Regulation of Gonadotropins
Summary and Keywords
The gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are glycoproteins produced by gonadotrope cells of the anterior pituitary gland. The two hormones act on somatic cells of the gonads in both males and females to regulate fundamental aspects of reproductive physiology, including gametogenesis and steroidogenesis. In males, LH stimulates testosterone production and sperm maturation. FSH also regulates spermatogenesis, though the importance of the hormone in this process differs across species. In females, FSH stimulates ovarian follicle maturation. Follicles are structures composed of oocytes surrounded by two types of somatic cells, granulosa and theca cells. FSH stimulates granulosa cells to proliferate and to increase their production of the aromatase enzyme. LH stimulates theca cells to make androgens, which are converted into estrogens by aromatase in granulosa cells. A surge of LH also stimulates ovulation of mature follicles.
Gonadotropin-releasing hormone (GnRH) from the brain is the principal stimulator of gonadotropin synthesis and secretion from the pituitary. The sex steroids (androgens and estrogens) that are produced by the gonads in response to the gonadotropins feedback to the brain and pituitary gland. In the brain, these hormones usually slow the release of GnRH through a process called negative feedback, which in turn leads to decreases in FSH and LH. The steroids also modulate the sensitivity of the pituitary to GnRH in addition to directly regulating expression of the genes that encode the gonadotropin subunits. These effects are gene- and species-specific. In females, estrogens also have positive feedback actions in the brain and pituitary in a reproductive cycle stage-dependent manner. This positive feedback promotes GnRH and LH release, leading to the surge of LH that triggers ovulation.
The gonadotropins are dimeric proteins. FSH and LH share a common α-subunit but have hormone-specific subunits, FSHβ and LHβ. The β subunits provide a means for differential regulation and action of the two hormones. In the case of FSH, there is a second gonadal feedback system that specifically regulates the FSHβ subunit. The gonads produce proteins in the transforming growth factor β (TGFβ) family called inhibins, which come in two forms (inhibin A and inhibin B). The ovary produces both inhibins whereas the testes make inhibin B alone. Inhibins selectively suppress FSH synthesis and secretion, without affecting LH. The pituitary produces additional TGFβ proteins called activins, which are structurally related to inhibins. Activins, however, stimulate FSH synthesis by promoting transcription of the FSHβ subunit gene. Inhibins act as competitive receptor antagonists, binding to activin receptors and blocking activin action, and thereby leading to decreases in FSH.
Together, GnRH, sex steroids, activins, and inhibins modulate and coordinate gonadotropin production and action to promote proper gonadal function and fertility.
The Hypothalamic-Pituitary-Gonadal Axis and Female Reproductive Cycles
GnRH and the Gonadotropins
Sexual reproduction in mammals depends on a complex endocrine (hormonal) feed forward-feedback system commonly referred to as the hypothalamic–pituitary–gonadal (HPG) axis (Fig. 1). The gonads (ovaries and testes) produce gametes (oocytes and sperm) in a hormone-independent fashion. The latter stages of gamete maturation and release, however, are driven by hormones from the brain (hypothalamus) and anterior pituitary gland. A small population of neurons in the preoptic area and/or mediobasal hypothalamus produces the decapeptide gonadotropin-releasing hormone (GnRH). GnRH cells project to the median eminence at the base of the brain where they release GnRH into the pituitary portal vasculature via fenestrated capillaries. GnRH then travels to the anterior pituitary gland where it binds to the GnRH receptor (GnRHR) on the plasma membrane of gonadotrope cells. There are five hormone-producing cell types in the anterior pituitary, and gonadotropes represent an estimated 10% of the total population. In response to GnRH, gonadotropes produce and secrete the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH and FSH travel via systemic circulation to the gonads, where they bind to receptors on distinct somatic cell types to regulate gamete maturation and sex steroid hormone synthesis.
In males, LH acts on interstitial Leydig cells, located between the seminiferous tubules, to stimulate testosterone production (Figs. 2A and 3A). Testosterone then acts in adjacent Sertoli cells to regulate the later stages of spermatogenesis (Smith & Walker, 2014). FSH acts directly on Sertoli cells, where it stimulates the production of androgen binding protein, which concentrates testosterone in the testes, and inhibin B, a hormone that selectively inhibits FSH synthesis by the pituitary (see Regulation of Gonadotropin Synthesis and Secretion by Activins and Inhibins section; Iliadou, Tsametis, Kaprara, Papadimas, & Goulis, 2015).
In females, LH has two main actions that are dictated by the cells that it targets (Richards & Hedin, 1988). First, it stimulates androgen (androstenedione and testosterone) production by the theca cells that surround ovarian follicles (Figs. 2B and 3B). Second, it stimulates ovulation (and associated events (see section on Ovarian Follicle Development and Steroid Feedback) via its actions on mural granulosa cells of mature follicles. Follicles are largely composed of oocytes surrounded by one or more layers of granulosa cells. The number of layers of these cells defines the stage of follicle development (Fig. 2B). Mature, preovulatory follicles possess two types of granulosa cells: cumulus cells, which surround the oocyte, and mural granulosa cells, which line the periphery of the follicle. FSH produces three main actions on granulosa cells: (a) proliferation, (b) synthesis of the aromatase enzyme, and (c) expression of LH receptors (specifically in mural granulosa cells of mature follicles; Richards, 1994). Granulosa cell proliferation is the major cause of follicle growth. Aromatase enables granulosa cells to convert theca-derived androgens into estrogens (Fig. 3B). LH action in mural granulosa cells is essential for the final stages of dominant (large, preovulatory) follicle growth and for several key periovulatory events. These include expansion of the cumulus granulosa cells that surround the oocytes, resumption of meiosis, luteinization of mural granulosa cells, and eventually follicle rupture. Luteinization refers to the process by which estrogen-producing granulosa cells differentiate into progesterone-producing luteal cells.
Ovarian Follicle Development and Steroid Feedback
Postpuberty, female mammals that ovulate spontaneously (as opposed to reflex ovulators) exhibit reproductive cycles, such as estrous cycles in many rodents and agricultural species (e.g., cows, sheep, and pigs) and menstrual cycles in primates, including humans (Fig. 4). Though there are important interspecies differences, there are several common characteristics of these cycles. In particular, increases in FSH levels drive the later stages of ovarian follicle development (Figs. 2B and 4). These increases occur during the follicular phase of the menstrual cycle in humans and on the early morning of estrus (the so-called secondary FSH surge) in rodents (Fig. 4). It is generally agreed that female mammals are born with their full complement of oocytes. These oocytes exist in a primordial stage in the cortex of the ovary, where they are surrounded by a thin layer of pregranulosa cells. These cells then develop/differentiate into bona fide granulosa cells at the primary follicle stage. Through mechanisms that still escape our understanding, primary follicles begin to grow through a process called recruitment. This involves both the growth of the oocyte and the proliferation of surrounding granulosa cells. Recruitment occurs independently of pituitary gonadotropins. Recruited follicles will continue to grow until they possess multiple layers of granulosa cells, or they will die through an apoptotic process known as atresia. Once growing follicles reach an advanced stage of maturation, they begin to form a fluid-filled cavity known as an antrum. It is at the early stages of antral follicle development that follicles become sensitive to FSH and can be selected for accelerated growth. That is, these follicles will mature rapidly (e.g., three to four days in rodents or about two weeks in humans) to the point that they will ovulate in response to a surge of LH from the pituitary, which acts on the mural granulosa cells that now express the LH receptor (Peng, Hsueh, LaPolt, Bjersing, & Ny, 1991; Richards & Ascoli, 2018). In response to FSH and (pulsatile) LH, dominant follicles (those “destined” to ovulate) produce high levels of estrogens that eventually stimulate the LH surge via actions in the brain and pituitary through a process called positive feedback.
During most of the reproductive cycle in females and all of the time in normal males, sex steroids such as 17β-estradiol (an estrogen) and testosterone (an androgen) have negative feedback actions on the brain and pituitary gland (Fig. 1). For example, in males, GnRH induces pituitary LH release, which stimulates testicular testosterone production. Testosterone then feeds back to the brain to slow GnRH secretion. A similar negative feedback mechanism, mediated via estrogens, exists in females. GnRH, like many neuropeptides, is released in a pulsatile fashion. The exact nature of the GnRH “pulse generator” is still the subject of intensive investigation. However, the available data strongly implicate a population of neurons in the hypothalamic arcuate nucleus as fundamental components of the pulse generation machinery (Clarkson et al., 2017). These cells, which are called KNDy neurons, make three important neuropeptides: kisspeptin, neurokinin B, and dynorphin (Moore, Coolen, Porter, Goodman, & Lehman, 2018). Kisspeptin is a potent GnRH secretagogue (Gottsch et al., 2004). Neurokinin B and dynorphin are, respectively, potent stimulators and repressors of KNDy neuron activity and thereby kisspeptin release (Navarro et al., 2009; Topaloglu et al., 2009). It is possible that rhythms of neurokinin B and dynorphin activity thereby regulate rhythmic kisspeptin and, consequently, GnRH release. Importantly, KNDy neurons express sex steroid receptors, and kisspeptin production in these cells is potently downregulated by steroid hormones, providing some insight into the steroid negative feedback mechanism (Smith, 2009). According to current dogma, GnRH neurons do not express sex steroid receptors and are therefore not thought to be directly regulated by estrogens or androgens (or progesterone). This has led to the assumption that most, if not all, steroid effects on GnRH are regulated indirectly via kisspeptin neurons. However, according to recent observations, estrogens may act via the estrogen receptor β in GnRH neurons to modulate their sensitivity to kisspeptin (Novaira et al., 2018). KNDy neurons innervate GnRH terminals at or near the median eminence (Moore, Prescott, et al., 2018; Yip, Boehm, Herbison, & Campbell, 2015) where they can regulate episodic (pulsatile) GnRH release (Fig. 1).
Mechanisms of sex steroid positive feedback have been most thoroughly characterized in rodents, where high levels of estrogens stimulate (rather than inhibit) kisspeptin synthesis in a second population of neurons in the anteroventral periventricular nucleus (AVPV, also known as the rostral periventricular region of the third ventricle; Smith, Cunningham, Rissman, Clifton, & Steiner, 2005; Fig. 1). These cells, which synapse on both GnRH cell bodies and terminals, are thought to mediate the surge release of GnRH before ovulation, which in turn stimulates the LH surge from the pituitary. Male rodents have a poorly developed population of kisspeptin neurons in their AVPV, which likely explains (in full or in part) their inability to demonstrate estrogen positive feedback (Clarkson & Herbison, 2006). Other mammalian species, including primates, also lack AVPV kisspeptin neurons but appear to possess a second population of estrogen-stimulated kisspeptin neurons in the preoptic area of the hypothalamus, which may mediate positive feedback in these species (Watanabe et al., 2014). Sex steroids also have negative and positive feedback actions at the level of the pituitary gland, and the relevant data are considered in a later section (see Regulation of Gonadotropin Synthesis and Secretion by Sex Steroids).
Figures 1 to 4 provide schematic representations of the HPG axis in males and females, and of estrous and menstrual cycles in female rats and humans, respectively. Though not all aspects of female reproductive cycles are described, the figures provide a general overview of the dynamics of hormone secretion and feedback in the axis. This background is needed to understand regulation of the gonadotropins (see the section “Regulation of Gonadotropin Synthesis and Secretion”).
Gene and Protein Structures
The gonadotropins (LH and FSH) are members of the glycoprotein hormone family, which also includes thyroid-stimulating hormone (TSH) and chorionic gonadotropin (CG). All four proteins are dimers composed of a shared α subunit (chorionic gonadotropin α, also known as the α gonadotropin subunit) noncovalently linked to hormone-specific β subunits (Pierce & Parsons, 1981). Production of the latter are generally considered rate-limiting in the synthesis of the mature hormones. β subunits also confer biological specificity to each ligand. All four glycoprotein hormones bind to G protein-coupled receptors (GPCRs). FSH and TSH bind to their eponymous receptors, whereas LH and CG both bind and signal via the LH receptor (referred to as the LHCG receptor; Casarini, Santi, Brigante, & Simoni, 2018).
The α subunit gene (CGA), which maps to chromosome (Chr.) 6 in humans, contains five exons and encodes a 116 amino acid precursor protein. The mature subunit is 92 amino acids and forms a cystine-knot through five intramolecular disulfide bonds. Indeed, all glycoprotein subunits, which are generally thought to have derived from a common precursor >500 million years ago (Kawauchi & Sower, 2006), are members of the cystine-knot family of growth factors (Roch & Sherwood, 2014). The FSHβ subunit is encoded by the FSHB (Fshb in rodents) gene on Chr. 11 in humans. Its three exons encode a 129 amino acid precursor protein. Upon cleavage of the signal peptide, the 111 amino acid FSHβ protein dimerizes with the α subunit to form the mature FSH hormone. Though the β subunits confer receptor binding specificity, both subunits contribute to the receptor binding interface of the dimeric ligand (Fan & Hendrickson, 2005). Mutations in the FSHB subunit gene that prevent dimer assembly cause sterility in both women and men, demonstrating the necessity for the hormone in human reproduction (Layman, 2000; Layman & McDonough, 2000). Similarly, Fshb knockout females are sterile because of a block in follicle maturation at the pre- or early antral stage (Kumar, Wang, Lu, & Matzuk, 1997). By contrast, male mice lacking FSH have reduced sperm counts but are still fertile. Therefore, FSH’s role in spermatogenesis appears to be species-specific. The 141 amino acid LHβ subunit (121 amino acids post-cleavage) is encoded by the LHB gene (Lhb in rodents) on Chr. 19 in humans. Loss of function mutations in the LHB subunit gene that block dimeric LH production lead to androgen-deficiency, anovulation, and infertility in humans and mice (Arnhold, Lofrano-Porto, & Latronico, 2009; Ma, Dong, Matzuk, & Kumar, 2004). Thus, both FSH and LH are essential for successful reproduction, and one hormone cannot compensate for the absence of the other. In general, the two hormones work in combination.
CGA, FSHβ, and LHβ are glycoproteins and possess two, two, and one site for asparagine-linked glycosylation, respectively (Bousfield, May, Davis, Dias, & Kumar, 2018; Wide & Eriksson, 2017). These modifications affect the stability (half-life) and activity of the FSH and LH ligands. In general, FSH is more heavily glycosylated than LH, which contributes to the former’s greater serum half-life and to differences in the ability to detect discernible LH versus FSH pulses. That is, as LH is rapidly eliminated, GnRH pulses generate clear LH pulses. Given that FSH is relatively stable in circulation, levels do not decline appreciably between GnRH pulses.
The nature (microheterogeneity) and extent (macroheterogeneity) of glycoprotein hormones has been studied extensively, particularly for FSH and TSH (Wide & Eriksson, 2017, 2018). In each FSH dimer, there are four potential sites for glycosylation, two in the α subunit and two in the β subunit. Both sites in α appear to be constitutively glycosylated. By contrast, the two sites in FSHβ can be glycosylated alone or together (Bousfield et al., 2018). Hypoglycosylated forms of FSH (with only one site glycosylated in β) have shorter half-lives than the hyperglycosylated forms (both sites in β glycosylated) but also appear to be more active at the FSH receptor in granulosa cells (Bousfield et al., 2018; Jiang et al., 2015; Wang, Butnev, Bousfield, & Kumar, 2016; Wang, May, et al., 2016). The mechanisms mediating this differential sensitivity have not yet been resolved.
CG, the so-called pregnancy hormone, is produced by trophoblast cells of the human placenta (as well as by the chorion of pregnant mares). It is a dimer of CGA and the CGβ subunit. CGβ is structurally related to (and evolutionarily derived from) LHβ, and the two hormones (CG and LH) share the same receptor (Casarini et al., 2018; Talmadge, Boorstein, Vamvakopoulos, Gething, & Fiddes, 1984; Talmadge, Vamvakopoulos, & Fiddes, 1984). An important difference between the two, however, exists at the carboxy-terminus of CGβ, which has a 29 amino acid extension. This peptide contains four sites for O-linked glycosylation (Lentz, Birken, Lustbader, & Boime, 1984). By virtue of this posttranslational modification, CG has a considerably longer half-life than LH (Matzuk et al., 1990). This kinetic difference has been exploited clinically, as CG can be used in place of LH, for example, in the context of in vitro fertilization (IVF) as a trigger for ovulation. This enables single as opposed to multiple drug administrations. The carboxy-terminal extension of CGβ has also been fused to FSHβ to make a longer acting form of FSH for ovarian stimulation in IVF (Fares et al., 1992; Fauser et al., 2010).
Regulated Versus Constitutive Release
In mammals, LH and FSH are generally produced by the same gonadotrope cells. There may be a small population of FSH-only cells in mice, but this has not been thoroughly characterized (Wen, Ai, Alim, & Boehm, 2010). Upon synthesis of the LH dimer, the majority of the protein is stored in dense core granules (McNeilly, Crawford, Taragnat, Nicol, & McNeilly, 2003), which fuse with the plasma membrane and secrete their contents in a calcium-dependent manner. This process is referred to as regulated hormone release. By contrast, FSH is largely (though not exclusively) secreted through the constitutive pathway (McNeilly et al., 2003; Muyan, Ryzmkiewicz, & Boime, 1994). Rather than being stored in dense core granules, FSH is principally secreted upon synthesis and dimerization of its two subunits. As a result, GnRH rapidly and potently stimulates LH release. By contrast, GnRH-stimulated FSH release is generally more modest in amplitude and delayed in time. The most notable exception to this pattern is just prior to ovulation when GnRH stimulates surges of both LH and FSH (Fig. 4).
The differential sorting of the two hormones is largely governed by a hydrophobic heptapeptide sequence at the carboxy-terminus of the LHβ subunit (Jablonka-Shariff, Pearl, Comstock, & Boime, 2008). Indeed, removal of this sequence from LHβ leads to preferential constitutive release of LH, as normally observed with FSH. By contrast, adding the LHβ heptapeptide to the carboxy-terminus of FSHβ drives FSH into the regulated pathway, like LH (Pearl, Jablonka-Shariff, & Boime, 2010). How the heptapeptide affects sorting has not yet been completely resolved, though a single leucine residue at position 118 appears to be particularly important (Jablonka-Shariff & Boime, 2011). This differential sorting may have important physiological consequences, as mice engineered to secrete FSH predominantly through the regulated pathway exhibit markedly altered folliculogenesis (Wang et al., 2014).
Regulation of Gonadotropin Synthesis and Secretion
GnRH is an essential regulator of gonadotropin synthesis and secretion. The decapeptide is produced by fewer than 2,500 neurons in the preoptic area (rodents) and/or mediobasal hypothalamus (primates; Moenter, DeFazio, Pitts, & Nunemaker, 2003). GnRH neurons are born outside of the brain in the olfactory placode and migrate into the hypothalamus during embryonic development (Schwanzel-Fukuda & Pfaff, 1989). Humans with Kallman’s syndrome suffer from hypogonadotropic hypogonadism (immature gonads due to gonadotropin deficiency) because of a failure of GnRH neuronal migration (Schwanzel-Fukuda & Pfaff, 1989). These individuals fail to go through puberty as, in the absence of GnRH, LH and FSH levels are extremely low (Mitchell, Dwyer, Pitteloud, & Quinton, 2011). Individuals with genetic mutations that prevent GnRH production or GnRH action (e.g., inactivating mutations in the GnRHR) are similarly hypogonadal because of gonadotropin deficiency (Stamou, Cox, & Crowley, 2016). These and related observations, in a variety of species (Mason et al., 1986; McDowell, Morris, Charlton, & Fink, 1982), clearly demonstrate the absolute necessity for GnRH in quantitatively normal LH and FSH synthesis and secretion.
GnRH is released from the hypothalamus in a pulsatile manner. Depending on the physiological condition, pulses occur at frequencies from every 30 minutes to every few hours. The GnRH pulse generator may reside in KNDy neurons in the arcuate nucleus. The pulsatile nature of GnRH release is essential for its function in gonadotrope cells. Continuous (as opposed to pulsatile) GnRH downregulates gonadotropin secretion (Belchetz, Plant, Nakai, Keogh, & Knobil, 1978; Marshall, Dalkin, Haisenleder, Griffin, & Kelch, 1993). The underlying mechanisms have not yet been fully described but do not appear to involve conventional homologous desensitization processes observed for other GPCRs (Davidson, Wakefield, & Millar, 1994; McArdle, Davidson, & Willars, 1999). The frequency of GnRH pulses also affects pituitary function. High pulse frequencies (e.g., one per 30 or 60 minutes) tend to preferentially stimulate LH secretion relative to FSH (Wildt et al., 1981). By contrast, low pulse frequencies (e.g., one per two to four hours) preferentially stimulate FSH relative to LH secretion. The mechanisms through which gonadotrope cells decode pulse frequency are unresolved (Tsutsumi & Webster, 2009).
GnRH binds to the GnRHR on the surface of gonadotrope cells (Fig. 5). The GnRHR is a GPCR, which preferentially couples to the so-called Gq/11 pathway. Gq and G11 are α subunits within heterotrimeric G protein complexes that bind to and mediate many of the actions of GPCRs. In their inactive state, Gα proteins are bound to guanosine diphosphate (GDP). Upon ligand binding, the GnRHR activates Gq and/or G11 by promoting the exchange of GDP for guanosine-5'-triphosphate (GTP; Hamm, 1998). The GTP-bound Gq and/or G11 dissociate from the Gβγ subunits and then activate plasma membrane-associated phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into two second messenger molecules: membrane-associated diacylglycerol (DAG) and soluble inositol 1,3,4-trisphosphate (IP3). DAG activates protein kinase C (PKC). PKC then stimulates mitogen activated protein kinase (MAPK) signaling, which is essential for LH synthesis. IP3, in contrast, stimulates the mobilization of intracellular calcium from the endoplasmic reticulum, which promotes LH release. PKC can also stimulate influx of calcium via membrane channels, which may contribute to LH synthesis (Jorgensen, Quirk, & Nilson, 2004; Naor, Benard, & Seger, 2000).
GnRH-mediated activation of MAPK signaling is critical for transcription of the LHβ subunit gene (LHB/Lhb). Specifically, activation of the extracellular regulated kinase (ERK) MAPK pathway promotes the expression of a transcription factor called early growth response 1 (EGR1). EGR1 works in concert with at least two additional transcription factors, steroidogenic factor 1 (SF1, also known as NR5A1) and paired-like homeodomain transcription factor 1 (PITX1), to drive Lhb transcription. The three proteins bind together to one or more regulatory sequences (cis-elements) in the proximal LHB/Lhb promoter in a variety of species, including humans (Dorn, Ou, Svaren, Crawford, & Sadovsky, 1999; Fortin, Lamba, Wang, & Bernard, 2009; Halvorson, Kaiser, & Chin, 1999; Tremblay & Drouin, 1999).
The mechanisms through which GnRH regulates FSH synthesis are, surprisingly, poorly understood. Several studies using cell line models suggested a role for GnRH-induced activator protein-1 transcription factors, but several in vivo mouse models failed to confirm these in vitro observations (Coss, Jacobs, Bender, & Mellon, 2004; Huang, Sebastian, Strahl, Wu, & Miller, 2001; Jonak, Lainez, Boehm, & Coss, 2018; Strahl et al., 1997; Strahl, Huang, Sebastian, Ghosh, & Miller, 1998; Wang et al., 2008). Other data suggest that, at low pulse frequencies, the GnRHR might couple to a different Gα subunit, namely Gs (Liu et al., 2002). Activated Gs stimulates the adenylate cyclase-cyclic adenosine monophosphate (cAMP)-protein kinase A-cAMP response element binding protein (CREB) pathway. In cell line models, low-frequency GnRH pulses can stimulate transcription of the FSHβ subunit gene (Fshb) in a CREB-dependent fashion (Thompson et al., 2013). However, the CREB binding element in the rat Fshb promoter is not conserved across species (Bernard, Fortin, Wang, & Lamba, 2010), and there are currently no published in vivo data that either confirm or refute this in vitro model. Several groups are actively investigating how GnRH regulates FSH in vivo, so the picture may come into clearer focus in the next few years.
By Sex Steroids
LH and FSH stimulate sex steroid production by the gonads (Fig. 3). These steroids have potent negative and positive feedback actions on the gonadotropins. Feedback is both direct (at the level of the pituitary) and indirect (at the level of the hypothalamus). In the case of negative feedback, the effects are predominantly indirect. That is, steroids reduce GnRH stimulation of the pituitary via their slowing of the pulse generator in the hypothalamus. Nevertheless, gonadotropes express androgen, progesterone, and estrogen receptors and therefore can be directly targeted by sex steroids (Mitchner, Garlick, & Ben-Jonathan, 1998; Turgeon & Waring, 2001; Wu et al., 2014).
In rodents (rats and mice), androgens, such as testosterone, directly stimulate FSH synthesis. This is most readily observed in cultured pituitaries (Gharib, Leung, Carroll, & Chin, 1990; Spady et al., 2004), as the negative feedback actions of androgens in the brain overshadow the stimulatory effects in the pituitary in vivo. Female mice lacking the androgen receptor specifically in the pituitary have reduced Fshb mRNA levels, further confirming a direct stimulatory role for androgens, via their canonical receptor, in FSH synthesis (Wu et al., 2014). Interestingly, LH synthesis and secretion are normal in the same animals, suggesting that androgens may not regulate the Lhb subunit gene at the pituitary level, at least not in mice (Curtin et al., 2001). In contrast to the situation in rodents, androgens have either no effect or suppress FSH synthesis and/or secretion in cultured pituitaries from primates (Kawakami, Fujii, Okada, & Winters, 2002). This difference may be explained by differences in hormone responsive regulatory elements in the FSHB/Fshb promoters of the different species. For example, a critical androgen response element in the murine Fshb promoter is absent in the human FSHB promoter (Bernard et al., 2010; Thackray, McGillivray, & Mellon, 2006). Testosterone does not affect LH secretion in primate pituitary cultures, consistent with the rodent data showing that androgens do not directly regulate LHB/Lhb.
Like androgens, progestogens (e.g., progesterone) stimulate FSH synthesis directly at the pituitary level in rodents but not humans (Thackray et al., 2006). In sheep, progesterone may inhibit FSH (Batra & Miller, 1985). The mechanisms underlying these species differences have not been fully resolved. However, the androgen responsive regulatory elements that are specific to the rodent Fshb promoters also appear to mediate progesterone effects via the progesterone receptor (Thackray et al., 2006). In rats, the secondary surge of FSH, which occurs on the morning of estrus (Fig. 4) and which stimulates the following wave of ovarian follicle development, is stimulated, at least in part, by progesterone (Knox & Schwartz, 1992). It is unlikely that increases in FSH observed during the follicular phase of the human menstrual cycle similarly depend on progesterone as the hormone is low at this stage of the cycle (Fig. 4).
Estrogens (e.g., 17β-estradiol) also regulate gonadotropins at the level of the pituitary, though the underlying mechanisms remain largely unresolved. In mice, estrogens regulate gonadotropins, and LH in particular, via the estrogen receptor α (also known as ESR1). Deletion of this receptor in the mouse pituitary can lead to increases in LH levels, suggesting that estrogens may negatively regulate LH pulse amplitude (pulse frequency is predominantly regulated in the brain; Gieske et al., 2008; Singh et al., 2009). FSH is unaffected in these same animals, indicating that estrogen’s negative feedback on FSH is centrally mediated in mice. By contrast, in several agricultural species, such as sheep, estradiol can inhibit basal FSH secretion from cultured pituitaries, indicating that estrogen regulation of FSH may be species-specific (Miller & Wu, 1981).
Though estrogens have negative feedback effects in the pituitary, it is clear that they also have important positive feedback actions at this level. For example, high estrogen levels, such as those occurring prior to ovulation, enhance the sensitivity of gonadotropes to GnRH (Libertun, Orias, & McCann, 1974). Though estradiol increases GnRHR expression in gonadotropes (Clayton & Catt, 1981), it is not clear that this alone explains the enhanced pituitary sensitivity to GnRH (Kim et al., 2011).
By Activins and Inhibins
The activins and inhibins are proteins in the transforming growth factor β (TGFβ) superfamily that selectively regulate FSH without affecting LH (Ying, 1988). In the context of reproduction, inhibins come in two forms: inhibin A and B. The two proteins are disulfide-linked dimers of the inhibin α subunit and one of two inhibin β subunits, inhibin βA and inhibin βB, which are the products of different genes (Fig. 6A). Both hormones are made in the ovary, whereas the testes of most mammalian species only produce inhibin B (Illingworth et al., 1996; Woodruff et al., 1996). Inhibin B is produced by granulosa cells in the ovary and Sertoli cells in the testes. Inhibin A is produced by growing antral follicles in rodents and some other mammalian species but is principally made by the corpus luteum in primates, including humans (de Jong, Grootenhuis, Klaij, & Van Beurden, 1990; Groome et al., 1996). This explains why inhibin A increases prior to ovulation in rodents and after ovulation (during the luteal phase of the menstrual cycle) in humans (Fig. 2). Inhibins are secreted by the gonads into systemic circulation. Once they reach the pituitary gland, they act directly on gonadotrope cells to suppress FSH synthesis and secretion (Fig. 1). According to the current model, inhibins produce these effects by antagonizing the actions of pituitary-derived activins (Lewis et al., 2000).
Activins are disulfide-linked dimers of the inhibin β subunits (Fig. 6A). Dimers of two βA subunits form activin A, whereas dimers of two βB subunits form activin B. Heterodimers of the two β subunits comprise activin AB. Gonadotropes express the βB, but not βA, subunit and therefore can only make activin B (Roberts et al., 1989). There is evidence that pituitary-derived activin B stimulates FSH synthesis and secretion in an autocrine or paracrine manner, at least in rats (Corrigan et al., 1991). Activins produce their actions in cells like other members of the TGFβ superfamily (Fig. 6B). They first bind to so-called type II receptors: activin type II receptors A or B (ACVR2A or ACVR2B). These are transmembrane proteins that bind ligand via their amino-terminal extracellular domains and transduce signals via their carboxy-terminal intracellular serine/threonine kinase domains. Upon binding, the ligand-type II receptor complex recruits a second set of receptors, called type I receptors. Activin B can bind and signal via at least two type I receptors, ACVR1B and ACVR1C, which are also transmembrane serine-threonine kinases (Tsuchida et al., 2004). The type II receptors trans-phosphorylate and activate the type I receptors. The type I receptors then phosphorylate signaling proteins in the homolog of Drosophila mothers against decapentaplegic (SMAD) family, specifically SMADs 2 and 3. In the case of FSH regulation, SMAD3 is most relevant (Li et al., 2017). Phosphorylated SMAD3 partners with SMAD4 in the cytoplasm and the complex translocates into and accumulates in the nucleus. SMAD3/4 complexes partner with a forkhead box transcription factor called FOXL2 and together bind to the proximal promoter of the Fshb subunit, driving its transcription (Bernard & Tran, 2013). Therefore, activins promote FSH synthesis by directly driving expression of the FSHβ subunit gene (Fshb).
Inhibins, in contrast, appear to suppress FSH by competitively binding to activin receptors. Specifically, they bind activin type II receptors, but this binding does not promote recruitment of type I receptors (Xu, McKeehan, Matsuzaki, & McKeehan, 1995). Therefore, inhibins not only fail to stimulate intracellular signaling but prevent activins from doing so. As a result, it is the loss of activin action that likely explains the ability of inhibins to suppress FSH. Inhibins bind to activin type II receptors with lower affinity than do activins. However, in the presence of a co-receptor called betaglycan (also known as the TGFβ type III receptor [TGFBR3]) inhibin A can form high affinity ternary complexes with activin type II receptors that impair binding by activins (Lewis et al., 2000; also see Fig. 6B). It is less clear that betaglycan mediates the actions of inhibin B in gonadotrope cells (Li et al., 2018; Makanji, Temple-Smith, Walton, Harrison, & Robertson, 2009).
Inhibins (and by inference activins) play essential roles in regulating FSH levels in females. In rodents, blocking inhibin actions during metestrus or diestrus leads to enhanced FSH levels and ovarian folliculogenesis (Rivier & Vale, 1989). How inhibins regulate FSH across the primate menstrual cycle is still an open question (Fraser & Tsonis, 1994). Some argue that estrogens regulate FSH during the follicular phase of the cycle. However, other data clearly implicate inhibin B in the control (or modulation) of follicular phase FSH secretion (Welt, 2004; see also Fig. 4). For example, FSH levels are elevated during the early follicular phase in perimenopausal relative to younger women. In fact, this is one of the first clinical signs of the menopausal transition. However, at the same time, estrogen and LH levels are normal, arguing for normal GnRH secretion. Inhibin B levels, in contrast, are reduced as the number of follicles (and inhibin B-producing granulosa cells) decline due to follicular exhaustion (Burger, Dudley, Robertson, & Dennerstein, 2002). It is likely that this loss of inhibin B negative feedback explains the observed increases in FSH. That women are sensitive to inhibins is also demonstrated in pathological states. For example, FSH levels are profoundly reduced in women with inhibin-hypersecreting ovarian tumors (Healy et al., 1993). Moreover, soluble forms of activin type II receptors, which bind and bioneutralize activins, suppress FSH levels in postmenopausal women, demonstrating that endogenous activins (or related TGFβ ligands) stimulate FSH synthesis and secretion (Ruckle et al., 2009) and that inhibins likely act by blocking these actions.
Inhibin B regulation of FSH in males appears to be both age- and species-specific. In rodents, FSH levels rise in early postnatal development, stimulating Sertoli cell proliferation and inhibin B production. This inhibin B provides a critical negative feedback signal driving decreases in FSH, limiting the time window for Sertoli cell proliferation (Sharpe et al., 1999). In adult rats and mice, inhibin B levels are relatively low and appear to play only a modest role in regulating FSH levels (Culler, 1990; Rivier, Cajander, Vaughan, Hsueh, & Vale, 1988). In male humans and non-human primates, however, inhibin B is the major testis-derived negative feedback regulator of FSH, even in adulthood (Medhamurthy, Abeyawardene, Culler, Negro-Vilar, & Plant, 1990).
The gonadotropins are essential regulators of reproductive physiology. Over the past century, we have learned a great deal about the mechanisms controlling their synthesis, secretion, and actions. Nonetheless, fundamental questions remain unanswered. For example, we lack a clear understanding of how GnRH regulates FSH synthesis. This gap in knowledge has contributed to our inability to decipher how gonadotropes decode GnRH pulse frequencies, which differentially regulate FSH and LH. This differential regulation is critical for driving reproductive cycles in females, and perturbations in this system may underlie reproductive disorders such as polycystic ovary syndrome.
Both LH and FSH are modified via glycosylation. The nature and extent of these modifications are regulated and affect the proteins’ activities. Yet, we do not yet understand with any real precision what factors regulate gonadotropin glycosylation or how they produce their actions. In addition, though we have a clear picture of how inhibin A regulates FSH, mechanisms of inhibin B action are currently unresolved.
Finally, in the past decade, noncanonical actions of gonadotropins have been described in a diverse array of tissues and cells, including bone, fat, and hematopoietic stem cells (Liu et al., 2017; Sun et al., 2006; Velardi et al., 2018). Whether these tissues/cells in turn regulate FSH and LH through novel or established feedback hormones has not been resolved but merits investigation.
This article is funded by Canadian Institutes of Health Research operating grants MOP-133394 and MOP-123447.
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