Evaluation of novel biomarkers for early pregnancy outcome prediction†.

OBJECTIVE: To assess performance and discriminatory capacity of commercially available enzyme-linked immunosorbent assays of biomarkers for predicting first trimester pregnancy outcome in a multi-center cohort. DESIGN: In a case-control study at three academic centers of women with pain and bleeding in early pregnancy, enzyme-linked immunosorbent assays of biomarkers were screened for assay performance. Performance was assessed via functional sensitivity, assay reportable range, recovery/linearity, and intra-assay precision (%Coefficient of Variation). Top candidates were analyzed for discriminatory capacity for viability and location among 210 women with tubal ectopic pregnancy, viable intrauterine pregnancy, or miscarriage. Assay discrimination was assessed by visual plots, area under the curve with 95% confidence intervals, and measures of central tendency with two-sample t-tests. RESULTS: Of 25 biomarkers evaluated, 22 demonstrated good or acceptable assay performance. Transgelin-2, oviductal glycoprotein, and integrin-linked kinase were rejected due to poor performance. The best biomarkers for discrimination of pregnancy location were pregnancy-specific beta-1-glycoprotein 9, pregnancy-specific beta-1-glycoprotein 1, insulin-like growth factor binding protein 1, kisspeptin (KISS1), pregnancy-specific beta-1-glycoprotein 3, and beta parvin (PARVB). The best biomarkers for discrimination of pregnancy viability were pregnancy-specific beta-1-glycoprotein 9, pregnancy-specific beta-1-glycoprotein 3, EH domain-containing protein 3, KISS1, WAP four-disulfide core domain protein 2 (HE4), quiescin sulfhydryl oxidase 2, and pregnancy-specific beta-1-glycoprotein 1. CONCLUSION: Performance of commercially available enzyme-linked immunosorbent assays was acceptable for a panel of novel biomarkers to predict early pregnancy outcome. Of these, six and seven candidates demonstrated good discriminatory capacity of pregnancy location and viability, respectively, when validated in a distinct external population. Four markers demonstrated good discrimination for both location and viability.

Kisspeptin as a new serum biomarker to discriminate miscarriage from viable intrauterine pregnancy.

OBJECTIVE: To validate the ability of serum kisspeptin-54 to discriminate between first-trimester viable pregnancies and miscarriages. DESIGN: Case-control study. SETTING: Academic medical centers. PATIENT(S): Women with confirmed viable intrauterine pregnancy (IUP) at estimated gestational age 6-10 weeks (n = 20), women with confirmed miscarriage (spontaneous abortion [SAB]) at estimated gestational age 6-10 weeks (n = 20), and nonpregnant women (n = 19). INTERVENTION(S): Collection of serum samples from women with confirmed IUP, SAB, and nonpregnant women for the measurement of serum kisspeptin and serum hCG levels. MAIN OUTCOME MEASURE(S): Serum kisspeptin and hCG. RESULT(S): The limit of detection was 0.024 ng/mL; intra- and interassay coefficients of variation were 5.1% and 8.6%, respectively. Kisspeptin levels differed between the pregnant and nonpregnant state and by viability. Kisspeptin levels were positively associated with gestational age. There was also a significant positive association with hCG in SAB, but not in IUP. CONCLUSION(S): Plasma levels of kisspeptin have been suggested as a biomarker for miscarriage. This study demonstrates kisspeptin assay stability in serum and its potential clinical utility as a biomarker for early pregnancy viability.

Antimüllerian hormone levels and antral follicle counts are not reduced compared with community controls in patients with rigorously defined unexplained infertility.

OBJECTIVE: To test the hypothesis that women with unexplained infertility demonstrate evidence of diminished ovarian reserve when compared with a population of community controls. DESIGN: Cross-sectional study. SETTING: Multicenter university-based clinical practices. PATIENT(S): Study participants included 277 healthy, normo-ovulatory female partners with rigorously defined unexplained infertility randomly selected from a multicenter trial (Assessment of Multiple Intrauterine Gestations from Ovarian Stimulation). Controls included 226 healthy, normo-ovulatory women not seeking treatment for fertility from a community-based cohort (Ovarian Aging study). INTERVENTION(S): Serum antimüllerian hormone (AMH) assay at a central laboratory, FSH, fasting serum metabolic testing, transvaginal ultrasonography for antral follicle counts (AFCs), anthropometric measurements. MAIN OUTCOME MEASURE(S): Average AMH, AFC, and AMH/AFC were compared between infertile and control women by age. Analyses of covariance compared these outcomes while controlling for confounders, including age, race, body mass index, smoking history, and study site. RESULT(S): In our models, AMH, AFC, and AMH/AFC ovarian reserve indices did not differ between infertile women and community-based controls, after controlling for age, race, body mass index, smoking history, and study site. CONCLUSION(S): Currently utilized predictors of ovarian reserve do not discriminate women with rigorously defined unexplained infertility from healthy community-based women of similar demographic characteristics. Contrary to our hypothesis, among women with FSH in the normal range (≤12 IU/L), women with unexplained infertility did not show evidence of decreased ovarian reserve as measured by AMH and AFC. Ovarian reserve markers in isolation may not serve as predictors of future fertility.

Acute Psychosocial Stress Inhibits LH Pulsatility and Kiss1 Neuronal Activation in Female Mice.

Psychosocial stress, such as isolation and restraint, disrupts reproductive neuroendocrine activity. Here we investigate the impact of psychosocial stress on luteinizing hormone (LH) pulses and gene expression and neuronal activation within Rfrp and Kiss1 cells in female mice. Mice were ovariectomized (OVX) and handled daily to habituate to the tail-tip blood collection procedure. Blood was collected every 5 minutes for 180 minutes for measurement of LH. After 90 minutes, stress animals were placed into restraint devices and isolated to new cages. No-stress control animals remained in their home cages. LH pulses occurred at regular intervals during the entire 180-minute sampling period in controls. In contrast, stress induced a rapid and robust suppression of pulsatile LH secretion. Stress reduced the frequency of pulses by 60% and diminished basal LH levels by 40%; pulse amplitude was unaffected. In a separate cohort of OVX females, brains were collected after 45, 90, or 180 minutes of stress or in no-stress controls. At all time points, stress induced a potent decrease in arcuate Kiss1 neuronal activation, using cfos induction as a marker, with a 50% to 60% suppression vs control levels, whereas Rfrp and cfos coexpression in the dorsal-medial nucleus was elevated after 45 minutes of stress. Although arcuate Kiss1 gene expression remained stable, Rfrp expression was elevated 20% after 180 minutes of stress. These findings demonstrate rapid suppression of LH pulsatile secretion by psychosocial stress, associated with reduced cfos induction in Kiss1 neurons and time-dependent increases in Rfrp neuronal activation and messenger RNA.

Age-stratified thresholds of anti-Müllerian hormone improve prediction of polycystic ovary syndrome over a population-based threshold.

OBJECTIVE: Due to its consistent elevation in polycystic ovary syndrome (PCOS) and correlation with polycystic ovarian morphology (PCOM), anti-Mullerian hormone (AMH) has been proposed as a marker of the syndrome. However, prior studies reporting thresholds of AMH for a PCOS diagnosis have been limited by small sample size, inappropriate controls, and heterogeneous AMH assays. We sought to evaluate the suitability of a standardized AMH assay as a biomarker of PCOS. DESIGN: Cross-sectional study at academic medical centres across the United States. PATIENTS: Women with PCOS were diagnosed by Rotterdam criteria and included 282 subjects from the multisite PPCOS II trial and 109 patients from a tertiary academic centre's multidisciplinary PCOS clinic. Controls included 245 participants in the ovarian ageing (OVA) study, a community-based cohort of ovulatory women not seeking treatment for fertility. MEASUREMENTS: Determination of AMH by a central laboratory. Receiver-operating characteristic (ROC) analyses were used to investigate the accuracy of AMH thresholds for prediction of PCOS diagnosis with stratification by age. RESULTS: The optimal threshold of AMH to distinguish PCOS from controls was 55.36 pmol/L (sensitivity: 0.82, specificity: 0.78, J: 0.60). When examining the population by age groups, the optimal AMH threshold decreased with increasing age. CONCLUSIONS: AMH is an effective biomarker of PCOS. Age-stratified thresholds more accurately predicted PCOS than an overall population-based threshold.

Letrozole versus clomiphene for infertility in the polycystic ovary syndrome.

BACKGROUND: Clomiphene is the current first-line infertility treatment in women with the polycystic ovary syndrome, but aromatase inhibitors, including letrozole, might result in better pregnancy outcomes. METHODS: In this double-blind, multicenter trial, we randomly assigned 750 women, in a 1:1 ratio, to receive letrozole or clomiphene for up to five treatment cycles, with visits to determine ovulation and pregnancy, followed by tracking of pregnancies. The polycystic ovary syndrome was defined according to modified Rotterdam criteria (anovulation with either hyperandrogenism or polycystic ovaries). Participants were 18 to 40 years of age, had at least one patent fallopian tube and a normal uterine cavity, and had a male partner with a sperm concentration of at least 14 million per milliliter; the women and their partners agreed to have regular intercourse with the intent of conception during the study. The primary outcome was live birth during the treatment period. RESULTS: Women who received letrozole had more cumulative live births than those who received clomiphene (103 of 374 [27.5%] vs. 72 of 376 [19.1%], P=0.007; rate ratio for live birth, 1.44; 95% confidence interval, 1.10 to 1.87) without significant differences in overall congenital anomalies, though there were four major congenital anomalies in the letrozole group versus one in the clomiphene group (P=0.65). The cumulative ovulation rate was higher with letrozole than with clomiphene (834 of 1352 treatment cycles [61.7%] vs. 688 of 1425 treatment cycles [48.3%], P<0.001). There were no significant between-group differences in pregnancy loss (49 of 154 pregnancies in the letrozole group [31.8%] and 30 of 103 pregnancies in the clomiphene group [29.1%]) or twin pregnancy (3.4% and 7.4%, respectively). Clomiphene was associated with a higher incidence of hot flushes, and letrozole was associated with higher incidences of fatigue and dizziness. Rates of other adverse events were similar in the two treatment groups. CONCLUSIONS: As compared with clomiphene, letrozole was associated with higher live-birth and ovulation rates among infertile women with the polycystic ovary syndrome. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others; ClinicalTrials.gov number, NCT00719186.).

The Pregnancy in Polycystic Ovary Syndrome II study: baseline characteristics and effects of obesity from a multicenter randomized clinical trial.

OBJECTIVE: To summarize baseline characteristics from a large multicenter infertility clinical trial. DESIGN: Cross-sectional baseline data from a double-blind randomized trial of two treatment regimens (letrozole vs. clomiphene). SETTING: Academic Health Centers throughout the United States. PATIENT(S): Seven hundred fifty women with polycystic ovary syndrome (PCOS) and their male partners took part in the study. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Historic, biometric, biochemical, and questionnaire parameters. RESULT(S): Females averaged 30 years and were obese (body mass index [BMI] 35) with ∼20% from a racial/ethnic minority. Most (87%) were hirsute and nulligravid (63%). Most of the women had an elevated antral follicle count and enlarged ovarian volume on ultrasound. Women had elevated mean circulating androgens, LH-to-FSH ratio (∼2), and antimüllerian hormone levels (8.0 ng/mL). In addition, women had evidence for metabolic dysfunction with elevated mean fasting insulin and dyslipidemia. Increasing obesity was associated with decreased LH-to-FSH levels, antimüllerian hormone levels, and antral follicle counts but increasing cardiovascular risk factors, including prevalence of the metabolic syndrome. Men were obese (BMI 30) and had normal mean semen parameters. CONCLUSION(S): The treatment groups were well matched at baseline. Obesity exacerbates select female reproductive and most metabolic parameters. We have also established a database and sample repository that will eventually be accessible to investigators. CLINICAL TRIAL REGISTRATION NUMBER: NCT00719186.

Estimation of estradiol in mouse serum samples: evaluation of commercial estradiol immunoassays.

The University of Virginia Center for Research in Reproduction Ligand Core performed an evaluation of nine commercial estradiol (E2) immunoassays for use with mouse serum. The evaluation had two components. 1) Recovery Studies: a mouse pool was spiked with E2 concentrations across the assay range, and percent recovery and parallelism to the assay standard curve were determined. 2) Correlation Studies: serum pools were collected from intact females, ovariectomized (OVX) and OVX-E2 treated mice and E2 assayed, then measured by gas chromatography/tandem mass spectrometry (GC/MSMS) for comparison to a gold standard method. Recovery results showed that E2 recovery from spiked mouse pools varied greatly (from <18% to >640%) among kits tested. However, three kits (DiaSorin Radioimmunoassay, Siemens Double Antibody RIA, and CalBiotech Enzyme Immunoassay) showed reasonable recoveries and parallelism. Data collected from the Correlation Study showed that values from intact, OVX and OVX-E2-treated mouse pools varied by several fold vs. GC/MSMS for most of the kits tested. The DiaSorin RIA and CalBiotech Enzyme Immunoassay Kits showed the best correlation to GC/MSMS. Unfortunately, while this evaluation was ongoing, the DiaSorin Kit was discontinued. In summary, the CalBiotech Kit was the only available assay tested that demonstrated good E2 parallelism to the assay standard curve and accuracy vs. a gold standard method (i.e. GC/MSMS). Also of note, the CalBiotech assay is sensitive and requires minimal sample volume. Therefore, based on these findings the CalBiotech E2 assay has been implemented for use in mouse serum samples within the Ligand Core.

GnRH pulse frequency differentially regulates steroidogenic factor 1 (SF1), dosage-sensitive sex reversal-AHC critical region on the X chromosome gene 1 (DAX1), and serum response factor (SRF): potential mechanism for GnRH pulse frequency regulation of LH beta transcription in the rat.

The issue of how rapid frequency GnRH pulses selectively stimulate LH transcription is not fully understood. The rat LHß promoter contains two GnRH-responsive regions: the proximal region has binding elements for SF1, and the distal site contains a CArG box, which binds SRF. This study determined whether GnRH stimulates pituitary SF1, DAX1 (an endogenous SF1 inhibitor), and SRF transcription in vivo, and whether regulation is frequency dependent. Male rats were pulsed with 25 ng GnRH i.v. every 30 min or every 240 min for 1-24 h, and primary transcripts (PTs) and mRNAs were measured by real time PCR. Fast frequency GnRH pulses (every 30 min) increased SF1 PT (threefold) within 1 h, and then declined after 6 h. SF1 mRNA also increased within 1 h and remained elevated through 24 h. Fast frequency GnRH also stimulated a transient increase in DAX1 PT (twofold after 1 h) and mRNA (1.7-fold after 6 h), while SRF mRNA rose briefly at 1 h. Slow frequency pulses did not affect gene expression of SF1, DAX1, or SRF. These findings support a mechanistic link between SF1 in the frequency regulation of LHß transcription by pulsatile GnRH.

Regulation of Lhb and Egr1 gene expression by GNRH pulses in rat pituitaries is both c-Jun N-terminal kinase (JNK)- and extracellular signal-regulated kinase (ERK)-dependent.

Pulsatile GNRH regulates the gonadotropin subunit genes in a differential manner, with faster frequencies favoring Lhb gene expression and slower frequencies favoring Fshb. Early growth response 1 (EGR1) is critical for Lhb gene transcription. We examined GNRH regulation of EGR1 and its two corepressors, Ngfi-A-binding proteins 1 and 2 (NAB1 and NAB2), both in vivo and in cultured rat pituitary cells. In rats, fast GNRH pulses (every 30 min) stably induced Egr1 primary transcript (PT) and mRNA 2-fold (P < 0.05) for 1-24 h. In contrast, slow GNRH pulses (every 240 min) increased Egr1 PT at 24 h (6-fold; P < 0.05) but increased Egr1 mRNA 4- to 5-fold between 4 and 24 h. Both GNRH pulse frequencies increased EGR1 protein 3- to 4-fold. In cultured rat pituitary cells, GNRH pulses (every 60 min) increased Egr1 (PT, 2.5- to 3-fold; mRNA, 1.5- to 2-fold; P < 0.05). GNRH pulses had little effect on Nab1/2 PT/mRNAs either in vivo or in vitro. We also examined specific intracellular signaling cascades activated by GNRH. Inhibitors of mitogen-activated protein kinase 8/9 (MAPK8/9 [also known as JNK]; SP600125) and MAP Kinase Kinase 1 (MAP2K1 [also known as MEK1]; PD98059) either blunted or totally suppressed the GNRH induction of Lhb PT and Egr1 PT/mRNA, whereas the MAPK14 (also known as p38) inhibitor SB203580 did not. In summary, pulsatile GNRH stimulates Egr1 gene expression and protein in vivo but not in a frequency-dependent manner. Additionally, GNRH-induced Egr1 gene expression is mediated by MAPK8/9 and MAPK1/3, and both are critical for Lhb gene transcription.

Regulation of intracellular signaling cascades by GNRH pulse frequency in the rat pituitary: roles for CaMK II, ERK, and JNK activation.

Pulsatile GnRH (GNRH) differentially regulates LH and FSH subunit genes, with faster frequencies favoring Lhb transcription and slower favoring Fshb. Various intracellular pathways mediate the effects of GNRH, including CaMK II (CAMK2), ERK, and JNK. We examined whether activation of these pathways is regulated by GNRH pulse frequency in vivo. GNRH-deficient rats received GNRH pulses (25 ng i.v. every 30 or 240 min for 8 h, vehicle to controls). Pituitaries were collected 5 min after the last pulse, bisected, and one half processed for RNA (to measure beta subunit primary transcripts [PTs]) and the other for protein. Phosphorylated CAMK2 (phospho-CAMK2), ERK (mitogen-activated protein kinase 1/3 [MAPK1/3], also known as p42 ERK2 and p44 ERK1, respectively), and JNK (MAPK8/9, also known as p46 JNK1 and p54 JNK2, respectively) were determined by Western blotting. The 30-min pulses maximally stimulated Lhb PT (8-fold), whereas 240 min was optimal for Fshb PT (3-fold increase). Both GNRH pulse frequencies increased phospho-CAMK2 4-fold. Activation of MAPK1/3 was stimulated by both 30- and 240-min pulses, but phosphorylation of MAPK3 was significantly greater following slower GNRH pulses (240 min: 4-fold, 30 min: 2-fold). MAPK8/9 activation was unchanged by pulsatile GNRH in this paradigm, but as previous results showed that GNRH-induced activation of MAPK8/9 is delayed, 5 min after GNRH may not be optimal to observe MAPK8/9 activation. These data show that CAMK2 is activated by GNRH, but not in a frequency-dependant manner, whereas MAPK3 is maximally stimulated by slow-frequency GNRH pulses. Thus, the ERK response to slow pulse frequency is part of the mechanisms mediating Fhb transcriptional responses to GNRH.

The regulation of FSHbeta transcription by gonadal steroids: testosterone and estradiol modulation of the activin intracellular signaling pathway.

Recent reports suggest that androgens increase FSHbeta transcription directly via the androgen receptor and by modulating activin signaling. Estrogens may also regulate FSHbeta transcription in part through the activin system. Activin signaling can be regulated extracellularly via activin, inhibin, or follistatin (FS) or intracellularly via the Smad proteins. We determined the effects of androgen and estrogen on FSHbeta primary transcript (PT) concentrations in male and female rats, and we correlated those changes with pituitary: activin betaB mRNA, FS mRNA, the mRNAs for Smads2, -3, -4, and -7, and the phosphorylation (p) status of Smad2 and -3 proteins. In males, testosterone (T) increased FSHbeta PT two- to threefold between 3 and 24 h and was correlated with reduced FS mRNA, transient increases in Smad2, -4, and -7 mRNAs, and a six- to 10-fold increase in pSmad2, and activin betaB mRNA was unchanged. In females, T also increased FSHbeta PT twofold and pSmad2 threefold but had no effect on activin betaB, FS, or the Smad mRNAs. Androgen also increased Smad2 phosphorylation in gonadotrope-derived alphaT3 cells. In contrast, estradiol had no effect on FSHbeta PT but transiently increased activin betaB mRNA and suppressed FS mRNA before increasing FS mRNA at 24 h and increased Smads2, -3, and -7 mRNAs and pSmad2 threefold. In conclusion, T acts on the pituitary to increase FSHbeta PT in both sexes and modulates FS mRNA, Smad mRNAs, and/or Smad2 phosphorylation. These findings suggest that T regulates FSHbeta transcription, in part, through modulation of various components of the activin-signaling system.

Stimulation of FSHbeta transcription by blockade of endogenous pituitary follistatin production: Efficacy of adenoviral-delivered antisense RNA in the rat.

This study investigated FSHbeta transcriptional responses to the suppression of endogenous follistatin (FST) production using FST antisense RNA (FST-AS) expressing adenovirus constructs in female rat pituitary cells in vitro. Adenoviral delivery systems were characterized and optimized using an adenovirus-green fluorescent protein construct, and maximal infection (85-90% of cells) was seen 48 h post adenovirus treatment. A 424 bp fragment, which included the translational start site and exons 1-3 of the rat FST gene, was subcloned in the reverse orientation into an adenovirus vector. Construct efficacy was tested using cultured rat pituitary cells infected with the adenovirus-FST-AS construct. Infection with adenovirus-FST-AS increased FST-AS mRNA expression in a dose-dependent manner, reduced FST protein expression to undetectable levels, and stimulated increases in FSHbeta primary transcript and FSH secretion. Treatment with testosterone alone stimulated FSHbeta primary transcript and FSH release, and responses were doubled in the presence of adenovirus- FST-AS. These results demonstrate the effectiveness of adenovirus FST-AS in suppressing pituitary FST protein expression and enhancing FSH biological responses at the transcriptional level. Thus, the FST-deficient rat gonadotrope cell is a model that allows for the investigation of factors regulating FSHbeta expression, which might otherwise involve the autocrine/paracrine actions of FST.

Pituitary follistatin gene expression in female rats: evidence that inhibin regulates transcription.

Follistatin (FS), along with the members of the transforming growth factor beta family activin and inhibin, are important regulators of FSH secretion and messenger RNA production. While activin and inhibin appear to function as tonic modulators of FSH (stimulatory and inhibitory, respectively), dynamic changes in FS are noted through the estrous cycle and under varying physiological experimental paradigms. This suggests that FS is a major contributor to the precisely coordinated secretion of FSH that maintains reproductive function. The aim of this study was to investigate changes in FS, in particular the early (<12 h) rise observed after ovariectomy (OVX), and to determine whether these changes were as a consequence of variations in gene transcription rates. FS primary transcript (PT) and mRNA were found to increase 3-fold 12 h post-OVX, indicating increased gene transcription during this time period. Replacement with estradiol and/or blockade of GnRH had only modest effects on FS PT concentration. Inhibin immunoneutralization of intact rats resulted in a 3-fold increase in FS PT 12 h after administration of inhibin alpha antisera. Significant increases in FS mRNA at both 2 and 12 h also suggested that inhibin also may have effects on message stability. After administration of recombinant human inhibin A, there was a prompt decline in both FS PT and mRNA. These results indicate that inhibin is a major regulator of FS, both by transcriptional and nontranscriptional mechanisms.

Regulation of luteinizing hormone-beta and follicle-stimulating hormone (FSH)-beta gene transcription by androgens: testosterone directly stimulates FSH-beta transcription independent from its role on follistatin gene expression.

The gonadotropin beta-subunit mRNAs are differentially regulated by androgens. Testosterone (T) suppresses LH-beta and increases FSH-beta. We aimed to determine whether androgens regulate LH-beta and FSH-beta transcription [as measured by changes in primary transcript (PT)] and to determine whether androgens act directly on FSH-beta or via the intrapituitary activin/follistatin (FS) system. In castrate + GnRH antagonist-treated rats, T increased FSH-beta PT between 3 and 48 h. In contrast, T suppressed LH-beta PT. The increases in FSH-beta mRNA and PT were associated with reduced FS mRNA. Activin betaB mRNA was modestly suppressed. The increase in FSH-beta PT after T was androgen specific. Both T and dihydrotestosterone (DHT) increased FSH-beta PT 2-fold and decreased both FS and betaB mRNA. Estradiol suppressed FSH-beta PT 3-fold and had no effect on FS or betaB mRNAs. LH-beta PT was suppressed by DHT. To determine whether T stimulation of FSH-beta PT reflected a decrease in pituitary FS, we gave androgen in the presence of exogenous FS in vitro. T and DHT increased FSH-beta PT 2- to 3-fold. FS alone decreased FSH-beta PT 40% but did not diminish the increase FSH-beta PT in response to T. T, DHT, and FS did not affect FS mRNA, betaB mRNA, or LH-beta PT. In conclusion, androgens acting directly on the pituitary increase FSH-beta and decrease LH-beta transcription. The increase in FSH-beta PT in response to T was androgen specific and occurs in the presence of excess FS, suggesting that T stimulates FSH-beta transcription independently of modulation of FS.

The calcium component of gonadotropin-releasing hormone-stimulated luteinizing hormone subunit gene transcription is mediated by calcium/calmodulin-dependent protein kinase type II.

Calcium influx plays a critical role in GnRH regulation of rat LH subunit gene transcription, but the site(s) of action are undefined. We investigated the potential of GnRH acting through calcium to activate calcium/calmodulin-dependent protein kinase type II (Ca/CaMK II) in mouse gonadotrope-derived LbetaT2 cells. GnRH stimulated Ca/CaMK II beta subunit activity 3-fold 2 min after treatment and returned to control values by 45 min. The Ca/CaMK II response to GnRH was blocked by administration of the Ca/CaMK II-specific inhibitor, KN-93. The calcium channel activator Bay K 8644 stimulated a 3-fold increase in Ca/CaMK II activity, similar to GnRH. Blocking calcium influx with nimodipine or depleting intracellular calcium storage pools with thapsigargin each resulted in a partial suppression of GnRH-induced activation of Ca/CaMK II, and in combination, completely suppressed the Ca/CaMK II response to GnRH. KN-93 and nimodipine also suppressed alpha-subunit and LHbeta promoter responses to GnRH by 40-60%. LHbeta promoter constructs containing either proximal or proximal and distal GnRH-responsive regions were sensitive to inhibition. These data show for the first time that Ca/CaMK II activation plays an important role in the transmission of GnRH signals from the plasma membrane to the LH subunit genes.

GnRH pulse frequency modulation of gonadotropin subunit gene transcription in normal gonadotropes-assessment by primary transcript assay provides evidence for roles of GnRH and follistatin.

We examined the time course of action of GnRH pulse frequency on gonadotropin subunit gene transcription and assessed the roles of GnRH, follistatin (FS), and activin on differential transcription of the LHbeta and FSHbeta genes. GnRH-deficient male rats were pulsed with 25 ng GnRH either every 30 min (fast frequency) or every 240 min (slow frequency) for 1-24 h. Both GnRH frequencies increased alpha primary transcript (PT) 5-fold within 6 h, but only fast frequency GnRH increased alpha mRNA. Only fast frequency GnRH pulses affected LHbeta PT, resulting in 6- to 9-fold increases between 1-24 h. Fast frequency GnRH pulses transiently increased FSHbeta PT at 1 and 6 h (4- and 2-fold, respectively); but by 24 h FSHbeta PT had returned to control levels and was correlated to a 5- to 9-fold increase in FS mRNA. In contrast, slow GnRH pulses increased FSHbeta PT 3- and 6-fold at 8 and 24 h, respectively, which was correlated with a decline in FS mRNA. Activin mRNA did not change significantly after either GnRH frequency, but tended to fall after fast pulses. To test whether activin was required for the effects of GnRH on FSHbeta transcription, rats were treated with GnRH pulses every 240 min for 8 h +/- FS. FS treatment alone markedly decreased basal FSHbeta PT. GnRH in the presence of FS increased FSHbeta PT 8-fold but did not restore FSHbeta transcription to control or GnRH alone values. In summary, whereas alpha-subunit transcription is independent of frequency, an increase in alpha mRNA requires fast frequency GnRH pulses. Fast frequency GnRH pulses increased both LHbeta and FSHbeta transcription, but the response of FSHbeta was transient. The sustained rise in FSHbeta transcription and mRNA expression required slow frequency GnRH pulses and was correlated to low FS mRNA. Neutralization of pituitary activin by exogenous FS markedly reduced basal FSHbeta PT and mRNA but did not prevent the stimulation of FSHbeta transcription by slow frequency GnRH pulses. These studies suggest that the frequency regulation of FSHbeta transcription involves both direct actions of GnRH and indirect effects, via changes in pituitary FS expression.