Guy Longepied

Sequence family variant loss from the AZFc interval of the human Y chromosome, but not gene copy loss, is strongly associated with male infertility

Background: Complete deletion of the complete AZFc interval of the Y chromosome is the most common known genetic cause of human male infertility. Two partial AZFc deletions (gr/gr and b1/b3) that remove some copies of all AZFc genes have recently been identified in infertile and fertile populations, and an association study indicates that the resulting gene dose reduction represents a risk factor for spermatogenic failure. Methods: To determine the incidence of various partial AZFc deletions and their effect on fertility, we combined quantitative and qualitative analyses of the AZFc interval at the DAZ and CDY1 loci in 300 infertile men and 399 control men. Results: We detected 34 partial AZFc deletions (32 gr/gr deletions), arising from at least 19 independent deletion events, and found gr/gr deletion in 6% of infertile and 3.5% of control men (p>0.05). Our data provide evidence for two large AZFc inversion polymorphisms, and for relative hot and cold spots of unequal crossing over within the blocks of homology that mediate gr/gr deletion. Using SFVs (sequence family variants), we discriminate DAZ1/2, DAZ3/4, CDY1a (proximal), and CDY1b (distal) and define four types of DAZ-CDY1 gr/gr deletion. Conclusions: The only deletion type to show an association with infertility was DAZ3/4-CDY1a (p = 0.042), suggesting that most gr/gr deletions are neutral variants. We see a stronger association, however, between loss of the CDY1a SFV and infertility (p = 0.002). Thus, loss of this SFV through deletion or gene conversion could be a major risk factor for male infertility.

TSPY, the Candidate Gonadoblastoma Gene on the Human Y Chromosome, has a Widely Expressed Homologue on the X - Implications for Y Chromosome Evolution

TSPY, a candidate gene for a factor that promotes gonadoblastoma formation (GBY), is a testis-specific multicopy gene family in the male-specific region of the human Y (MSY) chromosome. Although it was originally proposed that male-specific genes on the Y originated from a transposed copy of an autosomal gene (Lahn & Page 1999b), at least two male-specific genes (RBMY and SRY) descended from a formerly recombining X-Y identical gene pair. Here we show that a TSPY homologue with similar gene structure lies in conserved positions, close to SMCX, on the X chromosome in human (TSPX) and mouse (Tspx). TSPX is widely expressed and subject to X inactivation. TSPX and TSPY therefore evolved from an identical gene pair on the original mammalian sex chromosomes. This supports the hypothesis that even male-specific genes on the Y chromosome may have their origin in ubiquitously expressed genes on the X. It also strengthens the case for TSPY as a candidate for GBY, since independent functional studies link TSPX to cell cycle regulation.

Mouse H-Y encoding Smcy gene and its X chromosomal homolog Smcx.

SMCY is present on the Y in mouse, human, and most mammals. Importantly, it is present on the marsupial Y showing that SMCX/ SMCY diverged at least 120 Myr years ago (Agulnik et al. 1994a). Unlike many other Y-Chromosomal genes, SMCY is present in a single copy in mouse and man. Both Y and X Chr homologs are widely transcribed in all male tissues, and the X gene is expressed in all female tissues tested including preimplantation mouse embryos (Agulnik et al. 1994a). SMCX escapes X-inactivation in human and mouse (Agulnik et al. 1994b; Carrel et al. 1996; Jegalian and Page 1998; Sheardown et al. 1996; Wu et al. 1994a, 1994b), indicating that the X and Y copies are functionally interchangeable and that transcript dose is important for the expression of this gene. Although the biological role of the genes remains unknown, it has been established that human and mouse SMCY contain epitopes of the minor histocompatibility antigen, H-Y (Ehrmann et al. 1997; Markiewicz et al. 1998; Meadows et al. 1997; Scott et al. 1995; Wang et al. 1995). In this paper we report the complete cDNA sequence of the mouse SmcyandSmcxgenes. We have established the genomic structure of the Smcygene and show that the gene is composed of 26 exons spread over 47 kb of genomic DNA. The longest open reading frame encodes 1548 amino acids forSmcyand 1551 amino acids for Smcx.The genomic structure of SMCY is entirely conserved between mouse and human. As yet, its biological role is not understood, but the homology to RBP2 (retinoblastoma-binding protein 2) and the presence of a zinc-finger domain indicate a possible involvement of the SMCX/Y (SMCX and SMCY) proteins in DNA binding and transcriptional regulation. A 1.8-kb cDNA of the mouseSmcygene and 3 kb of the mouse Smcxgenes identified previously (Agulnik et al. 1994a,b) have been used in the present study to clone and sequence full-length transcripts of the genes. RT and RACE PCR, as well as conventional screening of testis cDNA libraries, have been employed to obtain a series of the overlapping cDNA fragments representing the missing 38 and 58 ends of both genes. The Smcysequence is 5316 bp, andSmcxis 5673 bp with open reading frames (ORF) of 4647 bp and 4656 bp respectively. The transcripts are predicted to encode polypeptides of 1548aa, MW 177 kDA ( Smcy), and 1551 aa, MW 175 kDa ( Smcx). The translation start codon in Smcyis situated in the context AACATGA, which is close to the optimal ACCATG present in the Smcxsequence (Kozak 1986). In the Smcy transcript, two consensus polyadenylation signals AATAAA are at 266 bp and 18 bp upstream of poly (A) tail. InSmcxthere is only one polyadenylation signal at 55 bp upstream of the poly(A) tail. During cDNA isolation we found several splice variants of the SmcyandSmcxtranscripts. Several splice variants of the SMCY gene were also recovered from human testis and lymphocyte polyA RNA (data not shown). Different transcripts apparently utilizing different splice sites have been also reported for the human SMCX gene (Wu et al. 1994a) and for the SMCY gene (cDNA KIAA0234, GeneBank accession # D87072). Specific differences between the amino acid sequence of the SMCY and SMCX genes, coupled with their widespread expression pattern (Agulnik et al. 1994a), form the basis of the H-Y male-specific minor antigen system. Thus, Smcyhas been shown to encode several H-Y antigen epitopes, the position of which are shown in Fig. 1 (Ehrmann et al. 1997; Markiewicz et al. 1998; Meadows et al. 1997; Scott et al. 1995; Wang et al. 1995). As shown in Fig. 1, proteins derived from the translation of the open reading frames of the human and mouse SMCX/SMCY genes are highly homologous to each other. Overall amino acid sequence similarities are as follows: Smcx/SMCX 96%; Smcy/ SMCY 84%; Smcy/Smcx 84%; Smcy/SMCX 83%; and Smcx/ SMCY 86%. In all proteins the C-terminal end encoded by the last exon is the least conserved. The similarity between the amino acid sequence of SmcxandSmcyin this region is only 32% compared with an overall value of 84%. The closest identified homolog of the SMCX/SMCY gene pair is the human retinoblastoma binding protein 2 (RBP2; Fataey et al. 1993). The overall identity of the latter protein to the products of the mouse X and Y genes is 58% (67% similarity) and 57% (65% similarity) respectively. The homology is higher at the N-terminal end of the sequence. At the C-terminal end, the middle part of the twenty-third exon ofSmcyencodes 57 amino acids, which are highly conserved among mouse and human SMCX/Y proteins and RBP2 (89% identity). The RBP2 protein contains two significant domains—a zinc-finger at the N-terminal end and a homeodomain similar to the engrailed family of homeotic genes in the middle part of the sequence (Fataey et al. 1993). The zinc-finger domain is well conserved between SMCX/Y products and RBP2 protein (70% identity), but the homeodomain is less conserved (53–57% identity). The retinoblastoma product (pRB) binding domain (a stretch of amino acids shared by all retinoblastoma-binding proteins) is not conserved in any SMCX/Y protein. At the nucleotide and amino acid level, database searching revealed that both genes have several homologs: yeast hypothetical 85.0 kDa protein (Accession # P47156); mouse jumonji protein (Q62315); yeast putative 90.2 kDa zinc-finger protein (P39956); and others. SMCX/Y proteins contain a zinc-finger domain as part of a highly conserved PHD finger, a cysteine-rich region encoded by the eighth exon of the gene. Such a domain is present in a number of putative proteins derived from yeast, mammals, and plants (Aasland et al. 1995). To obtainSmcygenomic clones, we have screened a mouse Correspondence to: A.I. Agulnik

Complete deletion of the AZFb interval from the Y chromosome in an oligozoospermic man

BACKGROUND Deletion of the entire AZFb interval from the Y chromosome is strictly associated with azoospermia arising from maturation arrest during meiosis. Here, we describe the exceptional case of an oligozoospermic man, 13-1217, with an AZFb + c (P5/distal-P1) deletion. Through the characterization of this patient, and two AZFb (P5/proximal-P1) patients with maturation arrest, we have explored three possible explanations for his exceptionally progressive spermatogenesis. METHODS AND RESULTS We have determined the precise breakpoints of the deletion in 13-1217, and shown that 13-1217 is deleted for more AZFb material than one of the AZFb-deleted men (13-5349). Immunocytochemical analysis of spermatocytes with an antibody against a synaptonemal complex component indicates synapsis to be largely unaffected in 13-1217, in contrast to 13-5349 where extended asynapsis is frequent. Using PCR-based analyses of RNA and DNA from the same testicular biopsy, we show that 13-1217 expresses post-meiotic germ cell markers in the absence of genomic DNA and transcripts from the AZFb and AZFc intervals. We have determined the Y chromosome haplogroup of 13-1217 to be HgL-M185. CONCLUSIONS Our results indicate that the post-meiotic spermatogenesis in 13-1217 is not a consequence of mosaicism or retention of a key AZFb gene. On the contrary, since the Hg-L Y chromosome carried by 13-1217 is uncommon in Western Europe, a Y-linked modifier locus remains a possible explanation for the oligozoospermia observed in patient 13-1217. Further cases must now be studied to understand how germ cells complete spermatogenesis in the absence of the AZFb interval.

Genetic diversity on the Comoros Islands shows early seafaring as major determinant of human biocultural evolution in the Western Indian Ocean

The Comoros Islands are situated off the coast of East Africa, at the northern entrance of the channel of Mozambique. Contemporary Comoros society displays linguistic, cultural and religious features that are indicators of interactions between African, Middle Eastern and Southeast Asian (SEA) populations. Influences came from the north, brought by the Arab and Persian traders whose maritime routes extended to Madagascar by 700–900 AD. Influences also came from the Far East, with the long-distance colonisation by Austronesian seafarers that reached Madagascar 1500 years ago. Indeed, strong genetic evidence for a SEA, but not a Middle Eastern, contribution has been found on Madagascar, but no genetic trace of either migration has been shown to exist in mainland Africa. Studying genetic diversity on the Comoros Islands could therefore provide new insights into human movement in the Indian Ocean. Here, we describe Y chromosomal and mitochondrial genetic variation in 577 Comorian islanders. We have defined 28 Y chromosomal and 9 mitochondrial lineages. We show the Comoros population to be a genetic mosaic, the result of tripartite gene flow from Africa, the Middle East and Southeast Asia. A distinctive profile of African haplogroups, shared with Madagascar, may be characteristic of coastal sub-Saharan East Africa. Finally, the absence of any maternal contribution from Western Eurasia strongly implicates male-dominated trade and religion as the drivers of gene flow from the North. The Comoros provides a first view of the genetic makeup of coastal East Africa.

Human and mouse ZFY genes produce a conserved testis-specific transcript encoding a zinc finger protein with a short acidic domain and modified transactivation potential

Mammalian ZFY genes are located on the Y chromosome, and code putative transcription factors with 12–13 zinc fingers preceded by a large acidic (activating) domain. In mice, there are two genes, Zfy1 and Zfy2, which are expressed mainly in the testis. Their transcription increases in germ cells as they enter meiosis, both are silenced by meiotic sex chromosome inactivation (MSCI) during pachytene, and Zfy2 is strongly reactivated later in spermatids. Recently, we have shown that mouse Zfy2, but not Zfy1, is involved in triggering the apoptotic elimination of specific types of sex chromosomally aberrant spermatocytes. In humans, there is a single widely transcribed ZFY gene, and there is no evidence for a specific role in the testis. Here, we characterize ZFY transcription during spermatogenesis in mice and humans. In mice, we define a variety of Zfy transcripts, among which is a Zfy2 transcript that predominates in spermatids, and a Zfy1 transcript, lacking an exon encoding approximately half of the acidic domain, which predominates prior to MSCI. In humans, we have identified a major testis-specific ZFY transcript that encodes a protein with the same short acidic domain. This represents the first evidence that ZFY has a conserved function during human spermatogenesis. We further show that, in contrast to the full acidic domain, the short domain does not activate transcription in yeast, and we hypothesize that this explains the functional difference observed between Zfy1 and Zfy2 during mouse meiosis.

Homozygous deletion of SUN5 in three men with decapitated spermatozoa

A recent study of 17 men with decapitated spermatozoa found that 8 carried two rare SUN5 alleles, and concluded that loss of SUN5 function causes the acephalic spermatozoa syndrome. Consistent with this, the SUN5 protein localises to the head-tail junction in normal spermatozoa, and SUN proteins are known to form links between the cytoskeleton and the nucleus. However, six of the ten SUN5 variants reported were missense with an unknown effect on function, and only one man carried two high confidence loss-of-function (LOF) alleles: p.Ser284* homozygozity. One potential exonic splice mutation, homozygous variant p.Gly114Arg, was not tested experimentally. Thus, definitive proof that loss of SUN5 function causes the acephalic spermatozoa syndrome is still lacking. Based on these findings, we determined the sequence of the SUN5 gene in three related men of North African origin with decapitated spermatozoa. We found all three men to be homozygous for a deletion-insertion variant (GRCh38 - chr20:32995761_32990672delinsTGGT) that removes 5090 base pairs including exon 8 of SUN5, predicting the frameshift, p.(Leu143Serfs*30), and the inactivation of SUN5. We therefore present the second case where the acephalic spermatozoa syndrome is associated with two LOF alleles of SUN5. We also show that the p.Gly114Arg variant has a strong inhibitory effect on splicing in HeLa cells, evidence that homozygozity for p.Gly114Arg causes acephalic spermatozoa syndrome through loss of SUN5 function. Our results, together with those of the previous study, show that SUN5 is required for the formation of the sperm head-tail junction and male fertility.

Mouse Y-Encoded Transcription Factor Zfy2 Is Essential for Sperm Head Remodelling and Sperm Tail Development.

A previous study indicated that genetic information encoded on the mouse Y chromosome short arm (Yp) is required for efficient completion of the second meiotic division (that generates haploid round spermatids), restructuring of the sperm head, and development of the sperm tail. Using mouse models lacking a Y chromosome but with varying Yp gene complements provided by Yp chromosomal derivatives or transgenes, we recently identified the Y-encoded zinc finger transcription factors Zfy1 and Zfy2 as the Yp genes promoting the second meiotic division. Using the same mouse models we here show that Zfy2 (but not Zfy1) contributes to the restructuring of the sperm head and is required for the development of the sperm tail. The preferential involvement of Zfy2 is consistent with the presence of an additional strong spermatid-specific promotor that has been acquired by this gene. This is further supported by the fact that promotion of sperm morphogenesis is also seen in one of the two markedly Yp gene deficient models in which a Yp deletion has created a Zfy2/1 fusion gene that is driven by the strong Zfy2 spermatid-specific promotor, but encodes a protein almost identical to that encoded by Zfy1. Our results point to there being further genetic information on Yp that also has a role in restructuring the sperm head.

Nuclear envelope remodelling during human spermiogenesis involves somatic B-type lamins and a spermatid specific B3 lamin isoform

The nuclear lamina (NL) is a filamentous protein meshwork, composed essentially of lamins, situated between the inner nuclear membrane and the chromatin. There is mounting evidence that the NL plays a role in spermatid differentiation during spermiogenesis. The mouse spermatid NL is composed of the ubiquitous lamin B1 and the spermatid-specific lamin B3, an N-terminally truncated isoform of lamin B2. However, nothing is known about the NL in human spermatids. We therefore investigated the expression pattern and localization of A-type lamins (A, C and C2) and B-type lamins (B1, B2 and B3) during human spermiogenesis. Here, we show that a lamin B3 transcript is present in human spermatids and that B-type lamins are the only lamins detectable in human spermatids. We determine that, as shown for their mouse counterparts, human lamin B3, but not lamin B2, induces strong nuclear deformation, when ectopically expressed in HeLa cells. Co-immunofluorescence revealed that, in human spermatids, B-type lamins are present at the nuclear periphery, except in the region covered by the acrosome, and that as the spermatid matures the B-type lamins recede towards the posterior pole. Only lamin B1 remains detectable on 33-47% of ejaculated spermatozoa. On spermatozoa selected for normal head density, however, this fell to <6%, suggesting that loss of the NL signal may be linked to complete sperm nucleus compaction. The similarities revealed between lamin expression during human and rodent spermiogenesis, strengthen evidence that the NL and lamin B3 have conserved functions during the intense remodelling of the mammalian spermatid nucleus.

Abnormal retention of nuclear lamina and disorganization of chromatin-related proteins in spermatozoa from DPY19L2-deleted globozoospermic patients

The aim of this study was to characterize the nuclear lamina (NL) and lamin chromatin-partners in spermatozoa from four DPY19L2-deleted globozoospermic patients. We tested for spermatid transcripts encoding lamins and their chromatin-partners emerin, LAP2α, BAF and BAF-L, by reverse transcriptase-PCR using spermatozoa RNA. We also determined the localization of lamin B1, BAF and BAF-L by immunofluorescent analysis of spermatozoa from all patients. In RNA from globozoospermic and control spermatozoa we detected transcripts encoding lamin B1, lamin B3, emerin, LAP2α and BAF-L, but not A-type lamins. In contrast, BAF transcripts were detected in globozoospermic but not control spermatozoa. The NL was immature in human globozoospermic spermatozoa: lamin B1 signal was detected in the nuclei of globozoospermic spermatozoa in significantly higher proportions than the control (P < 0.05; 56-91% versus 40%) and was predominantly observed at the whole nuclear periphery, not polarized as in control spermatozoa. Conversely, BAF and BAF-L were detected in control, but not globozoospermic spermatozoa. Our results strongly emphasize the importance of the NL and associated proteins during human spermiogenesis. In globozoospermia, the lack of maturation of the NL, and the modifications in expression and location of chromatin-partners, could explain the chromatin defects observed in this rare phenotype.