FSH Response Research Articles

Follicle-Stimulating Hormone Responses to Kisspeptin in the Female Rat at the Preovulatory Period: Modulation by Estrogen and Progesterone Receptors

Ovulation is triggered by the preovulatory surge of gonadotropins that, in rodents, is defined by the concomitant rise in circulating LH and FSH at the afternoon of proestrus (primary surge), followed by persistently elevated FSH levels at early estrus (secondary surge). In recent years, kisspeptins, products of the KiSS-1 gene that act via G protein-coupled receptor 54, have emerged as an essential hypothalamic conduit for the generation of the preovulatory LH surge by conveying positive feedback effects of estradiol onto GnRH neurons, an event that involves not only estradiol-induced transcription of the KiSS-1 gene at the anteroventral periventricular nucleus but also its ability to modulate GnRH/LH responses to kisspeptin. However, little is known about the potential modulation of FSH responsiveness to kisspeptin by sex steroids in the cyclic female. We report herein analyses on the consequences of selective blockade of estrogen receptors (ER)-alpha and -beta, as well as progesterone receptor (PR), on the ovulatory surges of FSH and their modulation by kisspeptin. Antagonism of ERalpha or PR equally blunted the primary and secondary surges of FSH and nullified FSH responses to kisspeptin at the preovulatory period. Conversely, selective blockade of ERbeta failed to induce major changes in terms of endogenous FSH surges, yet it decreased FSH responses to exogenous kisspeptin. In contrast, FSH responses to GnRH were fully conserved after ERbeta blockade and partially preserved after inhibition of ERalpha and PR signaling. Finally, secondary FSH secretion was rescued by kisspeptin in females with selective blockade of ERalpha but not PR. In sum, our results substantiate a concurrent, indispensable role of ERalpha and PR in the generation of FSH surges and the stimulation of FSH responses to kisspeptin at the ovulatory period. In addition, our data suggest that ERbeta might operate as a subtle, positive modulator of the preovulatory FSH responses to kisspeptin, a role that is opposite to its putative inhibitory action on kisspeptin-induced LH secretion and might contribute to the dissociation of gonadotropin secretion at the ovulatory phase in the cyclic female rat.

Acylated Ghrelin Inhibits Spontaneous Luteinizing Hormone Pulsatility and Responsiveness to Naloxone But Not That to Gonadotropin-Releasing Hormone in Young Men: Evidence for a Central Inhibitory Action of Ghrelin on the Gonadal Axis

Recent evidence suggests that ghrelin exerts a negative modulation on the gonadal axis. Ghrelin was reported to suppress LH secretion in both animal and human models. Moreover, acylated ghrelin (AG) also decreases the LH responsiveness to GnRH in vitro. The objective of the study was to evaluate the effects of AG infusion on spontaneous and stimulated gonadotropin secretion. In seven young healthy male volunteers (age mean +/- sem 26.4 +/- 2.6 yr), we evaluated LH and FSH levels every 15 min during: 1) iv isotonic saline infusion; 2) iv saline followed by AG; LH and FSH response to GnRH (100 microg iv as a bolus), 3) alone and 4) during AG infusion; LH and FSH response to naloxone (0.1 mg/kg iv as a slow bolus), 5) alone and 6) during AG infusion. Significant LH but not FSH pulses were recorded in all subjects under saline infusion. AG infusion inhibited LH levels [area under the curve((240-480)): 415.8 +/- 69.7 mIU/ml.min during AG vs. 744.6 +/- 120.0 mIU/ml.min during saline, P < 0.02] and abolished LH pulsatility. No change in FSH secretion was recorded. The LH and FSH responses to GnRH during saline were not affected by AG administration. However, AG inhibited the LH response to naloxone [area under the curve ((120-210)): 229.9 +/- 39.3 mIU/ml.min during AG vs. 401.1 +/- 44.6 mIU/ml.min during saline, P < 0.01]. FSH levels were not modified by naloxone alone or in combination with AG. AG inhibits both spontaneous LH pulsatility and the LH response to naloxone. Because AG does not affect the LH response to GnRH, these findings indicate that the ghrelin system mediates central inhibition of the gonadal axis.

Peripheral Kisspeptide Administration Elicits an Increase in Serum FSH in the Diestrous Mare.

Kisspeptin (KiSS) has been demonstrated to elicit LH and, in some cases, FSH release in several species. The proposed hierarchical role of KiSS stems from the ability of GnRH antagonists to mitigate a KiSS initiated pituitary release of LH and identification of KiSS immunoreactive neurons associated with GnRH neurons in hypothalamic nuclei considered key to reproductive neuroendocrinology. Elucidating the role of KiSS in a seasonal breeder such as the equine is not only important to reveal the underlying physiology of vernal transition, but could potentially impact a multi-million dollar industry. The objective of this study was to evaluate the change in peripheral FSH in diestrous mares (n=12) following three doses of rat KiSS decapeptide (1.0 µg, 0.5 mg, 1.0 mg KiSS-10, iv). To ensure pituitary responsiveness, a challenge of GnRH (250 µg, iv) was administered 4 h after treatment. Jugular blood samples were collected hourly from 2 h before to 4 h after treatment administration, and one hour after GnRH challenge. Serum concentrations of FSH were analyzed by RIA. Data were analyzed by ANOVA in a Latin Square design with repeated measures, such that each mare received each of the three treatments, allowing for a 48 h washout period between treatment days. KiSS-10 elicits (P < 0.05) a peak FSH response within one hour of treatment at doses of 0.5 mg (mean ± SEM, 185 ± 30 vs. 98 ± 9 ng/ml) and 1.0 mg (177 ± 41 vs. 97 ± 13 ng/ml), leading to almost a fold increase in serum FSH as compared to the pre-treatment period, respectively. However, the lowest dose of KiSS-10 (1.0 µg) did not lead to an increase in serum FSH as compared to the pre-treatment period (123 ± 18 vs. 120 ± 17 ng/ml). All mares were responsive to GnRH challenge (247 ± 25 ng/ml vs. 99 ± 8 ng/ml) and the magnitude of the serum FSH release following GnRH challenge was similar to that induced by 0.5 mg and 1.0 mg KiSS-10. The FSH data are consistent with our previous data demonstrating an increase in serum LH following peripheral KiSS-10 administration in the diestrous mare at the doses used in this study. Studies are currently underway to determine the potential effect of KiSS-10 on primary cultures of equine pituitary cells such that we may further our understanding of KiSSs role in the equine hypothalamic pituitary gonadal axis. This research was generously supported by the American Quarter Horse Foundation and the Preservation for Equine Genetics Foundation at Colorado State University.

Desensitization of gonadotropin responses to kisspeptin in the female rat: analyses of LH and FSH secretion at different developmental and metabolic states

Kisspeptins have emerged as potent elicitors of gonadotropin secretion and, therefore, putative targets for pharmacological intervention. In this context, desensitization of gonadotropin responses to continuous administration of kisspeptins has begun to be characterized, but information so far available is mostly restricted to LH responses in males, whereas the similar phenomenon in females, of obvious therapeutic interest, remains virtually unexplored. We report herein LH and FSH responses to continuous intracerebral administration of kisspeptin in female rats at different developmental and metabolic states. Infusion of kisspeptin-10 to adult female rats induced a transient elevation in serum LH concentrations, followed by a precipitous drop and normalization of LH levels thereafter. Elevation of LH after kisspeptin infusion was prolonged in underfed animals; a phenomenon mimicked by leptin administration. Conversely, FSH levels were persistently heightened along continuous kisspeptin infusion, but duration of this response was shortened by undernutrition. In pubertal females, LH and FSH levels remained elevated at the end of a 7-day infusion of kisspeptin; responses whose magnitude was augmented by subnutrition but not mimicked by leptin. In all settings, terminal gonadotropin-releasing hormone responses were fully preserved, suggesting that eventual desensitization must occur upstream from the pituitary. In summary, our current data document the pharmacological consequences of continuous administration of kisspeptin to female rats, with remarkable differences being detected between LH and FSH responses, in different developmental and metabolic states. These observations of potential pharmacological interest might help also to delineate the physiological roles of kisspeptins in the dynamic regulation of gonadotropin secretion in the female.

Developmental Programming: Impact of Prenatal Testosterone Excess on Pre- and Postnatal Gonadotropin Regulation in Sheep1

The goal of this study was to explore mechanisms that mediate hypersecretion of LH and progressive loss of cyclicity in female sheep exposed during fetal life to excess testosterone. Our working hypothesis was that prenatal testosterone excess, by its androgenic action, amplifies GnRH-induced LH (but not FSH) secretion and, thus, hypersecretion of LH in adulthood, and that this results from altered developmental gene expression of GnRH and estradiol (E2) receptors, gonadotropin subunits, and paracrine factors that differentially regulate LH and FSH synthesis. We observed that, relative to controls, females exposed during fetal life to excess testosterone, as well as the nor-aromatizable androgen dihydrotestosterone, exhibited enhanced LH but not FSH responses to intermittent delivery of GnRH boluses under conditions in which endogenous LH (GnRH) pulses were suppressed. Luteinizing hormone hypersecretion was more evident in adults than in prepubertal females, and it was associated with development of acyclicity. Measurement of pituitary mRNA concentrations revealed that prenatal testosterone excess induced developmental changes in gene expression of pituitary GnRH and E2 receptors and paracrine modulators of LH and FSH synthesis in a manner consistent with subsequent amplification of LH release. Together, this series of studies suggests that prenatal testosterone excess, by its androgenic action, amplifies GnRH-induced LH response, leading to LH hypersecretion and acyclicity in adulthood, and that this programming involves developmental changes in expression of pituitary genes involved in LH and FSH release.

Toward gene therapy of primary ovarian failure: adenovirus expressing human FSH receptor corrects the Finnish C566T mutation

Resistance ovarian syndrome is a heterogeneous disorder inherited as a Mendelian recessive trait and characterized by infertility, primary amenorrhea, normal karyotype and elevated serum FSH and LH levels. An inactivating mutation, C566T, in FSH receptor gene (FSHR) has been identified initially in Finland. We investigated if an adenovirus expressing a normal copy of human FSHR (Ad-hFSHR) has the ability to: (i) transfect granulosa cell lines, (ii) render the transfected cell lines responsive to FSH stimulation and (iii) transcomplement the malfunctioning form of human FSHR gene with C566T mutation. COS-7, JC-410, JC-410-P450-scc-luc and JC-410-StAR-luc cell lines were infected by Ad-hFSHR followed by treatment with FSH. Functional activity of the Ad-hFSHR was tested by measuring cyclic adenosine monophosphate (cAMP) or luciferase activity in response to FSH stimulation, and showed 2-4.6-fold increases in Ad-hFSHR transfected cells compared with untransfected or Ad-LacZ transfected cells, indicating that Ad-hFSHR is functionally active and expressing hFSHR. Generation of cAMP in cells expressing only mutated hFSHR-T566 showed minimal increase after FSH stimulation. Co-transfection of Ad-hFSHR in these cells carrying the malfunction form of human FSHR caused significant increases of 2.2-7.4-fold in FSH dependent cAMP generation (P = 0.0007). We concluded that adenovirus expressing a normal human FSHR can compensate the inactivating human FSHR-C566T mutation and restore FSH responsiveness.

Translational Fusion of Two β-Subunits of Human Chorionic Gonadotropin Results in Production of a Novel Antagonist of the Hormone

The strategy of translationally fusing the alpha- and beta-subunits of human chorionic gonadotropin (hCG) into a single-chain molecule has been used to produce novel analogs of hCG. Previously we reported expression of a biologically active single-chain analog hCGalphabeta expressed using Pichia expression system. Using the same expression system, another analog, in which the alpha-subunit was replaced with the second beta-subunit, was expressed (hCGbetabeta) and purified. hCGbetabeta could bind to LH receptor with an affinity three times lower than that of hCG but failed to elicit any response. However, it could inhibit response to the hormone in vitro in a dose-dependent manner. Furthermore, it inhibited response to hCG in vivo indicating the antagonistic nature of the analog. However, it was unable to inhibit human FSH binding or response to human FSH, indicating the specificity of the effect. Characterization of hCGalphabeta and hCGbetabeta using immunological tools showed alterations in the conformation of some of the epitopes, whereas others were unaltered. Unlike hCG, hCGbetabeta interacts with two LH receptor molecules. These studies demonstrate that the presence of the second beta-subunit in the single-chain molecule generated a structure that can be recognized by the receptor. However, due to the absence of alpha-subunit, the molecule is unable to elicit response. The strategy of fusing two beta-subunits of glycoprotein hormones can be used to produce antagonists of these hormones.

Differential Effects of Various Estradiol-17beta Treatments on Follicle-Stimulating Hormone Peaks, Luteinizing Hormone Pulses, Basal Gonadotropin Concentrations, and Antral Follicle and Luteal Development in Cyclic Ewes1

In a previous study, 10-day estradiol implant treatment truncated the FSH peaks that precede follicular waves in sheep, but subsequent ovine FSH (oFSH) injection reinitiated wave emergence. The present study's objectives were to examine the effects of a 20-day estradiol and progesterone treatment on FSH peaks, follicle waves, and responsiveness to oFSH injection. Also, different estradiol doses were given to see whether a model that differentially suppressed FSH peaks, LH pulses, or basal gonadotropin secretion could be produced in order to study effects of these changes on follicular dynamics. Mean estradiol concentrations were 11.8 +/- 0.4 pg/ml, FSH peaks were truncated, wave emergence was halted, and the number of small follicles (2-3 mm in diameter) was reduced (P < 0.05) in cyclic ewes given estradiol and progesterone implants (experiment 1). On Day 15 of treatment, oFSH injection failed to induce wave emergence. With three different estradiol implant sizes (experiment 2), estradiol concentrations were 5.2, 19.0, 27.5, and 34.8 (+/-4.6) pg/ml in control and treated ewes, respectively. All estradiol treatments truncated FSH peaks, except those that created the highest estradiol concentrations. Experiment 2-treated ewes had significantly reduced mean and basal FSH concentrations and LH pulse amplitude and frequency. We concluded that 20-day estradiol treatment truncated FSH peaks, blocking wave emergence, and reduced the small-follicle pool, rendering the ovary unresponsive to oFSH injection in terms of wave emergence. Varying the steroid treatment created differential FSH peak regulation compared with other gonadotropin secretory parameters. This provides a useful model for future studies of the endocrine regulation of ovine antral follicular dynamics.

Oxytocin is not important for the control of gonadotrophin secretion in the late follicular phase of the cycle

To study the role of oxytocin in basal and GnRH-induced gonadotrophin secretion in normal women. Normal women were studied in three cycles. When the diameter of the leading follicle was 15-16 mm, the women were infused intravenously (i.v.) for 3 h with normal saline (cycle 1), atosiban (cycle 2) or oxytocin (cycle 3). The study included 12 normally cycling women aged 23-38 years. After cessation of treatment, two injections of GnRH, 10 microg each, were administered i.v. 2 h apart and blood samples were collected every 30 min for a total of 240 min. The 30-min pituitary response (sensitivity) to a single GnRH injection (10 microg i.v.) was investigated thereafter every 12 h from the end of the 3-h infusion until the day of LH surge onset. No significant differences in LH and FSH response to GnRH (sensitivity and reserve) during the 240-min experiment were found between the three cycles. The time of LH surge onset from the initiation of the infusion was similar in the three cycles. Also similar in the three cycles were oestradiol (E2) and gonadotrophin levels as well as the 30-min response to GnRH for 48 h following the 3-h infusion. The present study demonstrates that neither exogenous oxytocin administration nor blockage of endogenous oxytocin action influences pituitary sensitivity to GnRH in cycling women.

Investigation of hypothalamo-pituitary-gonadal axis and glucose intolerance among the first-degree female relatives of women with polycystic ovary syndrome

To assess the glucose intolerance and the hypothalamo-pituitary-gonadal (HPG) axis in the first-degree relatives (FDRs) of patients with polycystic ovary syndrome (PCOS). Clinical study of the female FDRs of patients with PCOS. Outpatients at endocrinology department of a university hospital. Seventy FDRs of PCOS patients and 20 healthy women were evaluated. Basal hormonal investigations, buserelin test, and 75-g oral glucose tolerance test (OGTT) were performed. Basal hormone levels were measured. Additionally, insulinogenic index, glucose and insulin responses to OGTT, and FSH, LH, E(2), and 17-OHP responses to buserelin test were obtained. Four (5.7%) of the FDRs showed impaired glucose tolerance, and the FDRs had significantly higher fasting plasma glucose (P<.05) and basal insulin (P<.01) than control subjects. Peak and area-under-the-curve (AUC) insulin (P<.001) and AUC glucose (P<.05) responses to OGTT were also significantly higher in the FDRs than in control subjects. The FDRs showed higher insulinogenic index than the control subjects (P<.001). The FDRs had significantly (P<.05) higher LH and DHEAS levels and lower (P<.005) 17-OHP levels than the control subjects. Peak (P<.05) and AUC (P<.01) LH responses to buserelin testing were lower in the FDRs than in the control subjects while peak (P<.05) and AUC (P<.01) E(2) responses to buserelin were higher in the FDRs than in the control subjects. These data support the hypothesis that FDRs of PCOS patients may have insulin resistance and the HPG axis is more susceptible than in control subjects. The FDRs also have an increased prevalence of hyperandrogenism and high DHEAS levels compared with the background population.

Reversible Kallmann syndrome: report of the first case with a KAL1 mutation and literature review

Kallmann syndrome (KS) describes the association of isolated hypogonadotropic hypogonadism with hypo/anosmia. A few KS patients may reverse hypogonadism after testosterone withdrawal, a variant known as reversible KS. Herein, we describe the first mutation in KAL1 in a patient with reversible KS and review the literature. The proband was first seen at 22 years complaining of anosmia and lack of puberty. His brother had puberty at 30 years and a maternal granduncle had anosmia and delayed puberty. On physical examination, he was P(2)G(1), testes were 3 ml and bone age was 14 years. During 20 years of irregular testosterone replacement, he developed secondary sexual characteristics and testicular enlargement. At the age of 41 years, after stopping testosterone replacement for 5 months, his testes were 15 ml, serum testosterone, LH, and FSH responses to GnRH were normal, and his wife was pregnant. The molecular study revealed a cytosine insertion in exon 2 of KAL1, generating a frameshift at codon 75 and a premature stop at codon 85. The expected gene product is a truncated peptide with 85 of the 680 [corrected] amino acids present in the wild-type protein. Fourteen cases of reversible KS have been described but the genotype was only studied in a single case showing a heterozygous fibroblast growth factor receptor type 1 (FGFR1) mutation. Considering the low prevalence of mutations in KAL1 or FGFR1 in KS, it is possible that these genotypes are more prevalent in reversible KS than in other KS patients, but additional studies are necessary to confirm this hypothesis.

Oestradiol concentration as a predictor of ovarian response in FSH stimulated ewe-lambs

We investigated the prediction of ovarian response using oestradiol determination, in 37 gonadotrophin stimulated Karagouniko ewe-lambs. Ovarian stimulation was induced by serial FSH administrations, and laparoscopic follicular aspiration (OPU) was conducted 12 h after the last FSH injection. Oestradiol concentration was assessed in six blood samples collected prior to each FSH injection and in one sample collected prior to follicular aspiration. According to ovarian response, ewe-lambs were allotted in three groups: good, L1 ( n = 17); moderate, L2 ( n = 10); and poor, L3 ( n = 10). Based on the data obtained from 28 (75%) randomly selected animals, a statistical model was designed and tested on the remaining nine lambs for its ability to predict the probability of good ovarian response. From the 2nd sample, oestradiol concentration was constantly higher in group L1 in comparison with L3 lambs (all p-values for the contrasts were ≤0.02), while this difference between L1 and L2 lambs was significant only in the 6th and 7th sample (both p < 0.005). Using as criterion the oestradiol concentration of the 6th sample, the statistical model predicted all lambs that did not belong to group L1 and three of four lambs that belonged to group L1. Our results indicate that the moderate- and poor-donors could be safely predicted on the basis of oestradiol concentration 12 h prior to the scheduled follicular aspiration. Moreover, poor-donors could be identified—and rejected from further manipulations—on the basis of their inability to exhibit increased oestradiol levels in response to gonadotrophin stimulus.

Transcriptional Regulation of the Cholesterol Side Chain Cleavage Cytochrome P450 Gene (CYP11A1) Revisited: Binding of GATA, Cyclic Adenosine 3′,5′-Monophosphate Response Element-Binding Protein and Activating Protein (AP)-1 Proteins to a Distal Novel Cluster ofcis-Regulatory Elements Potentiates AP-2 and Steroidogenic Factor-1-Dependent Gene Expression in the Rodent Placenta and Ovary

The first and key enzyme controlling the synthesis of steroid hormones is cholesterol side chain cleavage cytochrome P450 (P450scc, CYP11A1). This study sought to elucidate overlooked modes of regulation of P450scc transcription in the rodent placenta and ovary. Transcription of P450scc requires two clusters of cis-regulatory elements: a proximal element (-40) known to bind either activating protein 2 (AP-2) in the placenta, or steroidogenic factor 1 in the ovary, and a distal region of the promoter (-475/-447) necessary for potentiation of the AP-2/steroidogenic factor 1-dependent activity up to 7-fold. In primary cultures of mouse trophoblast giant cells and rat ovarian granulosa cells, binding of trans-factors to the distal regulatory sequences generated transcriptional activity in a tissue-specific pattern: in the placenta, cAMP response element (CRE)-binding protein 1 (CREB-1) and GATA-2 binding generates promoter activity in a cAMP-independent manner, whereas in ovarian cells, CREB-1 and GATA-4 are required for FSH responsiveness. However, as ovarian follicles advance toward ovulation, elevated Fra-2 expression replaces CREB-1 function by binding the same CRE(1/2) motif. Our findings suggest that upon onset of follicular recruitment, CREB-1 mediates FSH/cAMP signaling, which switches to cAMP-independent expression of P450scc in luteinizing granulosa cells expressing Fra-2. In the placenta, the indispensable role of CREB-1 was demonstrated by use of dominant-negative CREB-1 mutant, but neither cAMP nor Ser133 phosphorylation of CREB-1 is required for P450scc transcription. These observations suggest that placental regulation of P450scc expression is subjected to alternative signaling pathway(s) yet to be found.

Validation of a prediction model for the follicle-stimulating hormone response dose in women with polycystic ovary syndrome

To validate a published model for the prediction of the individual FSH response dose for gonadotropin induction of ovulation in polycystic ovary syndrome (PCOS). Structured, complete, and carefully monitored patient-based data collection to test the external validity of the prediction model. Twenty-nine hospitals in The Netherlands. Eighty-five clomiphene citrate (CC)-resistant women with PCOS. Ovulation induction in a chronic low-dose step-up FSH regimen. Predicted individual FSH response dose, defined as follicle growth >10 mm in diameter on ultrasound. The model, using the women's body mass index, CC response, initial serum FSH level, and initial serum insulin-to-glucose ratio was studied in the validation sample. Overall, the FSH response dose predicted by the model was higher than the observed response dose. The predictive performance of the model was poor, with an R(2) of 0.11, and the average prediction error was 35 IU. The external validity of the model predicting the individual FSH response dose was inadequate in women with CC-resistant PCOS undergoing ovulation induction with recombinant FSH in a low-dose step-up regimen.

Effects of breed on kinetics of ovine FSH and ovarian response in superovulated sheep

Embryo production is a useful tool for ex situ conservation of endangered species and breeds, despite a high variability in the ovarian response to superovulatory treatments. The current study evaluated the incidence and mechanisms of genetic factors in such variability, by determining the pharmacokinetics and pharmacodynamics of a standard treatment with ovine FSH (oFSH) in two endangered Spanish sheep breeds (Rubia del Molar, R, and Negra de Colmenar, N) in comparison to Manchega ewes (M, control group). In the first experiment, pharmacokinetics of an i.m. single dose of 1.32mg of oFSH was evaluated in seven animals of each breed. Plasma FSH concentrations reached their maximum at 4h post-administration in all the ewes, but several of the kinetic parameters (plasma FSH concentration at 4h post-administration, maximum plasma FSH concentration, Cmax, and both the area under the plasma concentration-time curve extrapolated to the infinite, AUCinf, and to the last moment of sampling, AUClast) were higher in the N group. In the second trial, 10 animals of each breed were superovulated using eight decreasing doses of oFSH (3× 1.32mg, 2× 1.10mg, and 3× 0.88mg). The R group, when compared to N and M, showed both a higher number of corpora lutea (13.7±0.6 versus 10.0±0.4 in N and 9.8±0.6 in M, P<0.05 for both) and embryos (7.9±0.8 versus 4.3±0.4 in N, P<0.05, and 6.7±0.5 in M, n.s.). Evaluation of pharmacokinetic and dynamic parameters showed that, although there was a trend for a higher hormone availability in R sheep, mean FSH plasma concentrations were similar between breeds (0.54±0.08ng/ml for R, 0.45±0.05ng/ml for N and 0.35±0.05ng/ml for M). However, differences were found in the number of preovulatory follicles growing in response to the FSH treatment between R (24.4±2.2), M (18.9±1.5, n.s.) and N sheep (14.1±1.4; P<0.01). Thus, differences in embryo yields between breeds would be related to differences in the pattern of follicular growth in response to FSH treatment.

Follicle-stimulating hormone increases primordial follicle reserve in mature female hypogonadal mice

Ovarian primordial follicle reserve is considered hormonally independent or subject to depletion by FSH-driven follicle recruitment. To explore specific in vivo effects of FSH on early follicle populations in the absence of luteinizing hormone (LH) activity, we examined mature hypogonadal (hpg), gonadotrophin-deficient mice expressing transgenic (tg) human FSH. Sustained expression of tg-FSH (5.3 +/- 0.3 IU/l) increased ovary weights fourfold and significantly elevated total primordial follicle numbers twofold in tg-FSH hpg (4209 +/- 457) relative to non-tg hpg (2079 +/- 391) and wild-type (2043 +/- 195) age-matched ovaries. Absolute primary follicle numbers in tg-FSH hpg ovaries were similar to non-tg hpg and wild-type ovaries. Furthermore, tg-FSH quantitatively increased secondary and antral follicles in hpg ovaries to numbers equivalent to wild-type, but did not induce ovulation, indicating a selective FSH response without LH. Circulating inhibin B and inhibin A levels were significantly increased in tg-FSH hpg females compared with hpg controls, and inhibin B correlated with antral number, consistent with FSH-driven antral follicle formation. These findings revealed that sustained pituitary-independent FSH activity, in the absence of endogenous gonadotrophins, promotes an increase in primordial follicle reserve despite also stimulating follicular growth in mature females. Therefore, the tg-FSH hpg ovary presents a novel paradigm to evaluate specific gonadotrophin effects on follicle reserve and recruitment.

A Case of Suspected Lymphocytic Hypophysitis and Organizing Pneumonia during Maintenance Therapy for Autoimmune Pancreatitis Associated with Autoimmune Thrombocytopenia

A 75-year-old man with a medical history of autoimmune pancreatitis associated with autoimmune thrombocytopenia was emergently admitted to our hospital because of anorexia, vomiting, and transient loss of consciousness. Serum sodium was 115 mEq/l and the endocrinologic data indicated impaired secretion of ACTH, TSH, and gonadotropin, a preserved GH response, and increased PRL. Dynamic magnetic resonance imaging revealed marked swelling of the pituitary gland and stalk, with enhancement on early phase. The findings were consistent with lymphocytic hypophysitis according to the diagnostic criteria. Chest computed tomography revealed consolidation adjacent to the pleura in the right upper lobe, lower lobe, and left lower lobe. Because lymphocytic hypophysitis and associated organizing pneumonia were suggested, 50 mg of prednisolone was started and the dose was tapered. Swelling of the pituitary gland, lung lesion, and the LH and FSH response on the stimulation test were all markedly improved. Autoimmune pancreatitis, pituitary lesion, and organizing pneumonia might all be components of a systemic autoimmune fibrosclerosing disease in our case, although further studies are required to confirm this hypothesis.

Kallmann Syndrome Phenotype in a Female Patient with CHARGE Syndrome and CHD7 Mutation

We report on a 14 7/12-year-old Japanese female patient with CHARGE syndrome and CHD7 mutation who also exhibited Kallmann syndrome (KS) phenotype. She had poor pubertal development and apparently impaired sense of smell. A GnRH test showed severely compromised responses of LH (<0.5 --> <0.5 IU/L) and FSH (<0.5 --> 1.2 IU/L), and magnetic resonance imaging delineated hypoplastic olfactory bulbs. Mutation analysis revealed a heterozygous nonsense mutation at exon 33 of CHD7 (7027C>T, Q2343X). The results provide further support for the notion that KS phenotype can be included in the phenotypic spectrum of CHARGE syndrome, and indicate that CHARGE syndrome with KS phenotype is caused by a CHD7 mutation.

Differential response to estrogen challenge test in women with and without premenstrual dysphoria

This study tested the hypothesis that brain sensitivity to normal fluctuations in gonadal hormones is increased in women with premenstrual dysphoria. For this purpose, the effect of a common gonadal hormonal challenge on the sensitivity of the brain was investigated in 13 women with premenstrual dysphoria and 12 asymptomatic controls. The estrogen challenge test, comprising estradiolbenzoate 0.04 mg/kg, was given as an intramuscular gluteal injection between 0700 and 1000h on day 3 or 4 of the menstrual cycle; blood was sampled at 0, 0.6, 6.5, 24, 32, 48, 56, 72, 96, 120, and 144h and analyzed for estradiol, FSH and LH. Serum estradiol levels after the injection and the corresponding FSH responses were similar between the study groups; however, the LH responses were significantly different. Women with premenstrual dysphoria had a relatively stronger negative feedback response (p=0.014) up to the point of nadir LH levels (maximal negative feedback), but displayed higher LH levels at the nadir (p=0.01), more LH surge-like reactions (p=0.047), and a 50% higher area under the curve (AUC) for LH (p=0.03) than controls. The LH response in women with premenstrual dysphoria was related to the VAS-rated symptoms; the negative increment (AOC) correlated to luteal phase "bloating" (r(s)=0.73; p=0.0069) whereas the AUC of LH correlated to "irritability" (r(s)=0.58; p=0.040). A significant interaction term between study group and changes in LH during the negative feedback phase (32-0h), with regard to luteal phase "irritability" was found (test for interaction p=0.005). For the premenstrual dysphoria group, ratings of "depressed mood"were related to baseline FSH levels (r(s)=0.60; p=0.034), and to the AUC of FSH during the negative feedback phase (r(s)=0.58; p=0.043). Women with premenstrual dysphoria displayed a gonadotrophin response to estradiol challenge that differed from that of controls, and was correlated to symptom severity.

Protein kinases influence bovine oocyte competence during short-term treatment with recombinant human follicle stimulating hormone

The aim of this study was to investigate the effect of short-term treatment (first 2 or 6 h) with recombinant human follicle-stimulating hormone (r-hFSH) during in vitro maturation (IVM) on the developmental competence of bovine oocytes. The roles of protein kinase A (PKA) and protein kinase C (PKC) (possibly involved in FSH response), were investigated using activators (Sp-cAMPS, PMA) or inhibitors (Rp-cAMPS, sphingosine) of these two protein kinases, respectively. The developmental competence of bovine oocytes was measured by the rate of blastocyst formation after in vitro fertilization (IVF). Our results showed that when cumulus-oocyte complexes (COCs) were cultured with r-hFSH for the first 6 h, a highly significant (P < 0.0001) improvement is seen in blastocyst development rate as a proportion of oocytes in culture compared with those matured with r-hFSH for the first 2 or 24 h. A transient exposure (6 h) to the highest dose (100 microM) of forskolin (an activator of adenylate cyclase) increased (P < 0.05) the rate of blastocyst formation. But the PKA inhibitors (Rp-cAMPS) did not affect the stimulatory effects of r-hFSH on the blastocyst yield. However, stimulation of PKC by low doses of PMA (0.1-0.5 microM) during short-term treatment, enhanced (P < 0.0001) the developmental capacity of oocytes, while sphingosine (a specific inhibitor of PKC) inhibited (P < 0.05) the stimulatory effects of r-hFSH on the rate of blastocyst formation. Our results indicate that although the developmental capacity of bovine oocytes in vitro can be modulated by both the PKA, and the PKC pathways, the activation of PKC during short-term treatment can mimic the effect of r-hFSH on the cytoplasmic maturation in bovine oocytes in vitro.