Pituitary gonadotroph-specific patterns of gene expression and hormone secretion

Abstract

Pituitary gonadotrophs play a key role in reproductive functions by secreting luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The LH secretory activity of gonadotroph is controlled by hypothalamic gonadotropin-releasing hormone (GnRH) via GnRH receptors and is accompanied by only minor effects on high basal Lhb gene expression. The secretory profiles of GnRH and LH are highly synchronized, the latter reflecting a depletion of prestored LH in secretory vesicles by regulated exocytosis. In contrast, FSH is predominantly released by constitutive exocytosis, and secretory activity reflects the kinetics of Fshb gene expression controlled by GnRH, activin, and inhibin. Here is a review of recent data to improve the understanding of multiple patterns of gonadotroph gene expression and hormone secretion.

Introduction

Induction of puberty and subsequent reproductive abilities rely on the proper functioning of the hierarchic system known as the hypothalamic–pituitary–gonadal axis. A subpopulation of hypothalamic neurons secretes a decapeptide called gonadotropin-releasing hormone (GnRH) into the hypothalamic-pituitary portal system. GnRH in turn activates its receptors (GnRHRs) in the anterior pituitary gonadotrophs, which belong to the seven-transmembrane domain receptors, coupled to heterotrimeric Gq/11 proteins and phospholipase C signaling pathway, and may also cross-couple to Gs proteins to activate adenylate cyclase signaling pathway. This leads to modulation of the synthesis and secretion of two gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) [1].

Gonadotroph-produced LH and FSH are heterodimeric glycoprotein hormones formed by the association of a common glycoprotein alpha subunit (CGA) with glycoprotein beta subunits, LHB or FSHB. The genes coding for these subunits are Cga, also present in thyrotrophs, and Lhb and Fshb expressed only in gonadotrophs [2]. The unique LHB and FSHB subunits provide biological specificity and are a limiting component in dimer formations. Subunit-specific glycosylation levels determine the half-life of each subunit [3–5]. The assembly of LHB and FSHB subunits with the CGA subunit in the endoplasmic reticulum is a critical step for secretion [6] because it directs gonadotropins to different secretory vesicles [7]. Some FSH and all LH are packaged in chromogranin A- and Rab27-positive dense core granules [8, 9], which determines GnRH-regulated exocytosis for part of FSH and all LH, and constitutive exocytosis for most FSH [10]. Gonadotropins initiate gametogenesis and steroidogenesis in the ovaries and testes, and ovulation in females. Activation of LH receptor in Leydig cells of the testes and ovarian theca cells triggers the production of steroid hormones through the expression of steroidogenic enzymes. Androgens, estrogens, and progesterone not only contribute to the control of gonadal functions, but are also responsible for gonadal feedback at the level of the pituitary and hypothalamus. Outside the brief preovulatory period, gonadal steroids exert negative feedback on the hypothalamus and reduce GnRH secretion [11].

Here we briefly discuss new findings on the role of GnRH via GnRHRs in gonadotroph function, focusing on the mechanism by which these non-desensitizing receptors provide appropriate physiological responses in both males and females. These include variable native and experimentally/clinically determined patterns of GnRH release/application, coupling of GnRHRs to variable heterotrimeric G proteins, gene-specific basal expression and GnRH-evoked activation and desensitization patterns, modulatory role of activin and inhibin on Fshb but not Lhb expression, and hormone-specific use of two secretory pathways, regulated and constitutive exocytosis.

Patterns of GnRH secretion

GnRH-secreting cells are a small population of neurons distributed from the rostral preoptic area to the caudal hypothalamus, with most cells centered around the organum vasculosum laminae terminalis with nerve terminals located in the median eminence [12]. Although dispersed within the hypothalamus, these neurons secrete GnRH in a pulsatile manner, which is largely associated with pulsatile LH secretion, as determined by simultaneous sampling of pituitary and systemic blood in several species, including monkeys [13], rats [14], and sheep [15, 16]. This was first recognized by Knobil’s laboratory in 1970 and led to the concept of the GnRH pulse generator [17]. Recently, a neural network that forms a pulse generator has been characterized; kisspeptin-neurokinin B-dynorphin (KNDy) neurons were found to regulate distal projections of GnRH neurons, called dendrons, near the median eminence [18], and integrate the feedback effect of circulating levels of gonadal steroid hormones [19–21]. For details on the GnRH pulse generator, see an additional article in this issue of the Journal.

However, pulsatile GnRH release is not the only physiological secretory pattern of this neuropeptide. In females, the follicular phase ends with a large and sustained surge of GnRH, causing a preovulatory LH surge detected in rat [22], sheep [23] and monkey [24]. While direct detection of GnRH release in the hypothalamic-pituitary portal system is not possible in humans, GnRH and LH measurements in peripheral blood also supports the presence of a preovulatory GnRH surge [25] that precedes the LH surge [26]. The primary endocrine signal for the surge mode of LH secretion is an increase in estrogen production from the mature follicle(s), with estradiol showing positive feedback, but other triggers are also known [27]. Preovulatory LH surge lasts 3 – 4 h in mice [28] and rats [29], and can be pharmacologically evoked by a single intraperitoneal injection of GnRH or its synthetic analogs [30].

Except the large and sustained preovulatory surge of GnRH, continuous GnRH release was not observed under physiological conditions. From a pharmacological point of view, however, continuous GnRH application is clinically relevant for the treatment of variable reproductive diseases: endometriosis, precocious puberty, benign prostate hyperplasia, uterine leiomyomata, a polycystic ovary syndrome. It is also used in assisted reproduction, in vitro fertilization, and to minimize the gonadotoxic effects of chemotherapy of endocrine cancers in young women [31–36]. As discussed below, continuous application of GnRH is also often used in in vitro experimental work to answer specific questions regarding the role of GnRHRs in pituitary cell functions [30, 37].

GnRH and gonadotroph-specific genes

Recent single cell RNA sequencing studies conducted with freshly dispersed pituitary cells of various species, including fish [38], chicken [39], mice [40], rats [41, 42], and human embryonic pituitary [43], provide a broad view of anterior pituitary cell-type transcriptome profiles, including gonadotrophs. In general, all hormone-producing anterior pituitary cells and folliculostellate cells (FSCs), have the same origin as indicated by the joint expression of the Pitx1 and Pitx2 genes. A common feature of hormone-producing cells is the expression of dozen genes, including Resp18, Chga, Chgb, Scg2, Snap25, and Uchl1 [41, 42]. Contrary to the hypothesis of pituitary cell plasticity [44], gonadotrophs appear as a single cluster of homogeneous cells, uniquely expressing Fshb, Lhb, and Gnrhr. The dependence of Cga, Lhb, Fshb, and Gnrhr expression on GnRH has been shown in several laboratories (reviewed in [45]). Fshb transcription is also controlled by inhibin, activin, and follistatin [46–48].

Gonadotrophs also express other genes in a cell-specific manner, including Chrna4, Cnga1, Dmp1, Dusp15, Icam5, Lama1, Nhlh2, Nr5a1, Pitx3, Spp1, Tgfbr3l, and Vash2 [41, 42], Some of these genes are clearly expressed in a sex-specific manner, like Dmp1 and its sister gene Spp1 (23). In general, the specific roles of these genes in gonadotroph functions have not yet been elucidated. However, Nr5a1 is known to be required for stem cell differentiation into gonadotroph lineage, initiating the transcription of key gonadotroph genes, including Gnrhr and Lhb [49]. Gonadectomy in females and males increases GnRH release [50] accompanied with increase in Nr5a1 expression, and pulsatile GnRH administration in vivo in intact rats also stimulates its expression [51]. In contrast to these findings, in primary cultures of rat anterior pituitary cells, the expression of this gene is inhibited, while its expression increases in the absence of GnRH [30]. These observations points to indirect in vivo effects of GnRH on Nr5a1 expression.

Basal and regulated gonadotroph gene expression

If GnRH-dependent, gene expression in pituitary gonadotrophs after dispersion and cultivation in a GnRH-free medium should show a progressive decrease over time. In accordance with this hypothesis, the expression of Fshb, Gnrhr, and Cga decreased but was not abolished during the first 24 h of incubation of rat pituitary cells in GnRH-free medium; residual expression is called basal expression [30, 52]. However, we did not observe a decrease in Lhb expression in rat pituitary cells cultured in GnRH-free medium for 24 h after dispersion [18], and GnRH knockout mice also have detectable LH but not FSH in pituitary content [53]. This opposes the critical role of GnRH in the expression of this gene and emphasizes the physiological importance of high basal Lhb expression in LHB synthesis.

Data on epigenetic modulation of gonadotropin transcription come almost exclusively from an immortalized embryonic mouse gonadotroph cell line, but it becomes clearer that chromatin structure and dynamics in the regulatory regions affect both basal and regulated pituitary gonadotroph gene expression [54]. The same group recently suggested that mouse proliferating primary gonadotrophs could be used to study chromatin regulation in gonadotrophs [55], and such studies could help address some of the issues discussed below.

It is well established that pulsatile application of GnRH stimulates the expression of Lhb, Fshb, Gnrhr, and Cga [45] (and references within). We also observed an increase in Fshb, Cga, and Gnrhr expression, but not Lhb expression during pulsatile GnRH application [56], which probably reflects differences in experimental conditions. In vivo injection of GnRH agonist mimicking the surge-like LH profile also stimulated the expression of Lhb, Fshb, Gnrhr, and Cga [30]. Dmp1 expression was also observed during pulsatile [56] and surge-like GnRH applications [30].

Intrapituitary Fshb and circulating FSH levels are often highly correlated; that is, Fshb expression is a good indicator of the secretory FSH profile [7]. Unlike FSH, LH is predominantly secreted by regulated exocytosis, and GnRH is a very potent secretagogue [57], which is the best illustrated by simultaneous in vivo measurements of GnRH and LH, as discussed above. We also observed initial stimulation and subsequent decrease in LH secretion in vitro during the surge-like GnRH administration, reflecting almost complete depletion of the intrapituitary LH secretory pool despite elevated basal Lhb expression [30]. Therefore, inhibition of LH secretion occurs downstream of Lhb expression, probably at the level of de novo LH synthesis and/or the secretory pool maintenance. Consistent with this conclusion, translational control of gene expression could provide an explanation for some aspects of GnRH receptor signaling and secretion that cannot be explained by changes at the transcription level [58].

Gene expression and hormone secretion under continuous GnRH administration

As noted above, continuous GnRH release is frequently used in a clinical setup. It also provides an experimental tool for obtaining valid information on GnRH-induced gene expression and hormone release [30, 52]. In vitro, continuous GnRH stimulation induces different patterns of gene expression. GnRH treatment slightly desensitized high basal Lhb expression over time. Fshb expression is rapidly stimulated, reaching a peak response 2.5 h after the start of treatment, followed by exponential desensitization in gene expression below basal levels over an extended period [30]. Continuous GnRH treatment also transiently stimulates Gnrhr expression, reaching peak stimulation after 6 h of treatment and accompanied by a progressive decrease in gene expression toward basal level. However, no reduction in Gnrhr expression below basal levels was observed [52]. Finally, GnRH slowly stimulates Cga expression, but this response does not decrease over time [30]. These experimental observations raise two main questions. How does the same pattern of GnRH administration cause different gene expression profiles? What may be the physiological consequences of such gene response pattern?

The mammalian GnRHR is the only G protein-coupled receptor that does not have a carboxy-terminal tail and does not desensitize [59]. The existence of different patterns of GnRH stimulus-transcription coupling also argues against the receptor desensitization hypothesis. Therefore, GnRHR desensitization is not to blame for the suppression of Lhb and Fshb expression and LH and FSH secretion during exposure to continuous GnRH or at higher GnRH pulse frequency. Others have reported that sustained GnRHR activation leads to selective desensitization of signaling pathways and that evolution of this receptor benefits pituitary gonadotrophs and other tissues expressing this receptor [60]. In accordance with this view, the addition of carboxy-terminal tail to GnRHR impairs fertility in female mice [61]. The cross-coupling of GnRHRs to Gs signaling pathway may also contribute to the gene-specific expression pattern by GnRH [62].

Mechanistically, the GnRHR number is at least partially associated with Gnrhr transcription [63]. The presence of basal Gnrhr transcription in the absence and during the long-term clinical treatments with GnRH agonists preserves the ability of gonadotrophs to respond to endogenous pulsatile and surge patterns of GnRH release in the recovery phase. For example, basal Gnrhr transcription probably allows women with Kallman syndrome or hypothalamic amenorrhea to become pregnant with GnRH treatment [64]. It also explains the capacity of mice lacking Gq/11 in gonadotrophs to express ~50% Gnrhr mRNA [62]. Mice carrying the spontaneous GnRH mutation continue to show ~30% of GnRHRs compared to control mice and return to control values with exogenous GnRH [53].

Role of activin and inhibin in control of gonadotroph function

Rat pituitary cells express three inhibin subunit genes: Inha is expressed in all hormone-producing cell types and FSCs in the anterior pituitary and pituicytes in the posterior pituitary; Inhba is expressed only in FSCs and pituicytes, and Inhbb is expressed in gonadotrophs, corticotrophs, FSCs, pituicytes, and pituitary endothelial cells [41, 42]. The encoded protein subunits form homodimeric proteins: activin A composed of two INHBA subunits, activin B composed of two INHBB subunits, and activin AB composed of INHBA and INHBB. Heterodimeric proteins composed of a common INHA and specific INHBA or INHBB subunits are inhibin A or B, respectively. Thus, rat pituitary activin A, activin AB, and inhibin A can only be produced by FSCs and pituicytes, while activin B and inhibin B can be produced by gonadotrophs, corticotrophs, FSCs, pituicytes, and pituitary endothelial cells [41, 42].

Rat pituitary cells also express activin receptors; all cell types express Acvr1, Acvr1b, Acvr2a, and Acrvr2b, while all hormone-producing cells, but not FSCs and pituicytes, also express Acvr1c [41, 42]. Therefore, activins are potential agonists for all pituitary cells. When added to cultured rat pituitary cells, activins selectively stimulated FSH release without affecting LH, while inhibin suppressed FSH release. Activins produce these effects, in large part, by stimulating Fshb gene expression [65]. It has also been suggested that activins stimulate the synthesis and secretion of follistatin in the pituitary gland, which binds and neutralizes activins, suggesting that activins may stimulate the production of their own endogenous antagonists via short feedback loop [66]. The autocrine action of activin B on Fshb expression has been shown in vitro and in vivo with immunoneutralization in rats [67] and supported by FSH deficiency in gonadotroph-specific knockout mice for ACVR2A and ACVR2B receptors [68].

Activation of FSH receptor in Sertoli cells in testes and granulosa cells in ovaries stimulates inhibin secretion, which in turn reduces Fshb transcription in gonadotrophs, and pituitary inhibin may contribute to this action. Within pituitary cell types, inhibin receptors appear to be specifically expressed in gonadotrophs. Mice [40, 69, 70], rats [41, 42], and human normal and tumor gonadotrophs [71] express Tgfbr3l, which encodes an inhibin B coreceptor [70]. Rat gonadotrophs also express Tgfbr3 [41, 42], which is also required for suppression of FSH by inhibin. Female Tgfdbr3l knockout mice show increased FSH level and the litter size, while female mice lacking both Thfbr3 and Tgfbr3l are infertile [70].

Together, these findings imply that gonadotrophs hold tight control over Fshb expression, probably due to the constitutive nature of FSH secretion.

Conclusions

Gonadotrophs are a very homogeneous group of hormone-producing anterior pituitary cells expressing a larger number of cell-type-specific genes, several of them critical for reproductive functions. LH is secreted from prestored secretory vesicles by regulated exocytosis, and GnRH controls the secretion of this hormone via GnRHRs, as nicely documented by the synchronous release of GnRH and LH release in vivo. Preovulatory LH surge and continuous application of GnRH temporally deplete LH secretory pool, suggesting that pulsatile GnRH provides a mechanism for sustained LH release and high basal Lhb expression for replacement of released LH. FSH is predominantly secreted by constitutive exocytosis and reflects the kinetics of Fshb expression controlled by both hypothalamic GnRH and intrapituitary activins via activin receptors, also expressed in other cell types, and inhibin receptors specifically expressed in gonadotrophs. Pulsatile GnRH stimulates Fshb expression and continuous GnRH application shut-off the expression of this gene, effectively blocking reproduction. GnRH also stimulates and inhibits Gnrhr expression, depending on the pattern of secretion/application, but basal Gnrhr transcription is an intrinsic property of gonadotrophs that protect these cells from dedifferentiation during the continuous absence and presence of GnRH.

Schematic representation of gene expression and hormone secretion in pituitary gonadotrophs.

GnRH directly controls LH secretion through regulated calcium-dependent exocytosis and has a minor stimulatory and inhibitory effect on high basal Lhb expression. GnRH dependent and independent Lhb expression is necessary to provide sufficient pre-stored LH to respond to pulsatile and surge GnRH release. In contrast, FSH secretion reflects the expression status of Fshb, which is determined by GnRH and activin/inhibin; hormone is released predominantly through constitutive exocytosis. Therefore, the kinetics of Fshb expression define the pattern of FSH secretion. Cga expression is stimulated but not inhibited by GnRH, making this subunit unlimiting component in dimer formations.