Alan C. Dalkin

Evidence That Pituitary Adenylate Cyclase Activating Polypeptide Suppresses Follicle-Stimulating Hormone-β Messenger Ribonucleic Acid Levels by Stimulating Follistatin Gene Transcription1

There is accumulating evidence to suggest that pituitary adenylate cyclase-activating polypeptide (PACAP) may be an important modulator ofgonadotrope function. One of the actions of PACAP identified previously is to decrease FSHbeta messenger RNA (mRNA) levels. In the present series of experiments we demonstrate that PACAP-induced suppression of FSHbeta mRNA correlates with a rise in follistatin mRNA levels in primary pituitary cell cultures. Transient transfection of gonadotrope-derived alphaT3-1 cells with a rat follistatin promoter-luciferase reporter plasmid reveals that PACAP stimulates follistatin gene transcription. PACAP stimulation of LUC activity was maximal at concentrations as low at 1 nM. Furthermore, in alphaT3-1 cells PACAP activation of the follistatin promoter appears to be via the cAMP-dependent protein kinase A pathway. Accordingly, we propose that PACAP stimulates follistatin transcription, which neutralizes activin activity and thereby reduces FSHbeta mRNA. Since PACAP and follistatin are colocalized in multiple tissues including the brain, adrenals, and gonads, our findings may reflect a broadly distributed autocrine/paracrine mechanism for modification of activin effects that is under PACAP control.

Reproduction and Fertility

The control of reproduction includes a series of hormonal interactions between the central nervous system (hypothalamus and pituitary) and gonads. Gonadotropin-releasing hormone (GnRH) is a decapeptide produced by a few hundred hypothalamic neurons. These cells extend termini to the median eminence to secrete GnRH in a pulsatile fashion into the hypophyseal-portal venous system. GnRH is then carried to the anterior pituitary where the gonadotropes (which comprise 5–7% of the total cell population) respond to GnRH via high-affinity cell-surface GnRH receptors by effecting the synthesis and secretion of the gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). LH and FSH are dimeric peptides, comprised of a common α subunit and distinct β subunits, the latter conferring biological activity. Both LH and FSH are released in pulses into the systemic circulation, and act on the gonads in concert, with FSH predominantly regulating gamete development, but also being required to induce hormonal responses to LH. Thus, the combined actions of LH and FSH control both hormonogenesis (estradiol and progesterone from the ovary, testosterone from the testes, and inhibin, activin, and follistatin in both sexes) and gametogenesis. These steroid and peptide hormones have both local effects to promote gamete formation, and exert feedback regulation at the hypothalamus and pituitary.

Evidence that PACAP and GnRH down-regulate follicle-stimulating hormone-β mRNA levels by stimulating follistatin gene expression: effects on folliculostellate cells, gonadotrophs and LβT2 gonadotroph cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates alpha-subunit transcription and lengthens LH-beta mRNA transcripts, but reduces FSH-beta mRNA levels in rat pituitary cell cultures. PACAP also stimulates follistatin transcription, an effect which may explain the decrease in FSH-beta mRNA. To begin to investigate the cells in which PACAP activates the follistatin gene, quantitative in situ hybridization for follistatin mRNA combined with immunostaining for LHbeta and S100 protein was performed. In control cultures, follistatin mRNA was expressed in 70% of gonadotrophs and in 47% of folliculostellate cells (S-100+). PACAP increased (P<0.001) both the number of follistatin-expressing cells as well as the number of grains per cell in both gonadotrophs and folliculostellate cells, while GnRH only affected (P=0.01) gonadotrophs. Follistatin and FSH-beta gene expression in rat pituitary cultures were also measured by competitive quantitative RT-PCR and northern analysis, respectively. Both PACAP and GnRH increased (P<0.05) follistatin gene expression and suppressed (P<0.05) FSH-beta mRNA, and the effect of PACAP together with GnRH on follistatin exceeded that of GnRH alone. PACAP regulation of follistatin and FSH-beta gene expression was studied further in LbetaT2 cells that were found to express receptors for the specific PACAP receptor, PAC(1). Follistatin mRNA was undetectable in cultures exposed to control media, or stimulated with PACAP, GnRH or rh-activin-A. In contrast to the results in primary pituitary cultures, PACAP increased FSH-beta mRNA in these follistatin-deficient cells. Moreover, using transient transfection, PACAP stimulated transcription of ovine-FSH-beta-luciferase. GnRH likewise increased FSH-beta mRNA and stimulated FSH-beta gene transcription in LbetaT2 cells. Activin-A increased FSH-beta gene expression dose-dependently, and activin induction of FSH-beta mRNA was blocked completely by 3-fold excess follistatin. These results indicate that PACAP stimulates follistatin gene expression in both gonadotrophs and folliculostellate cells, and provide further evidence that follistatin is required for PACAP or continuous GnRH to down-regulate FSH-beta mRNA. These experiments suggest a mechanism by which PACAP influences FSH production selectively by an autocrine effect on gonadotrophs and by a paracrine mechanism through folliculostellate cells that involves follistatin.

Gonadotropin-Releasing Hormone Regulation of Gonadotropin Subunit Gene Expression in Female Rats: Actions on Follicle-Stimulating Hormoneβ Messenger Ribonucleic Acid (mRNA) Involve Differential Expression of Pituitary Activin (β-B) and Follistatin mRNAs1

GnRH is the primary stimulus in the regulation of gonadotropin subunit mRNA expression. Additionally, local (pituitary) production of activin and follistatin appear to modulate the expression of FSH β mRNA. The current studies aimed to determine whether GnRH regulation of pituitary activin (β-B) and follistatin mRNAs could play a role in the differential actions of GnRH pulse pattern on gonadotropin mRNA expression in female rats. In response to altered GnRH pulse amplitude, the expression of FSH β and follistatin mRNAs followed an inverse pattern. Only high dose GnRH increased expression of follistatin whereas, in contrast, β-B and FSH β expression were increased following lower doses of GnRH. To determine whether increased follistatin mRNA expression was correlated with FSH β mRNA responses, we examined their temporal relationship following high dose GnRH. Both FSH β and follistatin mRNAs were increased within 2 h and remained increased through 6 h. However, by 12 h FSH β mRNA levels returned to values ...

Regulation of Gonadotropin Subunit Gene Transcription by Gonadotropin-Releasing Hormone: Measurement of Primary Transcript Ribonucleic Acids by Quantitative Reverse Transcription-Polymerase Chain Reaction Assays1

GnRH regulates the synthesis and secretion of the pituitary gonadotropins LH and FSH. One of the actions of GnRH on the gonadotropin subunit genes (α, LHβ, and FSHβ) is the regulation of transcription [messenger RNA (mRNA) synthesis]. Gonadotropin subunit transcription rates increase after gonadectomy and following exogenous GnRH pulses. However, prior studies of subunit mRNA synthesis were limited by the available methodology that did not allow simultaneous measurement of gene transcription and mature mRNA concentrations. The purpose of the current studies was to: 1) develop a reliable and sensitive method for assessing transcription rates by measuring gonadotropin subunit primary transcript RNAs (PT, RNA before intron splicing); 2) investigate the PT responses to GnRH following castration or exogenous GnRH pulses; 3) characterize the half-disappearance time for the three PT species after GnRH withdrawal; and 4) correlate changes in PT concentration with steady-state gonadotropin subunit mRNA levels meas...

Gonadotropin Subunit Transcriptional Responses to Calcium Signals in the Rat: Evidence for Regulation by Pulse Frequency

Abstract Alterations in the frequency of calcium influx signals to rat pituitary cells can regulate the expression of gonadotropin subunit mRNAs in a differential manner, producing effects that are similar to those previously found for GnRH. The present study was conducted to investigate whether this reflects a transcriptional response to calcium pulse frequency, as determined by alterations in primary transcript (PT) expression. Perifused rat pituitary cells were given pulses of the calcium channel-activator Bay K 8644 (BK; with 10 mM KCl in the injectate) for 6 h. The response to alterations in pulse dose was examined by giving pulses of 1, 3, or 10 μM BK at 60-min intervals. Maximal increases in LHβ and FSHβ PTs were obtained with the 3-μM BK pulse dose and with the 10-μM dose for α. To investigate the effect of calcium pulse frequency, 3-μM BK pulses were given at intervals of 15, 60, or 180 min. Alpha PT was selectively stimulated by 15-min pulses and LHβ by 15- and 60-min pulses of BK. In contrast, FSHβ PT was maximally stimulated by the slower, 180-min pulse interval. These findings reveal that pulsatile increases in intracellular calcium stimulate α, LHβ, and FSHβ transcription in a differential manner. Thus, intermittent changes in intracellular calcium appear to be important in the transmission of GnRH pulse signals from the plasma membrane to the gene, and they may mediate the differential actions of pulse frequency on gonadotropin subunit gene expression.

Pituitary Adenylate Cyclase Activating Polypeptide Messenger RNA in the Paraventricular Nucleus and Anterior Pituitary During the Rat Estrous Cycle

Abstract The neuropeptide pituitary adenylate cyclase activating polypeptide (ADCYAP 1, or PACAP) has been demonstrated to enhance gonadotropin-releasing hormone (GnRH)-induced gonadotropin secretion and regulate gonadotropin subunit gene expression in cultures of anterior pituitary cells. In the present study, we used in situ hybridization and real-time polymerase chain reaction to examine the expression of Pacap mRNA within the paraventricular nucleus (PVN) and anterior pituitary throughout the estrous cycle of the rat. Levels of luteinizing hormone in serum and pituitary gonadotropin subunit mRNAs were evaluated and displayed cyclic fluctuations similar to those reported previously. Pacap mRNA expression in the PVN and pituitary varied significantly during the estrous cycle, with the greatest changes occurring on the day of proestrus. Pacap mRNA levels in the PVN declined significantly on the morning of diestrus. During proestrus, PVN Pacap mRNA levels significantly increased 3 h before the gonadotropin surge and then declined. Pituitary expression of Pacap mRNA also varied on the afternoon of proestrus with a moderate decline at the time of the gonadotropin surge and a significant increase later in the evening. Expression of the mRNA species encoding the 288 amino acid form of follistatin increased significantly following the rise in pituitary Pacap mRNA, at the termination of the secondary surge in follicle-stimulating hormone beta (Fshb) gene expression. These results suggest that PACAP is involved in events before and following the gonadotropin surge, perhaps through increased gonadotroph sensitivity to GnRH and suppression of Fshb subunit expression through increased follistatin, as previously observed in vitro.

Changes in Myocardial A1 Adenosine Receptor and Message Levels during Fetal Development and Postnatal Maturation

Previously we have shown myocardial adenosine A1 receptors are up-regulated during the newborn period. The timing of the increase or the mechanism of the changes are not known. The purpose

Differential Expression of the Pituitary Gonadotropin Subunit Genes During Male Rat Sexual Maturation: Reciprocal Relationship Between Hypothalamic Pituitary Adenylate Cyclase-Activating Polypeptide and Follicle-Stimulating Hormone β Expression

Abstract The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to differentially regulate the expression of the gonadotropin subunit genes in cultures of rat pituitary cells. PACAP is expressed within the hypothalamus, and concentrations of PACAP are 2- to 4-fold higher in the portal circulation than in the general circulation. Therefore, PACAP is a candidate regulator of pituitary function. In the present study, we examined the expression of PACAP mRNA within the paraventricular nucleus (PVN) during maturation (Days 20–60) in the male rat and compared this expression to the levels of the gonadotropin subunits, follistatin, and GnRH-receptor mRNAs within the anterior pituitary. Serum concentrations of FSH and LH confirm the established maturational pattern of divergent secretion of LH and FSH. Northern analysis of the gonadotropin subunit mRNAs revealed that FSHβ expression parallels FSH secretion whereas LHβ mRNA concentrations do not change during development. Expression of the GnRH receptor in the pituitary parallels that of FSHβ. In situ hybridization revealed a developmental pattern of PACAP mRNA within the PVN that is reciprocal to that of FSHβ. Competitive reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of total pituitary follistatin mRNA revealed no significant changes; however, semiquantitative RT-PCR analyses revealed the presence of two follistatin mRNA species, one of which, corresponding to follistatin-288, was developmentally regulated. These studies identified a reciprocal relationship between PVN PACAP and FSHβ gene expression in maturing rats. We propose that PACAP contributes to the selective regulation of FSHβ expression during maturation in the male rat, perhaps via regulation of follistatin.

Pituitary Follistatin Gene Expression in Female Rats: Evidence That Inhibin Regulates Transcription

Abstract Follistatin (FS), along with the members of the transforming growth factor β 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 α 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.