Pattern Of Luteinizing Hormone Release Research Articles

Female-specific pituitary gonadotrope dysregulation in mice with chronic focal epilepsy

Gonadotropin hormone release from the anterior pituitary is critical to regulating reproductive endocrine function. Clinical evidence has documented that people with epilepsy display altered levels of gonadotropin hormones, both acutely following seizures and chronically. Despite this relationship, pituitary function remains a largely understudied avenue in preclinical epilepsy research. Recently, we showed that females in the intrahippocampal kainic acid (IHKA) mouse model of temporal lobe epilepsy displayed changes in pituitary expression of gonadotropin hormone and gonadotropin-releasing hormone (GnRH) receptor genes. Circulating gonadotropin hormone levels, however, have yet to be measured in an animal model of epilepsy. Here, we evaluated the circulating levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), GnRH receptor (Gnrhr) gene expression, and sensitivity to exogenous GnRH in IHKA males and females. Although no changes in overall dynamics of pulsatile patterns of LH release were found in IHKA mice of either sex, estrus vs. diestrus changes in basal and mean LH levels were larger in IHKA females with prolonged, disrupted estrous cycles. In addition, IHKA females displayed increased pituitary sensitivity to GnRH and higher Gnrhr expression. The hypersensitivity to GnRH was observed on diestrus, but not estrus. Chronic seizure severity was not found to be correlated with LH parameters, and FSH levels were unchanged in IHKA mice. These results indicate that although there are changes in pituitary gene expression and sensitivity to GnRH in IHKA females, there may also be compensatory mechanisms that aid in maintaining gonadotropin release in the state of chronic epilepsy in this model.

SUN-483 High-Fat Diet Suppresses Basal Lh Pulsatile Levels in Female Mice at Estrus

Pubertal timing and reproductive maturation in females are known to be susceptible to obesogenic diets. Metabolic energy balance and reproduction are homeostatic processes that are tightly regulated, in part, by hypothalamic neurons in the arcuate nucleus (ARC) and high-fat diet is reported to steadily increase estradiol levels in rodents compared to standard chow. Estradiol regulates the hypothalamic-pituitary-gonadal axis via negative feedback mechanisms in estrogen-sensitive ARC neurons by modulating the pulsatile release of the gonadotropin luteinizing hormone (LH). However, it is unclear how exactly the circulating estradiol milieu of adult females interacts with a state of high caloric fat intake to alter the parameters of LH release, such as pulse frequency, pulse amplitude, and basal set points of pulsatile release. The objective of this study was to use a non-invasive method of measuring pulsatile LH release patterns at different stages of the estrous cycle in mice fed a high-fat diet, using an ultrasensitive mouse LH assay (1). Starting at weaning age (21 days old), female C57BL/6J mice were fed ad libitum with either regular chow diet (RD) or 60% kcal high-fat diet (HFD), and free access to water. After 12 weeks on either RD or HFD, estrous cyclicity was determined via examination of vaginal cytological smears. Blood samples were collected when mice were at diestrus, followed by one full estrous cycle, and then collected again at estrus. To examine LH pulsatile release during both estrous cycle stages, mice had tail tips snipped and whole blood samples were collected and immediately frozen in assay buffer at 10-second intervals for 3 hours (between 0900 and 1200 hours). Pulsatile LH release was analyzed for pulse frequency, basal mean LH levels, and pulse amplitude using the DynPeak algorithm program. Compared to RD controls, mice fed a HFD displayed significantly lower basal LH levels during estrus, but not diestrus. However, there were no differences in pulse amplitude between HFD and RD fed mice. Mice on HFD (at both diestrus and estrus) also showed a trend towards increased pulse frequency. These results suggest that endogenous circulating estradiol influences the impact that HFD has on regular pulsatile release of LH by changing the set point for basal LH levels, thereby modulating when obesogenic diet-induced changes may be most detrimental. This has implications on how diet may put women at risk of failing reproductive health when ovarian estradiol levels drastically change, for example during menopause or with hormone replacement therapy. 1) Steyn FJ et al. Development of a methodology for and assessment of pulsatile luteinizing hormone secretion in juvenile and adult male mice. Endocrinology 2013;154(12):4939-4945. doi:10.1210/en.2013-1502.

Developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin may alter LH release patterns by abolishing sex differences in GABA/glutamate cell number and modifying the transcriptome of the male anteroventral periventricular nucleus

Developmental exposure to arylhydrocarbon receptor (AhR) ligands abolishes sex differences in a wide range of neural structures and functions. A well-studied example is the anteroventral periventricular nucleus (AVPV), a structure that controls sex-specific luteinizing hormone (LH) release. In the male, testosterone (T) secreted by the developing testes defeminizes LH release mechanisms; conversely, perinatal AhR activation by 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) blocks defeminization. To better understand developmental mechanisms altered by TCDD exposure, we first verified that neonatal TCDD exposure in male rats prevented the loss of AVPV GABA/glutamate neurons that are critical for female-typical LH surge release. We then used whole genome arrays and quantitative real-time polymerase chain reaction (QPCR) to compare AVPV transcriptomes of males treated neonatally with TCDD or vehicle. Our bioinformatics analyses showed that TCDD enriched gene sets important for neuron development, synaptic transmission, ion homeostasis, and cholesterol biosynthesis. In addition, upstream regulatory analysis suggests that both estrogen receptors (ER) and androgen receptors (AR) regulate genes targeted by TCDD. Of the 23 mRNAs found to be changed by TCDD at least 2-fold (p<0.05), most participate in the functions identified in our bioinformatics analyses. Several, including matrix metallopeptidase 9 and SRY-box 11 (Sox11), are known targets of E2. CUG triplet repeat, RNA binding protein 2 (cugbp2) is particularly interesting because it is sex-specific, oppositely regulated by estradiol (E2) and TCDD. Moreover, it regulates the post-transcriptional processing of molecules previously linked to sexual differentiation of the brain. These findings provide new insights into how TCDD may interfere with defeminization of LH release patterns.

Sexual differentiation of the gonadotropin surge release mechanism: A new role for the canonical NfκB signaling pathway

Sex differences in luteinizing hormone (LH) release patterns are controlled by the hypothalamus, established during the perinatal period and required for fertility. Female mammals exhibit a cyclic surge pattern of LH release, while males show a tonic release pattern. In rodents, the LH surge pattern is dictated by the anteroventral periventricular nucleus (AVPV), an estrogen receptor-rich structure that is larger and more cell-dense in females. Sex differences result from mitochondrial cell death triggered in perinatal males by estradiol derived from aromatization of testosterone. Herein we provide an historical perspective and an update describing evidence that molecules important for cell survival and cell death in the immune system also control these processes in the developing AVPV. We conclude with a new model proposing that development of the female AVPV requires constitutive activation of the Tnfα, Tnf receptor 2, NfκB and Bcl2 pathway that is blocked by induction of Tnf receptor-associated factor 2-inhibiting protein (Traip) in the male.

Neuropeptide Y Gene Expression in Male Sheep: Influence of Photoperiod and Testosterone

The frequency of pulsatile release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) is high in the breeding season and low in the nonbreeding season. These alterations in the patterns of GnRH and LH release are due to an interaction of daylength and gonadal steroid negative feedback. A vast amount of data indicates that steroid-responsive neural systems may play a role in regulating seasonal changes in GnRH release. One candidate system is neuropeptide Y (NPY). To determine the independent and interactive influences of photoperiod and steroid exposure on NPY mRNA levels, we used hypothalamic tissue from four groups (n = 4 per group) of castrated male sheep that were simultaneously housed in photochambers and exposed to: (1) a 16L:8D photoperiod (LD); (2) LD and implanted with testosterone (LD + T); (3) a 10L:14D photoperiod (SD), and (4) SD + T. Circulating levels of T averaged 2.8 ± 0.2 ng/ml in implanted animals, but were undetectable in nonimplanted males. Mean LH levels were significantly reduced (p < 0.01) in the LD + T group as compared with the other groups which did not differ from each other. The silver grain area per NPY neuron in the arcuate nucleus, as assessed by in situ hybridization, was inversely related to mean LH values, with the grain area per cell being significantly greater (p < 0.05) for LD + T males than for all other groups which did not differ from each other. NPY cell numbers were not significantly different (p > 0.10) among the treatment groups. These results show that NPY mRNA expression is increased in male sheep during a LD photoperiod in a T-dependent manner. Our data are consistent with the idea that NPY is involved in the seasonal regulation of GnRH and LH release in the male sheep.

Lack of effect of intracerebroventricular infusion of H1 or H2 histamine receptor antagonists on luteinizing hormone secretion in fed or fasted rats

The effects of intracerebroventricular infusion of histamine receptor antagonists on pulsatile secretion of luteinizing hormone (LH) were examined in ovariectomized, estradiol-primed rats under conditions of normal feeding or fasting for 48 h. Blood samples for determination of the plasma LH concentration by radioimmunoassay were collected every 6 min for 3 h through an atrial indwelling catheter. Ten microliters of either the H1 receptor antagonist chlorpheniramine (3 mM), the H2 receptor antagonist ranitidine (3 mM), or artificial cerebrospinal fluid (vehicle) were infused into the third ventricle during the second 1-h period of blood sampling. The infusion of either receptor antagonist failed to restore the pattern of pulsatile LH release in fasted animals to that observed in fed rats. The plasma LH concentration as well as the pulse frequency and pulse amplitude of LH secretion in fed rats were also not affected by either receptor antagonist. These results indicate that H1 or H2 receptor antagonists did not affect on tonic LH secretion in fed or fasted rats.

TEMPORAL PATTERN OF LH SECRETION: REGULATION BY MULTIPLE ULTRADIAN OSCILLATORS VERSUS A SINGLE CIRCADIAN OSCILLATOR

The possibility that the 24h rhythm output is the composite expression of ultradian oscillators of varying periodicities was examined by assessing the effect of external continuously or pulsed (20-minute) Gonadotropinreleasing hormone (GnRH) infusions on in vitro luteinizing hormone (LH) release patterns from female mouse pituitaries during 38h study spans. Applying stepwise analyses (spectral, cosine fit, best-fit curve, and peak detection analyses) revealed the waveform shape of LH release output patterns over time is composed of several ultradian oscillations of different periods. The results further substantiated previous observations indicating the pituitary functions as an autonomous clock. The GnRH oscillator functions as a pulse generator and amplitude regulator, but it is not the oscillator that drives the ultradian LH release rhythms. At different stages of the estrus cycle, the effect of GnRH on the expression of ultradian periodicities varies, resulting in the modification of their amplitudes but not their periods. The functional output from the system of ultradian oscillators may superimpose a “circadian or infradian phenotype” on the observed secretion pattern. An “amplitude control” hypothesis is proposed: The temporal pattern of LH release is governed by several oscillators that function in conjunction with one another and are regulated by an amplitude-controlled mechanism. Simulated models show that such a mechanism results in better adaptive response to environmental requirements than does a single circadian oscillator. (Chronobiology International, 18(3), 399–412, 2001)

Gonadotropin-Releasing Hormone Fibers Innervate the Pituitary of the Male African Catfish (Clarias gariepinus) during Puberty

The development of the catfish gonadotropin-releasing hormone (cfGnRH) fiber network in the pituitary of male African catfish (Clarias gariepinus) was investigated in relation to puberty. Double immunolabeling studied by confocal laser scanning microscopy revealed a concomitant development of gonadotropes and of pituitary cfGnRH innervation during the first wave of spermatogenesis. Catfish GnRH-immunoreactive fibers in the proximal pars distalis (PPD) of the pituitary were initially observed at the age of 10 weeks (onset of spermatogonial proliferation) and gradually reached the adult pattern at the age of 20 weeks (spermatozoa present in the testis). The content of cfGnRH-associated peptide (cfGAP, part of the prohormone) in the pituitary similarly increased during puberty. At the electron microscopical level, fibers containing cfGAP-ir granules came into close proximity of the gonadotropes at 18 weeks of age. In vitro studies indicated a progressively increasing basal and cfGnRH-stimulated luteinizing hormone (LH) secretion during pubertal development. The LH secretion patterns were similar in response to exogenous cfGnRH (0.1 µM) or to endogenous cfGnRH, the release of which was induced by forskolin (1 µM). Castration experiments demonstrated that the innervation of the pituitary with cfGnRH fibers continued after surgery, accompanied by an increase in the cfGAP levels. However, gonadotrope development was retarded, suggesting a differential regulation of the two maturational processes. Since testosterone stimulates both processes, other testicular factors may also be involved. Puberty-associated changes in LH release patterns appear to reflect changes in the GnRH sensitivity and in the pool of releasable LH, while availability of cfGnRH does not appear to be a limiting factor.

The influence of gonadal steroids on pre-pro melanin-concentrating hormone mRNA in female rats.

Melanin-concentrating hormone (MCH) may have a regulatory role in the control of luteinizing hormone (LH) release. We have investigated if gonadal steroids induce changes in the expression of pre-pro MCH (ppMCH) that are associated with changes in the pattern of LH release. Using quantitative in-situ hybridization histochemistry we have determined the effect of administration of either oestradiol benzoate (5 microg/rat) or oestradiol benzoate followed 44 or 48 h later by progesterone (0.5 mg/rat) to ovariectomized rats on the expression of ppMCH in the medial and lateral zona incerta and the lateral hypothalamus. The prevalence of ppMCH transcripts in the intact female rat at 12.00 and 19.00 h on proestrus and the first day of dioestrus was also examined. Oestrogen reduced the intensity of hybridization signal for ppMCH mRNA and this was associated with both a decrease in the number of cells in which the message was detected in the medial zona incerta and a negative feedback effect on LH release in ovariectomized rats. Progesterone administration to oestradiol benzoate-primed rats did not alter the reduced expression in the medial zona incerta in spite of its positive feedback effect on LH release. We suggest that progesterone may act only on post-translational events. Expression in the MCH cell bodies of the lateral zona incerta were not affected but there was a transient decrease 4 h after progesterone treatment in the oestradiol benzoate-primed rats in expression in the lateral hypothalamus. No changes in ppMCH mRNA were seen in intact animals on proestrus or the first day of dioestrus indicating that gonadal steroids are not important in the modulation of ppMCH gene expression during the oestrous cycle. In other steroid-dependent physiological situations, however, oestrogen may influence the expression of ppMCH in a subpopulation of cell bodies in the medial zona incerta.

From Ultradian to Infradian Rhythms: LH Releases Patterns In Vitro

In the present study, we examined in vitro luteinizing hormone (LH) release patterns from pituitaries and from pituitary cell cultures (3 and 7 days in culture) to elucidate the endogenous period generated by the gonadotroph cell population and to evaluate the relationship between the basic period generated at the cellular level and the output pattern observed at the organ level. In addition, we examined the effect of photic environmental signals perceived by the animals on LH release patterns from pituitaries in vitro. When the animals were exposed to circadian photoperiodic signals, the in vitro LH release pattern from the pituitaries exhibited ultradian, circadian, and infra-dian frequencies. When the animals were exposed to continuous illumination, the in vitro patterns exhibited only ultradian and infradian frequencies. Furthermore, free running is a process, not a state. This process is driven by a change in the relative dominance of different frequencies that construct the pattern without changing the basic period length. Evaluation of the relative dominance of the different frequencies that construct the pattern indicates that, although infradian oscillators may take part in shaping the output pattern, the basic rhythm generated by the pituitary cells is in the ultradian domain. The results obtained from the examined system suggest that an endogenous oscillator is a cellular entity with ultradian periodicity, and that the rhythmic output of many biological variables is structured by various ultradian components that construct the circadian and infradian output rhythms.

Fasting suppresses pulsatile luteinizing hormone (LH) secretion and enhances orderliness of LH release in young but not older men.

Pulsatile gonadotropin secretion and sex-steroid concentrations are suppressed reversibly in young fasted or malnourished human subjects. In this study, we investigated the impact of age on the dynamic neuroendocrine mechanisms underlying this stress response in healthy young (age, 28 +/- 3 yr, n = 8) vs. older (age 67 +/- 2 yr, n = 8) men with similar body mass indices (mean, 26 +/- 0.6 vs. 26 +/- 1.3 kg/m2, respectively). Serum LH concentrations were measured by immunoradiometric assay (IRMA) in blood collected at 10-min intervals over 27 h on a control (fed) day and on the third day of a 3.5-day fast (water only) assigned in randomized order. After 24 h of basal sampling, GnRH (10 micrograms i.v. bolus) was administered to test gonadotrope responsiveness. Cortisol, dehydroepiandrosterone sulfate, androstenedione, testosterone, FSH, GH, and PRL were measured in 24-h pooled serum as positive and negative control hormones. Approximate entropy was used to quantitate the orderliness of LH release over 24 h, and a multiple-parameter deconvolution method was applied to quantify pulsatile LH secretion and LH half-life. In the fed state, older men exhibited elevated mean (24-h pooled) serum FSH and cortisol concentrations compared with young controls but equivalent serum LH concentrations and reduced serum GH, free testosterone, androstenedione, and dehydroepiandrosterone sulfate concentrations. Fed older men also manifested a lower frequency and amplitude of 24-h pulsatile LH secretion, and, by approximate entropy calculations, a more disorderly pattern of basal LH release than younger individuals. Three- and one-half days of fasting evoked 40% and 47% increases in mean (24-h) serum cortisol concentrations in young and older men, respectively (P < 0.01 vs. fed, but P = not significant for percentage rise in older vs. young men). Concurrently, fasting induced a 2.1-fold fall in the 24-h endogenous LH production rate in young men (fed 36 +/- 9.7 vs. fasted 17 +/- 2.0 IU/L of distribution volume/day, P < 0.01), but did not significantly affect the daily LH secretion rate in older men (fed 27 +/- 4.5 vs. fasted 21 +/- 3.4 IU/day). The reduced LH production rate in fasting young men was accounted for by a 1.7-fold decline in the mass of LH secreted per burst (fed 2.5 +/- 0.45 vs. fasted 1.5 +/- 0.16 IU/L, P < 0.05), whereas LH burst mass in older men remained unchanged (and low) during fasting. In addition, in young men, during the 3.5-day fast the number of computer-resolved LH secretory bursts per 24 h decreased (fed 15 +/- 0.7 vs. fasted 11 +/- 0.7, P < 0.01), and the interburst interval increased (fed 94 +/- 4.2 vs. fasted 125 +/- 8.7 min, P < 0.05). In contrast, in older men in the fed state, basal LH peak frequency and serum free testosterone concentrations were reduced compared with corresponding values in young men, and did not decline further with fasting. Whereas the orderliness of LH release patterns increased significantly during fasting in the young men, the approximate entropy measure failed to change significantly in unfed older subjects. By cosinor analysis, young men showed lower 24-h mesor (mean of nyctohemeral rhythm of) serum LH concentrations than older volunteers during fasting. Moreover, young but not older men manifested preserved 24-h variations in LH interpulse intervals when fasting. Exogenously stimulated LH release (mean 3-h serum LH concentration or calculated mass of LH secreted) following a single i.v. injection of 10 micrograms GnRH was independent of age and fasting status. We conclude that the metabolic stressor of short-term fasting unmasks specific age-related neuroendocrine contrasts in the stress-responsive control of both the pulsatile and nyctohemeral regulation of the male hypothalamo-pituitary-gonadal-axis.

Peptidases that degrade gonadotropin-releasing hormone: influence on LH secretion in the ewe.

Several peptidases have been postulated to degrade the hypothalamic peptide gonadotropin-releasing hormone (GnRH), but it is not known if such enzymes contribute significantly to the delivery of GnRH to the pituitary in vivo. Furthermore, the activity of GnRH-inactivating peptidases may vary in different reproductive states, such as across the estrous cycle. In the present study, specific fluorescent substrates were used to measure the activity of the two major GnRH-degrading enzymes, prolyl endopeptidase (PEP) and endopeptidase 3.4.24.15 (EP 24.15), in soluble extracts of the median eminentes (ME) of ewes during different phases of the estrous cycle. Levels of EP 24.15 and PEP activity in the ME did not vary significantly across the cycle, although PEP activity was lowest at the time of the preovulatory luteinizing hormone (LH) surge. However, a statistically significant decline in PEP activity (18%, P = 0.02) was observed in the ME of OVX ewes in which a surge was induced by estrogen when compared to oil-treated OVX controls, suggesting a possible negative regulation of PEP activity by this steroid. The effect of intracerebroventricular (i.c.v.) infusion of several peptidase inhibitors on the pulsatile release of LH in the conscious OVX ewe was also examined. No consistent changes in the pattern of LH release were observed with i.c.v. infusion of the EP 24.15 inhibitor N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP-AAY-pAB) or the angiotensin-converting enzyme (ACE) inhibitor captopril. Similarly, administration of the prolyl endopeptidase inhibitor bacitracin, or a more specific inhibitor of this enzyme, Z-Proprolinal (ZPP), did not alter LH release patterns. The results did not demonstrate a major role for changes in the activity of EP 24.15, PEP, or ACE in altering the pattern of GnRH secretion, but a minor reduction in PEP levels may occur at the time of the estrogen-induced LH surge.

A temporally intermediate mode of gonadotropin releasing hormone-induced desensitization of luteinizing hormone secretion

The classical mode of luteinizing hormone (LH) secretory desensitization in the rat appears after 3–6 h of continuous in vitro administration of gonadotropin (GnRH). A second mode has been reported to occur very rapidly (<2 min) after the onset of GnRH administration, and to reverse within 3 min after its withdrawal. Here, the existence of a third mode of desensitization is reported, occurring at 40–50 min after initiation of continuous GnRH administration. Rat pituitary cells were perifused with 10 −8 M GnRH for 6 h; 10 min samples were collected for LH measurements by radioimmunoassay. As expected, the pattern of LH release was biphasic: LH levels peaked in the first phase at 30 min, decreased at 40–50 min, increased in the second phase to maximal levels at 90–110 min, and then decreased in the classical desensitization mode to near-baseline values by 300–360 min. Static incubations of pituitary cells in Petri dishes in the presence of high (10 −8 M) or submaximal (10 −9 M) GnRH concentrations confirmed the decrease in LH secretion at 40–50 min. Measurement of LH by reverse hemolytic plaque assay (RHPA) confirmed the existence of this new mode of desensitization; since 93% of all gonadotropes had become secretory at 40–50 min, the possibility of two subpopulations of gonadotropes accounting for the two phases of LH secretion appears to be ruled-out. GnRH receptor binding studies demonstrated a ∼50% decrease in cell-surface binding in association with the desensitization at 40–50 min. These studies suggest the existence of a third mode of GnRH-induced LH secretory desensitization that is not due to gonadotrope subpopulations but may be causally associated with decreased GnRH receptor binding.

Pulsatile patterns of melatonin secretion in patients with gonadotropin-releasing hormone deficiency: effects of testosterone treatment.

Recently, we have demonstrated that male patients with gonadotropin-releasing hormone (GnRH) deficiency had increased nocturnal melatonin secretion that decreased to normal levels during testosterone treatment. The purpose of the current study was to examine if the abnormally increased melatonin levels in these patients were associated with pulsatile secretory patterns, and, if these were modified during testosterone administration. Characteristics of nocturnal melatonin and luteinizing hormone (LH) secretion were compared in six normal young males, six males with idiopathic hypogonadotropic hypogonadism (IGD), and in six males with constitutional delayed puberty (DP). Patients were examined in the untreated state and following the administration of 250 mg testosterone enanthate/month for 4 months. Serum samples for melatonin and LH levels were obtained every 15 min from 19.00 hr to 07.00 hr in a controlled light-dark environment. Pulse detection and pulse characteristics were determined by the program ULTRA. In comparison with normal controls, untreated IGD patients showed significantly higher pulse frequency, lower relative increments and shorter half-life times for melatonin. Similar findings were observed in DP patients, although statistically of borderline significance. Treatment with testosterone normalized melatonin pulse characteristics in both IGD and DP patients. The secretory pattern of LH release in these patients was characterized by significantly higher relative and absolute increments and shorter half-life time without any significant change in the number of LH pulses. Taken together, these data suggest that melatonin is secreted in a pulsatile pattern in normal adult males and in male patients with GnRH deficiency. The abnormally increased nocturnal melatonin secretion observed in these patients may indicate that the pineal pulse generator is expressing an altered activity pattern within its normal capabilities. Testosterone administration normalized melatonin secretory patterns in IGD and DP patients. The lack of relationship between the pulsatile LH and melatonin secretory patterns suggest an independent signal for the nocturnal pulsatile melatonin and LH secretions.

Effects of prenatal testosterone on sexual behavior, reproductive morphology and LH secretion in the female rat.

Sexual differentiation of many brain structures and functions is dependent on levels of testosterone (T) or its metabolites during certain 'sensitive' developmental periods. If T is present during these perinatal periods, masculinization and defeminization of sexual behavior occur; also, reproductive physiology, and central nervous system morphology and function are altered. The purpose of the present study was to characterize the influence of T at specific prenatal developmental intervals on offspring reproductive morphology, physiology, locomotor activity and sexual behavior during postnatal development. To avoid complications induced by endogenous testicular activity, only females were examined. Free T was used because of its relative short half-life, so that the effects induced by its administration on a specific gestational day (GD) could be evaluated. Pregnant rats received a single subcutaneous injection of either sesame oil (controls) or 5 mg of T on GD 16, 17, 18, 19, 20, 21, or 22. Female offspring of pregnant rats exposed to T displayed significant alterations in morphology and behavior. The anogenital distance, measured at 25 days postbirth, was significantly increased if T was administered on GD 16, 17 or 18. T treatment on GD 16 or each day thereafter through GD 20 significantly delayed the normal occurrence of vaginal opening (controls at 37.5 days vs. T treatment which ranged from 38.5 to 41.4 days). Abnormal vaginal morphology (enlarged clitoris) was also observed when T was injected during a similar prenatal interval (i.e. GD 16 to GD 22). Furthermore, prenatal T treatment on GD 18 (and each day thereafter), until GD 22 significantly decreased lordotic behavior compared to control values. However, exposure to T, on any prenatal GD did not alter the animals' ability to exhibit an induced luteinizing hormone (LH) surge. These results suggest that the onset for altered reproductive morphology occurs at least as early as GD 16, whereas the onset of sexual behavior sensitivity occurs precisely at GD 18, and that the normal pattern of adult LH release in females is not altered by prenatal androgen treatment using this specific paradigm.

Rhythmicity of luteinizing hormone secretion expressed in vitro.

In the present study we explored the possibility that the pituitary functions as an autonomous clock and is capable of generating rhythms of luteinizing hormone (LH) release independently of hypothalamic control. Pituitaries from estrous or diestrous day 1 female mice were perifused separately with Medium-199. Effluent samples were collected at 10-min intervals and assayed for LH levels. Fourier analysis and curve-fit analysis served to elucidate the presence of prominent periods whose significance was then determined by best-fit cosinor. The latter method was used to determine additional parameters for the significant rhythm. All perifused pituitaries exhibited LH release patterns that were composed of significantly long ultradian rhythms (approximately 16 and 8 h, p < 0.001). Continuous stimulation with gonadotropin-releasing hormone (GnRH) or estradiol did not alter the periods of the observed rhythms but affected other rhythm parameters. Gonadotropin-releasing hormone increased the mesor of the rhythm and estradiol increased the amplitude. The results indicate that pituitary gonadotropes are capable of producing rhythms of LH release for a long duration in vitro, in the absence of hypothalamic control. Both GnRH and estradiol affect different rhythm parameters but do not change the periods of these rhythms.

Negative feedback on luteinizing hormone secretion requires infrequent exposure to testicular androgens

Abstract The present study tested the hypothesis that a minimum frequency of testosterone pulses is required to exert negative feedback control of luteinizing hormone (LH). Effect of gonadal steroids on endogenous LH release in rams was investigated after injecting ovine LH (NIADDK-oLH-26, i.v.; 5 μg 100kg−1 BW) to induce testosterone pulses while rams were undergoing windows of LH suppression during the influence of a LH releasing hormone (LHRH) antagonist (SB-75; Bajusz et al., 1988, Int. J. Peptide Protein Res., 32: 425–435). Patterns of LH release were characterized as rams recovered from LH inhibition. Rams were exposed to artificial photoperiods of 12 weeks of long days (16L:8D) followed by short days (8L:16D) to initiate reproductive activity. The study began during Week 5 of short days. Serial plasma samples were collected from six postpubertal rams (five Suffolks and one Hampshire) at 15-min intervals for 69.5 h. All rams were given LHRH antagonist (10 μg kg−1 BW, s.c.) at Hour 24 during each of three collection periods which took place on alternating weeks in a Latin square design. Rams were given a different exogenous oLH treatment (no oLH, one dose at 12 h after antagonist or three doses at 6-h intervals with initial dose 6 h after antagonist) during each collection period. Exogenous oLH decreased the number of LH pulses during the recovery period (Hours 48 to 72 of experiment; 24 to 48 h after antagonist; P

Role of endogenous opioid peptides in central glucocorticoid Receptor (GR)-induced decreases in circulating LH in the male rat

While it has been shown that intracerebral administration of exogenous glucocorticoids diminishes pituitary luteinizing hormone (LH) release, it is not known if these hormones act directly on gonadotropin-releasing hormone (GnRH)-synthesizing neurons, or if their central inhibitory effects are mediated by specific neural substrates. In the present study, we examined whether the opioid receptor antagonist, naltrexone (NALT), alters patterns of LH release elicited by either systemic or intracerebroventricular (i.c.v.) delivery of GR agonists. Subcutaneous (s.c.) injection of the GR agonist, RU362 (2.5 mg/kg), promoted a significant reduction in circulating LH levels; pretreatment by i.c.v. injection of 1.0 μg NALT, however, attenuated this inhibitory hormonal response. It was also found that rats treated sequentially with NALT and RU362 exhibited significantly lower plasma LH levels compared to rats injected with NALT alone. In other experiments, intracranial delivery of the synthetic glucocorticoid, dexamethasone (DEX), into either the ventricular system or the hypothalamic ARC resulted in significantly decreased plasma LH concentrations; the central inhibitory effects of DEX on peripheral LH release were reversed, however, by i.c.v. pretreatment with NALT. In summary, the present studies show that opioid receptor blockade attenuates systemic, as well as intracerebral inhibitory effects of GR agonists on the GnRH-pituitary LH axis, suggesting that circulating glucocorticoids inhibit LH, in part, through central actions involving endogenous opioid receptors. The observed decline in peripheral plasma LH following intra-ARC injection of DEX suggests that local GR may be functional target sites for glucocorticoid effects on LH. Since i.c.v. administration of NALT reversed the inhibitory LH response to these localized effects of DEX, opioid receptors may reside within neural pathways mediating the inhibitory effects of ARC GR on pituitary LH.

Effects of peripheral versus central administration of the endogenous glucocorticoid, corticosterone, and the glucocorticoid receptor agonist, RU 28362, on LH release in male rats

The current studies evaluated the effects of the synthetic glucocorticoid receptor (GR) agonist, RU 28362, and the endogenous, non-selective receptor ligand, corticosterone (Cort), on pituitary luteinizing hormone (LH) secretion in male rats. Steroids were injected subcutaneously (s.c.) in animals previously implanted with intracardiac venous catheters, or administered intracerebroventricularly (i.c.v.) to other groups of animals. A dose-proportionate decrease in plasma LH was observed following either s.c. or i.c.v. administration of RU 28362; pretreatment with the GR antagonist, RU 38486, blunted the inhibitory impact of RU 28362 on circulating LH. In other experiments, s.c. injection of Cort elicited divergent, dose-dependent patterns of LH release. While the lowest peripheral dose (0.25 mg Cort/kg) promoted a transient elevation in plasma LH, higher doses exerted a progressively greater inhibitory effect on hormone release. The suppressive effects of the highest s.c. dose (2.5 mg Cort/kg) were reversed by pretreatment with the RU 38486, but not by the mineralocorticoid receptor antagonist, RU 26752. Plasma LH levels were transiently elevated following i.c.v. administration of graded doses of Cort. The lowest dose (0.1 μg Cort/rat) only facilatated LH release, but higher doses (1.0 and 10.0 μg/animal) elicited a biphasic LH response, which was characterized by an initial elevation, then subsequent reduction in plasma LH below preinjection baseline levels. Prior administration of the mineralocorticoid receptor antagonist, RU 26752, attenuated the stimulatory impact of i.c.v. Cort on LH release, while both RU 26752 and RU 38486 reversed the secondary decline in plasma LH. In summary, the present studies provide evidence that GR are inhibitory to LH release and that glucocorticoids may act to diminish LH, in part, through mechanisms initiated within the CNS. Systemic administration of the non-selective ligand, Cort, resulted in divergent, dose-dependent effects on LH release; the inhibitory effects of high doses of this steroid are apparently mediated by GR. In contrast to the bidirectional effects of s.c. Cort, i.c.v. delivery of the glucocorticoid resulted in a transient elevation in plasma LH; attenuation of this increase in hormone release by prior administration of RU 26752 supports the involvement of Cort-preferring mineralocorticoid receptors (CR) in central facilitory actions of Cort. The current results suggest that CR and GR mediate divergent effects of circulating glucocorticoids on LH release, and that the dose-dependent LH response to systemic injections of Cort may reflect specific patterns of occupancy of high affinity CR versus low affinity GR.

Age-related changes in the pattern of luteinizing hormone release induced in female rats by male rat urine

The release of luteinizing hormone (LH) in young (3–4 months) and aged (24–25 months) ovariectomized female Sprague-Dawley rats, s.c. implanted with a 17β-estradiol benzoate silastic capsule, was studied in the presence and absence of stimulation by exposure to male rat urine. After taking an initial blood sample at 12:00 (reference sample), either urine, collected from young adult male rats, or distilled water was poured into the female's cage. Blood samples were then collected hourly up to 18:00 via a catheterized jugular cannula. The concentration of LH in the plasma was measured by RIA. The basal plasma LH level in young control rats was found to increase significantly at 16:00 compared with the 12:00 reference sample while no statistically significant change in plasma LH concentration occurred in old controls over the same period. Male rat urine caused a significantly earlier (at 15:00) and prolonged (from 15:00 to 18:00) elevation of plasma LH in young rats compared with young controls. In contrast, exposure of old female rats to male rat urine resulted in no marked change in plasma LH levels. These results suggest that both basal LH release and the response to pheromonal stimulation by male rat urine may be modified with increasing age in female rats.