The Gonadotropin Hormones and Their Receptors

Physiological Function

The gonadotropins, LH, FSH, and human (h)CG play a critical role in the fundamental processes of development and reproduction. ^, LH and FSH are secreted by the gonadotrope cells of the pituitary gland, whereas hCG is a placental hormone. The effects of LH and hCG are mediated by their common receptor, LHCGR, while those of FSH are mediated by its receptor, FSHR. The major function of LH in the male is to stimulate LHCGR present specifically in testicular Leydig cells to produce testosterone that is essential for the development of puberty, male secondary sexual characteristics, and spermatogenesis. FSH targets its receptor present in Sertoli cells of the seminiferous tubule and supports their growth and differentiation, thereby indirectly supporting spermatogenesis. In the ovary, LHCGR is present in theca cells lining the follicles, mural granulosa cells of the preovulatory follicle, stromal cells, and luteinized cells. Consequently, LH regulates several functions within the ovary. LH-mediated activation of LHCGR in theca cells stimulates androgen production, while receptor activation in the mural preovulatory granulosa cells triggers autocrine and paracrine signaling pathways that lead to ovulation. Following ovulation, LH maintains progesterone production by the corpus luteum. FSH activates FSHR present exclusively in the granulosa cells of the follicle to stimulate the growth and maturation of the follicle, stimulate the production of aromatase for conversion of theca cell-produced androgens to estrogen, and induce LHCGR receptors in the mural granulosa cells of the preovulatory follicle. ^, In contrast to LH and FSH, hCG is essential for the initiation and maintenance of pregnancy and during fetal development. One of the major functions of placental hCG is to maintain progesterone production by the corpus luteum for the first few weeks of pregnancy before the transition to placental progesterone production. It also mediates multiple placental, uterine, and fetal functions including trophoblast invasion, development of syncytiotrophoblast cells, angiogenesis in the uterine endometrium, uterine growth and differentiation, placental development, and localized suppression of the immune system. ^, Another important physiological function of placental hCG is in male sexual development. hCG activates LHCGR in the fetal Leydig cells to produce testosterone that stimulates the growth and differentiation of male genitalia. The fetal ovary is not sensitive to hCG and female sexual differentiation is independent of gonadotropins.

Protein Structural Attributes

The three gonadotropins and TSH comprise the better characterized members of a family of complex proteins known as the glycoprotein hormones. They are noncovalently bound heterodimers composed of a common α-subunit and distinct β-subunits. The common α gonadotropin subunit (α-subunit) contains 92 amino acid residues, and LHβ, FSHβ, and hCGβ subunits are, respectively, 121, 110, and 145 amino acid residues in length. The additional length of the hCGβ subunit is due to a carboxy-terminal extension arising from a frameshift mutation in an ancestral LH β-subunit gene resulting in a read-through into an untranslated region of the LHβ subunit and an extension of the open reading frame. This extension is known as the carboxy-terminal peptide (CTP). The amino acid sequences of the human subunits are shown in Fig. 2.1 , and it can be seen that the α and β-subunits are relatively rich in Cys residues and that considerable homology exists in the β-subunits.

Crystal structures have been determined for partially active deglycosylated hCG, ^, glycosylated, antibody-bound hCG, a partially deglycosylated fully active hFSH, and a partially deglycosylated complex of a single chain hFSH bound to a large N-terminal fragment (residues 1-268) of the hFSHR ectodomain (ECD) and FSH-FSHR complex containing the entire ECD including the hinge region that is required for signal specificity. Fig. 2.2 shows the crystal structures of hCG and hFSH. Recently, the crystal structure of bovine (b) LH β was reported.

The conformations of hCG and hFSH are quite similar, each being highly elongated molecules with the two subunits intertwined one with another in a slightly twisted manner. Despite the absence of any striking sequence homology, the two subunits in both heterodimers have similar folds characterized by three major loops, and each subunit contains a cystine knot motif, consisting of three disulfides located in the core of each subunit. The α and β-subunits contain, in addition to the three disulfides in the cystine knot, two and three disulfides, respectively. A 20-amino acid residue region of the β-subunit denoted as the “seatbelt,” wraps around a portion of the α-subunit like a molecular seatbelt held in place with disulfide bonds. A major difference in the structures of hCG and hFSH is in the C-terminal portions of the seatbelts that exhibit distinct conformations. In both hCG and hFSH, the two subunits are associated in a head-to-tail arrangement ( Fig 2.2 ). While the structures of bLHβ and hCGβ are similar in the cysteine-knot core, the conformations of the extended loops show variation. Solution structures have also been obtained for deglycosylated human α-subunit ^, using NMR spectroscopy. The overall ensemble of structures determined for the α-subunit is similar to that obtained in the crystal structures of hCG and hFSH.

With crystal structures available for FSH and the FSH-FSHR ECD complex ^, (discussed elsewhere in this chapter), it is possible to delineate the conformational changes of the free heterodimer and that bound to a receptor. The unbound form of the hormone is more flexible than that of the bound form. It is the C-terminal region of the α-subunit; however, that undergoes the greatest change in conformation. In addition, the two C-terminal residues in the α-subunit are unordered in the crystal structure of FSH, but they are fully ordered in the complex with receptors.

Glycosylation

During their synthesis, gonadotropins are trafficked from the endoplasmic reticulum to the cis-Golgi and undergo glycosylation as they traverse the Golgi reaching the trans-Golgi, to yield the mature hormones. The human subunit primary sequences contain N-linked glycosylation sites (consensus sequence Asn-X-Ser/Thr, where X is any amino acid except proline): two on α-subunit at Asn52 and Asn78, two on hCG β-subunit (Asn13 and Asn30), two on FSHβ (Asn7 and Asn24) and one on LH β-subunit at Asn30. In addition, the hCG β-subunit contains four mucin-type O-linked glycans at serine 121, 127, 132, and 138, located on the CTP ( Fig. 2.3 ), resulting in a longer half-life of hCG as compared to LH. The carbohydrate moieties appear to be important in subunit assembly and stabilization, secretion, and circulatory half-life. Although earlier studies suggested a role of the N-linked glycan at Asn52 on α-subunit in receptor activation, evidence indicates that, in addition, the glycan acts as a conformational or stabilizing determinant of the protein. ^, Moreover, there is growing evidence that the particular type of glycosylation may influence biological activity. Of note is that the oligosaccharides on the α-subunit differ in a hormone-specific manner, apparently influenced by its cognate partner, since the characterization of the oligosaccharides released from the α-subunit can identify the β-subunit with which it was associated.

The biantennary N-linked glycans on hFSH and hCG terminate in sialic acid (and sulfate to a lesser extent), and the number of such moieties varies from 0 to 2, accounting in large part for the microheterogeneity of these glycoprotein hormones. In LH, the biantennary N-linked structures tend to terminate mainly in sulfate resulting in a decrease in its circulatory half-life compared to the sialic acid-containing hormones. This arises from a hepatic receptor that recognizes the terminal N-acetyl galactosamine-sulfate, rapidly removing it from circulation. Indeed, ablation of the gene encoding GalNAc-4-sulfotransferase, the enzyme responsible for modifying the terminal GalNAc in LH with sulfate, resulted in mice with increased half-life and circulating levels of LH.

These are but generalizations since, for example, hFSH also contains triantennary and tetraantennary N-linked glycans, and some hFSHβ-subunits lack N-linked structures completely. A variety of glycosyltransferases are responsible for N- and O-glycan biosynthesis; notably, sulfation in the pituitary requires N-acetylgalactosamine transferase and sulfotransferase, both of which are missing in the placenta. ^, Also, fucose is often found in the glycoprotein hormones. As summarized, nearly 50 different N-linked and O-linked glycans have been reported in hCG α and β preparations. The major gonadotropin N-linked and C-linked glycans are shown in Fig. 2.3 .

Folding and Assembly

Investigations into the kinetic folding pathways of the hCG subunits led to the interesting suggestion that disulfide exchange occurs during the maturation process and that subunit association occurred before completion of protein folding and disulfide formation. Moreover, it was posited that subunit association occurred before the seatbelt was latched by closure of Cys26 and Cys110 (wraparound model). In contrast to these reports, a different mechanism in which subunit assembly involved closure of the seatbelt latch, followed by threading of α loop 2, has been proposed (threading model). Others have also studied the folding patterns of hCG and reported that subunit association occurred between an almost completely folded α-subunit and an immature β-subunit. Another study suggests that the LH β-subunit is not completely folded prior to assembly with the α-subunit and that the α-subunit serves as a chaperone to facilitate the formation of the cysteine knot and the seatbelt latch.

In addition to the heterodimeric nature of the hormones, homodimers have also been found for the LH β-subunit ^, and for the α-subunit. ^{, ,} Whether these homodimeric forms of the glycoprotein hormones have any associated bioactivity remains to be shown, although it has been reported that the free α-subunit potentiates progesterone-mediated decidualization.

Site-Directed Mutagenesis

As discussed elsewhere in this chapter, only a limited number of naturally occurring mutations have been identified in α and β-subunits of the glycoprotein hormones. In contrast, there is a wealth of information available from site-directed mutagenesis followed by biological characterization of the mutant hormones. Few mutants have been described in which there was a significant increase in bioactivity; most mutations either have no effect or induce a loss of function, either disrupting folding, subunit assembly, or receptor binding/activation. Gain-of-function mutations in hCG were obtained by replacing single or multiple amino acid residues at the N-terminal region of the α-subunit with Lys. A twofold increase in potency of hCG was obtained with a single replacement of Phe with Thr at position 18 of α-subunit. Mutant forms of α-subunit missing the N-linked oligosaccharide at Asn52 are capable of associating with the hCG β-subunit or FSHβ-subunit giving a heterodimer that binds to the cognate receptor but has diminished signaling efficacy. It has been suggested that the role of N-linked glycosylation at Asn52 is to stabilize the active conformation of the heterodimer by formation of a hydrogen bond with a Tyr on the β-subunit. Mutations in the central region and at the C-terminus ^, of α-subunit yielded mutants that associated with the β-subunits of hCG and hFSH but displayed compromised functionality in receptor binding.

A large number of β-subunit mutations have been prepared and characterized. As with mutations in α, many interfere with folding, subunit assembly, or receptor binding. Overall, the data is consistent with the crystal structures of hCG and FSH. Deletion mutants at the N- and C-termini of the hCG β-subunit have also been reported by several groups ^{, , ,} and the shortest form that retains minimal functionality in subunit assembly and subsequent receptor binding and activation is a fragment consisting of residues 8–100. Unlike α-subunit mutants, however, there have been no reports of β-subunit mutants that retain the ability to form heterodimers and bind to receptors but do not signal. This suggests that the α-subunit may play a predominant role in the activation of gonadotropin receptors following the initial binding event.

Protein Engineering

Protein engineering has been used to produce a variety of chimeric and single-chain hormones yielding quite interesting results. A number of glycoprotein hormone chimeras have been characterized, providing useful information on specific amino acid residues involved in receptor binding and activation. In general, these results emphasize the role of the β-subunit seatbelt region in receptor binding, although different portions are important in receptor specificity.

A novel approach to studying gonadotropin structure-function relationships involved the design of single-chain (yoked or tethered) hormones, derived by fusion of α and β-subunits. In the configurations N-hCGβ-α-C or N-α-hCGβ-C, single-chain hCG with or without intervening peptide linkers were expressed and characterized, in many cases with interesting mutations in one or both subunits. From these studies, it was concluded that both subunit configurations were bioactive and, quite surprisingly, that, each disulfide of the subunits could be eliminated without a loss of activity. ^, Subsequently, similar fusion proteins of LH and FSH were also found to be bioactive. ^, The single-chain hormones displayed increased stability and heat resistance in vitro compared to their heterodimeric counterparts. Extending the approach of covalently linking the two subunits, disulfide-linked heterodimers and mini-gonadotropins with full bioactivity were designed and expressed. ^, These gonadotropin analogs substantiated the notion that α-Asn52 contributed to heterodimer stability and was not involved directly in signal transduction and led to suggestions that the C-terminus of the α-subunit is not required for LHR binding. This notion was dispelled by observations that some but not all single amino acid mutations in the C-terminus of the α—subunit in the context of heterodimeric hFSH only retained 10% or less of hFSH receptor-binding activity. The use of single-chain gonadotropins, particularly in the N-α-β-C configuration, also raises interesting questions about the role of the C-terminal region of the α-subunit in FSH where the structure of the hFSH-hFSHR ECD complex shows a large movement of α-subunit in the receptor complex compared to the heterodimer. This riddle is unlikely to be solved until the crystal structure of single-chain gonadotropin in complex with receptor is determined. Of great interest was the report that single-chain hCG β-β homodimers bind to LHCGR with an affinity about three times lower than wild type hCG but do not elicit a biological response and block hCG binding to LHCGR. A second single-chain hCG antagonist was designed by mutating three of the four N-linked glycosylation sites that are associated with LHCGR activation (Asn 13 and 30 in the β-subunit and Asn 52 in the α-subunit). This analog behaved as a competitive antagonist and suppressed ovarian hyperstimulation syndrome in rats.

The single-chain methodology has been extended to produce fusion proteins with dual and triple activities, although their mechanism of action is unclear. For example, a three-domain fusion protein of the form, N-FSHβ-hCG β-subunit-α-C, exhibited both LH and FSH activities. Disruption of heterodimer formation in this triple domain construct by mutation of either Cys10-Cys60 or Cys32-Cys84 did not eliminate bioactivity, suggesting that αβ contacts are not required for receptor binding and activation. Subsequently, a four-domain fusion protein, N-TSHβ-FSHβ-hCG β-subunit-α-C, although secreted inefficiently, was found to exhibit three distinct bioactivities both in cellular and whole animal studies. ^, These results raise intriguing questions regarding subunit association and conformation as manifested in receptor binding and activation. These selected results, along with others not covered here, indicate that single-chain glycoprotein hormones exhibit some properties distinct from those in heterodimers. The increased stability of the single-chain proteins, the single-chain hCG antagonists, and the analogs with dual activities make them interesting candidates for clinical utility.

The approach of single-chain gonadotropins was further expanded to produce fusion proteins of single-chain hCG with LHCGR, which when expressed, led to constitutive receptor activation in transfected cells and transgenic mice. This model also demonstrated that protein fusions of the individual subunits with LHCGR were devoid of bioactivity.

Gonadotropin Subunit Genes

The common α-subunit and the β-subunits of LH and FSH are each encoded by single genes; in contrast, the CG β-subunit, expressed in primates, is encoded by six genes. ^, In equids, however, the CG β-subunit and LH β-subunit are products of the same gene. It has been suggested that the glycoprotein hormone α and β-subunits diverged from a common ancestral gene over 900 million years ago, with the β gene undergoing duplications and mutations to yield the current family. In humans, the gene encoding the common α subunit, CGA, is on chromosome 6, which for FSHβF SHB ) on chromosome 11 and those for LHβ ( LHB ) and CGβ( CGB ) on chromosome 19 ( http://www.ensembl.org ). The CGA is 9.4 kb and contains four exons and three introns, FSHB is 4.2 kb with three exons and two introns, LHB is 1.1 kb with three exons and two introns, and the CGB genes are variable in length.

The one LHB and six CGB genes exist in a large cluster spanning about 52 kbp. ^, The six CGB genes (i.e., CGB, CGB1, CGB2, CGB5, CGB7, and CGB8 ) exist as tandem and inverted repeats. Detailed analysis of the CGB genes revealed that four of the genes, CGB, CGB5, CGB7, and CGB8 , exhibit 97% to 99% sequence identity while their identity with LHB gene is 92% to 93%. ^, These gene similarities lead to protein sequences that are 98% to 100% identical for the four CG β-subunits and 85% identical with the LH β-subunit.

Gonadotropin Subunit Transcripts

The available evidence indicates single transcripts for the gonadotropin genes, with the exception of the human FSHB gene for which four mRNA species have been described, arising from alternate splicing and the utilization of two polyadenylation sites. The CGB family is interesting in that the six genes appear to express transcripts of varying lengths, albeit sometimes without detectable protein production. CGB5 and CGB8 are highly expressed in the placenta. ^{, ,} CGB1 and CGB2 genes are expressed in placenta, ^, pituitary, testis, and in breast cancer, although no proteins have yet been identified for these genes. The predicted sizes of putative protein products of CGB1 and CGB2 are smaller than that of the hCG β-subunit; this observation, coupled with the distinct amino acid sequences predicted, suggests that, if biosynthesized, these proteins may have quite different functions than those of hCG. Using transgenic mice expressing a 36 kb cosmid insert that contained the six CGB genes, transcripts of CGB1 and CGB2 genes were found to be present in the brain at levels comparable to those of the other four CGB genes. The human LHB mRNA is 700 nucleotides in length, and depending upon the species, the CGA gene encodes an mRNA of 730 to 800 nucleotides.

Naturally Occurring Mutations

Mutations in the gonadotropin hormone genes, although rare, help in elucidating their physiological roles and defining the structural domains of the hormones. The only mutation reported in the CGA gene is that from a human carcinoma, Glu56Ala, resulting in a mutant form of α-subunit that does not associate with LH β-subunit. In contrast, there are several reports of mutations in the genes encoding the three gonadotropin β-subunits, resulting in loss of function and thus hypogonadism. ^{, ,}

The first report of a mutation in the LHB gene was that of a missense mutation in a male presenting with delayed puberty and hypogonadism. This mutant led to a replacement of Gln54 with Arg; while subunit assembly could occur, the heterodimer was unable to bind to LHCGR. Site-directed mutagenesis studies showed that LH β-subunit and hCG β-subunit with Gln54 replacements formed heterodimers with α-subunit, but these heterodimers exhibited reduced binding to LHCGR. ^, Another missense mutation reported in LH β-subunit was that of Gly36 to Asp, reported in a male with delayed puberty and infertility. Gly36 is part of the CAGYC sequence in the LH β-subunit that is critical to the formation of the cystine knot; presumably, an Asp at this position prevents at least one of the disulfides from forming.

The third identified mutation was a G-C substitution at the +1 position of intron 2 (a 5′ splice-donor site) that leads to a hypothetical aberrant protein with a 79 amino acid residue insert beginning after Met41 and a frameshift in exon 3, thus removing the essential seat belt loop of β and cysteines that participate in the cysteine knot motif. This suggested that the mutant LH-β subunit would not correctly assemble with the α-subunit and therefore would not be secreted. The offspring of consanguineous parents who were heterozygous for the mutation were analyzed. Three homozygous siblings presented with hypogonadism and infertility, undetectable levels of LH, and high levels of free α-subunit while their heterozygous siblings were fertile. The two homozygous males had elevated FSH and low testosterone. The homozygous female had FSH, estradiol, and progesterone values in the normal range and underwent normal pubertal development and menarche at age 13 years followed by oligomenorrhea and anovulation.

A 9 bp deletion in exon 2 resulting in deletion of amino acid residues 10 to 12 of LHβ was reported in a man and his sister. Both were homozygous for the deletion, while two additional unaffected siblings were heterozygous. In spite of undetectable levels of LH and concomitant low serum and intratesticular testosterone concentrations, the man had complete spermatogenesis and normal sperm count. Presumably, the low activity by the mutant LH detected in vitro was sufficient for normal spermatogenesis. The sister underwent normal puberty and menarche but subsequently had amenorrhea, infertility, ovarian cysts, and low estradiol levels.

A compound heterozygous mutation in a 31-year-old male with delayed puberty, azoospermia, and hypogonadism due to lack of LH was reported. The first mutation was identified as a 12-bp deletion in exon 2 of the LHB gene, causing a deletion of 4 leucine residues in the signal peptide and the second was a G to T mutation at the 5′ splice site of intron 2 resulting in aberrant RNA splicing. The patient’s 16-year-old sister harboring the same mutations had normal pubertal development but developed oligomenorrhea. A homozygous 3 bp deletion in the LHB gene resulting in the deletion of Lys40 in LHβ was identified in two brothers with LH deficiency and hypogonadism. The mutated LHβ-subunit was able to heterodimerize with α-subunit but was not secreted.

Two cases of male hypogonadism caused by premature termination of LHβ have been reported. ^, Homozygous deletion of a thymine nucleotide at position 325 in exon 3, predicted to result in frameshift and premature termination at codon 128 was identified in a 19-year-old man with delayed puberty and a homozygous mutation of c.84G>A[p.W28X] produces a truncated protein of seven amino acids.

Rare missense heterozygous mutations in the hCGβ subunit have been identified in a Northern European population. ^, A Val56Leu mutation in the CGB5 gene was identified in a patient with recurrent miscarriage (RM). Although the mutation impaired subunit assembly, it elicited a strong signaling response. An amino-terminal Arg8Trp mutation, also found in an RM patient, did not affect assembly. A Pro73Arg mutation found in 5 individuals (3 RM and 2 controls) resulted in altered conformation but did not affect biological activity. Individuals homozygous for these mutations have not been identified, perhaps because such genotypes would result in complete pregnancy failure and suggests that only mutations with mild functional consequences can be tolerated in the major CGB genes.

To date, 13 patients with mutations in exon 3 of FSHB have been identified and they present with an absence of pubertal development, amenorrhea, and infertility in females and delayed or normal puberty with azoospermia and infertility in males. ^{, ,} The first reported mutation was a homozygous 2 bp deletion at codon 61 (Val61X) causing a frameshift and premature termination of the β subunit in two women and later also identified in an 18-year-old male with delayed puberty. These observations were followed by a report of a compound heterozygous mutation, with one being the Val61X mutation and the other a missense mutation resulting in a Cys51Gly replacement. Other identified mutations include nonsense mutations (Tyr76X and R115X), a frameshift mutation at codon 79 (Ala79X) caused by a 1 bp deletion resulting in premature termination, and two missense mutations resulting in Cys82Arg and Cys122Arg replacements. ^, These mutations result in loss of bioactivity due to the production of a truncated protein or due to aberrant tertiary structure as a result of mutations in the cysteine residues involved in the cystine knot structure and inability to associate with the α-subunit. One case of hypoglycosylation, likely caused by altered conformation, was reported for FSH, resulting in a hormone with diminished activity.

Overall, the observed phenotypes associated with the naturally occurring mutations in LHB, FSHB, and CGB are consistent with the known structures and actions of the gonadotropins, although fertility in men does not always appear to be sensitive to some FSH mutations.

Polymorphisms

A well-characterized variant in the LHB gene (V-LHβ) appears in variable frequencies in ethnic groups throughout the world and results from two single nucleotide polymorphisms (SNPs) that are found together on one allele. One causes the replacement of Trp8 with Arg which resulted in altered immunoreactivity of the hormone. The other caused a substitution of Ile15 with Thr, which introduces an extra glycosylation site in the LHβ subunit. ^{, ,} V-LH demonstrates increased biopotency in vitro with an altered half-life in circulation. ^, The association of the V-LH with various clinical conditions has been assessed. ^{, ,} A number of studies have addressed the association between V-LH and various clinical conditions such as infertility, polycystic ovarian syndrome, and menstrual disorders. No clear association was found with PCOS. However, studies have found an association with female infertility, but not male.

Another LH β-subunit variant with a replacement of Gly102 with Ser and resulting in reduced LH biopotency in vitro has been associated with reproductive disorders in some populations. ^{, ,} The frequency of this polymorphism was recently reported to be higher in a population of Chinese Han women with PCOS but not in a population of Korean women. ^,

An unusual polymorphic variant of LH β-subunit involves an Ala to Thr replacement of three residues before the signal peptide cleavage site. ^, Using in vitro assays, it was found, rather surprisingly, that the mature protein from the variant appears less potent than wild type LH in cAMP production but more potent in inositol phosphate production. The SNP-related alteration may interfere with the proper processing of the β-subunit, although studies have not addressed this possibility.

A polymorphism in exon 3 of CGB5 , resulting in a Val79 replacement with Met in a random population in the United States, has been reported. This SNP results in a β-subunit with impaired ability to assemble with the α-subunit, although the biological activity of the variant is normal. The frequency and physiological consequences of this polymorphic variant are unknown; one sampling of just under 600 samples from four European groups failed to detect the polymorphism in this population. Other polymorphic variants have been detected, but these are silent or located in intron regions. ^, A case study analyzed CGB5 and CGB8 genes in RM and control fertile patients from Estonia and Finland. Seventy-one polymorphisms were identified, of which 48 were novel. A protective effect against RM was associated with two SNPs located at identical positions in CGB5 and CGB8 and with four CGB5 promoter variants. A follow-up study that included a Danish cohort with RM in addition to the Estonian and Finnish subjects confirmed that two SNPs in the CGB5 promoter region seemed to offer protection against RM, but variants in the CGB8 promoter region had no effect. These polymorphisms can be found in the dbSNP database ( http://www.ncbi.nlm.nih.gov/SNP/ ).

Only a few SNPs in the FSHB gene have been extensively studied. ^, An FSHB promoter polymorphism (rs10835638) (G/T) located 211 bp upstream of the transcription start site was identified in a cohort of European men and TT homozygous men have reduced serum FSH levels. This polymorphism has been associated with infertility in both sexes. The low-serum FSH levels associated with this SNP have been demonstrated to be due to reduced binding of the LHX3 transcription factor and reduced FSHB transcription. Recent genome-wide association studies (GWAS) identified an SNP (rs11031006) (G/A) associated with fertility and PCOS. This SNP was located approximately 26 Kb upstream of the FSHB transcriptional start site. ^, Surprisingly, functional studies revealed that the SNP resides within a conserved enhancer region and the minor (A) allele increased SF1 binding to the enhancer and increased FSHB transcription instead of decreasing expression as would be predicted in PCOS. The LHB and FSHB polymorphisms can be found in the SNP database ( www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?geneId=3972 and www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?geneId=2488 ).