Cytogenetic, Genomic, and Functional Characterization of Pituitary Gonadotrope Cell Lines.

LßT2 and αT3-1 are important, widely studied cell line models for the pituitary gonadotropes that were generated by targeted tumorigenesis in transgenic mice. LßT2 cells are more mature gonadotrope precursors than αT3-1 cells. Microsatellite authentication patterns, chromosomal characteristics, and their intercellular variation have not been reported. We performed microsatellite and cytogenetic analysis of both cell types at early passage numbers. Short tandem repeat (STR) profiling was consistent with a mixed C57BL/6J × BALB/cJ genetic background, with distinct patterns for each cell type. Spectral karyotyping in αT3-1 cells revealed cell-to-cell variation in chromosome composition and pseudodiploidy. In LßT2 cells, chromosome counting and karyotyping demonstrated pseudotriploidy and high chromosomal variation among cells. Chromosome copy number variation was confirmed by single-cell DNA sequencing. Chromosomal compositions were consistent with a male sex for αT3-1 and a female sex for LßT2 cells. Among LßT2 stocks used in multiple laboratories, we detected two genetically similar but distinguishable lines via STR authentication, LßT2a and LßT2b. The two lines differed in morphological appearance, with LßT2a having significantly smaller cell and nucleus areas. Analysis of immediate early gene and gonadotropin subunit gene expression revealed variations in basal expression and responses to continuous and pulsatile GnRH stimulation. LßT2a showed higher basal levels of Egr1, Fos, and Lhb but lower Fos induction. Fshb induction reached significance only in LßT2b cells. Our study highlights the heterogeneity in gonadotrope cell line genomes and provides reference STR authentication patterns that can be monitored to improve experimental reproducibility and facilitate comparisons of results within and across laboratories.

Single-cell stabilization method identifies gonadotrope transcriptional dynamics and pituitary cell type heterogeneity.

Immediate-early response genes (IEGs) are rapidly and transiently induced following an extracellular signal. Elucidating the IEG response patterns in single cells (SCs) requires assaying large numbers of timed samples at high accuracy while minimizing handling effects. To achieve this, we developed and validated RNA stabilization Buffer for Examination of Single-cell Transcriptomes (RNA-Best), a versatile single-step cell and tissue preservation protocol that stabilizes RNA in intact SCs without perturbing transcription patterns. We characterize for the first time SC heterogeneity in IEG responses to pulsatile gonadotropin-releasing hormone (GnRH) stimuli in pituitary gonadotrope cells. Our study identifies a gene-specific hierarchical pattern of all-or-none transcript induction elicited by increasing concentrations of GnRH. This quantal pattern of gene activation raises the possibility that IEG activation, when accurately resolved at the SC level, may be mediated by gene bits that behave as pure binary switches.

Modeling and high-throughput experimental data uncover the mechanisms underlying Fshb gene sensitivity to gonadotropin-releasing hormone pulse frequency.

Neuroendocrine control of reproduction by brain-secreted pulses of gonadotropin-releasing hormone (GnRH) represents a longstanding puzzle about extracellular signal decoding mechanisms. GnRH regulates the pituitary gonadotropin's follicle-stimulating hormone (FSH) and luteinizing hormone (LH), both of which are heterodimers specified by unique ß subunits (FSHß/LHß). Contrary to Lhb, Fshb gene induction has a preference for low-frequency GnRH pulses. To clarify the underlying regulatory mechanisms, we developed three biologically anchored mathematical models: 1) parallel activation of Fshb inhibitory factors (e.g. inhibin α and VGF nerve growth factor-inducible), 2) activation of a signaling component with a refractory period (e.g. G protein), and 3) inactivation of a factor needed for Fshb induction (e.g. growth differentiation factor 9). Simulations with all three models recapitulated the Fshb expression levels obtained in pituitary gonadotrope cells perifused with varying GnRH pulse frequencies. Notably, simulations altering average concentration, pulse duration, and pulse frequency revealed that the apparent frequency-dependent pattern of Fshb expression in model 1 actually resulted from variations in average GnRH concentration. In contrast, models 2 and 3 showed "true" pulse frequency sensing. To resolve which components of this GnRH signal induce Fshb, we developed a high-throughput parallel experimental system. We analyzed over 4,000 samples in experiments with varying near-physiological GnRH concentrations and pulse patterns. Whereas Egr1 and Fos genes responded only to variations in average GnRH concentration, Fshb levels were sensitive to both average concentration and true pulse frequency. These results provide a foundation for understanding the role of multiple regulatory factors in modulating Fshb gene activity.

Growth differentiation factor 9 (GDF9) forms an incoherent feed-forward loop modulating follicle-stimulating hormone ß-subunit (FSHß) gene expression.

Gonadotropin-releasing hormone (GnRH) is secreted in brief pulses from the hypothalamus and regulates follicle-stimulating hormone ß-subunit (FSHß) gene expression in pituitary gonadotropes in a frequency-sensitive manner. The mechanisms underlying its preferential and paradoxical induction of FSHß by low frequency GnRH pulses are incompletely understood. Here, we identify growth differentiation factor 9 (GDF9) as a GnRH-suppressed autocrine inducer of FSHß gene expression. GDF9 gene transcription and expression were preferentially decreased by high frequency GnRH pulses. GnRH regulation of GDF9 was concentration-dependent and involved ERK and PKA. GDF9 knockdown or immunoneutralization reduced FSHß mRNA expression. Conversely, exogenous GDF9 induced FSHß expression in immortalized gonadotropes and in mouse primary pituitary cells. GDF9 exposure increased FSH secretion in rat primary pituitary cells. GDF9 induced Smad2/3 phosphorylation, which was impeded by ALK5 knockdown and by activin receptor-like kinase (ALK) receptor inhibitor SB-505124, which also suppressed FSHß expression. Smad2/3 knockdown indicated that FSHß induction by GDF9 involved Smad2 and Smad3. FSHß mRNA induction by GDF9 and GnRH was synergistic. We hypothesized that GDF9 contributes to a regulatory loop that tunes the GnRH frequency-response characteristics of the FSHß gene. To test this, we determined the effects of GDF9 knockdown on FSHß induction at different GnRH pulse frequencies using a parallel perifusion system. Reduction of GDF9 shifted the characteristic pattern of GnRH pulse frequency sensitivity. These results identify GDF9 as contributing to an incoherent feed-forward loop, comprising both intracellular and secreted components, that regulates FSHß expression in response to activation of cell surface GnRH receptors.

Optimized amplification and single-cell analysis identify GnRH-mediated activation of Rap1b in primary rat gonadotropes.

Identifying the early gene program induced by GnRH would help understand how GnRH-activated signaling pathways modulate gonadotrope secretory response. We previously analyzed GnRH-induced early genes in LßT2 cells, however these lack GnRH self-potentiation, a physiological attribute of gonadotropes. To minimize cellular heterogeneity, rat primary pituitary cultures were enriched for gonadotropes by 40-60% using a sedimentation gradient. Given the limited number of gonadotropes, RNA was amplified prior to microarray analysis. Thirty-three genes were up-regulated 40 min after GnRH stimulation. Real-time PCR confirmed regulation of several transcripts including fosB, c-fos, egr-2 and rap1b, a small GTPase and member of the Ras family. GnRH stimulated rap1b gene expression in gonadotropes, measured by a sensitive single cell assay. Immunocytochemistry revealed increased Rap1 protein in GnRH-stimulated gonadotropes. These data establish rap1b as a novel gene rapidly induced by GnRH and a candidate to modulate gonadotropin secretion in rat gonadotropes.

Effects of triclocarban on intact immature male rat: augmentation of androgen action.

Triclocarban (TCC; 3,4,4'-trichlorocarbanilide) is an antimicrobial agent used widely in various personal hygiene products including soaps. Recently, TCC has been shown to enhance testosterone-induced effects in vitro and to enlarge accessory sex organs in castrated male rats. This study was designed to evaluate the effects of TCC on intact age-matched male rats and on human prostate LNCaP and C4-2B cells. Seven-week-old male Sprague-Dawley rats received either a normal diet or a diet supplemented with TCC (0.25% in diet) for 10 days. Triclocarban induced hyperplasia of accessory sex organs in the absence of significant qualitative histological changes. Serum luteinizing hormone (LH) and testosterone were not significantly altered by TCC treatment. In prostate cancer-derived LNCaP and C4-2B cells, TCC potentiated androgen actions via androgen receptor-dependent actions. In conclusion, TCC significantly affects intact male reproductive organs and potentiates androgen effects in prostate cancer cells.

Ca2+-activated K+ channels in gonadotropin-releasing hormone-stimulated mouse gonadotrophs.

GnRH receptor activation elicits release of intracellular Ca(2+), which leads to secretion and also activates Ca(2+)-activated ion channels underlying membrane voltage changes. The predominant Ca(2+)-activated ion channels in rat and mouse gonadotrophs are Ca(2+)-activated K(+) channels. To establish the temporal relationship between GnRH-induced changes in intracellular [Ca(2+)] ([Ca(2+)](i)) and membrane current (I(m)), and to identify specific Ca(2+)-activated K(+) channels linking GnRH-induced increase in [Ca(2+)](i) to changes in plasma membrane electrical activity, we used single female mouse gonadotrophs in the perforated patch configuration of the patch-clamp technique, which preserves signaling pathways. Simultaneous measurement of [Ca(2+)](i) and I(m) in voltage-clamped gonadotrophs revealed that GnRH stimulates an increase in [Ca(2+)](i) that precedes outward I(m), and that activates two kinetically distinct currents identified, using specific toxin inhibitors, as small conductance Ca(2+)-activated K(+) (SK) current (I(SK)) and large (big) conductance voltage- and Ca(2+)-activated K(+) (BK) current (I(BK)). We show that the apamin-sensitive current has an IC(50) of 69 pM, consistent with the SK2 channel subtype and confirmed by immunocytochemistry. The magnitude of the SK current response to GnRH was attenuated by 17beta-estradiol (E(2)) pretreatment. Iberiotoxin, an inhibitor of BK channels, completely blocked the residual apamin-insensitive outward I(m), substantiating that I(BK) is a component of the GnRH-induced outward I(m). In contrast to its suppression of I(SK), E(2) pretreatment augmented peak I(BK). SK or BK channel inhibition modulated GnRH-stimulated LH secretion, implicating a role for these channels in gonadotroph function. In summary, in mouse gonadotrophs the GnRH-stimulated increase in [Ca(2+)](i) activates I(SK) and I(BK), which are differentially regulated by E(2) and which may be targets for E(2) positive feedback in LH secretion.

Estradiol inhibition of voltage-activated and gonadotropin-releasing hormone-induced currents in mouse gonadotrophs.

We report the first study of voltage-activated and GnRH-induced plasma membrane currents and their modulation by estradiol (E2) in mouse gonadotrophs. In consideration of the pleiotropic effects of E2 on gonadotrophin secretion and the relationship between plasma membrane electrical excitability and secretion, our objective was to determine the role of E2 in modulating gonadotroph plasma membrane currents. We measured total voltage-activated and GnRH-induced currents using the perforated-patch configuration of the patch-clamp technique, which preserves signaling pathways, including GnRH-induced Ca2+ oscillations. We show that female mouse gonadotrophs are similar to those from other species in that the voltage-activated net current response exhibits an inward fast activating current that is inhibited by tetrodotoxin, which is characteristic of a Na+ current, and a larger magnitude outward current with a profile suggesting the presence of multiple K+ currents. Furthermore, in voltage-clamped mouse gonadotrophs, GnRH activates large amplitude current oscillations that are apamin sensitive and have a reversal potential of -90 mV, consistent with Ca2+-activated K+ currents. Significantly, E2 pretreatment for 2-5 d decreased the density of both the peak outward voltage-activated current and the peak GnRH-induced current. The specific linkage between the observed E2 effects on membrane currents and, ultimately, gonadotroph function remains to be established. However, because decreased K+ current density is associated with an increase in membrane electrical excitability, we postulate increased excitability is one of the modes of action of E2 in sensitizing the gonadotroph to GnRH, an event central to the regulation of cyclic gonadotrophin secretion.

Differential expression and regulation of progesterone receptor isoforms in rat and mouse pituitary cells and LbetaT2 gonadotropes.

Manipulation of endogenous progesterone receptor (PR) does not produce equivalent physiological effects in mouse and rat pituitary cells. To test whether this may be due in part to difference in PR isoform expression, we examined hormonally regulated pituitary PR-A and PR-B mRNA levels using quantitative real-time PCR. The LbetaT2 mouse gonadotrope line or pituitary cells from adult, ovariectomized rats or mice were cultured with or without 0.2 nM 17beta-estradiol (E(2)) for 3 days. PR-A was the predominant form expressed for all groups. For mouse cells, E(2) led to an increase in both isoforms without a change in the A:B ratio; for rat cells, the PR-B response to E(2) was more robust resulting in a decrease in the A:B ratio. Exposure of E(2)-treated pituitary cells to 200 nM progesterone for 6 h decreased both PR-A and PR-B levels in rat cells, but had no effect on PR isoform expression in mouse cells even when exposure was extended to 12 h. The low level of PR expression found in LbetaT2 gonadotropes was unaffected by E(2), alone or with progesterone. The weak PR expression and lack of responsiveness of LbetaT2 cells cannot be explained by a male phenotype as was shown by the more than tenfold higher PR mRNA level in primary cultures of male mouse pituitary cells, which responded to E(2) stimulation with a proportional increase in PR isoforms similar to female cells. Functionally, E(2)-stimulated changes in PR mRNA isoform ratios in rat, mouse or LbetaT2 cells correlated with the degree of progesterone augmentation of GnRH-stimulated LH secretion in these models. These results are consistent with the hypothesis that robust GnRH priming and progesterone augmentation of LH secretion in the rat compared to these events in the mouse are a consequence, in part, of differences in the E(2)-modulated ratio of PR isoforms.

Complex actions of sex steroids in adipose tissue, the cardiovascular system, and brain: Insights from basic science and clinical studies.

Recent publications describing the results of the Women's Health Initiative (WHI) and other studies reporting the impact of hormone therapy on aging women have spurred reexamination of the broad use of estrogens and progestins during the postmenopausal years. Here, we review the complex pharmacology of these hormones, the diverse and sometimes opposite effects that result from the use of different estrogenic and progestinic compounds, given via different delivery routes in different concentrations and treatment sequence, and to women of different ages and health status. We examine our new and growing appreciation of the role of estrogens in the immune system and the inflammatory response, and we pose the concept that estrogen's interface with this system may be at the core of some of the effects on multiple physiological systems, such as the adipose/metabolic system, the cardiovascular system, and the central nervous system. We compare and contrast clinical and basic science studies as we focus on the actions of estrogens in these systems because the untoward effects of hormone therapy reported in the WHI were not expected. The broad interpretation and publicity of the results of the WHI have resulted in a general condemnation of all hormone replacement in postmenopausal women. In fact, careful review of the extensive literature suggests that data resulting from the WHI and other recent studies should be interpreted within the narrow context of the study design. We argue that these results should encourage us to perform new studies that take advantage of a dialogue between basic scientists and clinician scientists to ensure appropriate design, incorporation of current knowledge, and proper interpretation of results. Only then will we have a better understanding of what hormonal compounds should be used in which populations of women and at what stages of menopausal/postmenopausal life.

Hormone therapy: physiological complexity belies therapeutic simplicity.

The results of the Women's Health Initiative, a study anticipated to provide definitive answers about health benefits and risks of postmenopausal hormone therapy, have generated debate and confusion among clinicians, researchers, and the lay public. The ovarian hormones estrogen and progesterone, which decline at menopause, normally elicit complex tissue-specific responses throughout the body. Major advances are providing a detailed molecular definition of how that differential action is achieved. Here we review estrogen and progestin actions, discuss how effectively knowledge of steroid hormone endocrinology has been incorporated into clinical studies, and consider the impact on modern hormone therapy protocols and pharmaceutical development.