Preovulatory Increase Research Articles

Oxytocin in Women's Health and Disease.

Oxytocin (OT) is a nonapeptide mainly produced in the supraoptic and paraventricular nuclei. OT in the brain and blood has extensive functions in both mental and physical activities. These functions are mediated by OT receptors (OTRs) that are distributed in a broad spectrum of tissues with dramatic sexual dimorphism. In both sexes, OT generally facilitates social cognition and behaviors, facilitates parental behavior and sexual activity and inhibits feeding and pain perception. However, there are significant differences in OT levels and distribution of OTRs in men from women. Thus, many OT functions in men are different from women, particularly in the reproduction. In men, the reproductive functions are relatively simple. In women, the reproductive functions involve menstrual cycle, pregnancy, parturition, lactation, and menopause. These functions make OT regulation of women’s health and disease a unique topic of physiological and pathological studies. In menstruation, pre-ovulatory increase in OT secretion in the hypothalamus and the ovary can promote the secretion of gonadotropin-releasing hormone and facilitate ovulation. During pregnancy, increased OT synthesis and preterm release endow OT system the ability to promote maternal behavior and lactation. In parturition, cervix expansion-elicited pulse OT secretion and uterine OT release accelerate the expelling of fetus and reduce postpartum hemorrhage. During lactation, intermittent pulsatile OT secretion is necessary for the milk-ejection reflex and maternal behavior. Disorders in OT secretion can account for maternal depression and hypogalactia. In menopause, the reduction of OT secretion accounts for many menopausal symptoms and diseases. These issues are reviewed in this work.

Subcortical structural changes along the menstrual cycle: beyond the hippocampus

Animal studies have robustly shown hormone related changes in spine density in various brain areas, specifically the hippocampus. Literature on hormone dependent gray matter volume changes in humans is however less consistent. While various areas have been reported to change along the menstrual cycle in women, many do not survive multiple-comparisons correction and only hippocampal changes have been replicated. We attribute these problems to small sample sizes and inconsistent definitions of menstrual cycle phases. In the present study a large sample of 55 women was scanned three times along their menstrual cycle in concisely defined time windows of hormonal changes. Accordingly this is the first study using a large enough sample size to assess menstrual cycle dependent changes in human brain structure with sufficient power. Results confirm a significant estradiol-dependent pre-ovulatory increase in gray matter volumes of the bilateral hippocampus, but also show a significant, progesterone-dependent increase in gray matter volumes of the right basal ganglia after ovulation. No other areas were affect by hormonal changes along the menstrual cycle. These hormone driven menstrual cycle changes in human brain structure are small, but may be the underlying cause of menstrual cycle dependent changes in cognition and emotion.

Enzyme immunoassays for water-soluble steroid metabolites in the urine and feces of Japanese macaques (Macaca fuscata) using a simple elution method.

A simple, non-alcoholic extraction method for measuring estrogen and progesterone metabolites in excreta using enzyme immunoassays (EIAs) was developed in Japanese macaques. The obtained detection limits of EIAs using estrone conjugates (E1C), pregnanediol glucuronide (PdG), and estriol glucuronide (E3G) polyclonal antibodies with cross-reactivity to urinary and fecal steroid metabolites were 6.6 pg/ml, 2.1 ng/ml and 0.35 ng/ml, respectively. These assays allowed the determination of E1C, PdG, and E3G from the excreta with good reproducibility and accuracy. Thereafter, urine and fecal samples of two menstrual cycles and six pregnancies from eight female Japanese macaques were assayed. A typical increase in urinary and fecal E1C in follicular phase and PdG in luteal phase were shown during non-conceptive menstrual cycles. Urinary E3G levels also showed a preovulatory increase; however, fecal E3G levels were very low throughout the non-conceptive menstrual cycles. Levels of E1C and PdG in the urine and feces of pregnant females were gradually increased until parturition, while fecal E3G levels were low and reached detectable levels after the mid-pregnancy period. Although the extraction rate of estrogen and progestogen metabolites by our method was lower compared to those of the previous extraction method using an alcohol-containing buffer, our method was simple, and the correlation coefficients for the relationship between two methods were found to be statistically significant. The results presented here are of great practical value for a non-invasive method of monitoring ovarian function and pregnancy in Japanese macaques.

Effect of premature serum progesterone rise on embryo transfer outcomes and the role of blastocyst culture and transfer in assisted reproductive technology cycles with premature progesterone rise

ObjectiveIn 1991, researchers reported that a modest preovulatory increase in serum progesterone levels is associated with lower pregnancy rates and higher incidence of pregnancy loss in in vitro fertilization (IVF). We wonder whether embryo transfer (ET) in assisted reproductive technology (ART) cycles in patients with premature progesterone rise (PPR) have a negative impact on the clinical pregnancy rates (CPRs) and/or live birth rates (LBRs) in our series. Consequently, will blastocyst transfer reverse the negative impact? Materials and methodsThis noninterventional, retrospective, observational tertiary center study was conducted between January 2010 and December 2012. All fresh ET cycles with serum progesterone levels measured (n = 599) on the day of hCG administration were analyzed. ResultsSera lutenizing hormone (LH), E2, and progesterone (P) were measured and analyzed. The CPRs of cycles in patients with p ≤ 1.5 ng/mL (low) versus those with p > 1.5 ng/mL (high) were 37.04% versus 41.03% [odds ratio (OR) = 1.18, 95% confidence interval (CI): 0.728–1.920; p = 0.50). The LBRs of cycles in patients with low progesterone level versus those with PPR were 30.52% versus 34.62% (OR = 1.21, 95% CI: 0.729–1.992; p = 0.47). No statistically significant association was detected. We further analyzed the outcomes according to different stages of ET and found that blastocyst (D5) ET significantly increase the LBRs as compared with cleavage stage (D2/D3) ET in the PPR group (44.44% versus 21.43%; p = 0.043). ConclusionPPR did not significantly compromise the clinical outcomes in this series. However, shifting to blastocyst transfer probably could increase the live birth in cycles with PPR.

Metabolic status, gonadotropin secretion, and ovarian function during acute nutrient restriction of beef heifers1,2

The effect of acute nutritional restriction on metabolic status, gonadotropin secretion, and ovarian function of heifers was determined in 2 experiments. In Exp. 1, 14-mo-old heifers were fed a diet supplying 1.2 × maintenance energy requirements (1.2M). After 10 d, heifers were fed 1.2M or were restricted to 0.4 × maintenance requirements (0.4M; d 0). Heifers received PGF2α (25 mg, intramuscularly) on d -10, 0, and 10 to synchronize ovulation. After 30 d, 1.2M and 0.4M heifers were realimented to 1.2 M for 100 d. Blood samples were collected every other day from d 0 to 14 then 3 times weekly thereafter. Heifers in Exp. 2 were managed as in Exp. 1 except that animals were fitted with an indwelling jugular catheter and blood samples were collected at 10-min intervals for 8 h on d 9, 10, and 11. Concentrations of progesterone in plasma were used to quantify ovarian luteal function. All 1.2M heifers ovulated, whereas only 30% of 0.4M heifers ovulated in Exp. 1. Concentrations of NEFA were greater and concentrations of thyroxine and IGF-I were less (P < 0.05) in plasma of 0.4M heifers compared with 1.2M heifers. The size of dominant follicles in Exp. 1 was reduced (P < 0.05) in 0.4M compared with 1.2M heifers. Concentrations of IGF-I were increased and anovulatory heifers resumed ovarian cycles an average of 35 d after realimentation. Concentrations of insulin were greater (P < 0.05) in plasma of 1.2M compared with 0.4M heifers in Exp. 2. The frequency of LH pulses was reduced (P < 0.05) in 0.4M heifers on d 9, and FSH in plasma on d 11 was not influenced by treatment. Reduced concentrations of IGF-I in plasma of nutrient-restricted heifers were associated with the reduced size of dominant follicles and indicated a local effect of growth factors on follicles. The decreased LH pulse frequency of 0.4M heifers before luteolysis indicates that restriction of nutrients decreased LH support of follicle growth. A preovulatory increase in estradiol in plasma and an ovulatory surge of LH were not detected in nutrient restricted heifers that did not ovulate. It is concluded that restricting beef heifers to 0.4 × maintenance energy requirements reduced the availability of metabolic fuel and decreased metabolic hormones, resulting in changes within the reproductive neuroendocrine-ovarian axis that compromised the ability of the dominant follicle to secrete sufficient concentrations of estrogen to stimulate an ovulatory surge of LH.

Effect of insulin on the relationship of estrous behaviors to estradiol and LH surges in intact ewes

The objective of this study was to determine whether acute stress alters the frequency of spontaneous estrous behaviors and temporal relationships with preovulatory increases in peripheral plasma estradiol and LH. Follicular phases of intact ewes were synchronized with prostaglandin administered at progesterone pessary withdrawal (PW). Twelve ewes served as controls and 12 were acutely stressed (insulin 4 IU/kg given at 30 and 32 h after PW). Ewes being near to ram(s) 21.3 ± 1.9 h after PW was the first precopulatory behavioral sign and rams nosed the perineal region of ewes after a further 9.0 ± 2.0 h ( P < 0.01), with ewes being nudged and mounted by rams 6.8 ± 2.3 h later still ( P < 0.01). Insulin did not affect the frequency or timing (relative to PW) of each behavioral sign of estrus. However, within each animal, estradiol values were more than 2 pg/ml lower ( P < 0.05) for 6 h following insulin, and the onset of the LH surge was delayed in insulin-treated ewes compared to controls (49.5 ± 3.3 versus 38.2 ± 2.6 h; P = 0.01). Consequently, the interval between the onset of being mounted and the LH surge was longer in insulin-treated ewes compared to controls (10.4 ± 3.0 versus 2.3 ± 0.7 h; P < 0.01). Maximum LH values were also 15 ng/ml lower after insulin ( P < 0.01). Thus, acute stress did not alter the timing or frequencies of estrous behaviors but it did reduce estradiol concentrations and delayed the onset and magnitude of the LH surge.

Transvaginal Doppler sonography for measuring blood flow in the pelvis

This is a report on our first experience with transvaginal Doppler sonography. In this way we can measure the different blood flow parameters in the internal iliac, ovarian and uterine arteries during the menstrual cycle. The detection of the particular vessels and their typical waves of flow are described in detail. In 279 measurements taken from 107 patients in the stimulated cycle we could show that the systolic blood flow after induction of ovulation or before ovulation increases significantly both in the internal iliac and in the ovarian artery. This experience made in the stimulated cycle could also be confirmed in the natural cycle where a preovulatory increase of systolic blood flow in the uterine artery was also noticed. During administration of a contraceptive pill only slight uncoordinated increases of blood flow could be registered. Any possible connections between the above mentioned changes with factors depending on the blood flow or on organic resistance on one hand or on endocrine parameters on the other, and possible practical applications, are discussed.

Gonadotropin Stimulation of Ovarian Fractalkine Expression and Fractalkine Augmentation of Progesterone Biosynthesis by Luteinizing Granulosa Cells

Recent studies indicated that ovarian functions are regulated by diverse paracrine factors induced by the preovulatory increases in circulating LH. Based on DNA microarray analyses and real-time RT-PCR, we found a major increase in the transcript levels of a chemokine fractalkine after human chorionic gonadotropin (hCG) treatment during the preovulatory period in gonadotropin-primed immature mice and rats. Although CX3CR1, the seven-transmembrane receptor for fractalkine, was also found in murine ovaries, its transcripts displayed minimal changes. Using tandem RT-PCR and immunohistochemistry, fractalkine transcripts and proteins were localized in cumulus, mural granulosa, and theca cells as well as the oocytes, whereas CX3CR1 was found in the same cells except the oocyte. Real-time RT-PCR further indicated the hCG induction of fractalkine transcripts in different ovarian compartments, with the highest increases found in granulosa cells. In cultured granulosa cells, treatment with fractalkine augmented hCG stimulation of progesterone but not estradiol and cAMP biosynthesis with concomitant increases in transcript levels for key steroidogenic enzymes (steroidogenic acute regulatory protein, CYP11A, and 3beta-hydroxysteroid dehydrogenase). In cultured preovulatory follicles, treatment with fractalkine also augmented progesterone production stimulated by hCG. Furthermore, treatment with fractalkine augmented the phosphorylation of P38 MAPK in cultured granulosa cells. The present data demonstrated that increases in preovulatory LH/hCG induce the expression of fractalkine to augment the luteinization of preovulatory granulosa cells and suggest the fractalkine/CX3CR1 signaling system plays a potential paracrine/autocrine role in preovulatory follicles.

Evidence that the preovulatory rise in intrafollicular progesterone may not be required for ovulation in cattle

Despite ample evidence pointing to an obligatory involvement of progesterone in ovulation, the mechanisms responsible for the ovulation promoting effects of intrafollicular progesterone are unclear. The objectives of this study were to determine if ovulation, luteinization and the gonadotropin surge-induced regulation of select extracellular matrix-degrading enzymes and their inhibitors, and mRNAs for prostaglandin (PG) biosynthesis and metabolizing enzymes are blocked following suppression of the intrafollicular increase in progesterone. Bovine preovulatory follicles were injected with the 3 beta-hydroxysteroid dehydrogenase inhibitor trilostane or diluent and collected at 0, 12, and 24 h after GnRH induction of the preovulatory LH surge. Intrafollicular trilostane administration blocked the preovulatory increase in follicular fluid progesterone resulting in concentrations similar to those observed at time 0 post-GnRH injection. The preovulatory increase in follicular fluid PGE(2) and PGF(2alpha) was reduced in trilostane-treated follicles and accompanied by upregulation of prostaglandin dehydrogenase mRNA in the granulosal and thecal cells. However, follicle rupture was not blocked by inhibition of the preovulatory rise in intrafollicular progesterone, and normal serum progesterone concentrations were observed during subsequent luteal development. Effects of trilostane administration on preovulatory changes in mRNA abundance and protein/activity in preovulatory follicles for most regulators of extracellular matrix remodeling examined were distinct from changes previously observed following the inhibition of intrafollicular prostaglandin synthesis. Results suggest that the preovulatory increase in intrafollicular progesterone may not be obligatory for bovine follicle rupture, luteinization, or regulation of prominent matrix-degrading proteinases and their inhibitors associated with ovulation.

Cyclic changes in messenger RNAs encoding inhibin/activin subunits in the ovary of the golden hamster (Mesocricetus auratus)

To elucidate changing patterns of inhibin/activin subunit mRNAs in the ovary of the golden hamster (Mesocricetus auratus) during the oestrous cycle, inhibin/activin subunit cDNAs of this species were cloned and ribonuclease protection assay and in situ hybridization were carried out. Inhibin alpha-subunit mRNA was localized in granulosa cells of primary, secondary, tertiary and atretic follicles throughout the 4-day oestrous cycle. It was also expressed in luteal cells on days 1 (oestrus), 2 (metoestrus) and 3 (dioestrus). betaA-subunit mRNA was localized in granulosa cells of large secondary (>200 microm) and tertiary follicles throughout the oestrous cycle. betaB-subunit mRNA was confined to granulosa cells of large secondary and tertiary follicles. Both alpha- and betaA-subunit mRNAs were also found in ovarian interstitial cells and theca interna cells of tertiary and atretic follicles in the evening of day 4 (pro-oestrus). A striking increase in betaA-subunit mRNA levels was also observed during the preovulatory period. The expression pattern of betaA-subunit mRNA during the preovulatory period is unique and not found in other species. An i.v. injection of anti-luteinizing hormone-releasing hormone (LHRH) serum before the LH surge abolished the expression of alpha- and betaA-subunit mRNAs in ovarian interstitial cells and theca interna cells. The treatment also abolished the preovulatory increase in betaA-subunit mRNA. Furthermore, administration of human chorionic gonadotrophin (hCG), which followed the injection of anti-LHRH serum, restored the expression patterns of alpha- and betaA-subunit mRNAs. The present study revealed that the golden hamster showed a unique expression pattern of betaA-subunit mRNA in response to the LH surge.

Fetal programming: testosterone exposure of the female sheep during midgestation disrupts the dynamics of its adult gonadotropin secretion during the periovulatory period.

Prenatal exposure of the female sheep to excess testosterone (T) leads to hypergonadotropism, multifollicular ovaries, and progressive loss of reproductive cycles. We have determined that prenatal T treatment delays the latency of the estradiol (E2)-induced LH surge. To extend this finding into a natural physiological context, the present study was conducted to determine if the malprogrammed surge mechanism alters the reproductive cycle. Specifically, we wished to determine if prenatal T treatment 1) delays the onset of the preovulatory gonadotropin surge during the natural follicular phase rise in E2, 2) alters pulsatile LH secretion and the dynamics of the secondary FSH surge, and 3) compromises the ensuing luteal function. Females prenatally T-treated from Day 60 to Day 90 of gestation (147 days is term) and control females were studied when they were approximately 2.5 yr of age. Reproductive cycles of control and prenatally T-treated females were synchronized with PGF2alpha, and peripheral blood samples were collected every 2 h for 120 h to characterize cyclic changes in E2, LH, and FSH and then daily for 14 days to monitor changes in luteal progesterone. To assess LH pulse patterns, blood samples were also collected frequently (each 5 min for 6 h) during the follicular and luteal phases of the cycle. The results revealed that, in prenatally T-treated females, 1) the preovulatory increase in E2 was normal; 2) the latencies between the preovulatory increase in E2 and the peaks of the primary LH and FSH surges were longer, but the magnitudes similar; 3) follicular-phase LH pulse frequency was increased; 4) the interval between the primary and secondary FSH surges was reduced but there was a tendency for an increase in duration of the secondary FSH surge; but 5) luteal progesterone patterns were in general unaltered. Thus, exposure of the female to excess T before birth produces perturbances and maltiming in periovulatory gonadotropin secretory dynamics, but these do not produce apparent defects in cycle regularity or luteal function. To reveal the pathologies that lead to the eventual subfertility arising from excess T exposure during midgestation, studies at older ages must be conducted to assess if there is progressive disruption of neuroendocrine and ovarian function.

Bayesian Modeling of Markers of Day-Specific Fertility

Cervical mucus hydration increases during the fertile interval before ovulation. Because sperm can only penetrate mucus having a high water content, cervical secretions provide a reliable marker of the fertile days of the menstrual cycle. This article develops a Bayesian approach for modeling of daily observations of cervical mucus and applies the approach to assess heterogeneity among women and cycles from a given woman with respect to the increase in mucus hydration during the fertile interval. The proposed model relates the mucus observations to an underlying normal mucus hydration score, which varies relative to a peak hydration day. Uncertainty in the timing of the peak is accounted for, and a novel weighted mixture model is used to characterize heterogeneity in distinct features of the underlying mean function. Prior information on the mucus hydration trajectory is incorporated, and a Markov chain Monte Carlo approach is developed. Based on data from a study of daily fecundability, there appears to be substantial heterogeneity among women in detected preovulatory increases in mucus hydration, but only minimal differences among cycles from a given woman.

Mechanisms for Ovarian Cycle Disruption by Immune/inflammatory Stress

This review summarizes highlights of our experiments investigating mechanisms, mediators and sites by which endotoxin disrupts reproductive neuroendocrine activity and interferes with the estrous cycle of sheep. Endotoxin, or lipopolysaccharide (LPS), is a commonly used model for immune and inflammatory stress. When administered to ovary-intact ewes, endotoxin interrupts the follicular phase of the cycle by interfering with several steps in the preovulatory chain of endocrine events. One such step is the development of high frequency LH pulses, which provide an essential stimulus for the preovulatory increase in estradiol secretion from the ovarian follicle. Follow-up experiments in ovariectomized ewes demonstrate that endotoxin inhibits pulsatile LH secretion at both the hypothalamic and pituitary levels, suppressing pulsatile GnRH secretion and reducing pituitary responsiveness to GnRH. This disruption of GnRH and LH pulsatility is mediated by pathways that include the synthesis of prostaglandins and cortisol, both of which are increased by endotoxin. It is postulated that a prostaglandin-mediated pathway disrupts the cycle during immune and inflammatory stress, whereas a separate cortisol-mediated pathway reinforces this disruption and also participates more generally in suppressing cyclicity during other stressful situations that activate the hypothalamo-pituitary-adrenal axis.

Progesterone on an oestrogen background enhances prolactin-induced apoptosis in regressing corpora lutea in the cyclic rat: possible involvement of luteal endothelial cell progesterone receptors.

Preovulatory surges of both prolactin (PRL) and progesterone have been suggested to be necessary for the induction of apoptosis in the regressing corpus luteum of the cyclic rat. The aim of these experiments was to study whether the administration of PRL and/or progesterone on the morning of pro-oestrus reproduces the regressive changes that happen in the cyclic corpus luteum (CL) during the transition from pro-oestrus to oestrus, and to analyse the temporal relationships between two characteristic features of structural luteolysis (luteal cell apoptosis and accumulation of macrophages). Cyclic rats (treated at 0900 h with an LHRH antagonist to block LH secretion) were injected at 1000 h with PRL and progesterone and killed at 0, 30, 60, 90 and 180 min after treatment. The number of apoptotic cells increased progressively from 60 min after treatment onward in hormone-treated rats, whereas the number of macrophages did not change throughout the period of time considered. Rats injected with PRL plus progesterone showed significantly greater numbers of apoptotic cells than those injected with PRL alone. The luteolytic effects of progesterone were in keeping with the presence of luteal endothelial cells showing progesterone receptor (PR) immunoreactivity in pro-oestrus. Treatment of rats during dioestrus and pro-oestrus with the specific antioestrogens LY117018 and RU58668 decreased the luteolytic effects of PRL and progesterone and the number of luteal endothelial cells immunostained for PR. These results strongly suggest that the preovulatory PRL surge and the preovulatory increase in progesterone together trigger structural regression of the corpus luteum. This seems to be dependent on oestrogen-driven cyclic changes in PRs in luteal endothelial cells.

The rat intraovarian interleukin (IL)-1 system: cellular localization, cyclic variation and hormonal regulation of IL-1β and of the type I and type II IL-1 receptors

An increasing body of evidence supports the possibility that intraovarian interleukin (IL)-1 plays an intermediary role in the periovulatory cascade. To gain further insight into the intraovarian IL-1 hypothesis, we studied the cellular localization cyclic variation and hormonal regulation of IL-1β, as well as of the type I and type II IL-1 receptors (IL-1R) in immature rats. In situ hybridization localized IL-1β and type I IL-1R transcripts to the granulosa cell compartment, the innermost layers of the theca interna and to the oocyte of the untreated immature ovary. Molecular probing of whole ovarian material in the course of a simulated estrous cycle revealed a progressive preovulatory increase in IL-1β and type I IL-1R transcripts to an in vivo peak at the time of ovulation (3.0- and 2.5-fold increases over untreated controls; P<0.05). Comparable efforts to localize and probe for type II IL-1R transcripts failed to elicit a detectable signal. The basal in vitro expression pattern of IL-1β and type II IL-1R transcripts by whole ovarian dispersates revealed an early (4 h) spontaneous increase to a peak (2.1- and 5.8-fold increases over time 0; P<0.05) followed by a gradual decline to a 48 h nadir. Treatment of whole ovarian dispersates with the IL-1 receptor antagonist (IL-1RA) or with IL-1β failed to alter the initial (4 h) burst of IL-1β or of type II IL-1R expression thereby suggesting IL-1-independence. Treatment with hCG proved equally ineffective. However, longer-term treatment of whole ovarian dispersates with IL-1β produced a significant secondary increase (5.9-fold over time 0; P<0.05) in IL-1β (but not type II IL-1R) transcripts by 48 h. This IL-1 effect was completely blocked by co-treatment with IL-1RA thereby suggesting mediation via a specific IL-1 receptor. Qualitatively comparable but quantitatively reduced results obtained for isolated granulosa cells. The basal in vitro expression pattern of type I IL-1R transcripts by whole ovarian dispersates revealed a progressive spontaneous increase (3.1-fold increase overall) over the 48 h culture. Treatment with IL-1β produced a significant ( P<0.05) increase (5-fold) in type I IL-1R transcripts by 48 h, an effect which was completely blocked by co-treatment with IL-1RA. Taken together, these observations: (1) localize IL-1β and its type I receptor to granulosa cells, the innermost layers of the theca interna and to the oocyte; (2) confirm their periovulatory in vivo expression pattern; (3) document their expression by untreated cultured whole ovarian dispersates; and (4) demonstrate their in vitro responsiveness to receptor-mediated/IL-1-driven autocrine amplification. The type II IL-1R was undetectable in vivo, its in vitro expression pattern proving IL-1- and hCG-independent. The periovulatory expression pattern of IL-1β and its receptor (type I) is compatible with the notion that the intraovarian IL-1 system may play an intermediary role in the ovulatory process.

Nutritionally induced anovulation in beef heifers: ovarian and endocrine function preceding cessation of ovulation.

Angus x Hereford heifers were used to determine endocrine and ovarian function preceding nutritionally induced anovulation. Six heifers were fed to maintain body condition score (M), and 12 heifers were fed a restricted diet (R) until they became anovulatory. Starting on d 13 of an estrous cycle, heifers were given PGF2alpha every 16 d thereafter to synchronize and maintain 16 d estrous cycles. Ovarian structures of M and R heifers were monitored by ultrasonography daily from d 8 to ovulation (d 1 of the subsequent cycle) until R heifers became anovulatory. Concentrations of LH and FSH were quantified in serum samples collected every 10 min for 8 h on d 2 and 15 (48 h after PGF2alpha), and estradiol and IGF-I were quantified in daily plasma samples from d 8 to 16 during the last ovulatory cycle (Cycle -2) and the subsequent anovulatory cycle (Cycle -1). During the last two cycles before anovulation, M heifers had 50% larger (P < .0001) ovulatory follicles than R heifers and 61% greater (P < .0001) growth rate of the ovulatory follicles. There was a treatment x cycle x day effect (P < .001) for concentrations of estradiol. The preovulatory increase in estradiol occurred in the R and M heifers during Cycle -2 but only in M heifers during Cycle -1. A treatment x cycle x day effect (P < .05) influenced LH concentrations. During Cycle -2, LH concentrations were similar for M and R heifers, but during Cycle -1, M heifers had greater LH concentrations than did R heifers. Concentrations of FSH were greater (P < .05) in R than M heifers after induced luteolysis when R heifers failed to ovulate. There was a treatment x cycle interaction (P < .05) for IGF-I concentrations, and M heifers had 4.7- and 8.6-fold greater IGF-I concentrations than did R heifers during Cycle -2 and -1, respectively. We conclude that growth rate and diameter of the ovulatory follicle, and concentrations of LH, estradiol, and IGF-I are reduced before the onset of nutritionally induced anovulation in beef heifers.

The use of a sensitive equilibrium dialysis method for the measurement of free testosterone levels in healthy, cycling women and in human immunodeficiency virus-infected women.

Measurements of total and free testosterone levels in women have lacked precision and accuracy because of limited assay sensitivity. The paucity of normative data on total and free testosterone levels in healthy women has confounded interpretation of androgen levels in women with human immunodeficiency virus (HIV) infection and other disease states. Therefore, the objectives of this study were to develop sensitive assays for the measurement of the low total and free testosterone levels in women to define the range for these hormones during the normal menstrual cycle and assess the total and free testosterone levels in HIV-infected women. By using a larger volume of serum, increasing the incubation time, and reducing the antibody concentration, the sensitivity of the total testosterone assay was increased to 0.008 nmol/L, and that of the free testosterone assay was increased to 2 pmol/L. The mean percent free testosterone was 1.0 +/- 0.1% of the total testosterone. Serum total and free testosterone levels in the follicular and luteal phases were not significantly different, but both demonstrated a modest preovulatory increase, 3 days before the LH peak. Serum total [0.50 +/- 0.32 (14.60 +/- 9.22) vs. 1.2 +/- 0.7 nmol/L (34.3 +/- 21.0 ng/dL); P < 0.0001] and free testosterone levels (5.56 +/- 2.70 (1.58 +/- 0.80) vs. 12.8 +/- 5.5 pmol/L (3.4 +/- 1.7 pg/mL); P < 0.0001) were significantly lower in HIV-infected women (n = 37) than in healthy women (n = 34). Serum total and free testosterone levels were also significantly lower in HIV-infected women who were menstruating normally. There were no significant differences in serum total and free testosterone levels between those who had lost weight and those who had not. Testosterone levels correlated inversely with plasma HIV ribonucleic acid copy number. Serum FSH, but not LH, levels were significantly higher in HIV-infected women than in controls. Using assays with sufficient sensitivity, we defined the range for total and free testosterone levels during the normal menstrual cycle. Serum total and free testosterone levels are lower in HIV-infected women and correlate inversely with plasma HIV ribonucleic acid levels. The hypothesis that androgen deficiency contributes to wasting in HIV-infected women remains to be tested.

Determination of oestrogen concentrations in bovine plasma by a recombinant oestrogen receptor-reporter gene yeast bioassay.

A recombinant cell yeast bioassay (RCBA) was applied to the generic measurement of bovine plasma oestrogen concentration. Samples were prepared by diethyl ether extraction of plasma following addition of [3H]17 beta-oestradiol as internal standard; organic and aqueous phases were separated by freezing (recovery 97.1 +/- 0.7%) and dried extract reconstituted in culture medium (recovery 31.4 +/- 4.5%). Plasma oestrogen concentrations were measured by incubation of extracts with yeast containing a stable human oestrogen receptor (hER) and a reporter construct comprising an hER response element regulating beta-galactosidase expression. The linearity of response for the analysis of spiked plasma samples using the RCBA, following corrections, is described by y = 0.8994x - 0.111 (r2 = 0.9776, P < 0.0001). Inter-assay variation for endogenous oestrogen was 11.5% for > 1 pg ml-1. Plasma oestrogen concentrations for intact (n = 5) and castrated (n = 3) males were < 0.5 pg ml-1, and 3.7 +/- 2.6 pg ml-1 for luteal phase females (n = 10). Analysis by RCBA of sequential samples from heifers during the reproductive cycle failed to detect the pre-ovulatory increase in plasma 17 beta-oestradiol as determined by radioimmunoassay (RIA) (maximal concentrations 2.09 +/- 2.1 pg ml-1 and 32.6 +/- 14.6 pg ml-1, respectively). Interestingly, when samples were hydrolysed using Helix pomatia glucuronidase the RCBA gave concentrations (29.5 +/- 8.9 pg ml-1) not significantly different to those obtained by RIA. These preliminary findings suggest that a substantial proportion of plasma oestrogen during the pre-ovulatory period may be conjugated. These data indicate the potential of the RCBA to measure biologically active and physiological levels of plasma oestrogens in cattle. One potentially valuable application of this generic oestrogen assay could be in surveillance programmes to detect illegal use of anabolic oestrogens in live-stock where the identity of the analyte may be unknown.

Changes in serum concentrations of growth hormone, insulin, insulin-like growth factor and insulin-like growth factor-binding proteins 1 and 3 and urinary growth hormone excretion during the menstrual cycle.

Few studies exist on the physiological changes in the concentrations of growth hormone (GH), insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP) within the menstrual cycle, and some controversy remains. We therefore decided to study the impact of endogenous sex steroids on the GH-IGF-IGFBP axis during the ovulatory menstrual cycle in 10 healthy women (aged 18-40 years). Blood sampling and urinary collection was performed every morning at 0800 h for 32 consecutive days. Every second day the subjects were fasted overnight before blood sampling. Follicle stimulating hormone, luteinizing hormone (LH), oestradiol, progesterone, IGF-I, IGFBP-3, sex hormone-binding globulin, dihydroepiandrosterone sulphate and GH were determined in all samples, whereas insulin and IGFBP-1 were determined in fasted samples only. Serum IGF-I concentrations showed some fluctuation during the menstrual cycle, with significantly higher values in the luteal phase compared to the proliferative phase (P < 0.001). Mean individual variation in IGF-I concentrations throughout the menstrual cycle was 13.2% (SD 4.3; range 0.1-18.3%). There were no cyclic changes in IGFBP-3 serum concentrations and no differences in IGFBP-3 concentrations between the luteal and the proliferative phases. Mean individual variation in IGFBP-3 concentrations throughout the menstrual cycle was 8.8% (SD 2.7; range 3.2-14.1). IGFBP-1 concentrations were inversely associated with insulin concentrations, and showed a significant pre-ovulatory increase that returned to baseline at the day of the LH surge. Fasting insulin concentrations showed large fluctuations throughout the menstrual cycle without any distinct cyclic pattern. No cyclic changes in urinary GH excretion during menstrual cycle were detected. We conclude that, although IGF-I concentrations are dependent on the phase of the menstrual cycle, the variation in IGF-I concentrations throughout the menstrual cycle is relatively small. Therefore, the menstrual cycle does not need to be considered when evaluating IGF-I or IGFBP-3 serum values in women suspected to have GH deficiency.

OVARIAN NITRIC OXIDE SYNTHASE (NOS) GENE EXPRESSION DURING PERIPUBERTAL DEVELOPMENT

Nitric oxide (NO) is generated from L-arginine by different isoforms of the enzyme nitric oxide synthase (NOS) and is known to be involved in mediating several biological functions, some of which are associated with reproduction. In this study, we examined the ability of the prepubertal ovary to express the inducible (i), as well as the neuronal-type constitutive (c) form of NOS and also, investigated whether either isoform undergoes changes in the ovary during peripubertal development. Results indicate that both forms of NOS were expressed in the ovary and that the iNOS mRNA transcripts were expressed without being exogenously induced. When compared with juvenile levels, iNOS, but not cNOS, mRNA increased (p<0.01) during the early proestrous phase of development. By the late proestrous phase, the levels of iNOS mRNA declined markedly (p<0.001) and remained low throughout both the first estrous and diestrous phases. Western blot analysis revealed both iNOS and cNOS protein expression in each phase of puberty with only iNOS showing a significant change during the peripubertal period. Specifically, there was an initial increase in the expression of iNOS protein during the late proestrous phase (p<0.05) which was accompanied by preovulatory increases in serum estradiol (p<0.01) and LH (p<0.001). The iNOS protein levels then dramatically increased to peak on the morning of first estrus (p<0.001), an event associated with declining (p<0.05) serum levels of estradiol. These data demonstrate developmental changes in the expression of ovarian iNOS mRNA and protein both before and after first ovulation; hence, suggesting a role for NO in the ovary during pubertal maturation and furthermore, providing compelling evidence at the gene level supporting the hypothesis that the NO/NOS system plays a physiological role in ovarian function.