Neurogenic bladder and neuroendocrine abnormalities in Pol III-related leukodystrophy.

BACKGROUND: Pol III-related leukodystrophies, including 4H leukodystrophy, are recently recognized disorders that comprise hypomyelination and various neurologic and non-neurologic clinical manifestations. We report the unique neurologic presentation of the micturition dysfunction in Pol III-related leukodystrophy and describe the novel endocrine abnormalities in this entity. CASE PRESENTATION: A 32-year-old Caucasian female exhibited chronic urinary incontinence that commenced at the age of 7 years and remained the unexplained symptom more than two decades before the onset of progressive neurologic decline. A transient growth failure and absent sexual development with hypoprolactinemia appeared in the meanwhile. Neurologic, endocrine, neuroradiologic, and genetic evaluation performed only in the patient's thirties, confirmed the diagnosis of 4H leukodystrophy as the only cause of the micturition disturbance. CONCLUSION: The report shows for the first time that an unexplained chronic bladder dysfunction should be evaluated also as a possible 4H leukodystrophy, thus alerting to the unexpected neurologic and endocrine features in 4H leukodystrophy.

Effect of polymeric nanoparticle poly(ethylene glycol)-block-poly(lactic acid) (PEG-b-PLA) on in vitro luteinizing hormone release from anterior pituitary cells of infantile and adult female rats.

OBJECTIVES: Polymeric PEG-b-PLA nanoparticles (NPs) were developed for delivery of poorly water-soluble drugs via blood brain barrier into brain parenchyma. We analyzed neuroendocrine disrupting effects of neonatal exposure of female rats to PEG-b-PLA NPs and diethylstilbestrol (DES) on the function of adenohypophyseal gonadotrophs of infantile or adult rats by examining in vitro luteinizing hormone releasing hormone (LHRH)-induced luteinizing hormone (LH) release. METHODS: Neonatal female Wistar rats were injected intraperitoneally, daily, from postnatal day (PND) 4 to PND7 with PEG-b-PLA NPs (20 mg.kg b.w.(-1)), DES (4 µg.kg b.w.(-1)) or vehicle. At the necropsy day (PND15 in infantile and the first estrus day after PND176 in adult rats), adenohypophyseal cells were isolated by enzymatic digestion, plated in 96-well plates (5×10(4) cells.well(-1)) in serum-supplemented medium and left to recover for 96 h. LHRH (10-7 mol.L(-1)) treatment was performed in serum-free medium for 60 min and LH levels in culture media were determined by radioimmunoassay. RESULTS: In all experimental groups, in vitro LHRH treatment significantly stimulated LH release from pituitary cells of infantile but not adult female rats. Neonatal DES treatment increased basal LH secretion from cultured pituitary cells of adult but not infantile rats. In both, infantile and adult rats, neonatal treatment with PEG-b-PLA significantly increased basal and LHRH-induced LH release from pituitary cells compared to corresponding controls and DES-treated group. CONCLUSIONS: Data indicate that neonatal exposure to PEG-b-PLA NPs may alter pituitary LH release, and thereby modify reproductive system development in infantile female rats leading to reproductive dysfunctions in adult age.

A factor analysis approach to examining relationships among ovarian steroid concentrations, gonadotrophin concentrations and menstrual cycle length characteristics in healthy, cycling women.

STUDY QUESTION: How are ovarian steroid concentrations, gonadotrophins and menstrual cycle characteristics inter-related within normal menstrual cycles? SUMMARY ANSWER: Within cycles, measures of estradiol production are highly related to one another, as are measures of progesterone production; however, the two hormones also show some independence from one another, and measures of cycle length and gonadotrophin concentrations show even greater independence, indicating minimal integration within cycles. WHAT IS KNOWN ALREADY: The menstrual cycle is typically conceptualized as a cohesive unit, with hormone levels, follicular development and ovulation all closely inter-related within a single cycle. Empirical support for this idea is limited, however, and to our knowledge, no analysis has examined the relationships among all of these components simultaneously. STUDY DESIGN, SIZE, DURATION: A total of 206 healthy, cycling Norwegian women participated in a prospective cohort study (EBBA-I) over the duration of a single menstrual cycle. Of these, 192 contributed hormonal and cycle data to the current analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Subjects provided daily saliva samples throughout the menstrual cycle from which estradiol and progesterone concentrations were measured. FSH and LH concentrations were measured in serum samples from three points in the same menstrual cycle and cycle length characteristics were calculated based on hormonal data and menstrual records. A factor analysis was conducted to examine the underlying relationships among 22 variables derived from the hormonal data and menstrual cycle characteristics. MAIN RESULTS AND THE ROLE OF CHANCE: Six rotated factors emerged, explaining 80% of the variance in the data. Of these, factors representing estradiol and progesterone concentrations accounted for 37 and 13% of the variance, respectively. There was some association between measures of estradiol and progesterone production within cycles; however, cycle length characteristics and gonadotrophin concentrations showed little association with any measure of ovarian hormone concentrations. LIMITATIONS, REASONS FOR CAUTION: Our summary measures of ovarian hormones may be imprecise in women with extremely long or short cycles, which could affect the patterns emerging in the factor analysis. Given that we only had data from one cycle on each woman, we cannot address how cycle characteristics may covary within individual women across multiple cycles. WIDER IMPLICATIONS OF THE FINDINGS: Our findings are generalizable to other healthy populations with typical cycles, however, may not be applicable to cycles that are anovulatory, extreme in length or otherwise atypical. The results support previous findings that measures of estradiol production are highly correlated across the cycle, as are measures of progesterone production. Estradiol and progesterone concentrations are associated with one another, furthermore. However factor analysis also revealed more complex underlying patterns in the menstrual cycle, highlighting the fact that gonadotrophin concentrations and cycle length characteristics are virtually independent of ovarian hormones. These results suggest that despite integration of follicular and luteal ovarian steroid production across the cycle, cycle quality is a multi-faceted construct, rather than a single dimension. STUDY FUNDING/COMPETING INTEREST(S): The EBBA-I study was supported by a grant from the Norwegian Cancer Society (49 258, 05087); Foundation for the Norwegian Health and Rehabilitation Organizations (59010-2000/2001/2002); Aakre Foundation (5695-2000, 5754-2002) and Health Region East. The current analyses were completed under funding from the National Institutes of Health (K12 ES019852). No competing interests declared.

Nimodipine, a calcium channel blocker, delays the spontaneous LH surge in women with regular menstrual cycles: a prospective pilot study.

BACKGROUND: Currently GnRH analogue injections are used to prevent premature LH surges in women undergoing assisted reproductive technology. This was a pilot study to determine the safety and effectiveness of nimodipine, an oral calcium channel blocker, to delay the mid-cycle spontaneous LH surge in women with regular menstrual cycles. METHODS: Eight women with regular menstrual cycles self-monitored three consecutive cycles for the day of an LH surge by daily urine assay. The first and third cycles were observatory. In the second cycle, subjects took nimodipine 60 mg by mouth three times daily for four days, starting two days prior to the expected LH surge day based on cycle one. RESULTS: The LH surge day in cycle 2 (nimodipine) was significantly delayed in comparison to both observatory cycle 1 (15.5+/-3.4 vs 14.0+/-2.8 days; p=0.033) and cycle 3 (15.1+/-3.5 vs 13.1+/-2.4 days; p=0.044). There was no difference in the LH surge day between the two observatory cycles (13.4+/-2.4 vs 13.1+/-2.4 days; p=0.457). Three patients experienced a mild headache. CONCLUSIONS: There was a statistically significant delay in the spontaneous LH surge day in the treatment cycle in comparison to both observatory cycles. Nimopidine should be further investigated as an oral alternative to delay a spontaneous LH surge.

In vitro growth and steroidogenesis of dog follicles are influenced by the physical and hormonal microenvironment.

The present study examined the influences of the physical and hormonal microenvironment on in vitro growth and steroidogenesis of dog follicles. Follicles were enzymatically isolated and individually encapsulated in 0.5% (w/v; n=17) or 1.5% (n=10) alginate and cultured with 0.5 IU/ml equine chorionic gonadotropin for 192 h. In a separate experiment, follicles were encapsulated in 0.5% alginate and cultured with 0 (n=22), 1 (n=23), 10 (n=20) or 100 (n=21) µg/ml FSH for 240 h. Follicle diameter and steroid production were assessed every 48 h in both studies. Follicles encapsulated in the 0.5% alginate grew faster (P<0.05) than those cultured in the 1.5% concentration. Oestradiol (E(2)) and progesterone (P(4)) increased consistently (P<0.05) over time, and follicles in the 1.5% alginate produced more (P<0.05) P(4) than those in the 0.5% solution. Follicles cultured in the highest FSH concentration (100 µg/ml) increased 100% in size after 240 h compared with 50 to 70% in lower dosages. E(2) concentration remained unchanged over time (P>0.05) across FSH dosages. However, P(4) increased (P<0.05) as culture progressed and with increasing FSH concentration. Results demonstrate that dog follicles cultured in alginate retain structural integrity, grow in size and are hormonally active. Lower alginate and increasing FSH concentrations promote in vitro follicle growth. However, the absence of an E(2) rise in follicles cultured in FSH alone suggests the need for LH supplementation to support theca cell differentiation and granulosa cell function.

Follicular-Stimulating Hormone, Luteinizing Hormone, and Prolactin Serum Level in Patients with Oral Lichen Planus in Comparison to Healthy Population.

MATERIALS AND METHODS: In this cross-sectional case control study, the serum level of LH, FSH, and prolactin of 40 women with lichen planus who have been referred to Shiraz Dental Faculty, Oral and Maxillofacial Disease Department during 2018-2019 has been evaluated in comparison to 40 healthy controls. Data were analyzed by SPSS version 18. Two-way ANOVA and Mann-Whitney test were used for data analysis. RESULTS: The mean serum level of FSH and LH was significantly higher in OLP patients while this difference was not reported for prolactin. Only FSH mean serum level was significantly higher in nonmenopausal OLP patients. The distribution of prolactin and FSH hormones' serum level was in normal range. CONCLUSIONS: The high serum level of FSH and LH can affect OLP pathogenesis by estrogen and progesterone modulation.

Assessment of the biopotency of follitropin alfa and lutropin alfa combined in one injection: a comparative trial in Sprague-Dawley rats.

BACKGROUND: The current study was designed to determine if follitropin alfa (recombinant human follicle-stimulating hormone; r-hFSH) and lutropin alfa (recombinant human luteinizing hormone; r-hLH) biopotencies were unchanged by reconstituting in sterile water for injection and mixing prior to injection. METHODS: The biopotencies of r-hFSH and r-hLH were determined following injection of female Sprague-Dawley rats with a mixture of follitropin alfa revised formulation female (RFF) and lutropin alfa (1:1, r-hFSH:r-hLH). Biopotencies of follitropin alfa RFF and lutropin alfa were measured using ovarian weight and ascorbic acid depletion assays, respectively, and compared with a reference standard. Stock mixtures of follitropin alfa RFF and lutropin alfa (1:1) were prepared within 1 h prior to each respective assay's injection and stored at 6 +/- 2 degrees C. Separate low dose (follitropin alfa RFF 1.5 IU/rat, lutropin alfa 2 IU/rat) and high dose (follitropin alfa RFF 3 IU/rat, lutropin alfa 8 IU/rat) treatments were prepared from stock mixtures or individual solutions by diluting with 0.22% bovine serum albumin saline solution and injected within 1 h of preparation. The main outcome measures were ovarian weight and ovarian ascorbic acid depletion. RESULTS: FSH bioactivities were similar (p > 0.10) between the individual follitropin alfa RFF test solution (84.2 IU) and follitropin alfa RFF/lutropin alfa (87.6 IU) mixtures prepared within 1 h of injection and stored at 6 +/- 2 degrees C. LH bioactivities were similar (p > 0.10) between lutropin alfa (94.7 IU) test solution and lutropin alfa/follitropin alfa RFF (85.3 IU) mixtures prepared within 1 h of injection and stored at 6 +/- 2 degrees C for not more than 1 h prior to injection. CONCLUSION: Mixing follitropin alfa RFF and lutropin alfa did not alter the bioactivity of either FSH or LH.

Influence of Hormones on Sjögren's Syndrome.

Sjögren's syndrome (SS) is an immune system oral disorder that is characterized generally by dry mouth and eyes. In this review, SS classification, presentation and pathogenesis are briefly discussed. Moreover, the epidemiology of SS regarding sex, age and association with other complications are also presented. This review also addresses the interactions between endocrine axes and SS, and the important findings up to regarding hormone treatment of this syndrome. The main hormones discussed in this review includes Adrenocorticotropic hormone (ACTH), Follicle-stimulating hormone (FSH), Luteinizing hormone (LH), Thyroid-stimulating hormone (TSH), and prolactin.

Ultrastructural and physico-chemical characterization of saliva during menstrual cycle in perspective of ovulation in human.

Human saliva is a potential diagnostic fluid and any alteration in body might be reflected in saliva so that saliva is considered as "mirror of the body". Variations in salivary hormone level, ultra structure, pH, flow rate, buffering capacity and electrolytes level are found during menstrual cycle in regard to ovulation. Thirty healthy volunteers were used for the assessment of physico-chemical changes in saliva. Reproductive cycle was categorized as pre-ovulation phase (5 to 12 days), ovulation phase (13 or 14 days) and post-ovulation phase (15 to 25 days) according to salivary arborization test and hormonal analysis. Estradiol and luteinizing hormone was gradually increased and attained peak at the level of 2.28 ± 0.20 pg/mL and 1.35 ± 0.41 mIU/mL respectively during the ovulation phase. The electrolytes result clearly indicates that the influx of common electrolytes is important for crystallization and help to induce clear ferning pattern in ovulation phase. Sodium (Na) and chloride (Cl) were found to be high during ovulation phase only. Average salivary pH was 7.5, 7.1, and 7.3 during ovulation, pre- and post-ovulation phases respectively. Buffering capacity of saliva was normal during pre- and post- ovulation phases. In contrast, in ovulation phase the buffer capacity was slightly higher. At the first time, the scanning electron microscopy (SEM) studies revealed the ultra structure difference of saliva during menstrual cycle. During ovulation phase a compact network-shaped mesh was appeared; such structure was not appeared in pre- and post ovulation phases. Additionally, we observed the saliva is arrayed as a fine mosaic-like structure during ovulation. Based on physico-chemical properties and hormonal levels may lead to develop a detection kit/sensor for detecting the ovulation phase in human.

Interleukin-1 Antagonism Decreases Cortisol Levels in Obese Individuals.

Context: Increased cortisol levels in obesity may contribute to the associated metabolic syndrome. In obesity, the activated innate immune system leads to increased interleukin (IL)-1ß, which is known to stimulate the release of adrenocorticotropin hormone (ACTH). Objectives: We hypothesized that in obesity IL-1 antagonism would result in downregulation of the hypothalamo-pituitary-adrenal axis, leading to decreased cortisol levels. Design and Participants: In this prospective intervention study, we included 73 patients with obesity (body mass index [BMI] ≥30 kg/m2) and at least one additional feature of the metabolic syndrome. Outcome Measures: The primary end point was change in morning cortisol from baseline to after the administration of the IL-1 receptor antagonist (anakinra/Kineret®, total dose 3 × 100 mg). Secondary end points were effects on salivary cortisol and ACTH. Results: Median age was 56 years, 50.7% of patients were female, and median BMI was 36.3 kg/m2. Median morning serum cortisol levels (nmol/L) decreased significantly after IL-1 antagonism [from baseline, 452 to 423; absolute difference, -38.7; 95% confidence interval (CI), -64 to -13.4; P = 0.0019]. Similar effects were found for salivary cortisol levels (-2.8; 95% CI, -4.4 to -1.3; P = 0.0007), ACTH levels (-2.2; 95% CI; -4.2 to -0.1; P = 0.038), systolic blood pressure (-5.2, 95% CI, -8.5 to -1.8; P = 0.0006), and heart rate (-2.9; 95% CI, -4.7 to -1.0; P = 0.0029). Conclusion: IL-1 antagonism in obese individuals with features of the metabolic syndrome leads to a decrease in serum cortisol, salivary cortisol, and ACTH levels along with a reduction in systolic blood pressure and heart rate.

A splice variant of the human luteinizing hormone (LH) receptor modulates the expression of wild-type human LH receptor.

We previously reported a splice variant form of human LH receptor [hLHR(exon 9)] that lacks exon 9, coding the N-terminal extracellular region close to the first transmembrane domain. Several recent studies suggest that G protein-coupled receptors are able to form dimerization or oligomerization of the receptor, suggesting an intermolecular interaction between hLHR(exon 9) and the wild-type LH receptor (hLHR). The aim of this study, using coimmunoprecipitation, is to examine whether hLHR forms an association with hLHR(exon 9). An interaction between hLHR(exon 9) with the immature band (68 kDa) of hLHR and not with the mature band (85 kDa) was seen. When hLHR and hLHR(exon 9) were coexpressed, the density of hLHR expression was significantly reduced, compared with hLHR expressed alone. The human chorionic gonadotropin-stimulated cAMP accumulation in the cells expressing hLHR(exon 9) was also impaired, compared with the cells expressing hLHR. In this study, we demonstrated that hLHR is capable of forming receptor complexes. Our findings may expand the possibility of a splice variant of hLHR specifically modulating the functional property of the wild-type hLHR.

Effects of progesterone on estrogen-induced gonadotropin release in male rhesus macaques.

The effects of progesterone (P) on estrogen (E)-induced gonadotropin release were studied in 10 adult male rhesus macaques castrated more than 2 yr earlier. The intent was to determine whether physiological levels of P (approximately 400 pg/ml) found in the systemic circulation of intact males would block E-induced gonadotropin release and whether the responses of castrated males were similar to those of castrated females with and without pretreatment with 17 beta-estradiol (E2). Different doses of P were administered in Silastic capsules (0.3, 4.0, and 5.0 cm) implanted sc. A 0.3-cm implant maintained serum P levels at about 400 pg/ml (equivalent to physiological levels in intact males); 5.0-cm implants produced serum levels of about 4.0 ng/ml (similar to luteal phase levels in females). In male monkeys treated for approximately 3 weeks with E2, only the highest dose (approximately 4.0 ng/ml) of P blocked FSH induced by estradiol benzoate (E2B). LH was blocked in one third of the animals thus treated. The same P dose was ineffective in blocking E2-induced LH release in spayed females pretreated with E2, but did block FSH release. Gonadectomized males and females not treated beforehand with E2 released LH in response to an E2B challenge, but FSH was not elevated in the peripheral circulation under these experimental conditions. These results suggest that luteal phase levels of P block E2-induced FSH release in gonadectomized males and females. With the same treatment regimens, P blocks E2 action in some males, but all females responded to E2B by releasing LH. These data also suggest that estrogen priming is necessary for FSH, but not LH, release in adult rhesus macaques of both sexes. The prerequisite of E treatment for the induction of positive feedback appears to be associated with the level of gonadotropin suppression before E2B treatment.

Evidence that neurotensin participates in the central regulation of the preovulatory surge of luteinizing hormone in the rat.

Neurotensin (NT) has been implicated in the central regulation of LH and PRL secretion in the rat. We investigated the importance of NT release to the neural events that trigger the preovulatory LH surge and coincident PRL surge, using as our animal model ovariectomized (OVX) rats treated with estrogen and progesterone to induce reliable and robust surges. To interfere with the action of endogenous NT in the basal forebrain, we administered a NT antiserum (NTAS) in a series of bilateral microinjections aimed at the anterior border of the medial preoptic area. One week after OVX, rats bearing cerebral guide cannulae received Silastic capsules (3 x 15 mm; sc) containing 17 beta-estradiol. Two days later, beginning at 0830 h, conscious rats were administered either NTAS or control serum bilaterally in a series of four 100-nl injections spaced at 30-min intervals. After an initial blood sample, rats received progesterone (4 mg, sc) at 1200 h; blood samples were then taken at 1-h intervals from 1400-2100 h. Blood samples were obtained from conscious, freely moving rats via a chronic atrial catheter implanted previously. Plasma levels of LH and PRL were measured by RIA, and the location of microinjection sites was verified histologically. Administration of NTAS caused a 66% reduction in the magnitude of the LH surge without altering its timing, whereas the PRL surge was unaffected. These results provide strong evidence that NT in the basal forebrain participates in the steroid-induced LH surge and suggest that NT plays a role in the preovulatory LH surge.

Development of gonadotrope desensitization to gonadotropin-releasing hormone (GnRH) and recovery are not coupled to inositol phosphate production or GnRH receptor number.

After initial GnRH pretreatment (10 nM, 5 h), subsequent GnRH-stimulated LH release from the gonadotrope was diminished (1 microM GnRH stimulated release of 36.4 +/- 1.4% total cellular LH over 3 h in cells initially pretreated with medium alone compared to 27.4 +/- 1.2% in GnRH-pretreated cells); however, inositol phosphate (IP) production in response to the releasing hormone remained unaffected (1 microM GnRH provoked IP accumulation of 161 +/- 9% above basal levels after 45 min in control cells and 162 +/- 11% in GnRH-pretreated cells). Pretreatment of pituitary cell cultures with NaF (a guanyl nucleotide binding protein activator, 10 mM, 3 h) also decreased subsequent GnRH-stimulated LH release, and in addition, provoked a decrease in GnRH receptor number, an increase in GnRH receptor affinity, reduction of GnRH-stimulated IP production to basal levels, and an increase in the amount of LH released in response to stimulation with the calcium ionophore A23187. In order to determine if the changes in LH release were a result of decreased IP production and/or decreased GnRH receptor binding, the time course of recovery to control levels of these processes was assessed. GnRH receptor binding continued to decrease after NaF pretreatment, reaching a nadir (62% of control) at 6 h after the pretreatment period and recovering at 48 h (90% of control). In contrast, GnRH-provoked IP accumulation did not return to control levels even after 48 h of recovery after NaF pretreatment (1 microM GnRH-stimulated IP accumulation in NaF-pretreated cells was 57% compared to control cells after 48 h of recovery). GnRH-stimulated LH release was inhibited immediately after NaF pretreatment (1 microM GnRH-stimulated LH release in NaF-pretreated cells was 65% of control levels). Cells began to recover within 3 h (80% of control) and were almost completely recovered by 6 h (90% of control). A23187-provoked LH release was enhanced immediately after NaF pretreatment (30 microM A23187-stimulated LH release in NaF-pretreated cells was 170% of control levels). Responsiveness to ionophore was 133% of control by 0.5 h, and complete recovery was measured within 1 h (100% of control). Furthermore, both NaF and GnRH pretreatment still provoked a decrease in gonadotrope responsiveness when IP production was inhibited by the phospholipase C inhibitor U-73122. The results suggest that the development of gonadotrope desensitization (by either NaF or GnRH pretreatment) can be uncoupled from changes in IP production.(ABSTRACT TRUNCATED AT 400 WORDS)

Increase in ubiquitin-immunoreactive nuclei in rat pituitary luteinizing hormone cells after castration.

Immunocytochemical detection of ubiquitin in the nucleus of rat LH cells and the effects of castration and testosterone replacement on the occurrence of immunoreactive ubiquitin in the nucleus were investigated. Immunoreactive ubiquitin occurred in certain nuclei, mostly belonging to identified LH cells. The concentration of testosterone in blood was altered by castration and implantation of testosterone into castrated rats, and the occurrence of ubiquitin was examined weekly for the following 4 weeks. In castrated rats, the proportion of LH cells with ubiquitin-immunoreactive nuclei was high throughout the experiment. In castrated rats implanted with testosterone, on the contrary, the proportion remained significantly lower. Ubiquitin may be involved in the cellular activity of LH cells in the rat pituitary.

A simple and rapid method using polyethylenimine-treated filters for assay of solubilized LH/hCG receptors.

Filtration through polyethylenimine (PEI)-treated membrane filters efficiently separates free radiolabeled human (h) LH or hCG from solubilized receptor-hormone complexes. Solubilized LH/hCG receptors of bovine, porcine, and rat origin are suitable for measurement by this method. Specific binding assayed by the PEI-filter technique was 106-138% of specific binding assayed by double polyethylene glycol precipitation, demonstrating that the method gave quantitative recovery of bound hormone. Nonspecific binding was consistently lower with PEI-treated filters. Intra- and interassay variability for the two different methods was similar. These results indicate that filtration through PEI-treated membrane filters is a simple and rapid method for assay of solubilized LH/hCG receptors.

Cholera toxin and pertussis toxin provoke differential effects on luteinizing hormone release, inositol phosphate production, and gonadotropin-releasing hormone (GnRH) receptor binding in the gonadotrope: evidence for multiple guanyl nucleotide binding proteins in GnRH action.

A growing body of evidence suggests a role for guanyl nucleotide binding proteins (G proteins) in GnRH action. G protein activation provokes LH release, inositol phosphate (IP) production, decreased gonadotrope responsiveness to GnRH, increased gonadotrope responsiveness to the calcium ionophore A23187, and decreased GnRH receptor binding. The specific G proteins involved in these actions, however, are not known. This study uses pertussis toxin (PTX) and cholera toxin (CTX), which affect the activity of a number of G proteins by ADP ribosylation of a Cys or an Arg residue, respectively, of the alpha-subunit. Although not an effective LH secretagogue in itself, CTX enhanced GnRH-, NaF-, and A23187-stimulated LH release after an 18-h pretreatment period. CTX pretreatment did not affect GnRH- or NaF-stimulated IP production. Conversely, 18 h pretreatment with PTX reduced GnRH- and NaF-provoked IP production compared to control values, but did not affect LH release. In addition, pretreatment with either CTX, PTX, or Bt2cAMP provoked a decrease in GnRH receptor binding compared to control. The results of this study suggest that: 1) GnRH stimulates IP production, but not LH release, through a PTX-sensitive G protein; 2) A distinct CTX-sensitive G protein appears to provoke gonadotrope sensitization by stimulating an increase in intracellular cAMP levels; and 3) there appears to be a distinct G protein, insensitive to PTX and CTX, capable of mediating LH release independent of IP production and cAMP.

Gonadotropin-releasing hormone-stimulated intracellular Ca2+ fluctuations and luteinizing hormone release can be uncoupled from inositol phosphate production.

In order to study the dependence of GnRH-stimulated LH release on inositol phosphate (IP) turnover, this study used an inhibitor of phospholipase C activity, 1-[6-[[17 beta-3- methoxyestra-1,3,5(10)-triene-17-yl]amino]hexyl]-1H-pyrrole-dione (U-73122) and an inactive analog 1-[6[[17 beta-3-methoxyestra-1,3,5(10)- triene-17-yl]amino]hexyl]2,5-pyrrolidine-dione (U-73343). U-73122 (10 microM) decreased GnRH-provoked (1 microM, 45 min) IP accumulation from 873 +/- 61 dpm to 365 +/- 50 dpm (basal accumulation also was decreased from 420 +/- 18 dpm to 207 +/- 16 dpm) while LH release was not inhibited (30.2 +/- 1.4% of cellular LH in control compared to 30.3 +/- 1.1% in U-73122 pretreated cells). GnRH provoked increased IP3 accumulation (123% of basal) after 15 sec of stimulation, IP2 accumulation (131% of basal) after 30 sec, and IP1 (121% of basal) after 1 min. Pretreatment with U-73122 blocked accumulation of IPs at these early timepoints. Sodium fluoride (NaF)-stimulated IP accumulation was also inhibited by U-73122 (from 1539 +/- 132 dpm to 414 +/- 21 dpm) while LH release increased from 22.9 +/- 1.4% total cellular LH to 28.0 +/- 2.2%. In contrast, GnRH- and NaF-stimulated IP accumulation were not significantly decreased in U-73343 pretreated cells (GnRH: 817 +/- 43 dpm compared to 873 +/- 61 dpm in control; NaF: 1133 +/- 74 dpm compared to 1539 +/- 132 dpm in control cells). Results of a perifusion study showed that U-73122 did not block the initial phase of GnRH-stimulated LH release or interfere with the development of desensitization to the releasing hormone. In addition, GnRH-stimulated intracellular Ca2+ fluctuations were similar in magnitude and duration in U-73122-pretreated compared to U-73343-pretreated cells. These results demonstrate that GnRH- as well as NaF-stimulated LH release can be uncoupled from IP production calling to question the role of IP3 as a second messenger for GnRH-stimulated LH release.

Steroid control of gonadotropin secretion and ovarian function in heifers.

The failure of anti-steroid treatments to induce multiple ovulations in cattle in a repeatable way, prompted us to examine the role of the gonadal steroids in the control of gonadotropin secretion in this species. A better understanding of the control of gonadotropin secretion in the cow would assist in the development of treatments to control prolificacy. Groups of six heifers were implanted with one of three sizes of estradiol (E2) implant on day 9 of a synchronized estrous cycle, and five control heifers received empty implants. All heifers were ovariectomized during the luteal phase of the subsequent estrous cycle and given progesterone-releasing intravaginal devices (PRID). Blood samples were taken every 10 min for 12 h with PRIDs and for 6 h after PRID withdrawal, for the measurement of LH and FSH concentrations. The two larger implant sizes (increasing E2 concentrations to 7.4 and 18.7 pg/ml plasma during the luteal phase of the cycle) decreased ovulation rate, the number of large follicles, and luteal weight. After ovariectomy, the three implant sizes produced E2 concentrations comparable with those during the luteal and follicular phases of the estrous cycle and at estrus (1.5, 4.4, and 10.5 pg/ml, respectively). E2 alone decreased mean LH and FSH concentrations and LH pulse amplitude, whereas progesterone alone reduced mean gonadotropin concentrations and LH pulse frequency. Only in the presence of progesterone did E2 decrease LH pulse frequency. Steroid concentrations which mimicked those of the luteal and follicular phases of the cycle produced luteal- and follicular-phase patterns of LH and FSH secretion. These results confirm that E2 and progesterone are important regulators of gonadotropin secretion in cattle, and question the role of inhibin in this respect.

Stimulation of luteinizing hormone release by sodium fluoride is independent of protein kinase-C activity and unaffected by desensitization to gonadotropin-releasing hormone.

GnRH stimulates secretion of pituitary LH by increasing intracellular calcium. Increased calcium may result from activation of phospholipase-C, since there is an increase in inositol phosphates and diacylglycerol, and a redistribution of protein kinase-C (PKC) from cytosolic to a particulate cell fraction in GnRH-stimulated pituitary cultures. A GTP-binding protein (G-protein) may mediate GnRH actions, since GTP stimulates LH release in permeabilized gonadotropes and decreases receptor affinity for a GnRH analog. In the present study we have used sodium fluoride, an exogenous activator of G-proteins, to investigate the possibility of a G-protein link between GnRH receptor activation, phospholipase-C activity, and LH release. Treatment of primary pituitary cell cultures from immature female rats with sodium fluoride stimulated the release of 20% total cellular LH and increased inositol phosphate accumulation. Sodium fluoride-stimulated LH release was insensitive to cholera toxin and pertussis toxin. Sodium fluoride-stimulated LH release was additive with a maximally effective concentration of phorbol 12-myristate 13-acetate and was not inhibited by depletion of cellular PKC, suggesting that PKC does not mediate sodium fluoride effects. Treatment of cultures with 3 mM EGTA and 10 nM GnRH for 5 and 16 h reduced pituitary responsiveness to subsequent treatment with GnRH, but had no effect on sodium fluoride-stimulated LH release. Although the precise mechanism of sodium fluoride-stimulated LH release remains to be described, our results support a role for a G-protein in regulation of LH release by the releasing hormone.