Which of the following hormones stimulates the release of a future egg cell by the ovaries?

3 GnRH

GnRH acts on the gonadotropic cells of the anterior pituitary to stimulate the release of LH and FSH (see Chapter 5). This polypeptide hormone must interact with a similar or identical receptor on two different cell types in the anterior pituitary, one which secretes primarily LH and the other FSH. On the other hand, there is evidence that both FSH and LH may be secreted from the same cell in response to GnRH. A period occurs in the ovarian cycle during which the estrogen from the developing follicle is usually high in blood and feeds back positively (rather than negatively) on the release of LH and FSH, but more intensively in terms of LH release. GnRH receptors may be more plentiful on the LH-containing cells than on the FSH-containing cells. There are other possible explanations, such as ovarian elaboration of inhibin, which is a specific inhibitor of FSH but not LH secretion. Ovarian estrogen could operate at the level of the anterior pituitary cell or on the release of GnRH.

A speculative mode of action of GnRH is shown in Fig. 3-14. P. Conn's laboratory has shown that ability to microaggregate receptors, presumably a minimum of 2 to within about 150 Å distance, even if accomplished by means other than using GnRH, is sufficient to promote GnRH agonist activity. GnRH-receptor complexes could thus move in the membrane to within a critical distance as a first step in hormone action following receptor occupation. This hypothetical movement is indicated by the opposing arrows in Fig. 3-14. Receptor internalization and turnover may follow. However, large-scale patching and capping or internalization may not be required for gonadotropin release. Prostaglandins do not seem to be implicated in the process of exocytosis, according to K. Catt's laboratory, so either arachidonic acid or some nohprostagladin product may be involved. Note that the mechanism of GnRH action proposed here differs from the one proposed for TRH action (Fig. 3-12).

There may be receptor sites for GnRH on gonadal cells. GnRH, in low concentrations in vitro, can alter responses of these cells to gonadotropes such as FSH so as to inhibit cyclic nucleotide accumulation. Such experiments would suggest an extrahypothalamic production of this releasing hormone, reflecting the well-known situation with somatostatin. Extrahypothalamic actions of GnRH, especially on the testis, are discussed in Chapter 12.

GnRH is released, perhaps indirectly, by Met-enkephalin, and it may be that Met-enkephalin operates through dopamine (Table 3-2) from dopaminergic neurons. GnRH suppresses dopamine synthesis in the rat, suggesting a negative feedback effect, and the fact that the dopaminergic neuron and the GnRH-secreting cell are adjacent geographically renders dopamine a possible candidate for a GnRIF. The number of GnRH receptors on gonadotrophic cells of the anterior pituitary is positively controlled by the level of GnRH itself. This is of significance in the middle of the ovarian cycle (see Chapter 13). There is a dramatic increase in LH (“LH spike”), which is initiated by high levels of estrogen from the developing ovarian follicle. Estrogen probably operates on the CNS to make GnRH more effective or increase its secretion by acting at the anterior pituitary level, or both.

Gonadotropins

These hormones are produced by the gonadotrophic cells of the anterior lobe of the pituitary gland. They act on the maturation and function of the ovaries and testes. Gonadotropins include FSH and LH, both of which are glycoproteins. FSH (∼34 kDa) contains 10% carbohydrates, while LH (∼25 kDa) has 15% carbohydrates. The hydrocarbon chains of both gonadotropins have mannose, galactose, fucose, glucosamine, galactosamine, and sialic acid. Gonadotropins consist of two different polypeptides, α and β. The α subunit is identical for both LH and FSH; the β chain is specific for each hormone. Although the activity depends on the β chain, the presence of the two subunits is necessary for the hormone’s biological functions.

FSH actions. FSH induces the maturation and development of the Graafian follicle in the ovary. Together with LH, it stimulates estrogen and progesterone synthesis in the ovary. The highest levels of FSH in plasma are attained before ovulation.

In testes, FSH promotes the development of the seminiferous tubules and is one of the factors involved in the initiation of spermatogenesis. It targets Sertoli cells to stimulate the production of estrogens from androgens, and, together with testosterone, it induces the synthesis of androgen-binding proteins in these cells, which helps maintaining high levels of testosterone locally, necessary for spermatogenesis. In men plasma, FSH levels are very constant.

LH actions. In females, LH controls the development of the corpus luteum and stimulates estrogen and progesterone secretion. It induces StAR protein and the synthesis of enzymes involved in pregnenolone and other steroid hormone precursors. Although the Graafian follicle development is mainly controlled by FSH, estrogen production depends on both corticotropins.

In males, LH is also called interstitial cell (Leydig cells)-stimulating hormone (ICSH). It promotes the production and secretion of testosterone, which in turn helps to maintain spermatogenesis and development of secondary sexual organs.

FSH and LH activate adenylate cyclase in the target cells. Levels of LH and FSH vary with age; they are low before puberty and elevated in postmenopausal women. Increased LH in males and cyclic FSH secretion in girls precede the onset of puberty. LH and FSH vary during the menstrual cycle. In the initial phase of the cycle, LH increases slowly and has a marked increase at midcycle, which triggers ovulation. FSH increases slightly at first, then drops, and at midcycle, increases parallel to that of LH occurs. The concentrations of both hormones fall after ovulation.

Secretion is controlled by GnRH; circulating sex steroids influence GnRH secretion by feedback.

Other paracrine factors of ovarian origin are activin and inhibin, proteins that have opposite effects on the gonadotrophic cells, and follicle statin, a peptide that regulates activin and inhibin.

Publisher Summary

Inhibins and activins were discovered by virtue of their effects on the gonadotropic cells of the anterior pituitary. Inhibin/activin subunits and their mRNAs have been detected in many tissues, including the ovary, testis, placenta, pituitary, central nervous system (CNS), adrenal, and bone marrow. Various tissues differ in the amounts of subunits, in the proportions of particular subunits, and in the processing of the precursors. It is possible that the proportions of subunits produced in a given cell determine the relative amounts of activin and inhibin generated. This chapter discusses the role of α2M in the delivery or clearance of inhibin and activin, similar to that proposed for the binding of transforming growth factor-β (TGFβ) and a number of other growth factors by α2M and other high molecular weight binding protiens. The activin–follistatin complex is, in contrast, biologically inactive and this binding protein plays an important role in limiting exposure of cells to activin. The activin is the only member within the TGF-β superfamily that has a functional antagonist formed by a dimer with a common subunit. The development of gonadal tumors in transgenic mice bearing deletions of the inhibin α subunit and, therefore, unable to make inhibin illustrates the importance of constraining activin to the survival/health of the animal. The initial step in the action of activin is to bind to plasma membrane receptors. Activin binds with high affinity to at least two classes of membrane proteins with molecular masses of ∼50 and ∼70 kDa referred to as the Type I and Type II receptors.

Abstract

Gonadotropin releasing hormone (GnRH) is secreted from the hypothalamus and stimulates gonadotropic cells in the anterior pituitary gland to release luteinizing hormone and follicle-stimulating hormone, which in turn regulate the gametogenic and steroidogenic functions of the gonads in male and female. This review summarizes the expression of GnRH and its receptor, mode of action and regulation of the native peptide. It highlights the clinical applications and preclinical investigation of the agonists, antagonists, and vaccine candidates for the treatment of hormone-dependent sex organ-related disorders. Additionally, examples of studies concerning the use of GnRH receptor ligands for targeted cancer therapy is discussed.

FSH/LH in the acute phase after aSAH

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are gonadotropins, glycoprotein polypeptide hormones released by the gonadotropic cells of the anterior pituitary gland. They modulate the reproductive system. Seven studies reported a range of FSH/LH impairment in the acute aSAH phase of between 7.7%–9.3% (Aimaretti et al., 2004; Lammert et al., 2011) to 61.5% (Dutta et al., 2012). Other investigations computed intermediate estimates, varying from 12%–58% (Klose et al., 2010) to 34%–43.1% (Pereira et al., 2013; Khajeh et al., 2015; Robba et al., 2020).

4.3.1 GnRH-FSH-LH signaling

Gonadotropin-releasing hormone (GnRH), a hormone produced by the hypothalamus, induces the release of FSH and LH from gonadotrophic cells in the anterior pituitary. Both LH and FSH travel through the circulatory system to the testis where they stimulate testosterone and glial cell line-derived neurotrophic factor (GDNF) production by Leydig and Sertoli cells, respectively. FSH binds to the FSH receptor in Sertoli cells, and stimulates GDNF synthesis (see below), as well as stimulates Sertoli cell proliferation in the prepubertal testis (Dierich et al., 1998; Haywood et al., 2003; Heckert & Griswold, 2002; O'Shaughnessy, Monteiro, Verhoeven, De Gendt, & Abel, 2010). Postpuberty, FSH has been implicated in meiosis maintenance; reduction in FSH levels during meiosis resulted in increased pachytene spermatocyte apoptosis and it has been postulated that FSH may act as an apoptosis suppressor (Ruwanpura, McLachlan, Matthiesson, & Meachem, 2008). On the other hand, LHR knockout (LHRKO) males were born phenotypically normal, with testes and genital structures indistinguishable from their wild-type littermates. Postnatally, testicular growth, external genital and accessory sex organ maturation were blocked in LHRKO males, and spermatogenesis was arrested at the round spermatid stage (Zhang, Poutanen, Wilbertz, & Huhtaniemi, 2001). Furthermore, the number and size of Leydig cells were dramatically reduced. Transplanting mesenchymal stem cells into the adult testis of LHRKO mice, restored serum testosterone levels and spermatogenesis (Lo, Lei, Rao Ch, Beck, & Lamb, 2004). Despite the importance of hormonal regulation for the development of the testis and the maintenance of spermatogenesis, the molecular mechanisms through which these hormones act are still poorly understood.

Androgen biosynthesis

The production of androgens is regulated through interactions between the nervous and endocrine system. The hypothalamus produces luteinizing hormone-releasing hormone (LHRH), which stimulates gonadotropic cells in the pituitary gland to release luteinizing hormone (LH) [14]. This hormone then induces testicular Leydig cells to synthesize testosterone. In addition to the testes, a relatively small amount of testosterone (~ 10% total serum) is also produced independent of LH by the adrenal cortex [15]. Given the transformative role of androgenic steroids, there are extensive feedback mechanisms to maintain cellular homeostasis. The classical androgen biosynthetic route occurs through a complex biochemical pathway whereby cholesterol is converted to pregnenolone, 17OH-pregnenolone, DHEA, androstenediol/androstenedione, and finally testosterone. When secreted into circulation, testosterone is proposed to passively diffuse into cells where it is reduced by 5-alpha reductase to the more potent dihydrotestosterone (DHT). In addition, androgenic steroids can also be produced by a so-called backdoor pathway [16]. This synthetic route bypasses DHEA and testosterone and directly produces androgenic DHT [17,18].

Etiology and Pathogenesis

The endocrine regulation of testicular or ovarian functions is performed by the gonadotropic hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), secreted by the gonadotropic cells of the anterior pituitary gland, the function of which is regulated via gonadotropin-releasing hormone (GnRH) released by the hypothalamus. In men, due to LH and FSH deficiency, the testicular functions of testosterone production and spermatogenesis are impaired under conditions of insufficient GnRH release (e.g., in Kallmann's syndrome and IHH). In women, the same applies to estrogen production and ovulation. For the X chromosomal recessive variant of the disease, the underlying mechanism of defective GnRH release has been described as impaired migration of GnRH neurons from the nasal olfactory epithelium to their proper anatomical location in the basal hypothalamus, a process that occurs during normal embryonic development. The gene responsible for this phenomenon is located on the short arm of the X chromosome (Xp22.3) and is called KAL-1. The corresponding protein seems to be an extracellular regulator of the directed outgrowth of axons and is called anosmin-1 (after the clinical feature of anosmia found in Kallmann's syndrome). Nevertheless, pedigrees of patients suggest an autosomal dominant and autosomal recessive inheritance as well, and most patients (65%) represent sporadic cases. Well-documented familial case reports demonstrate that Kallmann's syndrome, IHH, and isolated anosmia without concomitant deficient GnRH release can be regarded as manifestations within the spectrum of one underlying disease since these disorders are found in close relatives. Genes currently recognized to be involved in congenital hypogonadotropic hypogonadism include KAL-1, the GnRH receptor, DAX-1, and PROP-1. Furthermore, sporadic cases of mutations in ANK-1, SF-1, FGFR-1, LHX-3, and HESX-1, as well as the leptin (Ob) gene, the leptin receptor (Ob-R) gene, and the prohormone convertase-1 gene, have been reported. However, a genetic basis for IHH has been established in less than 30% of cases; thus, several autosomal and X-linked genes await description.

Regulation of Reproduction by the HPG Axis

The hypothalamus-pituitary-gonad axis regulates aspects of reproduction in vertebrates. In mammals, neurons within the hypothalamus release gonadotropin-releasing hormone (Gnrh) that then stimulates gonadotropic cells located within the anterior pituitary to synthesis and secrete the gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH; for review, see Plant, 2015). LH and FSH are heterodimeric glycoproteins composed of a shared alpha subunit that is encoded by the Glycoprotein hormone alpha gene, Cga, and a hormone-specific beta subunit encoded by the genes Fshb and Lhb, respectively (Gharib et al., 1990). LH and FSH are carried to the gonad by the circulatory system where they bind their respective G-protein coupled transmembrane receptors called Luteinizing Hormone/Choriogonadotropin Receptor (LHCGR) and Follicle Stimulating Hormone Receptor (FSHR), respectively (for more detailed review, see (Ascoli et al., 2002; Simoni et al., 1997). In mammals, FSHR is expressed by granulosa cells in the ovary and Sertoli cells in the testis, whereas LHCGR is expressed by Theca and granulosa cells in the ovary and Leydig cells in the testis. In mammalian females, FSH promotes the growth of the oocyte-follicle complex while LH functions to promote maturation of the fully grown oocyte and ovulation. Both LH and FSH promote the production of estrogen by the ovary. In mammalian males, FSH acts on the Sertoli cells to stimulate sperm production, while LH stimulates Leydig cells to produce testosterone. There are several differences between the reproductive strategies of eutherian mammals and fish that spawn their eggs. Nonetheless, recent genetic studies in zebrafish have uncovered both similarities and apparent differences in the roles the HPG axis plays in reproduction in teleost versus mammals. The phenotypes of zebrafish mutants in HPG axis components are summarized in Table 16.2.

Table 16.2. Summary of HPG axis mutant phenotypes.

Follicle-stimulating hormone (Fsh): Fsh appears to play a more prominent role in the ovary than the testis in zebrafish. Analysis of loss-of-function fshb mutants indicates that Fsh plays an essential role in promoting oocyte development (Chu et al., 2015; Zhang et al., 2015b). fshb mutant females have delayed ovary development during the juvenile stages (30–60 dpf), and oocyte development appears to be delayed at the transition from stage IA to stage IB (primary growth to follicle stage). However, mutant females are fertile at 90 dpf, suggesting that once the transition occurs, Fsh is not required for further oocyte development. Paradoxically, zebrafish mutant for the Fsh receptor, fshr, have a more severe phenotype then fshb mutants. Although male fshr mutants are fertile, the oocytes of female mutants arrest development at stage 1A, indicating that signaling through Fshr is necessary to promote the transition from stage 1A to stage IB. fshr mutant animals have a normal sex ratio during early juvenile stages (25–50 dpf), but the sex ratios become skewed toward male in late juvenile and adult stages (Zhang et al., 2015a). This suggests that mutant females are sex reverting to males. This latter result is consistent with the known role of oocytes in maintaining female sex determination in zebrafish (Dranow et al., 2013). Fsh is apparently dispensable for spermatogenesis in zebrafish. Mutant fshb and fshr males have a modest delay in testis development, but adults are fertile with a normal-sized testis (Chu et al., 2015; Zhang et al., 2015b).

Luteinizing hormone (Lh): Current evidence strongly argues that Lh plays an essential role in regulating oocyte maturation and ovulation in zebrafish but is not required for spermatogenesis. Wild-type zebrafish spawn soon after the vivarium lights come on (artificial sunrise). Under normal in vivo conditions oocyte maturation has been shown to initiate around 1.5 h before sunrise (i.e., lights on), as measured by assessing the transparency of fully grown oocytes: immature oocytes are opaque, while mature oocytes are more translucent (Shang et al., 2019). Correlated with visible indicators of oocyte maturation, expression levels of lhb peak at 2.5 h before dawn, as measured by RT-PCR (Shang et al., 2019). Finally, mutational analysis confirms that lhb function is required for oocyte maturation and ovulation, as lhb mutant ovaries have all stages of oocytes, from oogonia to Stage IV fully grown (FG) oocytes, but FG oocytes never undergo maturation or ovulation (Chu et al., 2014; Shang et al., 2019; Zhang et al., 2015b). As such, lhb mutant females are sterile, but their gonadal-somatic index gradually increases as their ovaries fill with FG oocytes. In contrast to lhb mutants, lhcgr mutant females are fertile (Chu et al., 2014; Zhang et al., 2015a). This result has led to the hypothesis that Lh can also signal through the Fsh receptor, a hypothesis consistent with the more severe phenotype of fshr versus fshb mutants described above. Finally, mutant analysis suggests that Lh signaling has a less important role in the testis than in the ovary. lhb and lhcgr mutant males are fertile with normal histology (Chu et al., 2014; Zhang et al., 2015a, 2015b). Furthermore, lhb mutants have wild-type levels of testosterone, suggesting that Lh is not necessary for the production of this hormone by Leydig cells, as it is in mammals (Shang et al., 2019).

Disrupting both Fsh and Lh signaling more severely disrupts gonad development than disrupting only one pathway. Double mutants for either fshb;lhb or fshr;lhcrg are all-male indicating failure of ovary development (Chu 2015; Zheng 2015a). A curious result is that fshb;lhb double mutants have a less severe testis phenotype then fshr;lhcrg double mutants. The ligand double mutant males have delayed testis development, and although their testes contain germ cells at all stages of spermatogenesis, including mature sperm, they have an overaccumulation of apparent spermatogonia (Chu et al., 2015; Zhang et al., 2015a). By contrast, the double receptor mutants are sterile males with testes that contain mostly apparent spermatogonia, few spermatozoa, and no mature sperm. It has been proposed that the differences between the ligand and receptor double mutant phenotypes are due to weak ligand-independent constitutive activity of the receptors, as has been demonstrated in heterologous tissue culture assays (Chu et al., 2015).

In summary, gonadotropin signaling in zebrafish plays an essential role in regulating germ cell development in both males and females, but in contrasts to mammals, current evidence supports more overlap in the expression and function of Fshr and Lhcrg in zebrafish than has been found in mammals. In addition, there is evidence that in fish, these receptors are not as specific for the Fsh and Lh ligands as are their mammalian counterparts. This apparent cross-talk may be explained by the evolution of this signaling pathway. For example, lamprey, a basal vertebrate, have only a single gonadotropin ligand that signals though probable homologs of Lhcgr and Fshr (Sower et al., 2009). In addition, Lhb and Fshb in teleost and mammals appear to have arisen by duplication of the beta subunit of this ancestral gonadotropin, which in fish appear to have retained their ability to activate both Lhcrg and Fshr receptors, but in mammals have further evolved to become receptor-specific.

Gonadotropin Releasing Hormone

Gonadotropin releasing hormone (GnRH) is a decapeptide secreted by hypothalamic neurons in a pulsatile manner into the capillary plexus of the median eminence, and affects the release of LH and FSH from gonadotropic cells of the anterior pituitary. Humans with Kallmann syndrome, characterized by the lack of GnRH, have hypogonadotropic (low levels of FSH and LH) hypogonadism (176,177). Kallmann syndrome is a genetically heterogeneous disorder with X-linked, autosomal dominant and autosomal recessive inheritance and occurs as a result of defective migration in GnRH producing neurons and olfactory neurons (178). One of the genetic loci responsible for Kallmann syndrome is located in the Xp22.3 region and encodes a KAL1 protein that shares functional domains with protease inhibitors and neural cell adhesion molecules. The absence of KAL1 prevents the embryonic migration and development of the neurons that are normally destined to secrete GnRH. However, since inheritance is X-linked, KAL mutations and deletions affect males more than females with 5:1 ratio, while carrier females are usually unaffected. The degree of GnRH deficiency is variable in women with Kallmann syndrome ranging from complete to partial deficiency in FSH and LH. Ovarian streak gonads were described in a 16-year-old woman with possibly autosomal dominant form of Kallmann syndrome (179). The cause of the ovarian streaks in this woman is unclear. A 32-year-old woman with Kallmann syndrome was also described as having primordial follicles on ovarian biopsy, but it is unclear whether more advanced follicular structures were present (180). Treatment with GnRH or FSH and LH can induce ovulation and fertility although response to the treatment is variable most likely due to the genetically heterogeneous nature of the disorder (180,181). Currently there are no published cases of women with mutations in the GnRH gene, therefore it is unclear what effect isolated deficiency of GnRH has on human folliculogenesis. A second GnRH peptide, GnRHII, and its receptor have been described in humans and primates although its role in ovarian folliculogenesis is not clear (182–185).

A spontaneous deletion in the hypogonadal (hpg) mouse removed 33.5 kb of the Gnrh locus leading to deletion of the last two exons but not the sequences encoding the signal sequence and the GnRH decapeptide (131). This partially deleted gene is transcriptionally active but GnRH cannot be detected by immunohistochemistry. Hpg mice have low or undetectable levels of GnRH and very low (but assayable) levels of FSH and LH. Ovaries from hpg mice at 4 weeks of age weigh 1/10th that of normal ovaries and lack antral follicles (133). In most cases, the oocyte is surrounded by two layers of granulosa cells (133). Further studies on follicular development revealed that at birth, hpg mice have 20% less follicles than wild type mice (132). Therefore, hpg mice are born with smaller endowment of follicles corresponding to types 1, 2, and 3a of the Pedersen and Peters scheme (13).

Gonadotropin releasing hormone receptor (GnRH-R) mutations in humans cause infertility (186). Several mutations have been described to date, and most are compound heterozygotes, with more severe phenotypes observed in homozygous mutations. Folliculogenesis is affected, but the degree of ovarian and follicle development is not clear due to difficulty in analyzing human ovaries. One affected woman had ovarian streaks at age 16 detected by laparoscopy (187). At age 22, the same woman underwent bilateral oophorectomy for benign ovarian tumors (seromucinous cystadenomas). Histologically, few primordial follicles were detected but no mention is made whether other follicular stages are present. Of interest is that ovarian tumors developed in this patient despite the low levels of gonadotropins. Since both GnRH and Gnrh-R are expressed in the human granulosa cells, disruption of signaling in the ovary may also play an important role in the phenotype (188). The phenotype in this particular woman also supports the thesis that gonadotropins may play a role in early folliculogenesis. No published Gnrhr knockout mouse model exists, and future studies are needed to assess contribution of GnRH autocrine signaling in ovarian folliculogenesis versus its central role in the hypothalamic–pituitary axis.