 h. Faecal progestin concentrations corresponded with the presence of functional CL and proved useful for monitoring luteal function, spontaneous and prostaglandin-F2alpha analogue-induced luteolysis and anovulation. In summary, faecal steroid monitoring is a practical, noninvasive method for characterising ovarian steroid excretion and has potential for facilitating the application of assisted reproductive technologies in scimitar-horned oryx.     

Metabolism and excretion of oestradiol-17beta and progesterone in the Sumatran rhinoceros (Dicerorhinus sumatrensis)

3H-labelled oestradiol-17beta and 14C-progesterone were injected i.v. into an adult female Sumatran rhinoceros (Dicerorhinus sumatrensis) and all urine and faeces collected over 4 days. Of the injected steroid, 68% of 3H-oestradiol and 89% of 14C-progesterone were recovered. Peak excretion in urine occurred on day 1 for both steroids, and for faeces on day 2 for 14C-progesterone, and between days 2 and 3 for 3H-oestradiol. Oestradiol metabolites were predominantly (nearly 70%) excreted into the urine, while progesterone metabolites were almost exclusively (> 99%) excreted into the faeces. The majority (> 70%) of urinary excreted oestrogens consisted of water-soluble (i.e., conjugated) forms, with > 90% of these being glucuronides. In contrast, > 75% of faecal oestrogen and progesterone metabolites were excreted as ether-soluble (i.e., unconjugated) forms. HPLC co-chromatography of oestrogens in hydrolysed urine indicated only one peak of radioactivity, co-eluting with authentic oestradiol-17beta, whereas two peaks of radioactivity were found after HPLC of faecal oestrogens, the major one co-eluting with oestrone and the less prominent one with oestradiol-17beta. Progesterone was excreted as numerous metabolites into the faeces. The three most abundant of these were identified using HPLC and gas chromatography mass spectrometry (GCMS) as 5beta-pregnane-3alpha,20alpha-diol, 5beta-pregnane-3alpha-ol-20-one, and a second pregnanediol, the exact structure of which could not be deduced. Measurement of urinary oestradiol-17beta and faecal immunoreactive pregnanediol and 5alpha-pregnane-3alpha-ol-20-one in daily samples enabled the first endocrine characterization of the ovarian cycle and indicated a cycle length of approximately 25 days.     

Follicular fluid hormone concentrations after ovarian stimulation using gonadotropin preparations with different FSH/LH ratios. I. Comparison of an FSH-dominant and a purified FSH preparation

OBJECTIVE: A small amount of LH is necessary for 17beta-estradiol production in the ovarian follicle. Human menopausal gonadotropin (hMG) contains equal amounts of FSH and LH activity, whereas recombinant FSH is a gonadotropin preparation without LH. The aim of the present randomized study was to investigate whether ovarian stimulation treatment with recombinant FSH or hMG resulted in different steroidal composition of follicular fluid. METHODS: Antral fluid from mature follicles was collected in in vitro fertilization cycles and concentrations of testosterone, androstenedione, estrone, estradiol, progesterone, FSH, and LH were determined. Seven patients (27 samples) were treated with hMG, 6 patients (22 samples) with recombinant FSH. RESULTS: Androgen, estrogen, progesterone, and FSH concentrations in follicular fluid tended to be lower in the group treated with recombinant FSH, but the variation was large and differences were statistically not significant. CONCLUSION: Treatment with a gonadotropin preparation containing no LH resulted in adequate androgen and estrogen levels in antral fluid of the ovarian follicle in women with normal endocrine profiles, even during pituitary suppression by a GnRH agonist. Apparently, the amount of endogenous LH was sufficient for steroid production within the follicle.     

Direct antagonism of ethanol''s effects on GABA(A) receptors by increased atmospheric pressure

We investigated hepatic blood flow, O2 exchange and metabolism in porcine endotoxic shock (Control, n = 8; Endotoxin, n = 10) with administration of hydroxyethylstarch to maintain arterial pressure (MAP)>60 mmHg. Before and 12, 18 and 24 h after starting continuous i.v. endotoxin we measured portal venous and hepatic arterial blood flow, intracapillary haemoglobin O2 saturation (Hb-O2%) of the liver surface and arterial, portal and hepatic venous lactate, pyruvate, glycerol and alanine concentrations. Glucose production rate was derived from the plasma isotope enrichment during infusion of [6,6-2H2]-glucose. Despite a sustained 50% increase in cardiac output endotoxin caused a progressive, significant fall in MAP. Liver blood flow significantly increased, but endotoxin affected neither hepatic O2 delivery and uptake nor mean intracapillary Hb-O2% and Hb-O2% frequency distributions. Endotoxin nearly doubled endogenous glucose production rate while hepatic lactate, alanine and glycerol uptake rates progressively decreased significantly. The lactate uptake rate even became negative (P<0.05 vs Control). Endotoxin caused portal and hepatic venous pH to fall significantly concomitant with significantly increased arterial, portal and hepatic venous lactate/pyruvate ratios. During endotoxic shock increased cardiac output achieved by colloid infusion maintained elevated liver blood flow and thereby macro- and microcirculatory O2 supply. Glucose production rate nearly doubled with complete dissociation of hepatic uptake of glucogenic precursors and glucose release. Despite well-preserved capillary oxygenation increased lactate/pyruvate ratios reflecting impaired cytosolic redox state suggested deranged liver energy balance, possibly due to the O2 requirements of gluconeogenesis.     

Effect of prostaglandin F2 alpha-and gonadotropin releasing hormone-induced luteinizing hormone releases on ovulation and corpus luteum function of beef cows

Luteinizing hormone (LH) concentrations were measured in suckled beef cows treated during the postpartum period with prostaglandin F2 alpha (5 mg Alfaprostol; PGF2 alpha) and then gonadotropin releasing hormone (100 micrograms Cystorelin 30 h after PGF2 alpha; GnRH). The objective was to determine if PGF2 alpha would cause a release of LH in the absence of progesterone and affect the GnRH-induced LH release and ovulation (Experiment 1). LH concentrations increased (P < 0.05) after PGF2 alpha treatment in both anestrous and cyclic cows but to a greater extent (P < 0.05) in anestrous cows. The GnRH-induced LH release and ovulation response in previously anestrous cows were greater (P < 0.05) when PGF2 alpha was administered 30 h earlier. In Experiment 2, 49 beef cows received PGF2 alpha (5 mg Alfaprostol) and GnRH (100 micrograms Cystorelin) 30 h later to determine if the profile of the preovulatory LH surge was associated with the occurrence of subnormal luteal phases in postpartum beef cows suckling calves. Cows that had normal luteal phases had a greater (P < 0.05) mean area under the GnRH-induced LH response curve and a greater (P < 0.05) mean GnRH-induced LH peak amplitude than cows that had subnormal luteal phases. In summary, results suggest that PGF2 alpha may exert a fertility effect by causing a LH release independent of progesterone withdrawal; administration of PGF2 alpha 30 h before GnRH elevated the GnRH-induced LH release and ovulation response. In addition, cows with subnormal luteal phases had GnRH-induced LH surges of less area and peak amplitude than cows with normal luteal phases.     

Effect of summer heat stress on serum luteinizing hormone and progresterone values in Holstein-Friesian cows in Arizona

Serum-luteinizing hormone and progesterone values were measured in lactating and nonlactating Holstein-Friesian cows to evaluate their relationship with reduced fertility observed during hot summer months in Arizona. The stress of hot weather had no effect on frequency of preovulatory increase of luteinizing hormone nor on the interval between the preovulatory increase and ovulation in either lactating or nonlactating cows. Progesterone values were significantly (P less than 0.05) increased in lactating cows which became hyperthermic during hot weather, yet progesterone values did not change in nonlactating cows which had remained homeothermic. A similar relationship was observed between fertility and serum progesterone values, and between fertility and degree of thermal stress caused by hot weather. Decreased fertility and increased serum progesterone values were associated with increased environmental temperature and hyperthermia.     

Involvement of ovarian steroids in the opioid-mediated reduction of insulin secretion in hyperinsulinemic patients with polycystic ovary syndrome

To evaluate the possible involvement of ovarian steroids on the opioid-mediated disorders of insulin in patients affected by polycystic ovary syndrome (PCOS), we studied 40 PCOS women. All patients underwent an oral glucose tolerance test (OGTT; 75 g) and basal hormone assay; based on the insulin response to OGTT, 26 women were classified as hyperinsulinemic and continued the study protocol. Patients were randomly divided into three groups characterized by different treatments: group A (nine patients) was treated with GnRH analog (one ampule every 28 days for 2 months), group B (eight patients) was treated with naltrexone (an oral opioid antagonist, 50 mg/day, orally) for 8 weeks, and group C (nine patients) was treated with GnRH analog plus naltrexone for 2 months. After continuation of treatment, all patients repeated the basal study in a second hospitalization. Naltrexone treatment significantly reduced the insulin response to OGTT, whereas GnRH analogue administration did not significantly change the insulin secretion after the glucose load. The GnRH analog/ naltrexone cotreatment was not able to influence the insulin secretory pattern; in fact, the insulin area under the curve was superimposable before and after therapy. These data could lead to the hypothesis that the opioidergic regulation of insulin secretion requires a normal steroidogenic pattern, thus suggesting that ovarian steroids modulate opioid activity also at peripheric districts.     

Estrogen, the ovary, and neutotransmitters: factors associated with aging

Our studies in the C57BL/6J mouse have been designed to examine the interactions of aging and the ovary, and their mutual effects on neuroendocrine function. In the pituitary, ovarian status and not age determines responsiveness to gonadotropin hormone releasing hormone (GnRH), but estrogen (E2) is an important mediator in CNS changes, and removal of the ovary (OVX) is deleterious to the neuroendocrine hypothalamus. OVX for just six days in young animals results in synaptic loss between noradrenergic terminals and gonadotropin hormone releasing hormone (GnRH) neurons. Long-term OVX, hypothesized to protect against neuroendocrine aging, fails to guard against any studied age-related changes. Some age-related changes occur as early as midlife. Although neuron number remains constant at middle age, opiatergic neurons undergo significant functional changes by producing opiate antagonist peptides. This change appears to be caused by alterations in the prohormone convertases, which cleave propeptide to peptide. Altered peptides may trigger the loss of reproductive capacity. The midlife shift in opiate peptide production is a component of natural developmental processes that begin in the neonate and continue through old age. In the cholinergic system, E2 mediates numbers of cholinergic receptors, cholinergic neurons, and cholinergic-modulated memory systems in both young and old animals. Regardless of age, ovarian steroids, if present at physiologic levels, are beneficial to the neuroendocrine CNS, and long-term deprivation from ovarian-produced factors is deleterious in the systems we have examined. Our studies have shown that deprivation from ovarian steroid hormones in the female appears to be a major factor in the health of the CNS and in events associated with aging.     

Interaction of estradiol, progesterone and corticosterone on uterine connective tissue degrading enzymes

The impact of ovarian hormones and corticosterone acetate on uterine connective tissue degrading enzymes were studied in mature albino rats. Ovariectomy resulted in a significant increase in the activities of alpha- and beta-galactosidases and glucosidases in the uterus. Administration of estradiol to ovariectomized rats brought back the activities of alpha-galactosidase and alpha-glucosidase to normalcy. While beta-galactosidase and beta-glucosidase were significantly decreased. Administration of progesterone to ovariectomized rats resulted in the increase of alpha- and beta-galactosidases and glucosidases. Administration of corticosterone to ovariectomized rats produced a further increase in alpha- and beta-galactosidases and glucosidases in the uterus. Adrenalectomy in ovary intact rats produced a decrease in alpha-galactosidase however, beta-glucosidase was significantly increased. Administration of corticosterone to ovary intact rats significantly increased the activities of alpha- and beta-galactosidases, while alpha- and beta-glucosidases were found to be decreased. Ovariectomy resulted in a significant increase in the activities of cathepsin-D and cathepsin-E. Administration of estradiol to ovariectomized rats brought back the activity of cathepsin-D to normalcy, whereas cathepsin-E was significantly increased. Administration of progesterone as well as estradiol to ovariectomized rats significantly increased the levels of cathepsin-E, however, cathepsin-D was brought back to normalcy. Administration of corticosterone to ovariectomized rats as well as ovariectomy + adrenalectomy significantly increased the activity of cathepsin-D and cathepsin-E. Adrenalectomy significantly decreased the activity of cathepsin-D, while administration of corticosterone increased the cathepsin-D and cathepsin-E in the uterus. Therefore, these results suggest that estradiol is a potent ovarian steroid protecting the extra cellular matrix components. The effect of progesterone appears to modulate and act hand in hand with estradiol. Corticosterone appears to have an opposite effect to that of estradiol.     

Sex specific action of insulin on the glucose metabolism of lymphatic tissue of rats and humans (author's transl)

Glucose metabolism of lymphoid cells isolated from thymus and spleen of Wistar rats, weighing 140-150 g, was found to be sensitive to insulin. The influence of insulin on glucose uptake by isolated cells from both lymphoid tissues displayed a significant sex specificity. Insulin at 0.1-10 nmol/1 stimulated glucose uptake of cells from male rats, whereas in cells from females, inhibition of glucose uptake was observed. However, lactate production was enhanced in lymphoid cells of both sexes. Cortisol (100 nmol/1) displayed a significant anti-insulin action on glucose uptake and lactate release by thymocytes of male rats. In contrast, in cells from females, cortisol and insulin both exhibited an inhibitory action on glucose uptake, whereas the hormones were found to antagonise lactate production. The sex specific stimualtory influence of insulin on glucose uptake by thymocytes of male rats was reversed and the hormone became inhibitory when animals were castrated. The action of insulin on glucose uptake was also shown to be age-dependent. In experiments with rats weighing either about 10 g or 40 g, sex-specific effects on glucose influx were found, that were similar to those of rats weighing 140-150 g. However, stimulation of glucose uptake was found with thymocytes from rats of both sexes weighing about 80 g. Comparable results were obtained with isolated thymocytes from immature humans (2 months-10 years old). Incubation of thymocytes from males with insulin (10 nmol/1) stimulated glucose uptake and lactate release, whereas insulin caused an inhibition of glucose uptake and an enhancement of lactate production in thymocytes from female. Age dependence and sex specificity of insulin action on glucose metabolism in lymphoid cells and tissues may explain the contradictory results reported by other authors.     

The insulin resistance in women with hyperandrogenism is partially reversed by antiandrogen treatment: evidence that androgens impair insulin action in women

To assess whether androgen excess per se might impair insulin action, insulin sensitivity was measured by a two-step (20 and 80 mU/m2.min) hyperinsulinemic euglycemic clamp combined with indirect calorimetry and tracer glucose infusion in 43 women (13 obese and 30 nonobese) with normal glucose tolerance and clinical evidence of increased androgen action (hirsutism and/or polycystic ovary syndrome) as well as 12 age- and body mass index-matched healthy controls. Hyperandrogenic women were studied basally and after 3-4 months of antiandrogen treatment with 3 different drugs: spironolactone (n = 23), flutamide (n = 10), or the GnRH agonist buserelin (n = 10). Six women given spironolactone were also reexamined after 1 yr of therapy. At baseline, insulin-mediated glucose uptake was lower in hyperandrogenic women than in controls (by ANOVA, F = 14.3; P < 0.001). Insulin resistance was observed in both ovarian and nonovarian hyperandrogenism, as distinguished by acute GnRH agonist testing. After antiandrogen therapy, insulin action on glucose metabolism significantly increased for both the patients as a whole (F = 7.4; P < 0.01) and each treatment group separately. However, insulin action remained lower than in controls and showed no further improvement in patients reevaluated after I yr of treatment. Increases in both oxidative and nonoxidative glucose metabolism accounted for the improvement in substrate disposal induced by antiandrogen drugs. The increase in the effectiveness of insulin was greater in the lean subjects, whereas the change was small and not statistically significant in the obese women. Response to treatment was more pronounced in women with nonovarian hyperandrogenism, particularly at the low insulin infusion rate. Endogenous glucose production in hyperandrogenic patients was similar to that in healthy women and was unaffected by therapy. In conclusion, antiandrogen treatment partially reversed the peripheral insulin resistance associated with hyperandrogenism regardless of which antiandrogen was used. These data strongly suggest that in women, androgen excess per se contributes to impairment of insulin action.     

Insulin stimulation of lactate accumulation in isolated human granulosa-luteal cells: a comparison between normal and polycystic ovaries

Polycystic ovary syndrome (PCOS) is often associated with hyperinsulinaemia and peripheral insulin resistance. Whether the ovary is resistant to insulin is a matter of controversy. The aim was therefore to study the effect of insulin on lactate accumulation, an indicator of glucose metabolism, in granulosa-luteal cells from women with PCOS and from women with normal ovarian function. The cells were obtained from women undergoing clinical in-vitro fertilization-embryo transfer, either from patients with normal ovarian function and tubal or male infertility, or from women with PCOS, with or without tubal factor. The patients were down-regulated with buserelin and stimulated with urofollitrophin and human chorionic gonadotrophin (HCG). Follicle aspiration was performed under ultrasound guidance. Following oocyte recovery the granulosa-luteal cells were isolated, washed and cultured (2-3 x 10(4) viable cells/well) in serum-free Eagle's minimal essential medium for 48 h. After washing, the cells were then cultured in medium containing HCG (0.1-10 IU/ml) or insulin (0.05-0.5 microg/ml) for 24-48 h. Lactate accumulation in the media and cellular protein were analysed. Basal lactate accumulation did not differ in granulosa-luteal cells obtained from normal or PCOS ovaries, and averaged 46 and 49 nmol/g protein/24 h, respectively. A significant stimulation (40-60%) was obtained by HCG in both groups. Insulin caused a dose-dependent increase in lactate in granulosa-luteal cells obtained from normal ovaries (control: 45.5 +/- 6.3; insulin 0.5 microg/ml: 77 +/- 10 nmol/microg protein). Lactate accumulation in granulosa-luteal cells from PCOS ovaries was not altered in the presence of insulin. These results suggest that granulosa-luteal cell glucose metabolism is resistant to insulin in PCOS.     

Secretion of progesterone and 20 alpha-dihydroprogesterone during pregnancy in goats

The relationships between the secretion of progesterone and 20 alpha-dihydroprogesterone and the sites of their production in pregnant goats were investigated. The progesterone concentration in the peripheral plasma increased after the goats mated, and was high from day 10 to day 140 of pregnancy. The 20 alpha-dihydroprogesterone concentration increased gradually from day 0 to day 140 after mating. The progesterone concentration before parturition decreased rapidly on day 1 prepartum, but the 20 alpha-dihydroprogesterone concentration remained high until the day of parturition and then began to fall. The ratio of progesterone to 20 alpha-dihydroprogesterone decreased and fell to less than one on day 1 prepartum. The plasma concentrations of progesterone in ovarian vein and of 20 alpha-dihydroprogesterone in the ovarian vein, umbilical vein, and umbilical artery were higher than those in the jugular vein. The ratio of progesterone to 20 alpha-dihydroprogesterone in placental tissue was less than one. These results suggest that in pregnant goats the main production site is the ovary for progesterone and the ovary and feto-placental unit for 20 alpha-dihydroprogesterone, and 20 alpha-dihydroprogesterone may take help to regulate progesterone production within the ovary and the feto-placental unit.