Sex chromosome DSD individuals with mosaic 45,X0 and aberrant Y chromosomes in 46,XY cells: distinct gender phenotypes and germ cell tumour risks§.

"Differences of Sexual Development (DSD)," individuals with rearranged Y chromosome breaks in their 46,XY cells are reported with male and female gender phenotypes and differences in germ cell tumour (GCT) risk. This raised the question of whether male or female gender and GCT risk depends on the site of the break and/or rearrangement of the individual´s Y chromosome. In this paper, we report molecular mapping of the breakpoint on the aberrant Y chromosome of 22 DSD individuals with a 45,X/46,XY karyotype reared with a different gender. Their Y chromosome breaks are found at different sites on the long and short Y arms. Our data indicate that gender rearing is, neither dependent on the site of Y breakage, nor on the amount of 45,X0 cells in the individuals' leukocytes. Most prominent are secondary rearrangements of the Y chromosome breaks forming di-centric Y-structures ("dic-Y"). Duplications of the short Y arm and the proximal part of the long Y arm are the results. A putative GCT risk has been analysed with immunohistochemical experiments on some dysgenetic gonadal tissue sections. With specific antibodies for OCT3/4 expression, we marked the pluripotent germ cell fraction being potential tumour precursor cells. With specific antibodies for DDX3Y, TSPY, and UTY we analyzed their putative Gonadoblastoma Y (GBY) tumour susceptibility function in the same specimen. We conclude GBY expression is only diagnostic for GCT development in the aberrant germ cells of these DSD individuals when strong OCT3/4 expression has marked their pluripotency.

FMR1 and AKT/mTOR Signaling in Human Granulosa Cells: Functional Interaction and Impact on Ovarian Response.

We aimed to determine whether a functional link with impact on female ovarian reserve exists between FMR1 expression and expression ratios of AKT/mTOR signaling genes in human granulosa cells in vivo, as suggested from prior in vitro data. Three hundred and nine women, who were classified as normal (NOR; n = 225) and poor (POR; n = 84) responders based on their ovarian reserve, were recruited during stimulation for assisted reproductive techniques. Expressions of FMR1 and of key genes of the AKT/mTOR and AKT/FOXO1/3 signaling pathways were comparatively analyzed in their granulosa cells. FMR1 expression in granulosa cells of NOR and POR correlated significantly with AKT1, TSC2, mTOR, and S6K expression. No correlation was found between FMR1 and FOXO1 in all, and FOXO3 expression in POR, patients. AKT1 expression was significantly higher and FOXO1 expression lower in POR samples, whereas AKT1 expression was lower and FOXO1 expression was higher in NOR samples. In human native granulosa cells, FMR1 expression significantly correlated with the expression of key genes of the AKT/mTOR signaling pathway, but not with the FOXO1/3 signaling pathway. Our data point to a functional link between FMR1 expression and expression of the AKT/mTOR signaling pathway genes controlling human follicular maturation.

FMR1 expression in human granulosa cells and variable ovarian response: control by epigenetic mechanisms.

In humans, FMR1 (fragile X mental retardation 1) is strongly expressed in granulosa cells (GCs) of the female germline and apparently controls efficiency of folliculogenesis. Major control mechanism(s) of the gene transcription rate seem to be based on the rate of CpG-methylation along the CpG island promoter. Conducting CpG-methylation-specific bisulfite-treated PCR assays and subsequent sequence analyses of both gene alleles, revealed three variably methylated CpG domains (FMR1-VMR (variably methylated region) 1, -2, -3) and one completely unmethylated CpG-region (FMR1-UMR) in this extended FMR1-promoter-region. FMR1-UMR in the core promoter was exclusively present only in female GCs, suggesting expression from both gene alleles, i.e., escaping the female-specific X-inactivation mechanism for the second gene allele. Screening for putative target sites of transcription factors binding with CpG methylation dependence, we identified a target site for the transcriptional activator E2F1 in FMR1-VMR3. Using specific electrophoretic mobility shift assays, we found E2F1 binding efficiency to be dependent on CpG-site methylation in its target sequence. Comparative analysis of these CpGs revealed that CpG 94-methylation in primary GCs of women with normal and reduced efficiency of folliculogenesis statistically significant differences. We therefore conclude that E2F1 binding to FMR1-VMR3 in human GCs is part of an epigenetic mechanism regulating the efficiency of human folliculogenesis. Our data indicate that epigenetic mechanisms may control GC FMR1-expression rates.

AZFa candidate gene UTY and its X homologue UTX are expressed in human germ cells.

The Ubiquitous Transcribed Y (UTY a.k.a. KDM6C) AZFa candidate gene on the human Y chromosome and its paralog on the X chromosome, UTX (a.k.a. KDM6A), encode a histone lysine demethylase removing chromatin H3K27 methylation marks at genes transcriptional start sites for activation. Both proteins harbour the conserved Jumonji C (JmjC) domain, functional in chromatin metabolism, and an extended N-terminal tetratricopeptide repeat (TPR) block involved in specific protein interactions. Specific antisera for human UTY and UTX proteins were developed to distinguish the expression of both proteins in human germ cells by immunohistochemical experiments on appropriate tissue sections. In the male germ line, UTY was expressed in the fraction of A spermatogonia located at the basal membrane, probably including spermatogonia stem cells. UTX expression was more spread in all spermatogonia and in early spermatids. In female germ line, UTX expression was found in the primordial germ cells of the ovary. UTY was also expressed during fetal male germ cell development, whereas UTX expression was visible only at distinct gestation weeks. Based on these results and the conserved neighboured location of UTY and DDX3Y in Yq11 found in mammals of distinct lineages, we conclude that UTY, such as DDX3Y, is part of the Azoospermia factor a (AZFa) locus functioning in human spermatogonia to support the balance of their proliferation-differentiation rate before meiosis. Comparable UTY and DDX3Y expression was also found in gonadoblastoma and dysgerminoma cells found in germ cell nests of the dysgenetic gonads of individuals with disorders of sexual development and a Y chromosome in karyotype (DSD-XY). This confirms that AZFa overlaps with GBY, the Gonadoblastoma susceptibility Y locus, and includes the UTY gene. LAY SUMMARY: AZFa Y genes are involved in human male germ cells development and support gonadoblastoma (germ cell tumour precursor cells) in the aberrant germ cells of the gonads of females with genetic disorders of sexual development. The AZFa UTY gene on the male Y chromosome is equivalent to UTX on the female X chromosome. These genes are involved in removing gene regulators to enable activation of other genes (i.e. removal of histone methylation known as epigenetic modifications). We wanted to learn the function of UTY and UTX in developing sperm and eggs in human tissues and developed specific antibodies to detect both proteins made by these genes. Both UTY and UTX proteins were detected in adult and fetal sperm precursor cells (spermatogonia). UTX was detected in egg precursor cells (primordial germ cells). UTY was detected in gonadoblastoma and dysgerminoma tumour cells (germ cell tumours originating from genetic disorders of sexual development due to having a Y chromosome). Based on our study, we conclude that UTY is not only part of AZFa, but also of GBY the overlapping gonadoblastoma susceptibility Y region.

FMR1 and AKT/mTOR signalling pathways: potential functional interactions controlling folliculogenesis in human granulosa cells.

Granulosa cells (GCs) play a major role in folliculogenesis and are crucial for oocyte maturation and growth. In these cells, the mTOR/AKT signalling pathway regulates early folliculogenesis by maintaining the dormancy of primordial follicles, while FSH induces their further differentiation and maturation. Because changes in number of CGG triplets in FMR1 exon 1 (below or beyond normal values of 26-34 triplets) affect ovarian reserve and pre-mutations containing >54 CGG triplets represent a known risk factor for premature ovarian insufficiency/failure, we investigated in the human GC model (COV434) how FMR1/FMRP and mTOR/AKT are expressed and potentially interact during GC proliferation. As FMR protein (FMRP) is expressed mainly in human ovarian GCs, we used these after inducing their proliferation using recombinant FSH (rFSH) and the repression of the mTOR/AKT signalling pathway. We showed that AKT and mTOR expression levels significantly increase after stimulation with rFSH, while S6K and FMR1 expression decrease. After inhibiting mTOR and AKT, FMR1 and S6K expression significantly increased. Only AKT inhibition led to decreased FMRP levels, as expected due to the known FMR1/FMRP negative feedback loop. But rFSH and the mTOR inhibition increased them, indicating a decoupling of this FMR1/FMRP negative feedback loop in our model system.

DDX3X, the X homologue of AZFa gene DDX3Y, expresses a complex pattern of transcript variants only in the male germ line.

DDX3X, the functional X homologue of the major AZFa gene, DDX3Y, belongs to the highly conserved PL10-subfamily of DEAD-box RNA helicase genes which are functionally conserved from yeast to man. They are mainly involved in cell cycle control and translation initiation control of gene transcripts with long 5'UTR extensions containing complex secondary structures. Interestingly, in humans both gene copies were found to be expressed at different phases of human spermatogenesis. Whereas DDX3Y transcripts are translated only in premeiotic male germ cells, the DDX3X protein is expressed only in postmeiotic spermatids. In this study, we found that the major class of DDX3X transcripts in human testis become activated first after meiosis and at a specific core promoter not active in somatic tissues and not present upstream of the DDX3Y homologue. Two alternative 5'UTR transcript lengths are subsequently produced by an additional testis-specific 5'UTR splicing event. Both transcripts are mainly processed for polyadenylation in their proximal 3'UTR. A minor transcript class starting at the same male germ line-specific core promoter produces primary transcripts with an extremely long 3'UTR (∼ 17 kb), which is subsequently spliced at distinct sites resulting in six short 3'UTR splice variants (I-VI). Comparative analyses of the DDX3X transcripts in mouse and primates revealed that this complex pattern of male germ line-specific transcript variants first evolved in primates. Our data thus suggest complex translational control mechanism(s) for the human DDX3X gene locus functioning only in the male germ line and resulting in expression of its protein only in the postmeiotic spermatids.

Single nucleotide polymorphism array analysis in men with idiopathic azoospermia or oligoasthenozoospermia syndrome.

OBJECTIVE: To identify copy number variations (CNVs) as a hint toward genes relevant for spermatogenesis and related to male factor infertility. DESIGN: Analysis of genomic DNA with high resolution Illumina SNP arrays (HumanOmni1-Quad Bead Chip). Sanger sequencing of the CLCA4 gene in all patients of the study. Analysis of CLCA4 expression in various human tissue samples. SETTING: University department. PATIENT(S): A total of 39 infertile men with idiopathic infertility ranging from oligoasthenoteratozoospermia to azoospermia. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Copy number variations more than 10 kb. RESULT(S): We detected a heterozygous deletion including exons 4-9 of the CLCA4 gene in one man with cryptozoospermia, as well as a total of 149 CNVs not yet reported in various databases and carrying 200 protein coding genes in the 39 men. CONCLUSION(S): According to our results CLCA4 is apparently expressed in postmeiotic germ cells and somatic cells. We therefore conclude that CLCA4 might be functional during human spermatogenesis after meiosis, most likely as a modifier of CFTR gene expression. CLCA4 can thus be considered as a novel dominant germ line gene potentially causing male factor infertility if functionally disrupted. Our study demonstrates the power of DNA arrays to identify novel CNVs carrying candidate genes causing male factor infertility.

Human Y chromosome microdeletion analysis by PCR multiplex protocols identifying only clinically relevant AZF microdeletions.

PCR multiplex assays are the method of choice for quickly revealing genomic microdeletions in the large repetitive genomic sequence blocks on the long arm of the human Y chromosome. They harbor the Azoospermia Factor (AZF) genes, which cause male infertility when functionally disrupted. These protein encoding Y genes are expressed exclusively or predominantly during male germ cell development, i.e., at different phases of human spermatogenesis. They are located in three distinct genomic sequence regions designated AZFa, AZFb, and AZFc, respectively. Complete deletion of an AZF region, also called "classical" AZF microdeletion, is always associated with male infertility and a distinct testicular pathology. Partial AZF deletions including single AZF Y genes can cause the same testicular pathology as the corresponding complete deletion (e.g., DDX3Y gene deletions in AZFa), or might not be associated with male infertility at all (e.g., some BPY2, CDY1, DAZ gene deletions in AZFc). We therefore propose that a PCR multiplex assay aimed to reduce only those AZF microdeletions causing a specific testicular pathology-thus relevant for clinical applications. It only includes Sequence Tagged Site (STS) deletion markers inside the exon structures of the Y genes known to be expressed in male germ cells and located in the three AZF regions. They were integrated in a robust standard protocol for four PCR multiplex mixtures which also include the basic principles of quality control according to the strict guidelines of the European Molecular Genetics Quality Network (EMQN: http://www.emqn.org). In case all Y genes of one AZF region are deleted the molecular extension of this AZF microdeletion is diagnosed to be yes or no comparable to that of the "classical" AZF microdeletion by an additional PCR multiplex assay analyzing the putative AZF breakpoint borderlines.

Sequence variation at the human FOXO3 locus: a study of premature ovarian failure and primary amenorrhea.

BACKGROUND: The forkhead transcription factor Foxo3 is a master regulator and potent suppressor of primordial follicle activation. Loss of Foxo3 function in the mouse leads to premature ovarian failure (POF) due to global follicle activation. METHODS AND RESULTS: Here, we show that the mouse Foxo3 locus is haploinsufficient, and that Foxo3-/+ females undergo early reproductive senescence consistent with an increased rate of primordial follicle utilization. Then, to determine if heterozygous or homozygous polymorphisms or mutations of the human orthologue FOXO3 contribute to POF or idiopathic primary amenorrhea (PA), we sequenced the exons and flanking splice sequences of the gene in a large number of women with idiopathic POF (n = 273) or PA (n = 29). A total of eight single-nucleotide polymorphisms (SNPs) were identified, revealing a substantial amount of genetic variation at this locus. Allelic frequencies in control samples excluded several of these variants as causal. For the remaining variants, site-directed mutagenesis was performed to assess their functional impact. However, these rare sequence variants were not associated with significant decreases in FOXO3 activity. CONCLUSIONS: Taken together, our findings suggest that, despite the potential for FOXO3 haploinsufficiency to cause ovarian failure, FOXO3 mutations or common SNPs are not a common cause of either POF or PA.

Polymorphisms associated with the DAZ genes on the human Y chromosome.

The human Y chromosome is unique in that it does not engage in pairing and crossing over during meiosis for most of its length. Y chromosome microdeletions, a frequent finding in infertile men, thus occur through intrachromosomal recombination, either within a single chromatid or between sister chromatids. A recently identified polymorphism associated with increased risk for spermatogenic failure, the gr/gr deletion, removes two of the four Deleted in Azoospermia (DAZ) genes in the AZFc region on the Y-chromosome long arm. We found the likely reciprocal duplication product of gr/gr deletion in 5 (6%) of 82 males using a novel DNA-blot hybridization strategy and confirmed the presence of six DAZ genes in three cases by FISH analysis. Additional polymorphisms identified within the DAZ repeat regions of the DAZ genes indicate that sister chromatid exchange plays a significant role in the genesis of deletions, duplications, and polymorphisms of the Y chromosome.

AZF deletions and Y chromosomal haplogroups: history and update based on sequence.

AZF deletions are genomic deletions in the euchromatic part of the long arm of the human Y chromosome (Yq11) associated with azoospermia or severe oligozoospermia. Consequently, it can be assumed that these deletions remove Y chromosomal genes required for spermatogenesis. However, these 'classical' or 'complete' AZF deletions, AZFa, AZFb and AZFc, represent only a subset of rearrangements in Yq11. With the benefit of the Y chromosome sequence, more rearrangements (deletions, duplications, inversions) inside and outside the classical AZF deletion intervals have been elucidated and intra-chromosomal non-allelic homologous recombinations (NAHRs) of repetitive sequence blocks have been identified as their major cause. These include duplications in AZFa, AZFb and AZFc and the partial AZFb and AZFc deletions of which some were summarized under the pseudonym 'gr/gr' deletions. At least some of these rearrangements are associated with distinct Y chromosomal haplogroups and are present with similar frequencies in fertile and infertile men. This suggests a functional redundancy of the AZFb/AZFc multi-copy genes. Alternatively, the functional contribution(s) of these genes to human spermatogenesis might be different in men of different Y haplogroups. That raises the question whether, the frequency of Y haplogroups with different AZF gene contents in distinct human populations leads to a male fertility status that varies between populations or whether, the presence of the multiple Y haplogroups implies a balancing selection via genomic deletion/amplification mechanisms.

Three new 46,XX male patients: a clinical, cytogenetic and molecular analysis.

BACKGROUND: XX males range phenotypically from completely masculinised individuals to true hermaphrodites and include a subset of SRY negative patients. The correlation between genotype (SRY+/-) and phenotype is still unclear. AIM: To report three new patients with this rare condition, one of whom was diagnosed prenatally and another was SRY negative, and to verify in our patients whether the presence of SRY results in a more masculinised phenotype. PATIENTS AND METHODS: We present two phenotypically normal XX male patients (10 and 13.5 years) and one 3.1 years old XX male with ambiguous external male genitalia Prader IV. The patients were diagnosed by clinical, hormonal, sonographic, genetic and histological criteria. RESULTS: Basal hormonal status was normal for phenotype but an excessive response to GnRH testing was noticed in the second patient together with insufficient hCG stimulation in all three patients. Pelvic ultrasound displayed male structures without Müllerian ducts; testicular biopsy, performed only in the intersex patient, showed Sertoli and Leydig cell hypoplasia. Chromosome analysis confirmed 46,XX karyotype. FISH analysis and molecular analysis by PCR were positive for Yp fragments/SRY gene on Xp in two patients and negative in the patient with ambiguous external genitalia. CONCLUSIONS: In our observation Y chromosome-specific material containing the SRY gene translocated to the X chromosome results in a completely masculinised phenotype. In the intersex patient, incomplete masculinisation without SRY suggests a mutation of one or more downstream non-Y testis-determining genes.

Azoospermia factor (AZF) in Yq11: towards a molecular understanding of its function for human male fertility and spermatogenesis.

The Y chromosomal azoospermia factor (AZF) is essential for human spermatogenesis. It has been mapped by molecular deletion analyses to three subintervals in Yq11, AZFa, AZFb, and AZFc, containing a number of genes of which at least some control, post-transcriptionally, the RNA metabolism of other spermatogenesis genes, functionally expressed at different phases of the spermatogenic cycle. Intrachromosomal recombination events between homologous large repetitive sequence block in Yq11 are now recognized as the major cause of the AZFa, AZFb and AZFc microdeletions, and an overlap of the AZFb and AZFc regions was revealed by sequence analysis of the complete Yq11 region. The increasing knowledge of the expression patterns of AZF genes in human germ cells suggests that the DBY gene is the major AZFa gene, the RBMY gene the major AZFb gene, although a functional expression of the other AZFa/b genes in the male germ line is also most likely. Genetic redundancy might exist in AZFc because a number of gene copies in the large P1 palindrome structure in distal AZFc were found to be deleted also in fertile men.

Genomic heterogeneity and instability of the AZF locus on the human Y chromosome.

The spermatogenesis locus azoospermia factor (AZF) in Yq11 has been mapped to three microdeletion intervals designated as AZFa, AZFb, and AZFc. They are caused by intrachromosomal recombination events between large homologous repetitive sequence blocks, and AZFc microdeletions are now recognised as the most frequent known genetic lesion causing male infertility. However, in the same Y-region, large genomic heterogeneities are also observed in fertile men, and only complete AZFa and AZFb deletions are associated with a specific testicular pathology. Partial AZF deletions are associated with variable pathologies and partial AZFc deletions may even have no impact on male fertility. This suggests a genetic redundancy of the multi-copy genes in AZFb and AZFc and a causative relationship between the occurrence of first microdeletions then macrodeletions in the repetitive structure of Yq11 where large palindromes are probably promoting multiple gene conversions and AZF rearrangements.

Developmental expression of POU5F1 (OCT-3/4) in normal and dysgenetic human gonads.

BACKGROUND: To investigate how long fetal germ cells retain pluripotency, which may be linked to their ability to transform into histologically variable tumours, we examined the expression of OCT-3/4 (POU5F1), a transcription factor essential for the maintenance of totipotency in embryonic stem cells. METHODS: The ontogeny of expression of OCT-3/4 was studied in 74 specimens of normal human gonads during development and in 58 samples of gonads from cases with testicular dysgenesis syndrome (TDS), including disorders of sex differentiation and malignant changes. RESULTS: OCT-3/4 expression was found in primordial germ cells during migration to the gonadal ridges and in the indifferent gonad. The expression in testes gradually decreased until approximately 20 weeks of gestation, and thereafter it was more rapidly down-regulated, but persisted in a few cells until 3-4 months of postnatal age, which coincides with the final differentiation of gonocytes into infantile spermatogonia. Subsequently, OCT-3/4 was not detected in normal testes. In the ovaries, OCT-3/4 was expressed in primordial oogonia, but was down-regulated in oocytes that formed primary follicles. The pattern of expression was heterogeneous in dysgenetic and intersex cases, with OCT-3/4-positive gonocytes detected in this series until 14 months of age. Visibly neoplastic gonadoblastoma and carcinoma in situ (CIS) expressed abundant OCT-3/4 regardless of the age. CONCLUSIONS: In the human ovary, OCT-3/4 is silenced at the onset of the first meiotic prophase, whereas in the testis, down-regulation of OCT-3/4 is a gradual process associated with differentiation of gonocytes. This normal pattern of expression is disturbed in dysgenetic gonads, especially in rare intersex cases, thus increasing the risk of malignant transformation. The high abundance of OCT-3/4 in CIS cells is consistent with their early fetal origin and pluripotency.

Molecular genetics of human male infertility: from genes to new therapeutic perspectives.

Genetic lesions causing human male infertility are manifold. Besides gross chromosomal aneuploidies and rearrangements, microdeletions and single gene defects can interfere with male fertility. Male fertility is not only dependent on genes controlling the male germ line but also on genes of the networks functional for male gonad development and male somatic development, respectively. It is popular to unravel these netweorks with mouse gene knock-out mutants displaying reproductive defects. However, substantial arguments can be given for more functional studies directly on the human genes, because multiple reproductive proteins evolve quickly most likely for adopting to the specific needs of the species class. Prominent examples are mutations of the FSHR gene causing different pathologies in mouse and human and the DAZ gene family not found in the mouse genome but in the human genome with an essential male fertility function. Therefore this review is focussed on a comprehensive overview of human genes known with mutations causing male infertility (AR; AZF gene families; CFTR, DM-1, DNAH gene family, FGFR1, FSHR, INSL3, KAL-1, LGR8- GREAT, LHR, POLG). Then some human genes are described well recognised as functional in spermatogenesis and male fertility although gene specific mutations causing infertility were not yet identified (CREM, CDY1, DAZL1, PHGPx, PRM-1, PRM-2). They are designated as "spermatogenesis phase marker" or "male fertility index" genes, because they are useful tools for diagnosing the patient's spermatogenesis disruption phase and for predicting the presence and quality of his mature sperms. Current therapeutic protocols for human male infertility do usually not cure the specific gene defect but try to bypass it using Artificial Reproductive Technology (ART). Putative imprinting defects in the early embryo probably associated with the used ART protocol and an increase of chromosome abnormalities in the ART offspring now strongly asks for a significant improvement of this outcome requesting urgently more basic research on the genes functioning in the human male germ line and during early human embryogenesis.

No AZF deletion in 160 patients with testicular germ cell neoplasia.

Testicular germ cell cancer is aetiologically linked to genital malformations and male infertility and is most probably caused by a disruption of embryonic programming and gonadal development during fetal life. In some cases, germ cell neoplasia is associated with a relative reduction of Y chromosomal material (e.g. 45,X/46,XY) or other abnormalities of the Y chromosome. The euchromatic long arm of the human Y chromosome (Yq11) contains three azoospermia factors (AZFa, AZFb, AZFc) functionally important in human spermatogenesis. Microdeletions encompassing one of these three AZF loci result in the deletion of multiple genes normally expressed in testis tissue and are associated with spermatogenic failure. The aim of our study was to investigate whether AZF microdeletions, in addition to causing infertility, predispose also to germ cell neoplasia, since subjects with poor spermatogenesis have an increased risk of testicular cancer. We screened for putative deletions of AZF loci on the Y chromosome in DNA isolated from white blood cells of 160 Danish patients with testicular germ cell neoplasia. Interestingly, although AZF microdeletions are found frequently in patients with idiopathic infertility, in all cases studied of testicular germ cell cancer the Yq region was found to be intact. We conclude that the molecular aetiology of testicular germ cell neoplasia of the young adult type most likely does not involve the same pathways as male infertility caused by AZF deletions. Malignant transformation of germ cells is thus caused by the dysfunction of some other genes that still need to be identified.

Polymorphic DAZ gene family in polymorphic structure of AZFc locus: Artwork or functional for human spermatogenesis?

Human spermatogenesis is regulated by a network of genes located on autosomes and on sex chromosomes, but especially on the Y chromosome. Most results concerning the germ cell function of the Y genes were obtained by genomic breakpoint mapping studies of the Y chromosome of infertile patients. Although this approach has the benefit of focussing on those Y regions that contain most likely the Y genes of functional importance, its major drawback is the fact that fertile control samples were often missing. In fertile men, molecular and cytogenetic analyses of the Y chromosome has revealed highly polymorphic chromatin domains especially in the distal euchromatic part (Yq11.23) and in the heterochromatic part (Yq12) of the long arm. In sterile patients cytogenetic analyses mapped microscopically visible Y deletions and rearrangements in the same polymorphic Y regions. The presence of a Y chromosomal spermatogenesis locus was postulated to be located in Yq11.23 and designated as AZoospermia Factor (ZF). More recently, molecular deletion mapping in Yq11 has revealed a series of microdeletions that could be mapped to one of three different AZF loci: AZFa in proximal Yq11 (Yq11.21), AZFb and AZFc in two non-overlapping Y-regions in distal Yq11 (Yq11.23). This view was supported by the observation that AZFa and AZFb microdeletions were associated with a specific pathology in the patients' testis tissue. Only AZFc deletions were associated with a variable testicular pathology and in rare cases AZFc deletions were even found inherited from father to son. However, AZFc deletions were found with a frequency of 10-20% only in infertile men and most of them were proved to be "de novo", i.e. the AZFc deletion was restricted to the patient's Y chromosome. Based mainly on positional cloning experiments of testis cDNA clones and on the Y chromosomal sequence now published in GenBank, a first blueprint for the putative gene content of the AZFc locus can now be given and the gene location compared to the polymorphic DNA domains. This artwork of repetitive sequence blocks called AZFc amplicons raised the question whether the AZFc chromatin is still part of the heterochromatic domain of the Y long arm well known for its polymorphic extensions or is decondensed and part of the Yq11.23 euchromatin? We discuss also the polymorphic DAZ gene family and disclose putative origins of its molecular heterogeneity in fertile and infertile men recently identified by the analyses of Single Nucleotide Variants (SNVs) in this AZFc gene locus.