Ageing in Drosophila: the role of the insulin/Igf and TOR signalling network.

A remarkable discovery of recent years is that, despite the complexity of ageing, simple genetic interventions can increase lifespan and improve health during ageing in laboratory animals. The pathways involved have often proved to sense nutrients and to match costly activities of organisms, such as growth, metabolism and reproduction, to nutrient status. For instance, the insulin/insulin-like growth factor and Target of Rapamycin signalling network has proved to play a function in ageing, from yeast to mammals, seemingly including humans. In the fruit fly Drosophila, altered activity of several components of this network can increase lifespan and improve locomotor and cardiac function during ageing. The fly brain, fat body (equivalent of mammalian liver and white adipose tissue) and the germ line are important in determination of lifespan, with considerable communication between different tissues. Cellular detoxification pathways, increased autophagy and altered protein synthesis have all been implicated in increased lifespan from reduced IIS/TOR activity, with the role of defence against oxidative stress unresolved. Reduced IIS/TOR signalling can alter or block the response of lifespan to dietary restriction. Reduced IIS can act acutely to lower death rate, implying that it may ameliorate the effects of ageing-related damage, rather than preventing it.

Mitochondrial DNA rearrangements in human oocytes and embryos.

Human mitochondrial DNA (mtDNA) rearrangements, including more than 150 deletions and insertions, accumulate with age and are responsible for certain neuromuscular diseases. Human oocytes, arrested for up to 50 years, may express certain mtDNA rearrangements possibly affecting function. Investigations have previously shown a single mtDNA rearrangement (dmtDNA(4977)) in human oocytes. Sequencing of other rearrangements and their correlation with maternal age have not been performed in human oocytes or embryos. Here we use a nested PCR strategy of long followed by short polymerase chain reaction (PCR) that amplifies two-thirds of the mitochondrial genome. mtDNA rearrangements were detected in 50.5% of the oocytes (n = 295) and 32.5% of the embryos (n = 197). This represents a significant difference in the percentage of mtDNA rearrangements between oocytes and embryos (P < 0.0001). Twenty-three novel mtDNA rearrangements with deletions, insertions and duplications were found. There was no significant age-related increase in the percentage of human oocytes or embryos that contained mtDNA rearrangements. Significant reductions in the number of oocytes containing mtDNA rearrangements occurred as oocyte development progressed from germinal vesicle to the mature metaphase II oocyte (P < 0.05). These findings are discussed as they relate to mitochondria, mtDNA, and ATP production in human oocytes and embryos.

Characterization of attenuated proestrous luteinizing hormone surges in middle-aged rats by deconvolution analysis.

Reproductive aging in female rats is associated with attenuated preovulatory LH surges. In this study, detailed analyses of the episodic characteristics of the proestrous LH surge were conducted in young and middle-aged regularly cyclic rats. On proestrus, blood samples were withdrawn at 3-min intervals for 6 h and analyzed for LH concentrations by RIA in triplicate. Deconvolution analysis of immunoreactive LH concentrations revealed that there was no difference in the detectable LH secretory burst frequency between young and middle-aged rats. However, in middle-aged rats with an attenuated LH surge on proestrus, the mass of LH secreted per burst and the maximal rate of LH secretion per burst were only one fourth (p < 0.01) of those in young and middle-aged rats with normal LH surges. Furthermore, middle-aged rats with attenuated LH surges had a 4-fold decrease (p < 0.01) in the maximal rate of LH secretion per burst compared to young and middle-aged females with normal LH surges. The apparent half-life of endogenous LH was similar among the 3 groups. The attenuated LH surges of middle-aged rats were related specifically to a decrease in LH burst amplitude with no change in pulse frequency. The orderliness of moment-to-moment LH release as quantified by the regularity statistic, approximate entropy, was comparable in the 3 groups. Our findings of a markedly decreased amount of LH released per burst and preserved orderliness of the LH release process strongly suggest that a deficient GnRH drive and/or reduced responsivity to the GnRH signal, rather than altered timing of the signal, accounts for the age-related decline in reproductive function in female rats as presaged by an attenuated proestrous LH surge in middle age.

Mitochondrial DNA deletion in human oocytes and embryos.

Mitochondrial DNA (mtDNA) deletions are present in both human oocytes and embryos. It has been found that these tissues contain a mtDNA mutation which is present in high amounts in patients with Kearns-Sayre syndrome (KSS) and progressive external ophthalmoplegia. In the present study, the frequency of this KSS deletion was investigated in human oocytes and embryos. Using a nested primer polymerase chain chian reaction (PCR) strategy, the frequency of the KSS deletion in 74 human oocytes and 137 embryos was found to be 32.8 and 8.0% respectively. Using a 'long PCR-short PCR' nested primer strategy, the frequency of the KSS deletion in 181 human oocytes and 104 embryos was found to be 47.0 and 20.2% respectively. There was no statistical correlation between the age of the patients at the time of oocyte retrieval and the presence of the deleted molecules. There was a statistical difference between the presence of the deleted molecules in oocytes versus embryos using either technique (P < 0.0001). The relevance of these findings to the accumulation of low levels of deleted mtDNA in both oocytes and embryos is discussed in this study.

Progesterone implants delay age-related declines in regular estrous cyclicity and the ovarian follicular reserve in Long-Evans rats.

The present study examined the effects of progesterone (P4) treatments on estrous cyclicity and the loss of ovarian follicles during aging. Young rats received repeated treatments with P4 or empty implants between 3.5 and 8 mo of age. At 8 mo, ovaries were obtained from some animals to determine the numbers of resting follicles, and estrous cycle patterns and hormone levels were determined from other groups of treated females. In contrast to the cyclic increases in P4, estradiol (E2), LH, and FSH in control animals, P4-implanted rats exhibited elevated serum P4 but low E2, LH, and FSH levels. After implant treatments, the follicular reserve was significantly (p < 0.05) larger in P4-treated females (2012 +/- 297 resting follicles per ovary, n = 5 rats per group) than in regularly cyclic control rats (713 +/- 226 follicles per ovary, n = 7). The effects of P4 implants on the follicular reserve were associated with a subsequently higher incidence of regular estrous cycles after P4 treatment. These results demonstrate that P4 prevents cyclic increases in E2 secretion and is associated with a conservation of the ovarian follicular reserve and the maintenance of regular estrous cycle patterns, indicating a protective effect of P4 on the age-related loss of ovarian follicles.

Characteristics of luteinizing hormone secretion in younger versus older premenopausal women.

OBJECTIVES: The objectives of this study were to document specific attributes of pulsatile luteinizing hormone secretion in middle-aged women before discernible alterations in their menstrual cycles and to compare the results to corresponding data obtained in younger women. STUDY DESIGN: After documenting normal cycle length, biphasic basal body temperatures, and normal midluteal progesterone in younger and middle-aged women during an initial cycle, daily blood samples and samples withdrawn at 10-minute intervals for 8 hours during the midfollicular phase were obtained during a subsequent cycle. RESULTS: Assessment of luteinizing hormone pulses with the pulse detection algorithm Cluster demonstrated a prolonged interpulse interval and increased pulse width in the older women. Assessment of luteinizing hormone secretory bursts and half-life with the deconvolution analysis procedure demonstrated a prolonged interburst interval and half-life in the older women. Appraisal of approximate entropy revealed greater orderliness of luteinizing hormone release in the older women. CONCLUSIONS: Middle-aged women exhibit alterations in hypothalamic-pituitary function that may account in part for age-related changes in reproductive potential.

Dehydroepiandrosterone reduces plasma plasminogen activator inhibitor type 1 and tissue plasminogen activator antigen in men.

Dehydroepiandrosterone (DHEA) may help prevent heart disease in men. To test the hypothesis that DHEA might exert its effects by enhancing endogenous fibrinolytic potential, a double-blind, placebo-controlled study was conducted that assessed the effects of DHEA administration on plasma plasminogen activator inhibitor type 1 (PAI-1) and tissue plasminogen activator (tPA) antigen. Eighteen men received 50 mg DHEA orally and 16 men received a placebo capsule thrice daily for 12 days. Serum DHEA-sulfate and plasma PAI-1 and tPA antigen were measured before and after treatment. In the DHEA group, serum DHEA-sulfate (from 7.5 +/- 1.2 micromol/L to 20.2 +/- 1.5 micromol/L (P < 0.0001), androstenedione (from 2.6 +/- 0.2 nmol/L to 4.0 +/- 0.4 nmol/L; P < 0.005) and estrone (from 172 +/- 21 pmol/L to 352 +/- 28 pmol/L; P < 0.005) increased, whereas plasma PAI-1 (from 55.4 +/- 3.8 ng/mL to 38.6 +/- 3.3 ng/mL; P < 0.0001) and tPA antigen (from 8.1 +/- 1.9 ng/mL to 5.4 +/- 1.3 ng/mL; P < 0.0005) decreased. In the placebo group, serum DHEA-sulfate declined slightly from 8.0 +/- 3.3 micromol/L to 7.3 +/- 3.4 micromol/L (P < 0.05), but no other measured steroid changed. Plasma PAI-1 and tPA antigen did not change in the placebo group. These findings suggest that DHEA administration reduces plasma PAI-1 and tPA antigen concentrations in men.

Luteinizing hormone secretion by dispersed anterior pituitary gland cells from middle-aged rats.

Several studies have shown that a certain subpopulation of middle-aged rats have an attenuated preovulatory LH surge, and that this is an early indicator of a general decline in reproductive cyclicity. The pituitary glands of middle-aged animals with attenuated LH surges have been shown to be significantly less responsive to GnRH than those from young (Y) animals. The present study was designed to investigate the relationship between the in vivo LH surge and the in vitro secretory capacity of dispersed anterior pituitary gland cells from the same animals. Individual LH-secreting cells were also studied with respect to their LH secretory capacity and proportionate numbers in the pituitary gland using reverse hemolytic plaque assay (RHPA). Two groups of animals, young (4-5 months old) and middle-aged (10-12 months old), were sampled on the afternoon of proestrus to determine the amplitudes of their preovulatory LH surges. The middle-aged animals were then classified as either normal (MAN) or attenuated (MAA) with regard to the peak levels of the surges. On the next proestrus, dispersed anterior pituitary gland cells were prepared for either perifusion or RHPA. Perifused anterior pituitary gland cells were exposed to three 2.5-min pulses of GnRH(30 nM) at 60-min intervals. In the RHPA, red blood cells were treated with protein-A to allow coating with rabbit antiovine LH-beta, and then co-incubated with anterior pituitary gland cells. The cells were then treated with one of several concentrations of GnRH (0.01, 1.0, 10, or 100 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

Coculture cells that express leukemia inhibitory factor (LIF) enhance mouse blastocyst development in vitro.

PURPOSE: Leukemia inhibitory factor is a cytokine that plays an important role in implantation and enhances mouse preimplantation embryo development in vitro. Since leukemia inhibitory factor enhances early embryo development, we tested the hypothesis that coculture cells that express leukemia inhibitory factor would enhance mouse blastocyst development in vitro. In this study, Northern analysis for leukemia inhibitory factor was performed on total RNA extracted from Vero cells, human embryonic fibroblasts, and human placental fibroblasts. METHODS: Two-cell mouse embryos were cultured to blastocyst in Ham's F-10 with 15% human cord serum (control) or with the coculture cells. Northern analysis demonstrated expression of LIF mRNA in Vero cells and embryonic fibroblasts but not in placental fibroblasts. Development to blastocyst was significantly enhanced in two-cell embryos cultured with Vero cells (86%, 140/163) and embryonic fibroblasts (83%, 118/142) when compared to controls (71%, 67/94, P < 0.05) or placental fibroblasts (71%, 95/134), P < 0.05). CONCLUSION: These data suggest that coculture cells that express leukemia inhibitory factor may be superior to nonleukemia inhibitory factor expressing cells for early preimplantation embryo development.

Relation of attenuated proestrous luteinizing hormone surges in middle-aged female rats to in vitro pituitary gonadotropin-releasing hormone responsiveness.

Prior to the cessation of regular cyclicity, middle-aged rats display pre-ovulatory luteinizing hormone (LH) surges of reduced magnitude. The present study was designed to identify whether middle-aged female rats with attenuated proestrous LH surges have alterations in pituitary responsiveness to gonadotropin-releasing hormone (GnRH). Young (4 months old) and middle-aged (10-12 months old) regularly cycling females were catheterized and sampled on proestrus to characterize their LH surge profiles. On the next proestrus (12.00 h), pituitaries were perifused individually and exposed to three pulses of GnRH (30 nmol/1). The patterns of the proestrous LH surges revealed that 12 of 22 middle-aged rats had attenuated surges (< 7 micrograms/l) while the remaining 10 middle-aged females had surges that were similar to those of young rats. Pituitaries perifused on the next proestrus showed similar basal LH release among the middle-aged and young females. However, the LH secretory rates following the second and third administration of GnRH, as well as the overall GnRH-stimulated LH secretory rates, were significantly decreased in middle-aged females with previously attenuated LH surges as compared to those from the young proestrous rats. In contrast, middle-aged rats with normal LH surges had pituitary LH responses that were no different from those of young females. These results indicate that a decrease in pituitary LH responsiveness to GnRH is only apparent in middle-aged rats that display attenuated proestrous LH surges.

Apparent absence of negative feedback in middle-aged persistent-estrous rats following luteinizing hormone-releasing hormone agonist treatment: relation to plasma inhibin and 17 beta-estradiol.

Reproductive aging in female rats is associated with a transition from regular estrous cyclicity to an anovulatory condition described as persistent estrous (PE). This PE condition is characterized by continued follicular development with elevated circulating levels of estrogen and FSH. In an attempt to investigate further the age-related changes in neuroendocrine function of PE rats, we have developed a model through which the return of hypothalamic-pituitary and ovarian function can be assessed following the withdrawal of chronic LHRH agonist suppression. Subsequent to withdrawal of continuous (2.5 micrograms/h for 12 days) LHRH agonist [DTrp6, Pro9-NHEt]-LHRH (LHRH-AG) treatment, circulating FSH concentrations in PE rats increase and remain elevated with an apparent absence of ovarian negative feedback, and these rats fail to return to estrous cyclicity. In the present studies, estrogen administration induced significant decreases in FSH secretion in PE rats following withdrawal of LHRH-AG treatment and ovariectomy (OVX), suggesting that the negative feedback response to estrogen is maintained in PE females. However, progesterone administration 2 days later failed to elicit a positive feedback response of gonadotropin secretion in PE females prior to LHRH-AG treatment, serum inhibin and 17 beta-estradiol (E2) concentrations were similar in middle-aged PE rats and young cyclic females on proestrus, while FSH levels were significantly greater in PE rats. After withdrawal of LHRH-AG treatment, plasma FSH concentrations remained elevated in PE rats as compared to young rats despite similar increases in E2. However, increases in plasma inhibin were delayed and significantly attenuated in PE rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in the postovariectomy increases in gonadotropin secretion in middle-aged persistent-estrous rats: correlation with pituitary gonadotropin subunit gene expression.

This study compared the changes in pituitary and serum levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) at various times following ovariectomy (OVX) between young cyclic and middle-aged persistent-estrous (PE) rats and related these to the relative gene expression of the pituitary gonadotropin subunits. In intact animals, both pituitary and serum levels of LH were similar between these two age groups, while the LH beta mRNA expression was significantly (p < 0.05) greater in young rats. Following OVX in young rats, the serum LH levels markedly increased (p < 0.05) beginning on day 7 and reaching a maximum fourfold increase by day 9. In contrast, the post-OVX increases in serum LH in middle-aged females were significantly delayed. OVX significantly (p < 0.05) increased pituitary LH contents of young rats by day 5, but had no effect on LH contents in middle-aged females until day 30 post-OVX. These changes were associated with increases in LH beta mRNA expression in both young and middle-aged females, but the levels were significantly (p < 0.05) lower in middle-aged females. Both pituitary and serum levels of FSH were significantly (p < 0.05) higher in middle-aged PE than in young rats prior to OVX, while the FSH beta mRNA expression was similar in both age groups. Following OVX in young rats, serum FSH levels rapidly increased (p < 0.05) on day 3 and attained tenfold higher values by day 30.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in gonadotropin secretion in middle-aged persistent-estrous rats following LHRH agonist treatment.

During aging female rats enter an anovulatory condition with chronically elevated circulating levels of estrogen described as persistent estrus (PE). Since this endocrine state is dramatically different from the fluctuating steroid milieu of young regularly cycling females, interpretations of altered neuroendocrine responses in aged rats have been difficult to attribute to aging per se. In the present study, young cyclic and middle-aged PE rats were treated with an LHRH agonist, [DTrp6,Pro9-NHEt]-LHRH, continuously (2.5 micrograms/h) for 12 days to suppress gonadotropin and ovarian steroid secretion. On the evening of the first day of LHRH agonist (LHRH-AG) treatment both young cyclic and middle-aged PE rats showed marked (p < 0.01) increases in plasma LH and FSH followed by progressive decreases in gonadotropin secretion which reached significantly (p < 0.05) lower levels than pretreatment values by day 7. LHRH-AG treatment significantly (p < 0.01) reduced circulating 17 beta-estradiol (E2) levels in both young and PE rats while progesterone concentrations did not change. Following LHRH-AG treatment, ovariectomy (OVX) resulted in increases of plasma LH and FSH that were delayed and attenuated in PE rats as compared to those of young females. When compared to nontreated rats, 12 days of LHRH-AG treatment in both young and PE females had a minimal and transient effect on the post-OVX gonadotropin responses, suggesting that the endocrine status immediately prior to OVX does not profoundly influence the post-OVX responses in young cyclic and middle-aged PE rats. Furthermore, LHRH-AG treatment does not appear to have permanent inhibitory effects on the hypothalamic-pituitary axis.(ABSTRACT TRUNCATED AT 250 WORDS)

Attenuated proestrous luteinizing hormone surges in middle-aged rats are associated with decreased pituitary luteinizing hormone-beta messenger ribonucleic acid expression.

OBJECTIVE: This study determined whether attenuated preovulatory luteinizing hormone surges in aging rats are associated with a decrease in pituitary luteinizing hormone content or luteinizing hormone beta-messenger ribonucleic acid expression on proestrus. STUDY DESIGN: Blood samples were taken every 90 minutes from 1:30 to 10:30 PM on proestrus in young (n = 8) and middle-aged (n = 12), regularly cyclic rats for plasma luteinizing hormone determination. On the next proestrus at 12 noon, rats were killed and the pituitaries were removed for luteinizing hormone content determination by radioimmunoassay and luteinizing hormone beta-messenger ribonucleic acid expression by dot blot analysis. Results were analyzed by one-way analysis of variance. RESULTS: Seven of the middle-aged rats had attenuated luteinizing hormone surges while the remaining five females had surges similar to those of young rats. On the next proestrus, all rats had similar quantities of pituitary luteinizing hormone. However, luteinizing hormone beta-messenger ribonucleic acid expression in middle-aged rats with attenuated luteinizing hormone surges was lower (p less than 0.05) than that of middle-aged and young rats with normal surges. CONCLUSION: Decreased luteinizing hormone beta-messenger ribonucleic acid expression, but not pituitary luteinizing hormone content at 12 noon on proestrus is correlated with attenuated luteinizing hormone surges in middle-aged rats.

Comparison of transdermal to oral estradiol administration on hormonal and hepatic parameters in women with premature ovarian failure.

Five women with premature ovarian failure were studied in a randomized cross-over design to compare the biochemical effects of transdermal to oral estradiol administration when used in doses appropriate for endometrial preparation in a donor oocyte program. Patients randomly received increasing dosages of oral micronized or transdermal estradiol for 4 week, with progesterone added in the last 2 weeks, to mimic a normal hormonal cycle. Serum samples were assayed throughout treatment and compared to those from normally cycling premenopausal controls. In general, serum estradiol remained within the normal range in both treatment groups, whereas peak serum estrone levels were 10-fold higher in the orally treated group than those in the transdermally treated group. Serum levels of sex hormone-binding globulin, thyroid binding globulin, and renin substrate were all significantly elevated by day 14 in the orally treated patients and unchanged in the transdermal subjects. While plasminogen was unaltered by either route of administration, antithrombin-III levels fell with both treatments. Changes in gonadotropin levels were similar in both groups, with suppression of FSH by the end of the simulated cycles, but not into the normal premenopausal range. In conclusion, both estrogen replacement regimens provided near-normal serum estradiol profiles. However, despite the relatively high doses necessary to mimic a hormonally normal cycle, the transdermal route did not significantly alter the hepatic parameters studied, suggesting that this route of administration may have less adverse hepatic effects.

Effects of sera from patients given various anesthetics on preimplantation mouse embryo development in vitro.

This study compared the effects of sera from patients given various anesthetics on in vitro mouse preimplantation embryo development. Patients electing bilateral laparoscopic tubal sterilization were subjected to general anesthesia with nitrous oxide (N2O) that included either isofluorane (ISO/N2O) as an inhalant or fentanyl or morphine (FEN/MOR/N2O). The addition of sera collected 1 hr after anesthetic induction significantly reduced the numbers of two-cell mouse embryos that developed to blastocyst in the ISO/N2O group as compared to that of preanesthesia sera. In contrast, no detrimental effects were revealed from sera of patients given FEN/MOR/N2O. Comparison of sera from patients given ISO/N2O and FEN/MOR/N2O for laparoscopic oocyte retrieval and from patients given spinal anesthesia and/or i.v. sedation for ultrasonic retrieval also revealed a decrease in mouse embryo development in the ISO/N2O group, but no differences were seen in the other anesthetic regimens. ISO/N2O anesthesia was also associated with a significantly decreased fertilization rate of mature oocytes retrieved. However, no significant effect of ISO/N2O anesthesia on IVF pregnancy rates could be demonstrated. These studies indicate that embryo toxic effects can be detected in sera from patients given ISO/N2O and that this anesthetic may be detrimental to the success of in vitro fertilization and embryo transfer.

A direct effect of hyperinsulinemia on serum sex hormone-binding globulin levels in obese women with the polycystic ovary syndrome.

To determine whether hyperinsulinemia can directly reduce serum sex hormone-binding globulin (SHBG) levels in obese women with the polycystic ovary syndrome, six obese women with this disorder were studied. Before study, ovarian steroid production was suppressed in each woman by the administration of 7.5 mg of a long-acting GnRH agonist, leuprolide depot, im, on days -56, -28, and 0. This resulted in substantial reductions in serum concentrations of testosterone (from 1.72 +/- 0.29 nmol/L on day -56 to 0.32 +/- 0.09 nmol/L on day 0), non-SHBG-bound testosterone (from 104 +/- 16 pmol/L on day -56 to 19 +/- 5 pmol/L on day 0), androstenedione (from 7.25 +/- 1.65 nmol/L on day -56 to 2.78 +/- 0.94 nmol/L on day 0), estrone (from 371 +/- 71 pmol/L on day -56 to 156 +/- 29 pmol/L on day 0), estradiol (from 235 +/- 26 pmol/L on day -56 to 90 +/- 24 pmol/L on day 0), and progesterone (from 0.28 +/- 0.12 nmol/L on day -56 to 0.08 +/- 0.02 nmol/L on day 0). Serum SHBG levels, however, did not change (18.8 +/- 2.8 nmol/L on day -56 vs. 17.8 +/- 2.6 nmol/L on day 0). While continuing leuprolide treatment, the women were administered oral diazoxide (300 mg/day) for 10 days to suppress serum insulin levels. Diazoxide treatment resulted in suppressed insulin release during a 100-g oral glucose tolerance test (insulin area under the curve, 262 +/- 55 nmol/min.L on day 0 vs. 102 +/- 33 nmol/min.L on day 10; P less than 0.05) and deterioration of glucose tolerance. Serum testosterone, androstenedione, estrone, estradiol, and progesterone levels did not change during combined diazoxide and leuprolide treatment. In contrast, serum SHBG levels rose by 32% from 17.8 +/- 2.6 nmol/L on day 0 to 23.5 +/- 2.0 nmol/L on day 10 (P less than 0.003). Due primarily to the rise in serum SHBG levels, serum non-SHBG-bound testosterone levels fell by 43% from 19 +/- 5 pmol/L on day 0 to 11 +/- 4 pmol/L on day 10 (P = 0.05). These observations suggest that hyperinsulinemia directly reduces serum SHBG levels in obese women with the polycystic ovary syndrome independently of any effect on serum sex steroids.

Suppression of serum insulin level by diazoxide does not alter serum testosterone or sex hormone-binding globulin levels in healthy, nonobese women.

Suppression of serum insulin levels with diazoxide is associated with a decrease in serum testosterone and an increase in serum sex hormone-binding globulin in obese women with the polycystic ovary syndrome. To determine whether physiologic insulin levels play a regulatory role in the androgen status of nonobese women with normal menses, the androgen status of five nonobese normal women was assessed on two occasions: during a control study and after 10 days of oral diazoxide (100 mg, three times daily) administration. Insulin release in response to 100 gm oral glucose administration decreased from 108.0 +/- 28.2 to 49.3 +/- 5.2 nmol.min/L (p = 0.05) after diazoxide administration. However, despite suppression of insulin release, diazoxide administration did not affect serum total testosterone (diazoxide, 0.73 +/- 0.10; control, 0.69 +/- 0.11 nmol/L; p = NS) or sex hormone-binding globulin (diazoxide, 79.7 +/- 16.6; control, 70.2 +/- 12.6 nmol/L; p = NS) concentrations. These observations suggest that physiologic insulin levels in nonobese healthy women do not regulate testosterone metabolism and that diazoxide does not exert a direct or independent effect on serum testosterone or sex hormone-binding globulin levels.

Gonadotropin-releasing hormone-stimulated luteinizing hormone secretion by perifused pituitary cells from normal, gonadectomized, and testicular feminized rats.

To elucidate further the manner in which gonadal steroids influence the secretion of LH, we examined the effects of gonadectomy and the absence of functional androgen receptors on GnRH-induced LH release from dispersed rat anterior pituitary cells. Intact and gonadectomized (GNX) normal rats and androgen-resistant, testicular feminized (Tfm) animals from the King x Holtzman strain (a mutant strain that possesses defective androgen receptors) were used. Dispersed pituitary cells were perifused with Medium 199 during a 4-h equilibration period and then subjected to eight 2.5-min pulses of GnRH introduced at 30-min intervals at concentrations ranging from 0.03-100 nM. Basal LH secretion by cells from intact male and female rats was indistinguishable (P = 0.79) and was substantially lower (P less than 0.0001) than that by cells from GNX male and female animals. Basal LH secretion by cells from Tfm rats was significantly higher (P less than 0.01) than that by cells from intact animals, but lower (P less than 0.005) than that by cells from GNX animals. In response to GnRH, perifused pituitary cells from animals representing all experimental groups demonstrated concentration-dependent LH release. Pituitary cells from intact female rats showed an overall greater (P less than 0.05) response to GnRH than cells from intact male rats. Pituitary cells from Tfm rats demonstrated a greater GnRH-stimulated LH mean response than cells from intact male (P less than 0.0001) or intact female (P less than 0.0001) rats. Gonadectomy of male rats resulted in an overall GnRH-stimulated LH release similar to that exhibited by cells from gonadectomized female rats (P = 0.61). Cells from Tfm animals released more LH in response to GnRH than those from gonadectomized male and female rats (P less than 0.001). These data demonstrate that the release of LH in response to GnRH by pituitary cells from intact male rats (i.e. in the presence of androgen and functional androgen receptors) is less than that seen by cells from intact females rats. Since circulating levels of testosterone and estradiol are known to be elevated in the testicular feminized rat, the heightened GnRH-stimulated LH release by cells from such animals may reflect either the long term lack of androgenic influence and/or the combined effects of androgen resistance and elevated levels of circulating estrogens.