Reproductive function in mammals is regulated by the pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH and FSH are secreted by the gonadotrope cell and act on the gonad in a sequential and synergistic manner to initiate sexual maturation and maintain cyclic reproductive function. The synthesis and secretion of LH and FSH are regulated mainly by the pulsatile release of the hypothalamic decapeptide hormone gonadotropin-releasing hormone (GnRH). The control of differential LH and FSH synthesis and secretion is complex and involves the interplay between the gonads, hypothalamus and pituitary. In this review, the transcriptional regulation of the gonadotropin subunit genes is discussed in a physiologic setting, and we aimed to examine the mechanisms that drive those changes.

Pulsatile GnRH release is essential to fertility and is modulated by gonadal steroids, most likely via steroid-sensitive afferents. Arcuate neurons coexpressing kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons) are steroid-sensitive and have been postulated to both generate GnRH pulses and mediate steroid feedback on pulse frequency. KNDy neurons are proposed to interact with one another via NKB and dynorphin to activate and inhibit the KNDy network, respectively, and thus alter kisspeptin output to GnRH neurons. To test the roles of NKB and dynorphin on KNDy neurons and the steroid sensitivity of these actions, targeted extracellular recordings were made of Tac2(NKB)-GFP-identified neurons from castrate and intact male mice. Single-cell PCR confirmed most of these cells had a KNDy phenotype. The neurokinin 3 receptor (NK3R) agonist senktide increased action potential firing activity of KNDy neurons. Dynorphin reduced spontaneous KNDy neuron activity, but antagonism of κ-opioid receptors (KOR) failed to induce firing activity in quiescent KNDy neurons. Senktide-induced activation was greater in KNDy neurons from castrate mice, whereas dynorphin-induced suppression was greater in KNDy neurons from intact mice. Interactions of dynorphin with senktide-induced activity were more complex; dynorphin treatment after senktide had no consistent inhibitory effect, whereas pretreatment with dynorphin decreased senktide-induced activity only in KNDy neurons from intact but not castrate mice. These data suggest dynorphin-mediated inhibition of senktide-induced activity requires gonadal steroid feedback. Together, these observations support the hypotheses that activation of NK3R and KOR, respectively, excites and inhibits KNDy neurons and that gonadal steroids modulate these effects.

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.

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 if activation of these pathways are regulated by GNRH pulse frequency in vivo. GNRH deficient rats received GNRH pulses (25ng iv every 30 or 240 min for 8h, 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 [PT]) and the other for protein. Phosphorylated (phospho) CAMK2, ERK (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. 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 post 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.

Estradiol feedback regulates gonadotropin-releasing hormone (GnRH) neurons and subsequent luteinizing hormone (LH) release. Estradiol acts via estrogen receptor α (ERα)-expressing afferents of GnRH neurons, including kisspeptin neurons in the anteroventral periventricular (AVPV) and arcuate nuclei, providing homeostatic feedback on episodic GnRH/LH release as well as positive feedback to control ovulation. Ionotropic glutamate receptors are important for estradiol feedback, but it is not known where they fit in the circuitry. Estradiol-negative feedback decreased glutamatergic transmission to AVPV and increased it to arcuate kisspeptin neurons; positive feedback had the opposite effect. Deletion of ERα in kisspeptin cells decreased glutamate transmission to AVPV neurons and markedly increased it to arcuate kisspeptin neurons, which also exhibited increased spontaneous firing rate. KERKO mice had increased LH pulse frequency, indicating loss of negative feedback. These observations indicate that ERα in kisspeptin cells is required for appropriate differential regulation of these neurons and neuroendocrine output by estradiol. The brain regulates fertility through gonadotropin-releasing hormone (GnRH) neurons. Ovarian estradiol regulates the pattern of GnRH (negative feedback) and initiates a surge of release that triggers ovulation (positive feedback). GnRH neurons do not express the estrogen receptor needed for feedback (estrogen receptor α [ERα]); kisspeptin neurons in the arcuate and anteroventral periventricular nuclei are postulated to mediate negative and positive feedback, respectively. Here we extend the network through which feedback is mediated by demonstrating that glutamatergic transmission to these kisspeptin populations is differentially regulated during the reproductive cycle and by estradiol. Electrophysiological and hormone profile experiments on kisspeptin-specific ERα knock-out mice demonstrate that ERα in kisspeptin cells is required for appropriate differential regulation of these neurons and for neuroendocrine output.

It is well established that cervical growth during rat pregnancy is relaxin dependent. The first objective of this study was to determine if relaxin also promotes vaginal growth in the pregnant rat. Finding that this is the case, the second objective of this study was to determine if cell proliferation accompanies relaxin-dependent vaginal and cervical growth during rat pregnancy. Primiparous pregnant rats were ovariectomized (O) or sham ovariectomized (group C) on day 9 (D9) of pregnancy, before relaxin (R) is detectable in the peripheral circulation. After ovariectomy, rats were treated continuously with progesterone (P) and estrogen (E, group OPE), or P, E, and porcine R (group OPER) in doses that restored normal pregnancy and parturition parameters. P and E were administered via silicon tubing implants. R was administered from miniature osmotic pumps. Vaginas and cervices were collected on D9 and D22 from group C, and on D22 from groups OPE and OPER (n = 6/group). Vaginas and cervices were weighed, frozen, and lyophilized until dry. Dried tissues were weighed, homogenized, and their DNA contents were determined. In sham-operated controls (group C), the wet weight, dry weight, and DNA contents of both the vagina and cervix increased 50-300% from D9-D22. On D22, vaginal and cervical wet and dry weights were significantly lower than controls in R-deficient group OPE; whereas, they were greater than controls in group OPER. Similarly, on D22, vaginal and cervical DNA content did not differ from D9 controls in group OPE; whereas they exceeded D22 controls in group OPER. In conclusion, this study demonstrates that vaginal growth during the second half of rat pregnancy is R dependent. Additionally, this study provides evidence that R may contribute to both vaginal and cervical growth by promoting cellular proliferation.

Acquisition of a mature pattern of gonadotropin-releasing hormone (GnRH) secretion from the CNS is a hallmark of the pubertal process. Little is known about GnRH release during sexual maturation, but it is assumed to be minimal before later stages of puberty. We studied spontaneous GnRH secretion in brain slices from male mice during perinatal and postnatal development using fast-scan cyclic voltammetry (FSCV) to detect directly the oxidation of secreted GnRH. There was good correspondence between the frequency of GnRH release detected by FSCV in the median eminence of slices from adults with previous reports of in vivo luteinizing hormone (LH) pulse frequency. The frequency of GnRH release in the late embryonic stage was surprisingly high, reaching a maximum in newborns and remaining elevated in 1-week-old animals despite low LH levels. Early high-frequency GnRH release was similar in wild-type and kisspeptin knock-out mice indicating that this release is independent of kisspeptin-mediated excitation. In vivo treatment with testosterone or in vitro treatment with gonadotropin-inhibitory hormone (GnIH) reduced GnRH release frequency in slices from 1-week-old mice. RF9, a putative GnIH antagonist, restored GnRH release in slices from testosterone-treated mice, suggesting that testosterone inhibition may be GnIH-dependent. At 2-3 weeks, GnRH release is suppressed before attaining adult patterns. Reduction in early life spontaneous GnRH release frequency coincides with the onset of the ability of exogenous GnRH to induce pituitary LH secretion. These findings suggest that lack of pituitary secretory response, not lack of GnRH release, initially blocks downstream activation of the reproductive system.

Pulsatile GNRH regulates the gonadotropin subunit genes in a differential manner, with faster frequencies favoring Lhb gene expression and slower favoring Fshb. Early growth response 1 (EGR1) is critical for Lhb gene transcription. We examined GNRH regulation of EGR1, and its two co-repressors 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 30min) induced Egr1 primary transcript (PT) and mRNA stably 2-fold (p<0.05) for 1–24h. In contrast slow GNRH pulses (every 240min) increased Egr1 PT at 24h (6-fold; p<0.05), but increased Egr1 mRNA 4–5 fold between 4 and 24h. Both GNRH pulse frequencies increased EGR1 protein 3–4 fold. In cultured rat pituitary cells, GNRH pulses (every 60min) increased Egr1 (PT = 2.5–3 fold; mRNA = 1.5–2 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 Kinases 8/9 (MAPK8/9 [aka JNK]; SP600125) and MAP Kinase Kinase 1 (MAP2K1 [aka MEK1]; PD98059) either blunted or totally suppressed the GNRH induction of Lhb PT and Egr1 PT/mRNA, whereas the MAPK14 (aka 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.