Preovulatory Luteinizing Hormone Surge Research Articles

In vitro regulation of local secretion and contraction of the bovine oviduct: stimulation by luteinizing hormone, endothelin-1 and prostaglandins, and inhibition by oxytocin.

The precise regulatory mechanisms of cyclic oviductal contraction in the cow are unclear. The purpose of this study was to evaluate the effect of luteinizing hormone (LH), steroids, prostaglandins (PGs) and peptides on the oviductal contraction and secretion of PGs and endothelin (ET-1). In addition, the cyclic expression of mRNA for ET-1 and its receptors (ET-R) was evaluated by reverse transcription-polymerase chain reaction (RT-PCR). In the in vitro microdialysis study, an infusion of LH alone or in combination with progesterone (P(4)), estradiol-17beta (E(2)) and/or ET-1 stimulated pronounced release of PGE(2), PGF(2alpha) and ET-1 in the oviducts from cows in the follicular and postovulatory phases. The addition of LH, LH+P(4)+E(2) and/or ET-1 to the medium increased the amplitude of oviductal contraction. However, oxytocin (OT) completely blocked the responses of oviductal secretion and contraction. In contrast, these substances did not show any effect in the oviducts from cows in the mid luteal phase. Similar expression patterns of mRNA encoding for ET-R type A and type B were found, which were highest during the postovulatory phase, lower during the luteal phase, with the lowest expression during the follicular phase. We suggest that the preovulatory LH surge, together with increasing E(2) levels from the Graafian follicle and a basal P(4) from regressing corpora lutea (CL), stimulates maximum oviductal production of PG and ET-1, resulting in oviductal contraction for a rapid transport of gametes. OT released from the newly-formed CL may block these mechanisms, and slow contractions for transport of the embryo to the uterus.

Proenkephalin and Opioid μ-Receptor mRNA Expression in Ovine Hypothalamus across the Estrous Cycle

The neural mechanism underlying the preovulatory surge of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) is thought to include reduced opioid inhibition of GnRH secretion (disinhibition). Possible mechanisms for disinhibition include reduced endogenous opioid peptide or receptor mRNA expression. Proenkephalin and opioid µ-receptor mRNA expression were measured by in situ hybridization using ³⁵S-labeled cRNA probes and computer-assisted grain counting in hypothalamic nuclei of ovary-intact ewes (n = 4) killed on day 10 of the luteal phase or 24 or 48 h into the follicular phase. In a second experiment, proenkephalin and µ-receptor mRNA expression were compared in ewes killed on day 10 of the luteal phase or during the preovulatory LH surge. Cells expressing proenkephalin mRNA were more widely distributed in ovine hypothalamus than previously described. In the periventricular nucleus, there was a significant reduction in the grain count per cell and the number of labeled cells during the follicular phase and during the LH surge, as compared to the luteal phase. In the ventromedial hypothalamus, there was a significant reduction in the grain count per cell during the follicular phase and LH surge as compared to the luteal phase, but no change in the number of labeled cells. No differences in proenkephalin mRNA expression were detected in the medial septum, diagonal band of Broca, preoptic area, anterior hypothalamic area or paraventricular nucleus across the estrous cycle. Cells expressing opioid µ-receptor mRNA were also widely distributed. No difference in µ-receptor mRNA expression was detected in the medial septum, diagonal band, medial preoptic area, anterior hypothalamus or bed nucleus of the stria terminalis across the cycle. We conclude that in sheep, proenkephalin gene expression in the periventricular nucleus and ventromedial hypothalamus is reduced during the follicular phase and at the time of the LH surge. This may be part of the neural mechanism underlying the GnRH/LH surge in this species.

Ovarian responses in gonadotrophin-releasing hormone-treated anoestrous ewes: follicular and endocrine correlates with luteal outcome.

The relationships between the development of antral follicles (growing from 3 to > or = 5 mm diameter), hormone secretion (luteinizing hormone (LH), follicle-stimlating hormone (FSH), oestradiol and progesterone), ovulation and the formation of luteal structures in response to gonadotrophin-releasing hormone (GnRH) were examined in 24 anoestrous Western White Face ewes (May-July). Ewes were monitored by transrectal ovarian ultrasonography for 34 days, commencing 15 days before the administration of GnRH. Following treatment with GnRH, 83% (20/24) of ewes ovulated. Twenty-five per cent of all ewes (6/24) subsequently had normal (full-life span) corpora lutea (CL), 37% (9/24) had inadequate CL, 17% (4/24) had both normal and inadequate CL, 17% (including three of four anovular ewes and one ewe with inadequate CL) formed luteinized follicles and only 4% (1/24) did not ovulate or produce any luteal structure. None of the variables of follicular growth (follicles reaching > or = 5 mm diameter) differed between follicles that either ovulated or failed to ovulate and there was no evident correlation between the age or stage of development of ovulatory sized antral follicles and the type of luteal structure formed, except for luteinized unovulated follicles; these follicles all emerged within 3 days of GnRH injection. Mean serum concentrations of FSH and oestradiol before treatment did not differ (P>0.05) between ewes with different ovarian responses, but peaks of fluctuations in serum concentrations of FSH in daily samples were higher in ewes that produced normal CL compared with ewes with inadequate CL. After GnRH treatment, oestradiol secretion was higher in ewes that formed luteinized unovulated follicles than in all ewes with inadequate CL (P<0.05). The peak concentration of the GnRH-induced LH surge was higher and the interval from GnRH to peak LH discharge was shorter in ewes with inadequate CL compared with ewes that had normal CL after ovulation (P<0.05). In conclusion, ovulatory sized antral follicles at a similar stage of their life span can give rise to either normal or inadequate CL and a proportion of these follicles do not ovulate in response to GnRH in seasonally anoestrous ewes. This suggests differences in ovarian follicular responsiveness to gonadotrophic stimuli. Both the amplitude of episodic elevations in daily serum FSH concentrations and the characteristics of the pre-ovulatory LH surge may be important for luteogenesis following ovulation.

Chapter 14 Neuroendocrine changes and fertility in breast-feeding women

Breast-feeding through the suckling stimulus suppresses fertility for a variable time after birth. Initially there is a period of pituitary gonadotroph recovery from the suppressive effects of the high steroid levels of pregnancy, followed by a period of suppressed ovarian activity associated with limited follicle growth. During this period of breast-feeding-induced amenorrhea, the pulsatile secretion of luteinizing hormone (LH), which reflects hypothalamic gonadotropin-releasing hormone (GnRH) release, is erratic and much slower than the one pulse per hour required in the normal follicular phase of the menstrual cycle to drive follicle growth. At some time the suckling stimulus drops below a threshold resulting in a resumption of reasonably organized pulsatile LH secretion, which is associated with development of follicles and some steroid secretion. However, positive feedback of estradiol which triggers the preovulatory LH surge and ovulation appears to be blocked by continued suckling, until suckling is reduced further and positive feedback and ovulation resumes. Often while women continue to breast-feed the first few ovulations and menses are associated with inadequate corpus luteum function, which would probably not support a pregnancy. Eventually normal menstrual cycles resume when suckling declines further. The duration of amenorrhea and subsequent period of inadequate luteal function varies greatly between mother-baby combinations, and in different societies. Exactly how the suckling stimulus reduces pulsatile secretion of GnRH/LH is not clear, although clinical studies do not support a role for opioids or dopamine. The role of prolactin remains uncertain since suckling releases both prolactin and suppresses GnRH release. Regardless of the precise mechanism, it is clear that breast-feeding in women can suppress fertility for prolonged periods, and women may proceed from pregnancy through lactation to another pregnancy and lactation with no menstrual period for several years.

The nuclear receptor co-repressor nrip1 (RIP140) is essential for female fertility.

Ovulatory dysfunction is the commonest cause of female infertility. Here we show that the co-repressor nuclear-receptor-interacting protein 1 (Nrip1; encoded by the gene Nrip1) is essential for ovulation. Mice null for this protein are viable, but female mice are infertile because of complete failure of mature follicles to release the oocyte at ovulation. In contrast, luteinization proceeds normally, resulting in a phenotype closely resembling that of luteinized unruptured follicle syndrome, often associated with infertility in women. Therefore, whereas the pre-ovulatory surge of luteinizing hormone induces both ovulation and luteinization, the ability to suppress the action of nuclear receptors is essential for the coordinated control of ovarian function with the essential process of oocyte release dependent on the activity of the transcriptional co-repressor Nrip1 (RIP40).

Involvement of Prolactin-Releasing Peptide in the Preovulatory Luteinizing Hormone and Prolactin Surges in the Rat

Prolactin (PRL)-releasing peptide (PrRP) is a novel hypothalamic peptide reported as a potent and specific stimulator of PRL secretion. In this study, we examined a possible role of PrRP in the ovarian steroid-induced PRL surge in the rat, simultaneously observing the change in luteinizing hormone (LH) surge. Experiments were performed on both normally-fed and three-day-fasted rats, which were ovariectomized and primed with estradiol and progesterone. From 11:00 to 18:00 h, blood was collected every 30 min to measure LH and PRL. All the following substances were given intracerebroventricularly at 11:00 h. Compared to control serum, anti-rat PrRP31 serum caused a significant reduction of the LH and PRL surges. The antiserum also delayed the onset of PRL surge. Fasted rats were devoid of significant surges of the hormones, while 3.0, but not 0.5 nmol of rat PrRP31 given to these animals produced a significant recovery of PRL surge. Although LH surge was not reinstated, basal LH secretion was transiently stimulated by 3.0 nmol of PrRP31. These results demonstrate for the first time a significant participation of PrRP in the preovulatory LH and PRL surges in the rat. Possible indirect pathways mediating this effect of PrRP were discussed, in view of the unique anatomical distribution of PrRP in the hypothalamus.

Apoptotic and proliferative changes during induced atresia of pre-ovulatory follicles in the rat

Atresia, a degenerative process through which many follicles are removed from the growing pool, involves apoptotic changes in the follicular granulosa cells. To identify histochemical markers of early stages of atresia, an in-vivo rat model was used which allowed the study of atresia of pre-ovulatory follicles in a synchronized and chronological order. By blocking the pre-ovulatory luteinizing hormone surge with a gonadotrophin-releasing hormone (GnRH) antagonist, ovulation of the pre-ovulatory follicles is prevented, after which these follicles became atretic. The first morphological sign of atresia (pyknotic granulosa cell nuclei) was found 27 h after injection of GnRH antagonist. Since the pre-ovulatory follicles gradually become atretic in a synchronous fashion, this model provided an opportunity to study and define markers of future atresia in pre-ovulatory follicles. Atresia involves apoptosis of granulosa cells, and therefore internucleosomal DNA fragmentation was examined. Using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labelling (TUNEL) assay it was found that the first sign of internucleosomal DNA fragmentation in granulosa cells of pre-ovulatory follicles was detectable 24 h after GnRH antagonist treatment. In order to find an upstream marker of atresia, the 5-bromo-deoxyuridine (BrdU) labelling index was used as a measure of proliferation. Already at 14 h after GnRH antagonist treatment, when morphological signs of atresia were not yet present, a clear decrease in BrdU labelling index was found in the granulosa cells.

Attenuation of Luteinizing Hormone Surges in Neuropeptide Y Knockout Mice

To clarify the role of neuropeptide Y (NPY) in the regulation of the reproductive axis, these experiments evaluated the extent to which reproductive hormone secretions may be compromised in the absence of NPY expression. In NPY knockout (NPY-KO) and wild-type (WT) mice, hormone secretions were analyzed under conditions of basal release, following ovariectomy (OVX), in proestrus, after estrogen treatments which induce gonadotropin surges and after injection of gonadotropin-releasing hormone (GnRH). Radioimmunoassays of serum from metestrous females revealed that basal luteinizing hormone (LH), follicular-stimulating hormone (FSH), estrogen and progesterone levels, as well as hypothalamic GnRH tissue concentrations, were not different between the two genotypes. The LH and FSH levels and GnRH tissue concentrations were likewise similar in WT and NPY-KO mice 5 and 10 days following OVX. Significant differences in LH levels were observed however when animals were exposed to pheromone stimulation (male mouse urine) to induce preovulatory LH surges. In proestrous animals, mean LH levels at 18.30–19.00 h were reduced by about 66% in NPY-KO versus WT mice (4.33 ± 1.12 ng/ml in the WT mice vs. 1.47 ± 0.42 ng/ml in the NPY-KO mice, p = 0.028). Despite diminishment of LH surges in NPY-KO mice, corpora lutea were equally abundant in the ovaries of NPY-KO and WT mice. In an additional experiment, a surge-inducing regimen of estradiol-17-β (E₂) and estradiol benzoate (E₂B) was administered to OVX animals. The LH surges in the NPY-KO animals treated in this manner were again diminished by approximately 50% compared to corresponding values in WT animals (WT mice 7.33 ± 0.97 ng/ml, NPY-KO mice 3.58 ± 0.74 ng/ml; p = 0.0063). To assess the contribution of altered pituitary responsiveness to the diminishment of LH surges, LH responses to a GnRH challenge (200 ng/kg subcutaneously) were determined; NPY-KO animals exhibited LH responses that were significantly reduced compared to values in WT mice (WT mice 4.88 ± 0.56 ng/ml, NPY-KO mice 3.00 ± 0.41 ng/ml; p = 0.013). Taken together, these observations do not support the idea that NPY plays a major role in the regulation of basal gonadotropin secretion or in mediating negative feedback actions of gonadal hormones. They demonstrate however that preovulatory NPY release is required for normal amplification of the LH surge that occurs on proestrus. Involvement of NPY in the generation of normal LH surges is partially mediated by the ability of the peptide to prime the anterior pituitary gland to GnRH stimulation.

Ovarian steroidogenic responsiveness to exogenous gonadotropin stimulation in young and middle-aged female rats.

Reproductive aging in the female rat is associated with gradual declines in LH secretion and ovarian progesterone (P) production. This study examined whether the influences of aging on P levels reflect decreased ovarian responsiveness to gonadotropin stimulation, as opposed to changes in gonadotropin release. Young and middle-aged regularly cyclic female rats received sodium pentobarbital to block endogenous proestrous luteinizing hormone (LH) surges, followed by administration of various doses of human chorionic gonadotropin (hCG). Similar treatments were performed in middle-aged acyclic persistent-estrous (PE) females. Injection of hCG resulted in equivalent plasma hCG levels in each treatment group. At the lowest hCG dose tested, a significant rise in plasma P levels was observed in middle-aged cyclic rats, but not in young cyclic or middle-aged PE females. This unexpected finding may reflect accelerated follicular development in middle-aged cyclic females, as suggested by a previous study. At the intermediate dose, young and middle-aged cyclic but not PE rats displayed significantly increased P in response to hCG. At the highest dose tested, all three groups of rats displayed increased P levels after hCG stimulation. However, P concentrations were significantly lower in middle-aged PE than regularly cyclic females. Northern and slot blot hybridization analyses revealed that ovarian mRNA levels for cytochrome P450 side-chain cleavage, the rate-limiting enzyme in P synthesis, were markedly reduced in PE rats following hCG stimulation. These findings indicate that ovarian responsiveness to gonadotropin stimulation is impaired in middle-aged PE, but not regularly cyclic rats, and suggest influences of cycle status on the biochemical and molecular mechanisms regulating ovarian steroid production. Furthermore, these findings reveal that attenuated P production in middle-aged proestrous rats is due to attenuated preovulatory LH surges, rather than decreased ovarian sensitivity to LH.

Impact of uremia on female reproductive cyclicity, ovulation, and luteinizing hormone in the rat

Impaired reproductive function accompanies chronic renal insufficiency (uremia) in both the human and experimental animal. Clinical hypogonadism occurs in both genders. The present studies were designed to investigate possible anti-ovulatory effects of uremia in the female rat, a species that produces multiple ova during the normal estrous cycle. Renal insufficiency (uremia) was induced by 5/6 nephrectomy. Two control groups comprised sham-operated animals fed ad libitum (sham) or pair-wise with the uremic animals (pair-fed). Estrous cycles were determined by cytology of vaginal lavage. We examined concomitant changes in the preovulatory luteinizing hormone (LH) surge by radioimmunoassay (RIA), immunoradiometric assay (IRMA), and bioassay. Repetitive LH measurements were made from blood samples taken by intra-atrial catheter throughout the afternoon and evening of proestrus. The following morning (estrus), ovaries were collected, and ova were enumerated per oviduct. Experimentally uremic animals manifested a threefold elevation of plasma creatinine and urea nitrogen and concomitantly a more than 50% impoverishment of ova production. Analyses of a large group of animals (N = 83) by RIA revealed uremia-associated attenuation of the preovulatory LH surge. Further measurements of the preovulatory LH surge by independent IRMA and LH bioassay (N = 26) corroborated this attenuation. Additional experiments indicated that these hormonal changes, but not changes in ovulation, might further reflect modulation of LH release by the anesthesia used in the preparative nephrectomy and catheterization surgeries. When normalized to body weight, the ovaries of uremic rats were found to weigh more than those of either the sham or pair-fed animals. The present experiments take advantage of an experimental uremic model to document a consistent decrease in the number of ova released during estrus in the uremic animal. Possible disruption of hypothalamic-pituitary-ovarian regulation is further highlighted by attenuation in the preovulatory LH surge. These results provide a basis for further studies of neuroendocrine pathophysiology in a rodent model of uremia-associated ovulatory disruption.

Circadian rhythm of the preovulatory surge of luteinizing hormone and its relationships to rhythms of body temperature and locomotor activity in turkey hens.

Simultaneous measurements of plasma LH, body temperature, and locomotor activity were made in laying turkey hens and are reported. Blood samples were remotely collected using a jugular cannula system, and body temperature and locomotor activity were remotely monitored using a radiotelemetry system in freely moving laying turkeys. Under a photoschedule of 14L:10D, the period for preovulatory surges of LH was 25.7 +/- 0.4 h while the periods for peak body temperature and onset of sustained locomotor activity were 24.9 +/- 0.4 and 25.7 +/- 0.5 h, respectively. During exposure to constant light, the periods for preovulatory surges of LH, peak body temperature, and onset of sustained locomotor activity increased to 27.9 +/- 0.9, 26.7 +/- 0.7, and 27.4 +/- 0.7 h, respectively. With the 14L:10D photoschedule, initiation of LH surges was restricted to the scotophase, but after 8 days of constant light, initiation of LH surges had dispersed throughout the 24-h subjective day and night. With constant light, the amplitude of the peak body temperature rhythm decreased, while the duration of the locomotor activity rhythm became broadened and, in some birds, disorganized. Peak body temperature and onset of locomotor activity rhythms and LH surges did not coincide, even though peak body temperature, onset of locomotor activity, and LH surges had similar periods. It is concluded that 1) the photoschedule influences the periods of the LH surge, peak body temperature, and onset of locomotor activity; and 2) a specific or direct relationship between the rhythms of LH surge, body temperature, and locomotor activity remains to be determined in laying turkey hens.

Variations in Plasma Levels of Substance P and Effects of a Specific Substance P Antagonist of the NK1 Receptor on Preovulatory LH and FSH Surges and Progesterone Secretion in the Cycling Cynomolgus Monkey

These studies investigated the role of substance P (SP) in the regulation of the hypothalamic-pituitary-ovarian axis in cynomolgus monkeys with normal menstrual cycles. Plasma concentrations of SP were determined in blood samples taken every morning in normally menstruating cynomolgus monkeys throughout the menstrual cycle. There was a significant decreasing linear trend of SP during the follicular phase (cycle day –13 to day 0) and a significant inverse relationship between SP plasma values and plasma 17β-estradiol (E₂) values from day –13 to day 0 of the adjusted cycle. Correspondingly, SP area under the curve was significantly greater during the follicular phase than the luteal phase. In a second experiment, plasma concentrations of E₂, luteinizing hormone (LH), follicle-stimulating hormone (FSH) and progesterone and length of cycles were measured after five daily intragastric administrations (10 mg/kg) of an NK₁ receptor (SP receptor) antagonist (RPR 100893; 10 mg/kg) initiated after serum E₂ concentrations had exceeded 125 pg/ml. There was a statistically significant reduction in the amplitude (41% of control) and the area under the curve (37% of control) of the preovulatory LH surge. In addition, there was a reduction of the duration of the LH surge (3 ± 0.1 days in controls vs. 2.1 ± 0.2 days in treated animals). The present results show for the first time that there are significant variations in plasma levels of SP, with a strong negative correlation with serum levels of E₂ during the follicular phase of the cynomolgus monkey, and that endogenous SP has a potentiating role in the interactive hypothalamo-anterior-pituitary mechanisms which lead to the preovulatory LH and FSH surges during the menstrual cycle in the monkey.

Neuroendocrine mechanisms for reproductive senescence in the female rat: gonadotropin-releasing hormone neurons.

Reproductive aging in female rats is characterized by profound alterations in the neuroendocrine axis. The preovulatory luteinizing hormone (LH) surge is attenuated, and preovulatory expression of the immediate early gene fos in gonadotropin-releasing hormone (GnRH) neurons is substantially reduced in middle-aged compared with young rats. We tested the hypothesis that alterations in GnRH gene expression may be correlated with the attenuation of the LH surge and may be a possible mechanism involved in neuroendocrine senescent changes. Sprague-Dawley rats ages 4 to 5 mo (young), 12-14 mo (middle-aged), or 25 to 26 mo (old) were killed at 10:00 AM or 3:00 PM on proestrus, the day of the LH surge, or diestrus I in cycling rats, and on persistent estrus or persistent diestrus in acyclic rats. RNase protection assays of GnRH mRNA and GnRH primary transcript were performed. GnRH mRNA levels increased significantly with age, whereas GnRH primary transcript levels, an index of GnRH gene transcription, decreased in old compared to young and middle-aged rats. This latter result suggests that an age-related change in GnRH mRNA levels occurs independently of a change in gene transcription, indicating a potential posttranscriptional mechanism. On proestrus, GnRH mRNA levels increased significantly from 10:00 AM to 3:00 PM in young rats. This was in contrast to proestrous middle-aged rats, in which this afternoon increase in GnRH mRNA levels was not observed. Thus, the normal afternoon increase in GnRH mRNA levels on proestrus is disrupted by middle age and may represent a substrate for the attenuation of the preovulatory GnRH/LH surge that occurs in rats of this age, prior to reproductive failure.

The reversible effects of raloxifene on luteinizing hormone levels and ovarian morphology in mice

Raloxifene is a selective estrogen receptor modulator that has estrogen agonist effects on bone and serum lipids and estrogen antagonist effects on breast and uterine tissues. This study assessed the effects of raloxifene hydrochloride (HCl) treatment on circulating luteinizing hormone (LH) levels and ovarian morphology in sexually mature, 15-week-old, female CD-1 mice. Mice were maintained on diets providing average daily doses of 0 or 233 mg/kg raloxifene for 2 weeks (Study 1) or 0, 7.9, or 236 mg/kg raloxifene for 4 weeks (Study 2). At the end of the treatment period, blood samples were collected every 2 hours for 24 h in Study 1 (5 mice per group) and at 10:00 a.m. and 10:00 p.m. in Study 2 (8 mice per group). Serum LH levels were measured by radioimmunoassay. Ovarian histomorphology was evaluated in the 10 mice per group (Study 1) and the 8 mice per group (Study 2). For the reversibility phase (Study 2), mice were fed untreated diets for 3 weeks; serum LH levels and ovarian histomorphology were then assessed. Raloxifene treatment at 233 mg/kg/day for 2 weeks (Study 1) significantly elevated circulating LH levels by 4- to 7-fold compared with control. Raloxifene-treated mice had elevated LH levels sustained over the 24-h sampling period and did not exhibit the preovulatory LH surge evident in some control mice at the 4:00 p.m., 6:00 p.m., and 8:00 p.m. time points. Mice treated with 236 mg/day raloxifene for 4 weeks (Study 2) had elevated LH levels (4.4-fold compared to control), whereas mice exposed to 7.9 mg/kg/day raloxifene had a slight, nonsignificant increase in LH (2-fold compared to control). In both dose groups, LH levels were indistinguishable from controls 3 weeks after raloxifene treatment was discontinued. The ovaries in six of the eight mice treated with 7.9 mg/kg/day raloxifene had dilated and/or anovulatory follicles. One mouse in this group had a single hemorrhagic follicle; however, corpora lutea distribution was normal, indicating that ovulation was occurring. Raloxifene-treated mice in Study 1 and mice treated with a comparable raloxifene dose (236 mg/day) in Study 2 had histomorphological changes in the ovary indicative of arrested follicular maturation, including anovulatory hemorrhagic follicles, some developing follicles, and very few corpora lutea. At the end of the reversibility phase, hemorrhagic follicles were no longer evident and follicular maturation and corpora lutea distribution were normal. Raloxifene treatment in mice produces a dose-dependent, sustained elevation in serum LH levels and is associated with changes in ovarian follicular morphology. These changes are reversible upon discontinuation of raloxifene treatment.

Evidence that Neuronal Nitric Oxide Synthase but Not Heme Oxygenase Increases in the Hypothalamus on Proestrus Afternoon

Nitric oxide (NO) has been implicated in the control of the proestrus luteinizing hormone (LH) surge in the rat but to date no studies have attempted to measure neuronal nitric oxide synthase (nNOS) or NO production on proestrus in the hypothalamus in order to determine if endogenous NO is increased on proestrus afternoon to activate gonadotropin-releasing hormone (GnRH) neurons. To address this deficit in our knowledge, we measured nNOS mRNA and protein levels as well as NOS activity levels in rat preoptic area (POA) and medial basal hypothalamus (MBH) fragments at 12.00, 14.00, 16.00, and 18.00 h of proestrus. Serum LH levels were also assessed to determine whether NOS changes correlate to the LH surge. To determine the specificity of observed changes we also measured mRNA levels for the enzyme heme oxygenase-2, which is responsible for production of another putative gaseous transmitter, carbon monoxide. In all studies a metestrus 12.00 h control group was included since steroid and LH levels would be basal at this time as compared to proestrus. The results revealed that nNOS mRNA and protein levels, as well as NOS activity did not change significantly in the MBH on proestrus. In contrast, nNOS mRNA levels were significantly elevated in the POA at proestrus 12.00 and 14.00 h, as compared to metestrus 12.00 h. Likewise, at the protein and activity level, nNOS protein levels in the POA were significantly elevated on proestrus at 14.00 and 16.00 h, with NOS activity significantly increased at 16.00 h on proestrus. The elevation of nNOS protein and activity levels in the POA occurred at the time of initiation of the LH surge. The elevation of nNOS was specific as mRNA levels for the CO-synthetic enzyme heme oxygenase-2 did not change significantly on proestrus in the POA or MBH. As a whole, the current studies provide new evidence that nNOS is elevated in the POA on proestrus, and thus could play a role in the activation of GnRH neurons to produce the preovulatory LH surge.

Ovarian function in ewes at the onset of the breeding season.

Transrectal ultrasonography of ovaries was performed each day, during the expected transition from anoestrus to the breeding season (mid-August to early October), in six Western white-faced cross-bred ewes, to record ovarian antral follicles > or = 3 mm in size and luteal structures. Jugular blood samples were collected daily for radioimmunoassay (RIA) of follicle-stimulating hormone (FSH), oestradiol and progesterone. The first ovulation of the breeding season was followed by the full-length oestrous cycle in all ewes studied. Prior to the ovulation, all ewes exhibited a distinct increase in circulating concentrations of progesterone, yet no corpora lutea (CL) were detected and luteinized unovulated follicles were detected in only three ewes. Secretion of FSH was not affected by the cessation of anoestrus and peaks of episodic FSH fluctuations were associated with the emergence of ovarian follicular waves (follicles growing from 3 to > or = 5 mm). During the 17 days prior to the first ovulation of the breeding season, there were no apparent changes in the pattern of emergence of follicular waves. Mean daily numbers of small antral follicles (not growing beyond 3 mm in diameter) declined (P < 0.05) after the first ovulation. The ovulation rate, maximal total and mean luteal volumes and maximal serum progesterone concentrations, but not mean diameters of ovulatory follicles, were ostensibly lower during the first oestrous cycle of the breeding season compared with the mid-breeding season of Western white-faced ewes. Oestradiol secretion by ovarian follicles appeared to be fully restored, compared with anoestrous ewes, but it was not synchronized with the growth of the largest antral follicles of waves until after the beginning of the first oestrous cycle. An increase in progesterone secretion preceding the first ovulation of the breeding season does not result, as previously suggested, from the ovulation of immature ovarian follicles and short-lived CL, but progesterone may be produced by luteinized unovulated follicles and/or interstitial tissue of unknown origin. This increase in serum concentrations of progesterone does not alter the pattern of follicular wave development, hence it seems to be important mainly for inducing oestrous behaviour, synchronizing it with the preovulatory surge of luteinizing hormone (LH), and preventing premature luteolysis during the ensuing luteal phase. Progesterone may also enhance ovarian follicular responsiveness to circulating gonadotropins through a local mechanism.

Plasma Gonadotropin Concentrations in the Cyclic Female Brushtail Possum (Trichosurus vulpecula)

Changes in plasma concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), and their relationship to antral follicle development and ovulation, were determined in female brushtail possums (Trichosurus vulpecula) in experiments in which pouch young were removed (RPY) from lactating females to promote ovarian activity. In Experiment 1 (n=8), the development of preovulatory follicles and ovulation was monitored by laparoscopy. In Experiment 2 (n=15) estrus and mating were monitored by cytology of urine. Ovulation occurred in 4/8 (Experiment 1) and 9/16 (Experiment 2) possums, and in these animals, plasma FSH concentrations fell progressively over the period of preovulatory follicle development and returned to pretreatment levels after ovulation. With the exception of samples taken at the time of the preovulatory gonadotropin surge, mean plasma LH levels remained basal. In those possums that failed to ovulate, plasma FSH concentrations were elevated while plasma LH concentrations were low; these patterns remained unchanged throughout the sampling period. It was not possible to distinguish between animals that would ovulate and those that would not ovulate after RPY on the basis of gonadotropin profiles at the time of RPY. A further group of possums (Experiment 3, n=10) were blood-sampled at hourly intervals for 48 h to characterize preovulatory gonadotropin surges, using laparoscopy to monitor preovulatory follicular development and predict ovulation. A preovulatory LH surge (max. conc. 10.2–43.5 ng/ml, duration 7–9 h) was recorded in 4 animals, with a coincident preovulatory FSH surge (max. conc. 1.4–21.4 ng/ml, duration 3–11 h) observed in 3 of these possums. The patterns of gonadotropin secretion in the cycling brushtail possum conform to those reported for eutherians that ovulate spontaneously and appear to be regulated by similar mechanisms.

Prostaglandin E2, prostaglandin f2α and endothelin-1 production by cow oviductal epithelial cell monolayers: Effect of progesterone, estradiol 17β, oxytocin and luteinizing hormone

The optimal oviductal environment, including contractile activity for gamete transport, fertilization and early embryonic development, is mediated by physiological and anatomical changes in the oviduct during the estrous cycle. Oviductal epithelial cell culture was utilized to investigate the effect of ovarian steroids (progesterone [P4]and estradiol 17β[E2]), oxytocin (OT) and luteinizing hormone (LH) on the local production of prostaglandin E2 (PGE2), prostaglandin F2α (PGF2α) and endothelin-1 (ET-1) in the cow oviduct. Epithelial cells isolated from oviducts collected during the follicular phase were cultured in M199 under standard culture conditions until monolayer formation. Then the cells were trypsinized and plated at a density of 3 x 10 4 /mL/well and cultured again until subconfluency, at which time the cells were incubated for 4 or 24 h with M199 only (control), high P4 (H-P4;1μg/mL), low P4 (L-P4;10 ng/mL), E2 (1 ng/mL), LH (10 ng/mL),OT (10 −9 M) ET-1 (10 −9 M), PGE2 (10 −8 M) PGF2a (10 −9 M) or their combination (H-P4+E2, L-P4+E2, LH+E2, ET-1+E2, L-P4+E2+LH and H-P4+E2+LH). The production of both PG and ET-1 was increased by E2+low P4 and LH+E2+low P4 (P<0.05), while LH+E2 enhanced the production of PGF2a and ET-1 (P<0.05). Moreover, E2+ET-1 stimulated PG production (P<0.05). However, OT had no effect on the production of any of these substances. These results suggest that the preovulatory LH surge, together with locally re-circulated high levels of E2 from the Graafian follicle and basal P4 from regressing corpus luteum (CL), induces the maximum stimulatory effect on oviductal PGE2, PGF2a and ET-1 production during the periovulatory period. Consequently, the elevated local ET-1 concentration during periovulatory period may induce the high contractile activity of the oviduct and, at the same time, the stimulation of PG production. Thus, ET-1 may act as a local amplifier for oviductal PG production stimulated by LH and ovarian steroids.

17β-Estradiol regulation of melanin-concentrating hormone and neuropeptide-E-I contents in cynomolgus monkeys: a preliminary study

Melanin-concentrating hormone (MCH) and neuropeptide-E-I (NEI) regulate several behaviors and neuroendocrine functions in rats. Possible influence of these peptides on sexual behavior and reproduction in mammals other than rodents prompted us to investigate: 1) The sites of synthesis of MCH and NEI in the brain of a non-human primate ( M. fascicularis); 2) The effect of 17β-estradiol (E 2) benzoate (E 2B) on pro-MCH-derived peptide concentrations in the hypothalamus of the ovariectomized (OVX) cynomolgus monkeys ( M. fascicularis). Expression of MCH mRNA and peptides was examined by Northern blotting, RT-PCR and RP-HPLC/RIA. Our results demonstrate that the MCH gene is predominantly expressed in hypothalamus of macaque. E 2B exposure of OVX monkeys provoked parallel phasic variations in the MCH-immunoreactivity (IR) and NEI-IR. NEI-IR and to a lesser extent MCH-IR, showed a transient increase (associated with the estradiol peak) at 30 h with a final rise of both MCH-IR and NEI-IR observed at the time (72 h post E 2B) of the luteinizing hormone (LH) surge. RP-HPLC analysis of peptide extracts revealed the presence, in addition to mature MCH and NEI, of different MCH-IR and NEI-IR forms in the hypothalami of control and E 2B-treated monkeys. Taken together, our results indicated that hypothalamic MCH and NEI contents are regulated after E 2B treatment and they suggest the possible involvement of these peptides in the regulation of the pre-ovulatory midcycle LH surge in primates.

Locus ceruleus lesions block pulsatile LH release in ovariectomized rats

Luteinizing hormone (LH) secretion during the reproductive cycle and in ovariectomized (OVX) rats is pulsatile and this pattern of secretion is determined by intermittent discharges of LH-releasing hormone (LHRH) into the hypophysial portal vessels. LHRH secretion is probably controlled by prior pulsatile norepinephrine (NE) release. The locus ceruleus (LC) is an important source of NE to the LHRH neurons. We have shown previously that LC lesions block the preovulatory LH surge and ovulation and also cause a decrease in plasma LH concentrations in OVX rats. The possible role of the LC in regulating pulsatile LH release has not been explored. Therefore, the aim of this work was to investigate, in OVX rats, the effects of LC lesions on pulsatile LH secretion. LC lesions were produced in adult female rats three weeks after OVX. On the next morning, the jugular vein was catheterized and, on the afternoon of the same day, blood samples (0.3 ml) were withdrawn every 5 min, during 90 min, from conscious freely moving rats. Plasma LH was measured by radioimmunoassay. LC lesions greatly suppressed pulsatile LH secretion by decreasing both LH pulse frequency and amplitude. The basal as well as total secretion of LH were also decreased. This inhibitory effect of the lesions was observed only when at least 50% of the nucleus was destroyed. Data from sham-operated animals as well as those with less than 50% destruction of the LC did not differ from those of the control rats without brain lesions. Since LC lesions induce a decrease in NE content in the preoptic area and median eminence, the inhibition of pulsatile LH release in ovariectomized rats with LC lesions occurs presumably as result of decreased pulsatile NE release into these areas of the brain that decreases both the frequency and the amount of LHRH released per pulse.