The genetic basis of infertility in men

BaillieÁre's Clinical Endocrinology and Metabolism Vol. 14, No. 3, pp. 363±388, 2000

doi:10.1053/beem.2000.0085, available online at http://www.idealibrary.com on

3 The genetic basis of infertility in men Shalender Bhasin

MD

Professor of Medicine UCLA School of Medicine Chief Division of Endocrinology, Metabolism, and Molecular Medicine, Charles R. Drew University of Medicine and Science, Los Angeles, CA 90059, USA

Con Mallidis

PhD

Post-doctoral Fellow

Kun Ma

PhD

Assistant Professor of Medicine Division of Endocrinology, Metabolism, and Molecular Medicine, Charles R. Drew University of Medicine and Science, Los Angeles, CA 90059, USA

Subfertility in men is a heterogeneous syndrome, its pathophysiology remaining unknown in the majority of a€ected men. A large number of genes and loci are associated with sterility in experimental animals, but the human homologues of most of these genes have not been characterized. A British study suggested that, in a large proportion of men with idiopathic infertility, the disorder is inherited as an autosomal recessive trait; this provocative hypothesis needs con®rmation. Because normal germ cell development requires the temporally and spatially co-ordinated expression of a number of gene products at the hypothalamic, pituitary and testicular levels, it is safe to predict that a large number of autosomal, as well as X- and Y-linked, genes will probably be implicated in di€erent subsets of male subfertility. Key words: male infertility; Y-chromosome deletions; genetics; autosomal loci for infertility; CFTR mutations; CREM mutations; intracytoplasmic sperm injection.

The process of spermatogenesis is divided into three stages1: spermatogonial replication, meiosis and spermiogenesis. Normal spermatogenesis requires complex interactions between germ cells and various somatic cells such as Sertoli and Leydig cells2, as well as synergistic actions of the pituitary gonadotrophins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH, after binding to its G-protein-coupled receptor, stimulates the production of testosterone by the Leydig cells, a high intratesticular testosterone concentration being essential for the initiation and maintenance of spermatogenesis. FSH initiates function in immature Sertoli cells by stimulating the formation of the blood±testis barrier and the secretion of a wide range of proteins and growth factors, such as androgen-binding protein, inhibin, activin, stem cell factor, plasminogen activator, transferrin and sulphated glycoproteins.2 A failure of 1521±690X/00/030363‡26 $35.00/00

c 2000 Harcourt Publishers Ltd. *

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spermatogenesis can result from the impaired secretion or action of LH and FSH1,3±7 or from intrinsic defects in spermatogenesis within the testis. A number of testis-speci®c gene products are required for the orderly completion of germ cell development, and inactivating mutations of many such genes have been described. Single-gene mutations can, however, account for spermatogenic failure in only a small subset of infertile men. This chapter will highlight the known genetic syndromes associated with infertility in man, mouse and Drosophila. A signi®cantly larger database exists in the mouse and Drosophila; a careful study of the Drosophila and mouse sterility loci can therefore provide useful clues to candidate genes that are associated with defects of germ cell replication, meiosis or spermiogenesis in man. GENETIC DISORDERS ASSOCIATED WITH INFERTILITY IN MAN Genetic disorders associated with impaired gonadotrophin secretion or action The disorders a€ecting gonadotrophin secretion and action can be broadly classi®ed into three categories: 1. disorders of hypothalamic gonadotrophin-releasing hormone (GnRH) secretion or action; 2. primary disorders of pituitary LH and FSH secretion and action; 3. disorders of pituitary development. Because LH and FSH are trophic hormones for the testes and ovaries, the impaired secretion of these gonadotrophins (hypogonadotrophism) results in hypogonadism. Clinically, patients with hypogonadotrophic hypogonadism may present with one or both of the following: . symptoms and signs of sex steroid (androgen in the male and oestrogen in the female) de®ciency; . infertility resulting from impaired germ cell development The symptoms and signs of androgen de®ciency depend on its time of onset and the degree of gonadotrophin de®ciency. Androgen de®ciency during fetal life may result in a failure of the wolan structures to develop, ambiguity of the external genitalia because of a failure of fusion, hypospadias, microphallus or a combination of these features. In patients with isolated hypogonadotrophism, placental human chorionic gonadotrophin (hCG) stimulates the fetal testis to produce sucient androgen in early fetal life. Therefore, most of the patients with congenital GnRH de®ciency have normal wolan structures and external genitalia. During the second half of pregnancy, however, the fetal gonad is under the control of fetal pituitary LH and FSH. Severe LH and FSH de®ciency during this period may thus result in undescended testes and microphallus since testicular descent is partly androgen dependent. If androgen de®ciency occurs after birth but before puberty, sexual development is delayed or arrested; these children present with delayed adolescence. Other androgen-dependent events that occur in the peri-pubertal period, such as the epiphyseal fusion of the long bones and the calci®cation of the laryngeal cartilages, are also delayed. A delay in fusion of the epiphyses results in the continued growth of the long bones, causing a reversal of the upper segment to lower segment ratio and eunuchoidal proportions (span greater than height by more than 10 cm). Men with pre-pubertal androgen de®ciency retain their high-pitched voice and do not develop the male pattern temporal recession of the hairline.

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Androgen de®ciency acquired after the completion of puberty is characterized by a regression of the secondary sex characters, an impairment of libido and sexual function, a loss of muscle mass, increased fat mass and infertility. However, these changes occur insidiously so that many years may elapse before patients seek medical attention. This may partly explain why men with prolactin-secreting pituitary adenomas usually have a much larger tumour (macroadenoma) at the time of initial presentation. The early interruption of the menstrual cycle in women, on the other hand, alerts them to seek medical advice earlier, leading to an earlier diagnosis (at the microadenoma stage). Disorders associated with hypogonadotrophic hypogonadism can be classi®ed into congenital and acquired disorders. Acquired disorders are much more common than congenital disorders and may result from functional abnormalities in GnRH secretion or from organic conditions such as neoplastic, in¯ammatory or in®ltrative diseases. In this chapter, however, we will only discuss the congenital disorders of gonadotrophin secretion or action.

Hypothalamic disorders associated with impaired GnRH secretion or action Idiopathic hypogonadotrophic hypogonadism. Kallmann et al8±10 ®rst described a syndrome characterized by delayed or arrested sexual development and anosmia. These patients have selective gonadotrophin de®ciency resulting from an isolated defect in GnRH secretion.8±10 The primary pathogenetic defect in these patients is hypothalamic, the impaired gonadotrophin secretion being secondary to the hypothalamic abnormality in GnRH secretion.10±11 Although anosmia and hyposmia are the most well-known and the ®rst associations described for this syndrome, a number of other somatic abnormalities have been recorded.8±12 The more common associations include colour blindness, cleft lip and palate, cranial nerve defects (including eighth nerve deafness), horseshoe-shaped kidneys, cryptorchidism and optic atrophy. There is considerable heterogeneity in the clinical presentation of idiopathic hypogonadotrophic hypogonadism (IHH).10±13 The phenotype is, to a large degree, determined by the severity of GnRH de®ciency. Those with the most severe de®ciency may present with a complete absence of pubertal development, sexual infantilism and, in some cases, varying degrees of hypospadias and undescended testes. Patients with partial GnRH de®ciency may have a varying degree of impairment in their sexual development, in proportion to the severity of the gonadotrophin de®ciency. Spratt et al11 studied the secretory pro®les of LH and FSH in men and women with IHH; these studies revealed that patients with IHH are quite heterogeneous in their LH secretory pro®le.11 The largest subset comprises patients who display no pulsatile LH secretion at all. This apulsatile group represents one extreme, characterized by the most severe GnRH de®ciency. A smaller subset displays low-amplitude pulses. Another subset of patients has LH pulses at a markedly reduced frequency. A fourth subset is characterized by sleep-entrained pulses reminiscent of the pattern seen in the early stages of puberty; these patients can be considered to su€er from a `developmental arrest'. Two variants of IHH are particularly interesting. The term `fertile eunuch syndrome' has been used to describe patients with eunuchoidal proportions and delayed sexual development who have normal-sized testes. Another variant with predominantly FSH de®ciency has also been described14, although these patients are rare. Such individuals appear to have sucient gonadotrophins to raise intratesticular testosterone level

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suciently to initiate spermatogenesis, but not enough testosterone secretion into the systemic circulation to virilize the peripheral tissues. From a genetic perspective, IHH is a heterogeneous disorder.11±12 Only a third of IHH patients have a positive family history.12 Of those with a positive family history, approximately 20% have an X-linked pattern of inheritance, one-third have an autosomal recessive one, and one-half an autosomal dominant mode of inheritance.11±12 Physical and genetic mapping studies15±16 have assigned the locus for the Xlinked form of Kallmann's syndrome to chromosomal region Xp22.3. Two groups17±18 independently cloned from this region an adhesion molecule-like protein encoded by the KALIG-1 (Kallmann's syndrome interval-1) gene. The protein product of the KALIG1 gene presumably regulates the migration of the GnRH and olfactory neurones and their morphogenesis. Deletions of the KALIG-1 gene have been reported in only a small number of men with IHH.19±20 Other groups have described point mutations in the KALIG-1 gene, most of which have been located in the ®bronectin-III domain of the KALIG-1 gene.21±26 The mutations in the KALIG-1 gene account for only a small fraction of patients with the X-linked form of IHH, and it is certain that additional, as yet unidenti®ed, X-linked genes are implicated in other subsets of Kallmann's syndrome. Schwanzel-Fukuda and Pfa€27 studied the migration of the GnRH neurones in the mouse embryo. These neurones ®rst appear in the epithelium of the olfactory placode in the mouse embryo and then migrate to the forebrain and ®nally to their ultimate hypothalamic location. Such observations suggest that IHH may be a developmental defect resulting from an abnormal migration of LH-releasing hormone neurones, much like DiGeorge syndrome, which results from the abnormal migration of the branchial arches. Further support for this hypothesis comes from magnetic resonance imaging studies, which show that the olfactory bulbs and sulci are poorly developed in patients with IHH who have anosmia or hyposmia.28 Mutations of the DAX-1 gene. The product of the DAX-1 gene is an orphan nuclear receptor.29±36 The mutations in the C-terminal end of the DAX-1 gene have been associated with X-linked hypogonadotrophic hypogonadism and adrenal insuciency (adrenal hypoplasia congenita). Patients with DAX-1 mutations usually have erratic LH pulses. Mutations in the DAX-1 gene are, however, an unusual cause of IHH, accounting for fewer than 1% of cases.29±36 These patients typically have a normal testosterone response to hCG, indicating normal Leydig cell function. A contiguous gene syndrome characterized by an interstitial deletion of Xp has been reported in a man with Duchenne's muscular dystrophy, hypogonadotrophic hypogonadism and adrenal insuciency. Mutations of the GnRH receptor gene. Several families with hypogonadotrophic hypogonadism caused by mutations of the GnRH receptor have been reported.37±40 The GnRH receptor is a G-protein-coupled receptor with an extracellular N-terminus, seven transmembrane regions and a truncated intracellular C-terminus. Hormone binding to the extracellular elements of the receptor results in the intracellular activation of phospholipase C and an increased mobilization of the intracellular calcium. De Roux et al38 described a family with compound heterozygous mutations of the GnRH receptor. One mutation was in the ®rst extracellular loop of the GnRH receptor and was associated with decreased GnRH binding to its receptor. The second mutation that was located in the third intracellular loop did not alter GnRH binding to the receptor but decreased the activation of phospholipase C.

Genetic basis of infertility in men 367

Patients with GnRH receptor mutations have a normal sense of smell.37±40 In these families, the pattern of inheritance of IHH is autosomal recessive; GnRH receptor mutations account for almost 40% of cases in families with an autosomal recessive pattern of inheritance and 10% of sporadic cases. Consanguinity is often present in these families. The male:female distribution among a€ected individuals is equal. Deletion of the GnRH gene in the hypogonadotrophic mouse. The GnRH gene has been characterized in many species, including the rat, mouse and human.41±44 In the rat and the mouse, the GnRH gene has been mapped to chromosome 841 and consists of four exons and three introns. The ®rst exon codes for the 50 untranslated sequences, the second for the signal peptide, the GnRH decapeptide and the ®rst 12 amino acids of the GnRH-associated peptide (GAP), the third for amino acids 13±41 of the GAP, and the fourth for amino acids 42±54 of the GAP and the 30 untranslated sequences. The hypogonadotrophic (hpg) mouse is an interesting model of GnRH de®ciency.43 These mice have small testes and accessory sex organs, are infertile43 and fail to respond to a single bolus of GnRH by an increase in LH and FSH secretion. However, the repeated administration of GnRH normalizes LH and FSH secretion. Similarly, grafts of the hypothalamic pre-optic area restore gonadotrophin secretion, consistent with the premise that the primary abnormality in hpg mice lies in the hypothalamic GnRH neurones.43 Molecular studies of the GnRH genes in hpg mice have revealed a large deletion encompassing the third and the fourth exons44; the nucleotide sequences encoding the GnRH decapeptide are not deleted. Interestingly, in situ hybridization studies have demonstrated that the mRNA coding for the GnRH decapeptide is transcribed in the hypothalamus. The GnRH peptide is not, however, translated, suggesting that sequences in the 30 untranslated region or in the introns may be required for the translation of the GnRH mRNA. Mason et al44 established the critical role of this deletion in the causation of the reproductive disorder by demonstrating that transgenic hpg mice bearing a wildtype GnRH transgene showed a restoration of the reproductive processes.41,44 These studies in hpg mice had raised hopes that patients with IHH might also have a somewhat analogous molecular defect. Several investigators45±46 have examined the structure of the GnRH gene in patients with IHH, using Southern blot and polymerase chain reaction (PCR)-based analyses. These studies have failed to uncover major deletions or rearrangements of the GnRH gene in patients with IHH by Southern blot analysis or the sequencing of PCR products. However, more subtle defects within the GnRH gene have not been completely excluded.45±46 Mutations of known genes can not account for the majority of cases of IHH. Known mutations of the KALIG-1, GnRH receptor and DAX-1 genes can account for only 25±30% of cases of hypogonadotrophic hypogonadism (KALIG-1 10±15%, GnRH receptor 15% and DAX-1 51%). Therefore, additional autosomal and X-linked genes are likely to be implicated in other cases of IHH. Prader±Willi Syndrome. A syndrome consisting of obesity, hypotonic musculature, mental retardation, hypogonadism, short stature and small hands and feet has been well described.47±49 Hypogonadism, cryptorchidism and micropenis are common.47 Histologically, the testis is immature, without germinal cells but with Sertoli cells and diminutive tubules.47 The LH response to a single bolus of GnRH is subnormal in comparison to that of obese controls.47 The degree of gonadotrophin de®ciency in

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these patients is variable. A few patients with hypergonadotrophic hypogonadism have also been described. Clomiphene has been shown to turn on the pituitary gonadal axis of individuals of either sex with Prader±Willi syndrome, leading to the secretion of gonadotrophins and gonadal steroids. Prader±Willi syndrome is a disorder of genomic imprinting that commonly results from deletions of the proximal portion of paternally derived chromosome 15q.47,48 The maternally derived copies of genes responsible for the Prader±Willi syndrome, in the proximal region of chromosome 15q, are normally silent.47,48 Therefore, the deletion of the paternally derived copy of the normally active genes produces the disease. Prader±Willi syndrome can also result if both copies of the gene are derived from the mother because the maternal copies are inactivated, presumably by DNA methylation; this condition is known as uniparental disomy. Structural abnormalities of the imprinting centre can also produce Prader±Willi syndrome. The genes responsible for Prader±Willi syndrome have not been identi®ed. Allele-speci®c methylation at locus D15S63 can be detected by a PCR method and has been used as a diagnostic test for this syndrome.47,48 Laurence±Moon±Biedl syndrome. This condition is characterized by obesity, hypogonadism, mental retardation, polydactyly and retinitis pigmentosa.49 Renal abnormalities are common and include glomerular sclerosis, mesangial proliferation and cyst formation. The syndrome is inherited as an autosomal recessive disorder. Delayed adolescence is a common feature. In adult patients, however, the prevalence of hypogonadism is seen in about half the patients.49 Retinal degeneration is seen early in life, between 4 and 10 years of age. Miscellaneous congenital hypogonadotrophic disorders. A large number of congenital defects and syndromes have been described in association with hypogonadotrophic hypogonadism. An extensive list of these disorders can be found in textbooks of genetic disorders.49 Primary disorders of pituitary LH and FSH secretion or action Mutations in the genes encoding LH b- and FSH b-subunits. Hypogonadism can be caused by mutations in the FSH b-subunit gene. Inherited mutations of the FSHb genes are uncommon but have been reported to produce male hypogonadism and delayed puberty in boys.50±53 FSH-de®cient male mice are fertile although they have small testes and subnormal spermatogenesis.54 Female mice de®cient in the FSH b-subunit, produced by the embryonic stem cell technology, are infertile, with a block in folliculogenesis prior to antral follicle formation.54 A 46,XX patient, homozygous for an FSHb point mutation, presented with primary amenorrhoea, infertility and a low serum FSH level.52,53 An analysis of the FSHb gene revealed a two-nucleotide deletion that resulted in a frame shift of subsequent codons and premature termination.52,53 A relative of the index case was post-menopausal and had a subnormal FSH level. The two point mutations in the FSHb gene observed in this family were both located in exon 3 at codons 51 and 61.52,53 The mutant cDNAs (Val61X and Cys51Gly) were stably transfected into Chinese hamster ovary (CHO)cells, and the FSH concentration in the medium was measured by immunoradiometric assay. The CHO cells transfected with the mutant FSHb genes secreted very little FSH into the medium compared with cells transfected with the wildtype gene.52

Genetic basis of infertility in men 369

Hypogonadism is also associated with inactivating mutations of the LHb gene. A single patient with a mutation of the LH b-subunit gene has been reported55; this individual presented with delayed pubertal development.55 He had an increased serum immunoreactive LH level but a decreased bio-active LH concentration. The mutant LH in this patient had a homozygous substitution of glycine in position 54 with arginine (G54R). The mutant LH, expressed in CHO cells, showed decreased receptor binding activity. The male individuals in the family who were heterozygous for this mutation had a lower testosterone level.55 Heterozygous females had regular menstruation and were fertile. A polymorphic variant of LH has been reported in Finland and Japan.56 The variant LH has two amino acid substitutions, W8R and I15T, that are associated with increased bio-activity and a reduced serum half-life.56 The clinical signi®cance of this polymorphism is unknown. Inactivating mutations of LH and FSH receptor genes. Inactivating mutations of the LH receptor gene are associated with hypogonadism and Leydig cell hypoplasia. A number of families with resistance to LH action resulting from inactivating mutations of the LH receptor have been reported.57±59 Men with LH receptor mutations present with a spectrum of phenotypic abnormalities ranging from feminization of the external genitalia in 46,XY males to Leydig cell hypoplasia, primary hypogonadism and delayed sexual development.57±59 In a patient with Leydig cell hypoplasia and hypogonadism, a T to A mutation in position 1874 of the LH receptor gene was found.57 Testicular histology in this man revealed the absence of mature Leydig cells in the interstitium; the seminiferous tubules had thickened basal lamina and spermatogenic arrest at the elongated spermatid stage. Female members of the kindred with LH receptor mutation revealed a normal development of secondary sex characteristics, an increased LH level and amenorrhoea. Inactivating mutations of FSH receptor gene can also occur. In one report60, an inactivating mutation of the FSH receptor gene was associated with familial ovarian dysgenesis and primary amenorrhoea. Related men in the family had a variable alteration of spermatogenesis and fertility. Activating mutations of LH and FSH receptor genes. Activating mutations of the LH receptor gene are associated with gonadotrophin-independent sexual precocity. Activating or gain-of-function mutations of the LH receptor are associated with gonadotrophin-independent, sexual precocity in boys but do not produce a discernible phenotype in females.61±63 An analysis of the LH receptor in this patient showed a C to T substitution in exon 11 that resulted in an Ala to Val change. When the COS-7 cells were transiently transfected with the wildtype or Ala373Val mutant LH receptor cDNA, the mutant cDNA construct had higher basal and hCG-stimulated cAMP accumulation.61 Only a single case of an activating mutation of the FSH receptor is on record; this patient was fertile even after surgical hypophysectomy that had lowered his FSH immunoreactivity to an undetectable level.63,64 It has been hypothesized that activating mutations of the LH and FSH receptors might cause the development of ovarian and testicular neoplasms. Activating mutations of the FSH receptor have not been reported in ovarian tumours. A single patient with LH receptor mutation and seminoma has been reported, although a cause-and-e€ect relationship between LH receptor mutation and the testicular tumour has not been established.

370 S. Bhasin et al Table 1. Developmental disorders associated with abnormalities of pituitary development. Homeodomain gene

Phenotype

Pit-1 Lhx3

De®ciencies of GH, TSH and prolactin De®ciencies of GH, TSH, prolactin, LH and FSH Rigid cervical spine De®ciencies of LH and FSH, GH, TSH and prolactin ACTH secretion preserved Septo-optic dysplasia Extreme dwar®sm, sexual infantilism and increased perinatal mortality Candidate gene for Rieger syndrome, an autosomal dominant disorder with variable craniofacial, dental, eye and pituitary anomalies

Prop1 or POU1F1 Hesx1 Gsx1 Ptx2a and Ptx2b

GH ˆ growth hormone; TSH ˆ thyroid-stimulating hormone; LH ˆ luteinizing hormone; FSH ˆ follicle-stimulating hormone; ACTH ˆ adrenocorticotrophic hormone.

Developmental disorders of the pituitary caused by mutations of the homeodomain transcription factors (Table 1) Mutations in the Pit-1 homeodomain transcription factor have been associated with the failure of several di€erentiated cell types to develop within the pituitary gland and with de®ciencies of growth hormone (GH), prolactin and thyroid-stimulating hormone (TSH), but normal gonadotrophins.65±67 These patients have either small or normally sized pituitary glands. Pit-1 induces the transcription of the GH and prolactin genes and is also necessary for the control of TSHb gene transcription. Both autosomal dominant and recessive forms of inheritance have been described, depending on the DNA-binding properties of the mutant protein. Patients with mutations of Prop1 have de®ciencies of LH and FSH in addition to de®ciencies of GH, prolactin and TSH.68,69 Adrenocorticotrophic hormone (ACTH) secretion is normal at birth, but corticotropes may degenerate secondarily. These patients have normal, small or sometimes large pituitary glands. Gsx-1, an orphan homeobox gene, is required for normal pituitary development. Homozygous mutations of the Gsx-1 gene are associated with extreme dwar®sm, sexual infantilism and increased perinatal mortality.70 The pituitary gland from a€ected individuals is small and hypocellular, with a reduced number of GH- and prolactinproducing cells. Another homeobox gene, Hesx1, encodes a pituitary transcription factor that is ®rst expressed at gastrulation in the mouse embryo. Mutations of the Hesx1 gene have been described in humans and mice with septo-optic dysplasia.71 A novel homeodomain gene that is transcribed as two alternately spliced mRNAs encoding for two separate proteins, Ptx2a and Ptx2b, is a candidate gene for Rieger syndrome, an autosomal dominant disorder with variable craniofacial, dental, eye and pituitary anomalies. Combined pituitary hormone de®ciency has been linked to missense mutations in the Lhx3 gene, which encodes a member of the LIM class of homeodomain proteins. Netchine et al72 identi®ed homozygous mutations in Lhx3 in members of two unrelated consanguineous families; the a€ected persons had de®ciencies of multiple pituitary hormones except ACTH and a rigid cervical spine. Two patients had small, hypoplastic pituitaries, and one had an enlarged anterior pituitary.

Genetic basis of infertility in men 371

Primary defects of spermatogenesis Sex chromosome disorders Approximately 5% of infertile men carry chromosome abnormalities; of these, the majority (4% on average) involve the sex chromosomes, 1% involving the autosomes.73±76 The prevalence of sex chromosomal and autosomal abnormalities in infertile men is 15 and 6 times higher respectively than in the general population.77,78 Klinefelter's syndrome. Klinefelter's syndrome is the most common chromosomal disorder associated with male infertility, being found in between 1 in 500 and 1 in 1000 liveborn males.77,78 The most frequent karyotype in men with Klinefelter's syndrome is 47,XXY (93%), but 46, XY/47,XXY; 48,XXXY; 48,XXYY; and 49,XXXXY karyotypes have also been reported.79±81 The XXY chromosomal constitution has been described in other mammals such as the mouse, Chinese hamster, cat, dog, sheep, ox and pig, and is also associated with sterility. The testes of 47,XXY animals are devoid of germ cells.80,81 Azoospermia is the rule in men with Klinefelter's syndrome who have the 47,XXY karyotype. Men with mosaicism may have germ cells in their testes, especially at a younger age. Testicular histology in men with Klinefelter's syndrome shows a hyalinization of the seminiferous tubules and the absence of spermatogenesis.81,82 Patients with mosaicism may have normal size testes and spermatogenesis at puberty. However, a progressive degeneration and hyalinization of the seminiferous tubules takes place after puberty. In some men, this tubular dysgenesis is patchy, degenerating tubules being interspersed with apparently normal tubules. The Leydig cells appear to be increased in number, although their function is impaired. The 47,XXY karyotype in patients with Klinefelter's syndrome results from nondisjunction during the ®rst meiotic division in one of the parents. Non-disjunction of the maternal chromosomes is the cause of the 47,XXY karyotype in two-thirds of a€ected men, advanced maternal age being a risk factor for non-disjunction. The mechanism by which an extra X chromosome renders patients infertile is not known. In male germ cells, the inactivation of the single X chromosome in the primary spermatocytes of heterogametic males is necessary for spermatogenesis to proceed through meiosis. Noonan's syndrome, the `male Turner's syndrome'. These patients have a 46,XY karyotype, male external genitalia and the clinical stigmata of Turner's syndrome.83 Testis size is reduced and Leydig cell function impaired. Sterility and cryptorchidism are common. Mixed gonadal dysgenesis. Patients with mixed gonadal dysgenesis usually have a 45,X/ 46,XY karyotype, a testis on one side and a streak gonad on the other.85 Some degree of ambiguity of the genitalia is usual. Phenotypic males with mixed gonadal dysgenesis often have an abdominal testis with normal Leydig cells but without any germ cells.85 The dysgenetic gonad is at a high risk of neoplastic degeneration. XX males. These patients have a male phenotype but are azoospermic and have high LH and FSH levels.86,87 The portion of the Y chromosome that contains the Sry may be translocated onto the X chromosome or an autosome; a few patients may be mosaics and carry some 46,XY cell lines. Because they lack other Y-speci®c genes required for spermatogenesis, however, they are sterile.

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Y chromosome microdeletion syndrome. Large deletions of the Y chromosome that can be seen under the microscope in late prophase and hence detectable on routine karyotyping, are uncommon in infertile men. Submicroscopic deletions of the long arm of the Y chromosome, which are not detectable on karyotyping and hence called microdeletions, are, however, present in 5±10% of azoospermic men.88±100 These microdeletions can be detected by PCR-based, sequence-tagged site mapping or by Southern hybridization. The initial studies focused on infertile men with a severe defect of spermatogenesis, i.e. those with azoospermia. More recent studies have, however, shown that Y deletions are also present in oligozoospermic men.95,97 Most infertile men with Y deletions have a severe defect of spermatogenesis, i.e. either azoospermia or severe oligozoospermia.91,93,94 Although the total number of infertile men with Y deletions that have been studied in detail is small, most of these patients have had a testicular volumar of less than 15 ml and an elevated FSH level. The testicular histologies in the small number of reported cases of Y deletion have revealed either Sertoli cell only- or germ cell arresttype phenotypes. The limited number of patients in whom testicular histology has been examined has not allowed a correlation between the location and size of the deletion and the histological phenotype. However, Vogt et al89 have reported that three loci can be identi®ed in Yq, termed AZFa, AZFb, and AZFc, wherein deletions are associated with speci®c testicular histopathology. Two Y-speci®c candidate gene families have been cloned by the deletion mapping of infertile men with Yq deletions and proposed as candidates for the putative AZF locus; these are the RBM (RNA Binding Motif containing)96,101 and the DAZ (deleted in azoospermia) gene families.93,102±104 Both are multiple-copy gene families101,104 that contain the RBM. The RBM gene family has more than 30 copies spread throughout the Y chromosome, most of which are located in deletion intervals 6A and 6B. At least two members of the RBM gene family, RBM-1 and RBM-2, are expressed in the testis.101 The presence of the RBM in the predicted protein sequence suggests that these genes play a role in RNA processing; the precise role of the RBM protein(s) in germ cell development remains, however, unclear. The DAZ gene family is also a multiple-copy gene family.104 The mouse and Drosophila homologues of the DAZ have been mapped to chromosomes 17 and 3 respectively.102,105 An autosomal homologue of the DAZ has also been identi®ed in the human and mapped to chromosome 3.107,108 The homologues of the autosomal DAZlike gene, DAZL1, are present in all mammalian species, DAZ homologues being present only on the Y chromosomes of the great apes and Old World monkeys. Mutations of the DAZL1 gene in Drosophila, boule, are associated with meiotic arrest and azoospermia.102 Similarly, DAZL1 mutations in knockout mice are associated with sterility, providing further evidence for the role of this gene product in germ cell development.109 In infertile men with DAZ deletions, both meiotic arrest and Sertoli cell-only phenotypes have been described; it is possible that germ cell degeneration may occur secondarily. The precise physiological function and role of the RBM and DAZ gene families in human spermatogenesis remains unclear. The RNA molecules that are the targets of these RNA-binding proteins have not been identi®ed. Using DAZ as bait in a twohybrid system, Tsui et al110 identi®ed two novel proteins, DAZ-associated proteins (DAZAPs) 1 and 2, which interact with DAZ and DAZL1. The DAZAP genes have been mapped to chromosomal regions 19p13.3 and 2q33-q34. Although deletions involving the DAZ gene(s) appear to be the most frequent, a large proportion of Y deletions lie outside the DAZ region, some involving the RBM gene. Additional candidate gene families, including BPY2, CDY1, PRY and TTY2, have

Genetic basis of infertility in men 373

been identi®ed in the AZFc region of the Y chromosome.111 The role of these additional Y-speci®c gene families in germ cell development and infertility is not understood. It is also unclear how deletions of one or two copies of the RBM or DAZ genes can explain infertility when there are multiple copies of these genes elsewhere on the Y chromosome. A signi®cant proportion of infertile men with DAZ deletions are oligozoospermic rather than azoospermic. Furthermore, only 5±10% of infertile men have Y deletions. These data suggest that additional Y-speci®c and autosomal genes may be involved in other infertility phenotypes.111 Length of the polyglutamine tract in the androgen receptor protein and infertility The length of the CAG trinucleotide repeat in exon 1 of the androgen receptor gene that encodes for the polyglutamine tract in the androgen receptor protein is polymorphic in humans.112±115 In vitro studies have demonstrated that the transactivational activity of the androgens receptor protein is inversely related to the length of the polyglutamine tract. Individuals with very long tracts have spinobulbal muscular atrophy (Kennedy's disease), a degenerative disease of the spinal cord neurones. However, several reports indicate that men with idiopathic oligozoospermia have a higher likelihood of having longer polyglutamine tracts than fertile men.112±114 In one study of 153 infertile men and 72 healthy controls, for example, Yong et al114 reported that 20% of infertile men have reduced androgenicity because of a long CAG length in exon 1 of the androgen receptor gene. These authors have proposed that long polyglutamine tracts are associated with an increased risk of infertility and a reduced risk of prostate cancer. Conversely, they assert that short polyglutamine tracts are associated with increased transactivational activity, an increased risk of prostate cancer and a reduced risk of infertility. Dadze et al115, on the other hand, found no relationship between the CAG repeat length and impaired spermatogenesis in a sample of infertile men of German origin. The validity of this hypothesis therefore remains to be veri®ed. Autosomal gene defects and male infertility Cyclic adenosine 30 ,50 -monophosphate response element modulator gene expression and spermatogenic arrest. The cyclic AMP response element modulator (CREM) gene codes for transcription factors that are expressed in post-meiotic germ cells and are important for the physiological regulation of the balance between di€erentiation and apoptosis during normal germ cell development.116±120 Mice that are null for CREM protein are sterile and show spermatogenic arrest at the ®rst step of spermiogenesis.120 Late spermatids are absent, and there is an increase in the number of apoptotic germ cells in the seminiferous tubules of CREM-mutant mice.120 Normal spermatogenesis in fertile men is characterized by a switch from CREM repressors to CREM activator isoforms in the post-meiotic germ cells. In situ hybridization studies on testicular biopsies obtained from infertile men with germ cell arrest have demonstrated the absence of activator isoforms of CREM in post-meiotic germ cells and increased apoptosis.116±119 These data suggest that germ cell arrest could result from a failure of this normal transition from repressor to activator isoforms of CREM in the seminiferous tubule. Bilateral congenital absence of the vas deferens and cystic ®brosis transmembrane conductase regulator gene mutations. Mutations in the coding region of the cystic ®brosis

374 S. Bhasin et al

transmembrane conductance regulator gene may result in congenital absence of the vasa without causing the classical pulmonary disease.121 Fifty to seventy per cent of men with congenital absence of the vas deferens harbour mutations of this gene. About 50% are homozygous for a common cystic ®brosis gene abnormality such as F508, and some have compound heterozygosity. Gonadal dysfunction associated with sickle cell disease and beta-thalassaemia. A signi®cant proportion of men with sickle cell disease have a low testosterone level. The majority of men with sickle cell disease who have a low testosterone level su€er from primary testicular dysfunction.122 It is assumed that testicular dysfunction results from microinfarcts in the testis because of the vaso-occlusive disease. Hypogonadotrophic hypogonadism caused by hypothalamic-pituitary dysfunction has, however, been reported in men with sickle cell disease. The pituitary and gonadal dysfunction of thalassaemia occurs as a result of iron deposition in these tissues.123 Hypogonadotrophic hypogonadism is the predominant form of androgen de®ciency syndrome in men with thalassaemia and can be treated e€ectively with gonadotrophin replacement therapy. Pituitary and testicular overload and the resulting hypogonadism can be prevented by prophylactic iron-chelating therapy. Testicular dysfunction in myotonic dystrophy. Myotonic dystrophy is an autosomal dominant disorder associated with CTG repeats in the dystrophin gene. Testicular atrophy occurs in 75% of these men, primarily because of degeneration of the seminiferous tubules. Although the Leydig cells are preserved, the serum testosterone level is low in many patients as a result of primary testicular failure.124 MALE-STERILE MUTATIONS IN DROSOPHILA Male-sterile mutations occur frequently in natural populations of Drosophila (Table 2).125±127 Spermatogenesis in Drosophila is extremely sensitive to many metabolic stresses, leading to a high frequency of mutations that can cause sterility by pleiotropic e€ects. For example, 30% of temperature-sensitive lethal mutations do not actually cause death but render males sterile at a restrictive temperature after development at a permissive temperature.125 It has been estimated that the number of male-sterile mutations in Drosophila could be as high as 2400.126,128 This number seems very high, and the assumptions behind this estimate have been questioned. As shown in Table 2, sterility in male ¯ies can result from abnormal testicular development, a reduced number of germ cells, meiotic arrest and defects in postmeiotic di€erentiation, as well as problems with mating behaviour. Y chromosome abnormalities in Drosophila The Y chromosome plays a key role in regulating spermatogenesis in Drosophila. The lack of the Y chromosome in X0 males of D. melanogaster leads to meiotic arrest at or before metaphase I.128±130 The male D. melanogaster with X±Y translocations carrying either the long arm, XYL/0, or the short arm, XYS/0, of the Y chromosome is sterile. Stern131 proposed a genetic map in which the Drosophila Y-associated fertility complexes are subdivided into seven fertility loci, ®ve (kl-1 to kl-5) on the long arm and two (ks-1, ks-2) on the short arm. The deletions of the regions containing kl-5, kl-3

Genetic basis of infertility in men 375

and ks-1 prevent the formation of the outer dynein arm of the axoneme. The deletion of ks-2 disturbs the proper apposition of axoneme and nebenkern, leading to a complex phenotype with nuclear crystal formation and abnormal meiosis.132 A lampbrush loop-like structure is formed from the Y chromosome in the primary spermatocyte of D. melanogaster and all other Drosophilids studied.133,134 Each of the ®ve pairs of lampbrush loops observed in D. hydie has a speci®cally de®ned morphology, is ordered linearly on the Y chromosome and is essential for male fertility.133,134 The inactivation of one or more loops renders the males carrying them sterile.134 The fertility genes in D. hydie are composed mainly of complex, locus-speci®c repetitive DNA sequences that are transcribed stage-speci®cally in the primary spermatocyte nucleus as continuous long transcription units with a length ranging from 260 kb to up to 4000 kb.135 The fertility locus on the short arm of the Y chromosome of D. hydei, known as locus Q, which forms the lampbrush loop nooses, reveals no open reading frames.136 However, these Y-speci®c sequences show a high capacity to form secondary structures as a result of repeats, leading to the speculation that these sequences may be involved in DNA±protein interaction.136 Sequences homologous to the Drosophila fertility gene have been mapped to deletion interval 6 of the human Y chromosome.

MOUSE MODELS OF STERILITY (TABLES 3 AND 4) In mice, all the reciprocal X±autosome translocations described arrest spermatogenesis at the pachytene/metaphase I (MI) stage of the primary spermatocyte and render the males sterile.137 The Y±autosome translocations in mice are often but not always male-sterile137±139, with a breakdown at diakinesis or MI. Approximately 20% of autosomal reciprocal translocations in the mouse are associated with male sterility. Many cases of human infertility associated with autosomal chromosome aberrations, including translocations and insertions, have been reported140, although chromosomal translocations are not a common cause of infertility in man. The mouse orthologue of the human X-linked gene A1S9 encodes the ubiquitinactivating enzyme E1.141 Two copies of this gene, A1s9Y-1 and A1s9Y-2, are present on the mouse Y chromosome. The deletion of the functional copy, A1s9Y-1, is associated with sterility, leading to speculation that A1s9Y-1 may be a candidate for the spermatogenesis gene, Spy. Mice with inactivating mutations at the W and Sl loci that code for the c-kit and stem cell factor genes are sterile in addition to having defects in haematopoesis, neural development and skin pigmentation.142 Stem cell factor plays a critical role in primordial germ cell proliferation, migration and survival.142 We do not know whether mutations of the SCF gene or its receptor are responsible for a subset of men with the Sertoli cell-only phenotype. Mice homozygous for the Mus spretus allele of a t complex gene called Hst-1 have abnormalities of sperm ¯agellar curvature and are sterile.143 The gene(s) responsible for ¯agellar curvature have not been cloned. The principal morphological abnormality appears to be a failure to form an axoneme. Defects of the t haplotype are also associated with defective sperm±egg interaction. DNA sequences homologous to the mouse t locus have been described on human chromosome 6. However, human genes that subserve a similar function have not yet been characterized.

Mutation symbol

heph

Hephaestus

Infertility owing to meiotic defects Always early mutation Cannonball Meiosis Arrest Degenerative spermatocyte

aly can mia des

Infertility due to a reduced number of germ cells Chikadee chic Diaphanous dia

Abnormalities of male sexual di€erentiation Sex-lethal sxl Transformer, transformer-z Tra, tra-z RBP1 Intersex ix Double sex dsx

cue

Cueball

Infertility caused by defects in gonadogenesis and germ Serpent, Hucklebein Columbus, heartless Abdominal A, abdominal B Abd-A, Abd-B Zfh-1 Bocce boc

Common name of the mutation

All result in meiotic arrest. These genes are required for entry into the ®rst division and progression into spermatogenesis. The post-meiotic stages of spermatogenesis are absent. Mutations result in infertility in males Failure to initiate meiotic chromosomal condensation

Results in testes with reduced germinal content: males are sterile Males are infertile; those at least 5 days old have empty testes. In females, ovaries have reduced egg chambers, rendering them semi-sterile

Male lethality due to inadequate X-dosage compensation Aberrant tra mRNA splicing leading to female di€erentiation Aberrant splicing of mRNA of the SR gene XX female intersex mutants Intersex mutants, female repressor

cell migration Both associated with abnormal gene cell migration Abnormal mesoderm migration Abnormal germ cell association with gonadal mesoderm Attachment of germ cells to somatic cells Males have small testes and a variable nuclear size in spermatocyte cysts. They are infertile. Females are semi-sterile, the number of eggs laid being reduced In males, the testis is short with a defective sheath. Spermatocytes contain cytoplasmic abnormalities. Females are semi-sterile; ovaries are small and misshapen Tip of the testes is enlarged in circumference to approximately twice that of wildtype

Phenotype

Table 2. Naturally occurring sterile mutants in Drosophila.

376 S. Bhasin et al

ken

Ken and Barbie

Data from References 5, 6, 136, 140, 162 and 163.

ptl

Pointless

Infertility resulting from behavioural defects Cuckold cuc Fruitless fru

cas disd e€ hal sat tho stc dbf

shk

Shank

Cashews Dispersed E€ete Halley Scattered Thousand points of light Scratch Double fault

bol

fbl 1(2)26ab

Boule

Defects in post-meiotic di€erentiation Fumble 1(2)26Ab

Twine (cdc25 homologue), cdc2 Pelota pelo

Males are semi-sterile due to failure to court females Males are infertile because they court both females and males, but fail to mate because the abdominal musculature is reduced Males are semi-sterile with wildtype levels of motile sperm in seminal vesicles. Little or no sperm is transferred to females External genital structures are absent in some males and females. Aristae are sparse and unpigmented. Breakdown in courting. Mutation is semi-lethal, and both males and females are semi-sterile

Needle-shaped crystals accumulate through developing germ line, resulting in male sterility In males, spermatid nuclei fail to change shape, rendering them infertile

In males, spermatid nuclei fail to elongate, and the mutation is lethal Elongated spermatid nuclei are dispersed

In males, testes contain degenerating spermatids with large nebenkerne and micronuclei at onion stage Testes are short and ®lled with cysts of 16-cell spermatocytes, which degenerate prior to completion of growth phase, resulting in infertility in males Some 16-cell cysts resemble pelota; others contain nuclei in addition to the abnormally large nebenkerne Spermatids contain two or four nuclei associated with a single large nebenkerne, resulting in infertility in males. Females are semi-sterile In the males of these Drosophila mutants, the elongated spermatid nuclei are dispersed

Meiosis is skipped and spermatid di€erentiation proceeds abnormally Males are infertile because of meiotic arrest

Genetic basis of infertility in men 377

378 S. Bhasin et al Table 3. Mutations a€ecting fertility in male mice. Gene symbol

Gene name

Hpg

Hypogonadotrophic mouse

Chromosome E€ects A large deletion of exons 3 and 4 of the GnRH gene resulting in de®ciency of LH and FSH and hypogonadotrophic hypogonadism

Bc

Blind-sterile

±

Abnormal spermiogenesis

c3H/C6H

Albino-deletion heterozygotes

±

Abnormal spermiogenesis

Hop

Hop-sterile

±

Polydactyly. Sperm tails absent or aberrant

Hpy

Hydrocephalic polydactyly

6

Polydactyly. Sperm abnormal and immotile

Olt

Oligotriche

±

Azoospermia

pbs

p-Black-eyed sterile

7

Coat colour diluted. Sperm abnormality

6H

25H

p ,p

S

Pink-eyed, sterile

7

Abnormal spermiogenesis

Pcd

,p

Purkinje-cell degeneration

±

Abnormal spermiogenesis

Qk

Quaking

17

Defects of myelination and spermiogenesis

tx/tx

t-haplotypes

17

Abnormal spermatids, few spermatozoa

tx/ty

t-haplotypes

17

Spermiogenic defects, failure of sperm function

Sl locus mutation

SCF mutations

Anaemia, fur pigmentation defect, infertility

W locus mutation

c-kit mutation

Anaemia, fur pigmentation defect, infertility

An (Hertig's anaemia)

Anaemia, infertility

At (atrichosis) Wr

Decreased hair density, infertility Wobbler

Abnormal spermiogenesis

Data from references 5, 6, 136, 140 and 162. GnRH ˆ gonadotrophin-releasing hormone; LH ˆ luteinizing hormone; FSH ˆ follicle-stimulating hormone.

Mouse sterility associated with insertional mutagenesis and over-expression of genes144,150 The introduction of a transgene into the genome of the host animal can occasionally disrupt the expression of a functional gene. The insertional mutations that are associated with sterility are of interest because they provide clues to the genes that are essential for spermatogenesis. A partial list of these insertional mutations is provided in Table 3. The over-expression of some gene products in the testis can adversely a€ect spermatogenesis.144,146 The transgenic male mice that over-express human GH have larger testes than wildtype mice, but these animals have a higher incidence of infertility.145 The male mice that over-express interferon gene product in the testis

Genetic basis of infertility in men 379

experience a degeneration of spermatogonia and the atrophy of their seminiferous tubules.145 The over-expression of murine interleukin-2 is also associated with testicular atrophy and spermatogenic arrest.145,146

Null mutations and infertility in knockout mice With the availability of stem cell technology, investigators have produced null mutations in a number of gene (see Table 4).151±160 The phenotype resulting from the `knockout' can provide useful clues to the function of the gene. Gene knockout sometimes produces no discernible phenotype, presumably because the function of the disrupted gene is taken over by another. In addition, the presence of sterility in knockout mice produced by embryonic stem cell technology does not necessarily establish a role for its gene product in spermatogenesis. Although we assume that the embryonic stem cells are totipotent and can di€erentiate into all cell types in the body, including germ cells, these cells may, after multiple passages, lose their ability to di€erentiate into germ cells, leading to the absence of germ cells in the testis. Mouse mutations associated with androgen de®ciency A disruption of some genes can be associated with impaired testosterone secretion. Mice null for insulin-like growth factor-1 expression have impaired mating behaviour because of a lower testosterone level.151 Targeted mutations of hepatocyte nuclear factor-1a, Sp4 and colony-stimulating factor-1 also result in decreased testosterone secretion and a failure to copulate. Mouse mutations associated with a reduced number of germ cells Handel (1987)162 provides a detailed list of references for further reading. Disruption of the FAC (Fanconi anaemia complementation) gene, the DAZ gene and the TIAR gene is associated with a reduced number or absence of germ cells in the testis of these animals.162 The TIAR mutation is associated with the reduced survival of primordial germ cells, which migrate to the genital ridge around embryonic day 11.5; spermatogonia consequently fail to develop.162 Mice with null mutations of the DAZLA gene experience a loss of germ cells and a complete absence of gamete production.162 FAC knockout mice su€er chromosomal breakage, have a reduced number of germ cells in the testes and are sterile.162,163 Hoxa 11 knockout disrupts spermatogenesis because of a failure of testicular descent and malformation of the vas deferens. Mouse mutations associated with defects in meiosis Targeted mutations of the A-myb, Bclw, Bmp 8A, ataxia-telangiectasia and Hsp70-2 genes are associated with meiotic defects.152±160 The testis of mice null for the A-myb gene show germ cells entering the meiotic prophase but arresting at pachytene.150 Knockout of the ataxia-telangiectasia gene results in meiotic arrest at the zygotene or pachytene stages, because of abnormal chromosomal synapses and fragmentation.155 The failure of meiosis in Hsp70-2 mutant mice is associated with increased spermatocyte apoptosis; these mice lack post-meiotic spermatids and sperm.

E€ect of the targeted mutation/phenotype

A-myb

Pms 2

Mutations associated with meiotic defects Mlh 1

Spermatocytes exhibit a high level of prematurely separated chromosomes and arrest in ®rst division of meiosis; microsatellite instability Abnormal chromosome pairing in meiosis; microsatellite instability Predisposition to tumours Germ cells enter meiotic prophase and arrest at pachytene Growth defects

Mutations associated with abnormal germ cell development and/or de®ciency of germ cells in the testis TIAR Reduced survival of primordial germ cells that migrate to the genital ridge around embryonic day 11.5. Failure of development of spermatogonia Zfx Reduction in number of primordial germ cells prior to gonadal sex di€erentiation. Subfertile owing to reduced sperm count Dwarf; less viable FAC (fanconi anaemia complementation) Reduced number of germ cells Normal neonatal viability and gross morphology Cells have chromosome breakage and DNA cross-linear sensitivity. Progenitor cells hypersensitive to interferon gamma Bmp 8B Failure or reduction of germ cell proliferation. In adults, a signi®cant increase in programmed cell death of spermatocytes, leading to sterility

Mutations associated with impaired Leydig cell function and reduced testosterone level HNF-1a (hepatocyte nuclear factor-a) Reduced testosterone level and sterility Growth retardation and non-insulin-dependent diabetes mellitus IGF-1 Failure of androgenization owing to reduced testosterone level Testes reduced in size; spermatogenesis sustained at only 18% of normal Impaired mating behaviour and sterility Dwarf (growth retardation) Sp4 Reduced testosterone level leading to impaired mating behaviour Growth retardation Two-thirds die within ®rst few days of birth

Mutations associated with de®cient gonadotrophin secretion CSF-1 (colony stimulating factor-1) Reduced testosterone level owing to low serum luteinizing hormone Disruption of the normal testosterone negative feedback response of the hypothalamus Reduced mating ability and low sperm number

Gene name

Table 4. Targeted gene mutations in knock-out mice associated with male sterility.

380 S. Bhasin et al

Meiosis arrested at the zygotene/pachytene stage of prophase 1 owing to abnormal chromosomal synapses and chromosome fragmentation Growth retarded Majority develop thymic lymphomas and die before 4 months of age Failure of meiosis coincident with dramatic increase in spermatocyte apoptosis. Lack post-meiotic spermatids and mature sperm

Data from references 3, 4, 136, 140, 151±160, 162 and 163.

Insucient information available to accurately classify cause of infertility DAZLA (deleted in azoospermia) Loss of germ cells and complete absence of sperm in homozygous mice. Heterozygotes have few sperm that are abnormal. Phenotype di€erent from the Drosophila boule that shows meiotic arrest Bax Disordered seminiferous tubules with an accumulation of atypical pre-meiotic germ cells, but no mature haploid sperm. Hyperplasia or hypoplasia Bmp 8A (bone morphogenetic protein) Disruption of spermatogenesis. Degeneration of germ cells and epididymal epithelium ER (oestrogen receptor) Disruption of spermatogenesis and degeneration of seminiferous tubules. Reduced mating frequency. Low sperm number. Defective sperm function

Mutations associated with post-testicular defects in sperm maturation, fertilization or embryonic development Testis ACE (angiotensin-converting enzyme) Sperm show defects in transport within oviducts and binding to zona pellucida; reduced fertility Apo-B (apolipoprotein B) Sperm show abnormal binding to the egg after fertilization. Reduced sperm motility, survival time and sperm count; reduced fertility C-ros Defective epididymis. Defective sperm maturation and fertilization. Reduced fertility PC4 In vivo fertility or spermatozoa of males severely impaired in absence of evidence of spermatazoan abnormality. Egg fertilized by sperm fails to grow to the blastocyst stage

Mutations associated with post-meiotic defects in spermiogenesis Bclw Spermatogenesis is blocked during late spermatogenesis in young adults. Gradual depletion of all stages of germ cells by 6 months of age. Later, Sertoli cells are lost from the seminiferous tubules, and the Leydig cell population is reduced RXR beta Failure of spermatid release within germinal epithelium. Epididymis contains few spermatozoa, these exhibiting abnormal acrosomes and tail.s 50% die before or at birth; the 50% who survive are sterile Sprm-1 Sperm functionally compromised owing to interruption of the regulatory function of the haploid Spermatids; subfertile compared with heterozygous and wildtype Normal testes morphology; normal sperm production hHR6B Derailment of spermatogenesis during post-meiotic condensation of chromatin in spermatids CREM (cyclic AMP responsive element Developing spermatids fail to di€erentiate into sperm. Post-meiotic gene expression in the testis declines modulator) dramatically. No sperm production

Hsp70-2

ATM (ataxia-telangiectasia)

Genetic basis of infertility in men 381

382 S. Bhasin et al

Mouse mutations associated with defects in post-meiotic di€erentiation Mutations of the hHR6B157 and Sprm-1161 genes are associated with a defect of postmeiotic di€erentiation. There is a failure of post-meiotic chromatin condensation in mice mutant for the hHR6B gene.156 Sprm-1 mutations result in the production of functionally impaired sperm.161 Some mutations are associated with sterility even though they produce no apparent abnormality in spermatogenesis.

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