The Hypothalamo-Pituitary Unit, Testis, and Male Accessory Organs

Overview: Ensemble Nature of Reproductive System

Male reproductive hormones comprise the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH), pituitary gonadotropins, luteinizing, and follicle-stimulating hormone (LH and FSH), testicular steroids, testosterone, and estradiol (Te and E ₂ ), and putatively systemically active gonadal peptides like inhibin B. Integration requires repeated incremental and decremental feedforward (stimulatory) and feedback (inhibitory) signaling among neural and hormonal components of the axis ( Fig. 13.1 ) . The negative-feedback system can be exploited to regulate male fertility by suppressing spermatogenesis with exogenous sex hormones. Gonadotropin and Te deficiency can result in infertility or subfertility, impaired sexual maturation, reduced muscle and bone mass, insulin resistance, visceral fat accumulation, diminished erythropoiesis, libido, potency, and general well being.

Essential Roles of GnRH and Upstream KiSS1 Neurons

In the adult, specialized neurons (approximately 1200 in number in the human) located in the arcuate-nucleus of the mediobasal hypothalamus secrete GnRH in a pulsatile fashion into a portal microvasculature system. GnRH pulses stimulate gonadotropes located in the anterior pituitary to secrete LH and FSH. In the embryo, migration of GnRH neurons from the olfactory placode into the upper diencephalon is essential to establish the anatomic proximity required for effectual hypothalamo-pituitary signaling. Several genes ( KAL1, KAL2 and PROK2 ) are critical for both olfactory-bulb morphogenesis and proper GnRH neuronal migration. KAL1, KAL2 and PROK2 are located on chromosomes X, 8 and 3, and respectively encode anosmin-1, fibroblast growth-factor (FGF) receptor 1 and prokineticin 2. Inactivating mutations of any of these genes, including FGF8 which is the ligand for the FGF receptor 1, and less commonly neuroembryonic LHRH factor (NELF) cause Kallmann syndrome. The syndrome is characterized clinically by hyposmia or anosmia and isolated gonadotropin deficiency. Inheritance patterns are respectively X-linked, autosomal dominant and recessive for defects in anosmin, FGF-receptor 1 and prokineticin or its receptor. Recently identified mutations in the chromodomain helicase DNA binding protein-7, which cause CHARGE syndrome, may also present with Kallmann syndrome and idiopathic hypogonadotropic hypogonadism. ^, Additional pleiotropic genes, which cause reproductive and nonreproductive phenotypes, manifest mutations identified in at least 25 separate loci. Next generation sequencing and other modern methods will continue to unveil novel mutations. These mutations will ultimately need to be functionally validated experimentally or bioinformatically.

Pulsatile GnRH release is both intrinsic to clusters of GnRH neurons and essential to stimulate LH secretion over the long term. Continuous GnRH delivery downregulates gonadotrope GnRH receptors and signaling pathways, which forms the basis for treating prostatic cancer and precocious puberty with long-acting GnRH agonists. Episodic GnRH secretion begins during fetal development when GnRH neurons first populate the mediobasal hypothalamus. A second surge of high-amplitude LH pulsatility emerges in infant boys during the first several months of age. GnRH secretion declines during childhood only to be reinstated at markedly higher amplitude and slightly higher frequency in puberty. ^,

The kisspeptin-kiss1R system controls the timing of puberty, in part through epigenetic factors, and may also regulate GnRH secretion in other epochs of life. Kiss1R (formerly known as GPR54) is a G-protein coupled receptor expressed on GnRH neurons. KiSS-1 is the gene that encodes kisspeptin-121, which is proteolytically cleaved to a 54 amino-acid peptide named kisspeptin-54 (also known as metastin) and kisspeptin-14 and 13 and possibly further modified by endopeptidase 24.15. Kisspeptin-54 is the natural ligand for the receptor Kiss1R. Inactivating mutations of either KiSS-1 or Kiss1R in mice and of KiSS1 in men result in normosmic hypogonadotropic hypogonadism, thus establishing that kisspeptin and cognate receptor are necessary upstream activators of GnRH neurons. Mutations in the genes TAC3 and TACR3, which encode the peptide neurokinin B and its receptor, respectively, also result in hypogonadotropic hypogonadism. Furthermore, specific neurons (KNDy neurons) in the arcuate nucleus of the hypothalamus, which coexpress kisspeptin, neurokinin B, and the endogenous opioid peptide, dynorphin, have been identified in sheep and rodents, but not yet conclusively in humans. These KNDy neurons are critical for sex-steroid feedback regulation of GnRH secretion, and the proximate control of kisspeptin neurons and Kiss1R receptors by sex steroids and neurotransmitters is under intensive study (see Fig. 13.2 ) . The stimulatory role of neurokinin B and the inhibitory role of dynorphin autosynaptically synchronize KNDy neurons, resulting in coordinated Kiss1 secretion, which ultimately increases both the frequency and amplitude of pulsatile secretion of GnRH and LH. Table 13.1 highlights some of a myriad of GnRH regulators, whose site(s) of action require more precise definition. Regulators include seasonal, appetitive, anorexigenic, stress-associated, and anabolic (trophic) factors.

Sex steroids, acting in part via estrogen-receptor alpha, regulate KiSS1, neurokinin B, and dynorphin gene expression in a brain region-specific manner. Estrogen and androgen repress both KiSS1 and dynorphin in KNDy neurons in the arcuate nucleus, thereby inferably transducing sex-steroidal inhibition of GnRH outflow. This is not true in the anteroventral periventricular nucleus, which directs “positive feedback” by estrogen in female rodents ( Fig. 13.2 , left panel ). The latter site contains few KiSS1 neurons in the male. Other neurotransmitters may be important, since the administration of antihypertensives (particular alpha-adrenergic blockers, selective serotonin-reuptake inhibitors, gamma-aminobutyric acid agonists and dopamine) can influence LH secretion. However, whether such effects are direct or indirect with respect to KiSS1 and GnRH neurons remains largely unexplored.

Differential Control of LH and FSH Secretion

GnRH preferentially stimulates FSH secretion in childhood and LH secretion in infancy, puberty, and adulthood. Like GnRH, kisspeptin stimulates LH release 2-fold more than FSH release in healthy men. The putative bases for these distinctions include feedforward and feedback factors. With respect to feedforward, slower GnRH pulse frequencies prior to puberty favor gonadotrope biosynthesis of specific FSH-beta over LH-beta subunits. ^{, ,} In addition, the dimeric glycoprotein, activin A, may stimulate FSH secretion in the prepubertal rat and nonhuman primate. At a second level of feedforward, blood-borne FSH acts on Sertoli cells in spermatogenic tubules to drive inhibin B and E ₂ secretion, and LH acts on Leydig cells in the testis interstitium to cause Te production. There is some indirect evidence that FSH can also increase Te production, possibly via Sertoli cell-secreted nonsteroidal factors, ^, but the effect is quantitatively small. In relation to resultant feedback, pubertal elevations in inhibin B and E ₂ concentrations would tend to restrict FSH responses to GnRH. Elevated Te concentrations would likewise tend to repress the rise in LH concentrations. The fact that the latter does not occur suggests muting of Te’s feedback and/or strong central GnRH drive. Both mechanisms are inferable from mathematical feedback analyses performed in transpubertal cohorts of boys.

Te’s negative feedback on LH is mediated primarily but not exclusively via local aromatization to E ₂ . Significant feedback occurs at the pituitary level. Sex steroids repress GnRH secretion, but inhibition is not likely to be direct because GnRH neurons do not express the androgen receptor (AR) or estrogen receptor alpha (ERα). Although ER-β is detectable and might enhance Ca ²⁺ -dependent synchrony of GnRH neurons transgenic silencing of AR or ERα rather than ERβ is needed to elevate LH and Te concentrations in the adult animal. ^, These data favor a feedback role of KiSS1 neurons, which express both AR and ERα. In addition, nongenomic actions of Te and E ₂ transduced via membrane-dependent signaling may contribute to feedback in some species.

GnRH and GnRH Receptors

More than 23 different GnRH peptides exist among species. Human GnRH decapeptide exists in at least two forms designated GnRH I and GnRH II, cloned initially in mammals and chickens, respectively. The genes encoding these peptides are located on chromosome 8p11–21 and 20p13, respectively. Rapid and slow GnRH half-lives in men are about 2.6 and 5.2 min. GnRH I is the predominant agonistic peptide regulating gonadotropin secretion in humans. In animals, GnRH II seems to stimulate sexual arousal and courting behavior, whereas GnRH III (lamprey) induces significant FSH release. Truncational mutation of the GnRH-I gene occurs in the hpg/hpg mouse but has not been described in man.

At least 23 GnRH-receptor types have been cloned to date. GnRH receptors are calcium-dependent G-protein coupled receptors (GPRs), ^, which control the activity of intracellular heterotrimeric G proteins by promoting guanine-nucleotide exchange and activation of adenylyl cyclase, transmembrane Ca ²⁺ influx or intracellular Ca ²⁺ mobilization. GPRs contain seven transmembrane segments that form α-helices connected by three extracellular and three intracellular loops. Receptor activation causes a conformational change in the associated G protein α-subunit, promoting the release of GDP and binding of the activator nucleotide, GTP. The GTP-bound α-subunit dissociates from the receptor leaving a stable βγ-dimer. Dissociated subunits modulate cellular signaling as proximate messengers in the cyclic AMP-protein kinase A (α subunit) and phospholipase C-protein kinase C (βγ dimer) pathways. High intrahypothalamic concentrations of GnRH may mediate autoinhibition via a G _i subunit.

Two distinct GnRH receptors have been identified in man. GnRH I receptor is the hypophyseal receptor coupled to LH and FSH synthesis, which is encoded by a gene located on chromosome 4. Two splice variants of the GnRH I receptor exist. Both GnRH I (secreted) and GnRH II (brain) peptides can activate the GnRH I receptor. The GnRH II receptor gene is located on chromosome 1, but is silenced in the human, chimpanzee, cow, and sheep. GnRH regulates neurotransmission and sexual behavior in the rodent via the GnRH II receptor. Truncational, nonsense, and missense mutations in the GnRH receptor I gene or homeobox genes (LHX3) can lead to nonexpression, inactivation, misfolding, and misrouting of nascent receptors, and thereby autosomal recessive hypogonadotropic hypogonadism without anosmia. ^, Activating mutations of the GnRH receptor could result in isosexual precocious puberty, but have not yet been identified. Other nonGnRH signals (e.g., IGF-I, galanin, activin A, inhibin B, follistatin, cytokines, gonadotropin-inhibitory hormone, phoenixin) may potentiate or inhibit GnRH action in the pituitary in animal models. ^, The physiological relevance of some of these signals for the regulation of the gonadal axis of humans seems to differ according to sex. Recent first-in-human studies of gonadotropin-inhibitory hormone administration have shown some immediate inhibition of GnRH-stimulated LH secretion in women, but no attenuation of kisspeptin-stimulated LH secretion in men. ^, Other neuropeptides also show sexually dimorphic responses: phoenixin potentiates GnRH-stimulated LH release only from primary cell cultures from female, and not male, pituitary rats.

Gonadotropins and Cognate Receptors

Gonadotropins comprise lutropins (LH and human chorionic gonadotropin, hCG) and follitropin (FSH). Gonadotropins and thyroid-stimulating hormone (TSH) are oligosaccharide-modified dimeric proteins with a molecular mass of 30 to 40 kDa. All four corresponding genes are expressed in the human pituitary gland, albeit minimally in the case of hCG. Each contains a common α subunit and a hormone-specific β subunit. Subunits are associated by noncovalent interactions, but cysteine residues (10 in the α subunit and 12 in the β subunit) permit disulfide linkages within subunits. The α subunit is encoded by a single gene localized at 6q12.21. Mutations of this gene have not been identified, possibly because simultaneous loss of all glycoprotein hormones may be lethal in utero . The α subunit gene is larger than the β subunit gene because of a noncoding exon 1 and a long first intron, but the translated proteins are comparable in size. Beta-subunit genes are located on chromosomes 19q13.32 (LH/hCG cluster) and 11p13 (FSH). The human LH/hCG cluster consists of one LHβ and six hCGβ genes and pseudogenes. Glycosylation and terminal sialylation or sulfation via critical N- and O-linked sites within α (two sites) and β (one site in LH and two in FSH and TSH) subunits influence in vivo bioactivity, receptor binding, and metabolic clearance. In particular, four O-linked glycosylation linkages present in an extended C-terminal portion of hCGβ (but not LHβ) increase intact hCG half-life to 24 to 30 hours compared with 1 hour for LH, and augment biopotency by limiting hCG-receptor dissociation.

Exonic polymorphisms and mutations have been described for each gonadotropin β subunit (hCG, LH, and FSH) gene. In mice, knockout of LHβ results in decreased production of Te and fewer elongated spermatids. One polymorphism of LHβ, termed variant LHβ, occurs in as many as 30% of some Northern European and Australian aboriginal populations, and contains two amino-acid transversions (Ile15Thr and Trp8Arg) and a supernumerary consensus glycosylation site (Asn13-Ala-Thr) reminiscent of hCGβ. Variant LH is more biopotent, but has a shorter in vivo half-life than wild-type LH putatively due to resistance to peptidases or impaired sialylation and sulfation of terminal oligosaccharides. A single nucleotide polymorphism (SNP) in the promoter of the FSHβ gene (-211G>T) is present in about 15% of Baltic men. The T allele is associated with lower serum FSH and semen sperm concentrations, and smaller testis volume. In contrast, frank mutations of the FSHβ gene are rare in men. There are only a few reported cases, each being associated with undetectable FSH and azoospermia ^, and one with hypoandrogenism.

The LH/hCG receptor is a G-protein coupled receptor that is encoded by an 11-exon gene located on chromosome 2p21. The LH/hCG receptor and the FSH receptor (as well as the TSH receptor) are characterized by a large extracellular leucine-rich-repeat hormone binding domain and possess a common internal peptide sequence near the C-terminal part of the extracellular domain that is critical for signal transduction. Unlike the FSH receptor, the lutropin receptor is widely expressed outside the gonad including in the brain, leading to speculative extragonadal actions of LH or hCG. ^, Men with inactivating LH-receptor mutations present with male pseudohermaphroditism and a phenotype ranging from micropenis and hypospadias to complete feminization, in each case without an extragonadal phenotype. Activating mutations of the LH/hCG receptor become manifest as gonadotropin-independent (familial) male sexual precocity or testotoxicosis. LH-receptor polymorphisms have also been described (such as Asn291Ser and Asn312Ser) in association with undermasculinization and Leydig cell hypoplasia. A rare splice variant (deleted exon 9) is marked by inhibitory properties. Thus, structural analyses of interactions among LH receptor, ligand, and relevant G proteins should lead to targeted ligand discovery.

The FSH receptor is also a G-protein coupled receptor, but is encoded by a 10-exon gene located on chromosome 2p21. The crystal structure has been determined, revealing a hand-clasp interaction between the receptor and FSH (follitropin) that orients the glycoprotein hormone perpendicular to the long axis of the ligand-binding domain. The follitropin receptors appear to be expressed exclusively on Sertoli cells located within the blood-testis barrier formed by tight junctions. For this reason, FSH is believed to regulate spermatogenesis primarily. However, a few controversial reports exist of FSH-receptor expression elsewhere, such as on spermatogonia, adipocytes, or skeletal osteoclasts. ^, Furthermore, blocking FSH action with monoclonal or polyclonal antibodies directed at FSH inhibits bone resorption and bone loss, and stimulates bone formation in mice ; inhibits osteoclast formation in cell culture ; and decreases fat mass and promotes beiging of white adipose tissue in mice. Nevertheless, direct evidence from interventional studies showing that FSH regulates bone or fat metabolism in humans is lacking.

Polymorphisms of the FSH receptor (e.g., FSHR Asn680Ser) occur. The Ser allele is associated with lower testis volume and FSH concentrations. ^, However, the effect of this allele is small (−1.40 mL of testis volume) and was detected only after stratifying for the FSHβ (-211G>T) polymorphism. These data show a newly recognized interplay between the FSHβ and FSH receptor genes under physiological conditions. Inactivating mutations of the FSH receptor in five men were associated with subfertility (three cases) and fertility (two cases) with phenotypic variations ranging from oligozoospermia to euspermia. Such data suggest that very low receptor activity is sufficient for spermatogenesis and/or that other endocrine or intratesticular factors may compensate to varying degrees. An activating mutation of the FSH receptor was reported, but functional significance has not been corroborated.

In young men, FSH seems necessary for the formation of spermatogonia type B and pachytene spermatocytes, and LH/hCG for round spermatids. In contrast, LH is the dominant agonist of steroidogenesis.

Aromatization, 5α-Reduction, and Inactivation of Te

Untransformed Te exerts potent anabolic effects. Te also functions as a prohormone, which undergoes conversion to other steroids, such as E ₂ and DHT. Converted steroids can mute or amplify, and thus diversify, Te’s actions through ligand-receptor and coactivator specificities ( Fig. 13.5 ). In particular, DHT binds Erα negligibly, whereas E ₂ binds AR with an affinity one order of magnitude less than that of Te. Testosterone directly stimulates AR, but with a potency of 2 to 30 times less than that of DHT depending upon target tissue. Thus, by being reduced or aromatized, Te may oppose or potentiate the actions of E ₂ on certain genes.

Estradiol acts via two primary (and multiple variant) ERs, whereas Te and DHT act via a single AR. Heterogeneity of tissue responses is presumably conferred via cellular differences in the expression of AR and important corepressors and coactivators. The existence of tissue-selective modulators of nuclear AR action confers a basis for designing pharmacologically selective AR modulators (SARMs) to target anabolism in osteoblasts, erythropoietic precursors, stellate cells in skeletal muscle and brain while exerting little or no effect on adipocytes or the prostate gland. A subset of SARMs may be suitable for use in male contraception. ^{, ,} Compared with Te, DHT is more potent due to both higher affinity for and slower dissociation from AR. DHT is considered a pure androgen in that it is nonaromatizable. Only 4% of blood Te is converted to DHT, ^{, ,} less than 1% is transformed to E ₂ , ^, and approximately 2% to androstenedion ( Fig. 13.5 ). Although systemic transformation is minimal, tissue-specific conversion of androstenedione to Te and of Te to DHT or E ₂ may be critical in organs expressing AR or ER. For example, systemic Te administration elevates intraprostatic DHT concentrations by providing substrate for local 5α-reductase type II, whereas exogenous DHT lowers prostatic DHT concentrations by suppressing LH and thereby Te secretion. Other hormones such as vitamin D, glucocorticoids, and progesterone also modify 5α-reductase under pharmacological conditions.

The CYP19 gene, located on chromosome 15q21.1, encodes the enzyme aromatase, which converts Te to E ₂ and androstenedione to estrone. ^, Aromatase is widely distributed in the testis (Leydig cells, Sertoli cells, spermatid lineage), bone, brain, pituitary, liver, intestine, skin fibroblasts, adipocytes, and breast stromal cells. Systemic aromatization predominantly (>60%) proceeds in extraabdominal adipose tissue. ^, The coexistence of CYP19 and ER in the brain, pituitary, and testis suggests that locally produced E ₂ is physiologically important in these key sites. Evidence favors a role for E ₂ and Erα in stimulating libido and mating frequency especially in animals, inhibiting testis descent into the scrotum by repressing the INSL-3 gene, opposing germ-cell apoptosis in older animals, decreasing LH secretion, inhibiting Leydig-cell growth and differentiation, maintaining efferent spermatogenic ductule water reabsorption and endocytosis, and stimulating male breast anlagen ( Table 13.2 ). Moreover, transgenic silencing and rare human mutations of CYP19 or Erα have unmasked important roles for E ₂ in bone-mineral density, fat-deposition patterns, appetitive behavior, and feedback on LH and FSH. Experiments in normal men using pharmacological agents that block aromatase activity have confirmed many of these findings. ^, Splice variants and polymorphisms of CYP19 exist, ^, which might modify the risks of prostatic cancer or osteoporosis. ^,

Two isoforms (type I and type II) of 5α-reductase are present in the human, encoded by separate genes located on chromosomes 5p15 and 2p23. ^, Type I is found mainly in skin, liver, and brain, and type II primarily in male accessory sex glands, liver, and brain. The isoenzymes share approximately 50% structural peptide homology. Polymorphisms of both isoenzymes have been reported, some of which (e.g., V89L) alter enzymatic activity or correlate with sperm output (A49TT and V89LV). Genetic variations in DHT synthesis or catabolism were thought to contribute to interindividual variability in spermatogenic suppression by Te in male contraceptive regimens. However, prospective assessments of this postulate did not verify the original finding.

Testosterone and DHT are inactivated in the liver, kidney, and prostate by mixed-function oxidoreductases (e.g., 3α- and 3β-hydroxysteroid dehydrogenases) or glucuronidases (conjugation reactions). Three-alpha reduction of DHT decreases its affinity for AR by a factor of 100,000-fold. The oxidoreduced and conjugated metabolites are lost into urine and bile, respectively. Certain metabolites of DHT, such as 5 alpha-androstane-3 beta, 17 β-diol may mimic E ₂ action via ERβ or modulate neurotransmission ( Fig. 13.5 ).