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.

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.

SPINK2 deficiency causes infertility by inducing sperm defects in heterozygotes and azoospermia in homozygotes.

Azoospermia, characterized by the absence of spermatozoa in the ejaculate, is a common cause of male infertility with a poorly characterized etiology. Exome sequencing analysis of two azoospermic brothers allowed the identification of a homozygous splice mutation in SPINK2, encoding a serine protease inhibitor believed to target acrosin, the main sperm acrosomal protease. In accord with these findings, we observed that homozygous Spink2 KO male mice had azoospermia. Moreover, despite normal fertility, heterozygous male mice had a high rate of morphologically abnormal spermatozoa and a reduced sperm motility. Further analysis demonstrated that in the absence of Spink2, protease-induced stress initiates Golgi fragmentation and prevents acrosome biogenesis leading to spermatid differentiation arrest. We also observed a deleterious effect of acrosin overexpression in HEK cells, effect that was alleviated by SPINK2 coexpression confirming its role as acrosin inhibitor. These results demonstrate that SPINK2 is necessary to neutralize proteases during their cellular transit toward the acrosome and that its deficiency induces a pathological continuum ranging from oligoasthenoteratozoospermia in heterozygotes to azoospermia in homozygotes.

Clinical data and parenthood of 63 infertile and Y-microdeleted men.

OBJECTIVE: To collect follow-up data for infertile men with Y microdeletion. DESIGN: Retrospective, observational survey. SETTING: Multicenter IVF units associated with genetics laboratories. PATIENT(S): Sixty-three patients with Y microdeletion. INTERVENTION(S): Karyotype analysis, Y microdeletion screening, and assisted reproductive technology. MAIN OUTCOME MEASURES: Medical history, karyotype, nature of the AZF deletion, semen parameters, testis biopsy results, choice of assisted reproductive technology, and results of intracytoplasmic sperm injection (ICSI). RESULTS: Abnormal karyotypes were found in 8 men (12.7%), who were azoospermic except 1. Of these 8 men, 5 presented a combined AZFb+c deletion, and 3 had a deletion in AZFc only. Most men (39 of 63) were azoospermic, 3 were cryptoazoospermic, and 19 had extreme oligozoospermia (sperm concentration

Lack of association between genetic polymorphisms in enzymes associated with folate metabolism and unexplained reduced sperm counts.

BACKGROUND: The metabolic pathway of folate is thought to influence DNA stability either by inducing single/double stranded breaks or by producing low levels of S-adenosyl-methionine leading to abnormal gene expression and chromosome segregation. Polymorphisms in the genes encoding enzymes in the folate metabolism pathway show distinct geographic and/or ethnic variations and in some cases have been linked to disease. Notably, the gene Methylenetetrahydrofolate reductase (MTHFR) in which the homozygous (TT) state of the polymorphism c.665C>T (p.A222V) is associated with reduced specific activity and increased thermolability of the enzyme causing mild hyperhomocysteinemia. Recently several studies have suggested that men carrying this polymorphism may be at increased risk to develop infertility. METHODOLOGY/PRINCIPAL FINDINGS: We have tested this hypothesis in a case/control study of ethnic French individuals. We examined the incidence of polymorphisms in the genes MTHFR (R68Q, A222V and E429A), Methionine synthase reductase MTRR; (I22M and S175L) and Cystathionine beta-synthase (CBS; G307S). The case population consisted of DNA samples from men with unexplained azoospermia (n = 70) or oligozoospermia (n = 182) and the control population consisted of normospermic and fertile men (n = 114). We found no evidence of an association between the incidence of any of these variants and reduced sperm counts. In addition haplotype analysis did not reveal differences between the case and control populations. CONCLUSIONS/SIGNIFICANCE: We could find no evidence for an association between reduced sperm counts and polymorphisms in enzymes involved in folate metabolism in the French population.

Preliminary study on the role of the human IZUMO gene in oocyte-spermatozoa fusion failure.

We studied the IZUMO gene 9 coding exons sequence in four groups of patients including those with fertilization failure by conventional IVF. We observed in our populations two combinations of four polymorphisms that appeared to be preferentially linked (CGG-CG and TAA-TT) without any significant difference between different genotype repartitions.

Testicular sperm extraction in a single cancerous testicle in patients with azoospermia: a case report.

OBJECTIVE: To optimize the management of patients with cancer in a single testis and azoospermia. DESIGN: Case report. SETTING: Reproductive biology department. PATIENT(S): Two male patients referred in an emergency context for gamete cryopreservation before castration for cancer in a single testis; the patients were azoospermic before any sterilizing treatment. INTERVENTION(S): Testicular sperm extraction (TESE) was carried out synchronously with orchiectomy, allowing the cryopreservation of testicular spermatozoa. MAIN OUTCOME MEASURE(S): One year after the end of the cancer treatment, thawed testicular spermatozoa were successfully used in an intracytoplasmic sperm injection procedure. A twin pregnancy was obtained, and a healthy boy and girl were born. CONCLUSION(S): TESE and cryopreservation could be offered to every patient with cancer in a single testis and azoospermia.

Two fast methods for detection of Y-microdeletions.

OBJECTIVE: To test two recently available commercial kits: the new Promega Y Chromosome Deletion Detection System 2.0 kit and the Bird-Set kits (Y Chromosome UE and Extension). DESIGN: A comparative technical study. SETTING: Male infertility clinic. PATIENT(S): Twelve various Y chromosome microdeleted patients were included in the present study: two AZFa deleted, two AZFb deleted, four AZFb+c deleted, three AZFc deleted, and one AZF a+b+c deleted. INTERVENTION(S): DNA samples were tested with both the Promega kit and the Bird-Set kits. MAIN OUTCOME MEASURE(S): Electrophoresis and analysis comparison on a bioanalyzer. RESULT(S): Both kits (Promega 2.0 and Bird Y Chromosome UE) confirmed the 12 Y deletions. Both kits (Bird Extension and Promega 2.0) were able to determine the length of the 12 microdeletions with various differences. The new Promega 2.0 kit was considerably improved, with the addition of two sequenced tag sites (STS) in AZFa (sY86 and sY84) and the deletion of some (sY239 and sY153) but not all supernumerary inadequate STS. CONCLUSION(S): The two new commercially available kits use two different protocols to detect Y chromosome microdeletions. The Promega kit is a one-step diagnostic test. The Bird diagnostic test uses a two-step protocol. Both kits offer relevant methods such as standardization and ready-to-use mixes. However, both kits need further evaluation under routine conditions using nontested DNA samples.

Y chromosome microdeletion screening in infertile men in France: a survey of French practice based on 88 IVF centres.

Y chromosome microdeletion screening is advised in cases of severely impaired spermatogenesis. Improvements in molecular biological techniques have made diagnosis more accessible in routine analysis. However, Y chromosome microdeletions are not diagnosed in all IVF centres. The aim of the present study was to determine the regulatory (indications, financing) and performance (methods, invoicing) conditions required for this analysis, in France. Microdeletion detection was found to be spreading fast and consistantly. It therefore seems necessary for a consensus to be reached on indications, with a view to a standardized technique, with a common effort of experts in the field. Financial management by the French Health Insurance bodies (Sécurité Sociale) would be an essential step towards routine adoption. Lastly, the answers to our questionnaire revealed a strong demand for information concerning this analysis.