A new deletion of the mouse Y chromosome long arm associated with the loss of Ssty expression, abnormal sperm development and sterility

The mouse Y chromosome carries 10 distinct genes or gene families that have open reading frames suggestive of retained functionality; it has been assumed that many of these function in spermatogenesis. However, we have recently shown that only two Y genes, the testis determinant Sry and the translation initiation factor Eif2s3y, are essential for spermatogenesis to proceed to the round spermatid stage. Thus, any further substantive mouse Y-gene functions in spermatogenesis are likely to be during sperm differentiation. The complex Ssty gene family present on the mouse Y long arm (Yq) has been implicated in sperm development, with partial Yq deletions that reduce Ssty expression resulting in impaired fertilization efficiency. Here we report the identification of a more extensive Yq deletion that abolishes Ssty expression and results in severe sperm defects and sterility. This result establishes that genetic information (Ssty?) essential for normal sperm differentiation and function is present on mouse Yq.     

The influence of the deletion on the long arm of the Y chromosome on sperm motility in mice

The multicopy region on the long arm of the mouse Y chromosome contains four known genes. There are evidences that deletions in this region lead to decrease of sperm quality in mutant mice. Male mice completely lacking this region are infertile. Here we report results obtained by using the computer assisted semen analysis system (CASA), describing the movement parameters of spermatozoa from mutant males with partial deletion on the long arm of the Y chromosome (B10. BR-Y(del)). First we have determined that genes necessary for spermiogenesis and located in this region are still active in mutants, than we have compared the sperm movement of mutants and control animals. This analysis revealed that the Yq deletion affects: velocity parameters (VAP, VCL, VSL), parameters describing sperm head activity during movement (ALH and BCF) and linearity (LIN) of movement. Our findings indicate that sperm movement is controlled by genes located in the long arm of the Y chromosome.     

A protein encoded by a member of the multicopy Ssty gene family located on the long arm of the mouse Y chromosome is expressed during sperm development

Multicopy Y-chromosomal genes in human and mouse have been postulated to play a role in spermatogenesis. The mouse Y long arm (Yq) carries hundreds of supposedly intronless copies of Ssty, for which no protein has hitherto been identified; mice lacking Yq are sterile with grossly abnormal sperm. We have now identified an Ssty-encoded protein (Ssty1) that is expressed in spermatids. The protein is absent from spermatids of mice that lack Yq, but is not reduced in mice with a two-thirds reduction of Ssty copies, implying that most do not produce this protein. Furthermore, no protein was produced by a strongly transcribed intronless Ssty transgene, raising doubts as to the protein-encoding potential of these intronless genes. We have now identified an intron-containing copy that is also present in multiple copies on Yq. One or more intron-containing copies are retained in the Ssty-deficient mice and may be the source of the Ssty1 protein.     

The relationship between Y chromosome DNA haplotypes and Y chromosome deletions leading to male infertility

Microdeletions on the short arm of the Y chromosome have defined three non-overlapping regions (AZFa, b, c) recurrently deleted among infertile males. These regions contain several genes or gene families involved in male germ-cell development and maintenance. Even though a meiotic origin for these microdeletions is assumed, the mechanisms and causes leading to microdeletion formation are largely unknown. In order to assess whether some Y chromosome groups (or haplogroups) are predisposed to, or protected against, deletion formation during male meiosis, we have defined and compared Y chromosome haplogroup distribution in a group of infertile/subfertile males harbouring Yq deletions and in a relevant Northwestern European control population. Our analyses suggest that Y chromosome deletion formation is, at least in the study populations, a stochastic event independent of the Y chromosome background on which they arise and may be caused by other genetic and/or environmental factors.     

Deficiency in mouse Y chromosome long arm gene complement is associated with sperm DNA damage

Background  

Mice with severe non-PAR Y chromosome long arm (NPYq) deficiencies are infertile in vivo and in vitro. We have previously shown that sperm from these males, although having grossly malformed heads, were able to fertilize oocytes via intracytoplasmic sperm injection (ICSI) and yield live offspring. However, in continuing ICSI trials we noted a reduced efficiency when cryopreserved sperm were used and with epididymal sperm as compared to testicular sperm. In the present study we tested if NPYq deficiency is associated with sperm DNA damage - a known cause of poor ICSI success.     

Types of Y chromosome deletions and their frequency in infertile men

Deletions of Y chromosome AZF locus were analyzed during a large-scale andrological and genetic examination of 810 infertile men. The search for Yq microdeletions was carried out according to the standard EAA/EMQN guidelines. The breakpoints were mapped for the deletions in AZF locus. The Y chromosome macro- and microdeletions were detected in 61 (7.5%) infertile men. The frequencies of AZF deletions during azoospermia and severe oligozoospermia amounted to 12.2 and 8.1 %, respectively. On the whole, the frequencies of Yq microdeletions and the genophenotypic correlations characteristic of various AZF deletion types comply with the relevant published data. However, spermatozoids in the ejaculate sediment of men with completely deleted AZFa region or AZFb+c deletions (from solitary spermatozoids to several dozens) were detected for the first time. It was demonstrated that the breakpoints were localized between AZFa and AZFb regions proximally to AZFb+c microdeletions for the majority of cytogenetically detectable deletions in the Y chromosome long arm. This indicates that the mechanisms underlying Yq macro- and microdeletions are somewhat different. The issues related to the role of Y chromosome deletions in the origins of monosomy for X chromosome and X/XY mosaicism are discussed.     

Types of Y chromosome deletions and their frequency in infertile men

Y chromosome deletions in the AZF locus were analyzed during a large-scale andrological and genetic examination of 810 infertile men. The search for Yq microdeletions was carried out according to the standard EAA/EMQN guidelines. The breakpoints were mapped for the revealed AZF deletions. The Y chromosome macro-and microdeletions were detected in 61 (7.5%) infertile men. The frequencies of AZF deletions in patients with azoospermia and severe oligozoospermia amounted to 12.2 and 8.1%, respectively. On the whole, the frequencies of Yq microdeletions and the genophenotypic correlations characteristic of various AZF deletion types agree with the relevant published data. However, spermatozoa in the ejaculate sediment of men with completely deleted AZFa region or AZFb+c deletions (from solitary spermatozoa to several dozens) were detected for the first time. It was demonstrated that the breakpoints were localized between AZFa and AZFb regions proximally to AZFb+c microdeletions for the majority of cytogenetically detectable deletions of the Y chromosome long arm. This indicates that the mechanisms underlying Yq macro-and microdeletions are somewhat different. The issues related to the role of Y chromosome deletions in the origins of X chromosome monosomy and X/XY mosaicism are discussed.     

Bidirectional transcription of a novel chimeric gene mapping to mouse chromosome Yq

Background  

The male-specific region of the mouse Y chromosome long arm (MSYq) contains three known highly multi-copy X-Y homologous gene families, Ssty1/2, Sly and Asty. Deletions on MSYq lead to teratozoospermia and subfertility or infertility, with a sex ratio skew in the offspring of subfertile MSYqdel males     

Cytogenetic and molecular analysis of male infertility

Reduced male fertility and subfertility can be caused by genetic factors that affect both germ cell development, differentiation, and function; in particular, chromosome abnormalities and Yq microdeletions are a possible cause of spermatogenetic impairment in males as shown by their higher frequency in infertile men than in the general male population. Microdeletion of the long arm of the Y chromosome (Yq) are associated with spermatogenic failure and have been used to define three regions on Yq (AZFa, AZFb, and AZFc) that are critical for germ cell development. With the advent of assisted reproductive technology and intracytoplasmic sperm injection, knowledge about the various factors leading to spermatogenic impairment is one of the most important aspects of scientific research. Therefore, this study was designed to identify the frequency of cytogenetic and submicroscopic interstitial deletions in azoospermia factor loci in infertile Indian males. One hundred and eighty males with nonobstructive oligozoospermia and azoospermia were included in this study. Semen analysis was done in each case to determine the spermatogenic status. Individuals were subjected to detailed clinical examination, family history, and endocrinological and cytogenetic study after consent from the patient. Peripheral blood cultures were set up according to standard protocols and 30 G-banded metaphases were analyzed in each case. Numerical and structural chromosomal abnormalities were detected in 40 infertile cases. Fluorescence in situ hybridization analysis was done in some cases to identify the percentage of mosaic cell lines and any cryptic or low-level mosaicism. Polymerase chain reaction microdeletion analysis was done in 140 cytogenetically normal cases. Of the 140 cases, 8 showed deletion of at least one of the sequence-tagged site markers. Review of literature has shown that the overall frequency of microdeletions varies from 1 to 55%. In the present study, the frequency of microdeletion was 5.8%, and deletions were identified in cases with undescended testis and varicocele and cases with bilateral severe testiculopathy.     

Chromosome abnormalities in sperm of individuals with constitutional sex chromosomal abnormalities

The most common type of karyotype abnormality detected in infertile subjects is represented by Klinefelter's syndrome, and the most frequent non-chromosomal alteration is represented by Y chromosome long arm microdeletions. Here we report our experience and a review of the literature on sperm sex chromosome aneuploidies in these two conditions. Non mosaic 47,XXY Klinefelter patients (12 subjects) show a significantly lower percentage of normal Y-bearing sperm and slightly higher percentage of normal X-bearing sperm. Consistent with the hypothesis that 47,XXY germ cells may undergo and complete meiosis, aneuploidy rate for XX- and XY-disomies is also increased with respect to controls, whereas the percentage of YY-disomies is normal. Aneuploidy rates in men with mosaic 47,XXY/46,XY (11 subjects) are lower than those observed in men with non-mosaic Klinefelter's syndrome, and only the frequency of XY-disomic sperm is significantly higher with respect to controls. Although the great majority of children born by intracytoplasmic sperm injection from Klinefelter subjects are chromosomally normal, the risk of producing offspring with chromosome aneuploidies is significant. Men with Y chromosome microdeletions (14 subjects) showed a reduction of normal Y-bearing sperm, and an increase in nullisomic and XY-disomic sperm, suggesting an instability of the deleted Y chromosome causing its loss in germ cells, and meiotic alterations leading to XY non-disjunction. Intracytoplasmic injection of sperm from Y-deleted men will therefore transmit the deletion to male children, and therefore the spermatogenic impairment, but raises also concerns of generating 45,X and 47,XXY embryos.     

Offspring derived from intracytoplasmic injection of transgenic rat sperm

The objective of the present study was to produce rat offspring by intracytoplasmic sperm injection (ICSI) using a Piezo-driven micromanipulator. Transgenic male rats carrying a green fluorescent protein gene (GFP: homozygous) were used as sperm donors. The epididymal spermatozoa were suspended and sonicated in m-KRB medium and were frozen in the same medium at –20°C until use. When the sperm heads were aspirated into injection pipettes 7–10m in diameter and introduced into oocytes from the Wistar strain, no offspring resulted from the transfer of 59 eggs. In contrast, the sperm heads were hung on the tip of injection pipettes 2–4m in diameter and introduced into the oocytes, use of Piezo resulting in the production of 18 transgenic offspring carrying the GFP gene from 181 eggs transferred. The oocytes from the Sprague–Dawley strain also supported full-term development following ICSI with three offspring resulting from 163 transferred eggs. In an additional ICSI trial, spermatozoa from infertile transgenic rats carrying human lactalbumin with the thymidine kinase gene (LAC3: heterozygous) were used. The spermatozoa of the LAC3 transgenic rats appeared to be defective and immotile because of the expression of thymidine kinase in the testes, and no ICSI offspring resulted from 218 transferred eggs. These results suggest that ICSI is applicable in rats when Piezo-driven smaller pipettes are used to inject sperm heads together with a limited amount of the surrounding medium and that the ability of isolated sperm heads to participate in normal embryo development is maintained under the cryopreservation conditions employed.     

Chromosome abnormalities in sperm from infertile men with normal somatic karyotypes: oligozoospermia

Recently, intracytoplasmic sperm injection (ICSI) has been extremely successful for the treatment of male infertility. However, transmission of cytogenetic defects to offspring is a great concern. There are two types of cytogenetic problems in patients seeking ICSI; one is the transmission of genetic defects from patients with constitutional chromosomal abnormalities and the second is the generation of de novo defects in infertile men. Generally about 5.1% of infertile men have chromosomal abnormalities. Among such infertile men, men with severe spermatogenesis defects, including oligozoospermia and azoospermia, are subjects for ICSI. Therefore it is very important to obtain cytogenetic information in these infertile patients. Furthermore, oligozoospermic men with a normal somatic karyotype also have increased frequencies of sperm chromosome abnormalities. Oligozoospermia is usually associated with other sperm alterations, for example oligoasthenozoospermia, oligoteratozoospemia and oligoasthenoteratozoospermia. In this review, the relationship between sperm concentration and sperm aneuploidy frequencies has been analyzed. The inverse correlation between the frequency of sperm aneuploidy and concentration has been reported in extensive studies. Especially in severe oligozoospermia, a significantly higher frequency of sex chromosome aneuploidy has been observed and this has been corroborated in recent clinical outcome data of ICSI.     

Chromosomal aberrations, Yq microdeletion, and sperm DNA fragmentation in infertile men opting for assisted reproduction

Male infertility is a multi‐factorial disorder, and identification of its etiology in an individual is critical for treatment. Systematically elucidating the underlying genetic causes (chromosomal and Yq microdeletion) and factors, such as reactive oxygen species (ROS) levels and total antioxidant capacity (TAC), which contribute to sperm DNA damage, may help to reduce the number of men with idiopathic infertility and provide them with the most suitable therapeutics and counseling. This study was done to comprehensively investigate genetic and oxidative stress factors that might be the etiology of a large percentage of men with idiopathic infertility. One hundred twelve infertile men and 76 fertile controls were screened for chromosomal aberrations and Yq microdeletions. ROS, TAC, and sperm DNA damage were assessed in cytogenetically normal, non‐azoospermic men with intact Y chromosome (n = 93). ROS was assessed in neat and washed semen by chemiluminescence; seminal TAC with a commercially available kit; and sperm DNA damage by the comet assay. Two men had cytogenetic abnormalities and seven men harbored Yq microdeletions. ROS levels in neat and washed semen of infertile men were significantly higher (P < 0.01) than controls. Infertile men had significantly lower (P < 0.01) TAC levels (1.79 mM), whereas sperm DNA fragmentation in infertile men was significantly higher (P < 0.01) than controls. Genetic factors and oxidative stress cumulatively account for large number of idiopathic infertile cases. Unlike, genetic causes, which cannot be cured, timely identification and management of oxidative stress may help to reverse/reduce the effects on induced DNA damage, and improve the outcomes for infertile males. Mol. Reprod. Dev. 79: 637–650, 2012. © 2012 Wiley Periodicals, Inc.     

Sperm analysis in a subfertile male with a Y;16 translocation, using four-color FISH.

Sperm analysis was performed in a male with oligoasthenoteratozoospermia (OAT) and a reciprocal t(Y;16) (q11. 21;q24), using four-color FISH. Intracytoplasmic sperm injection (ICSI) treatment in this patient had resulted in the birth of one chromosomally balanced and two chromosomally normal children. To assess the risk of having a chromosomally unbalanced conception after ICSI, morphologically normal spermatozoa were studied with a set of probes allowing detection of all segregation variants. There were 51% normal or balanced sperm cells. The fraction of sperm products resulting from alternate and adjacent I segregation was 87%, 12% were products of 3:1 disjunction, and the other 1% had other types of aneuploidy. If morphologically abnormal cells were also included in the FISH analysis, nearly 90% of all the spermatozoa were unbalanced. We conclude that although the majority of males with a Y/autosome translocation are infertile due to azoospermia, our patient produces sufficient morphologically and chromosomally normal spermatozoa to have chromosomally normal or balanced offspring after ICSI. Assuming that ICSI with an unbalanced spermatozoon from this patient would result in a nonviable embryo in many cases, the combination of in vitro and subsequent in vivo selection probably results in a risk of unbalanced offspring of much less than 50%. Hence, FISH studies on the sperm of translocation carriers are useful for estimating the risk of having unbalanced offspring after ICSI and in understanding the mechanisms underlying infertility in such carriers.     

AZF microdeletions on the Y chromosome of infertile men from Turkey

Intervals V and VI of Yq11.23 regions contain responsible genes for spermatogenesis, and are named as "azoospermia factor locus" (AZF). Deletions in these genes are thought to be pathogenetically involved in some cases of male infertility associated with azoospermia or oligozoospermia. The aim of this study was to establish the prevalence of microdeletions on the Y chromosome in infertile Turkish males with azoospermia or oligozoospermia. We applied multiplex polymerase chain reaction (PCR) using several sequence-tagged site (STS) primer sets, in order to determine Y chromosome microdeletions. In this study, 61 infertile males were enrolled for the molecular AZF screening program. In this cohort, one infertile male had 46,XX karyotype and the remaining had 46,XY karyotypes. Forty-eight patients had a diagnosis of azoospermia and 13 had oligozoospermia. Microdeletions in AZFa, AZFb and AZFc (DAZ gene) regions were detected in two of the 60 (3.3%) idiopathic infertile males with normal karyotypes and a SRY translocation was determined on 46,XX male. Our findings suggest that genetic screening should be advised to infertile men before starting assisted reproductive treatments.     

Does Rbmy have a role in sperm development in mice?

The Y(d1) deletion in mice removes most of the multi-copy Rbmy gene cluster that is located adjacent to the centromere on the Y short arm (Yp). XY(d1) mice develop as females because Sry is inactivated, probably because it is now juxtaposed to centromeric heterochromatin. We have previously produced XY(d1)Sry transgenic males and found that they have a substantially increased frequency of abnormal sperm. Staining of testis sections with a polyclonal anti-RBMY antibody appeared to show a marked decrease of RBMY protein in the spermatids of XY(d1)Sry males compared to control males, which led us to suggest that this may be responsible for the increase in sperm anomalies. In the current study we sought to determine whether augmenting Rbmy expression specifically in the spermatids of XY(d1)Sry males would ameliorate the sperm defects. An expressing Rbmy transgene driven by the spermatid-specific mouse protamine 1 promotor (mP1Rbmy) was therefore introduced into XY(d1)Sry males. This failed to reduce the frequency of abnormal sperm. In the course of this study, a new RBMY antibody was generated that, in contrast to the original antibody, failed to detect RBMY in spermatid stages by immunostaining. The lack of RBMY was confirmed by western blotting of lysates from purified round spermatids and elongating spermatids. The implications of these results for the proposed role for RBMY in sperm development are discussed.     

严重寡精症ICSI精子供体的DAZ基因拷贝缺失研究

DAZ基因拷贝缺失与人类的生精障碍有关。为了解中国正常生精男性和ICSI中严重寡精症精子供体DAZ基因拷贝缺失的分布, 探讨DAZ基因拷贝数检测在严重生精障碍精子供体遗传缺陷筛查中的意义, 本研究运用多重PCR和PCR-RFLP技术, 对128例严重寡精症ICSI精子供体和287个正常生精男性的DAZ基因缺失进行了研究。发现DAZ1/DAZ2、DAZ3/DAZ4和全部4个拷贝缺失等3种拷贝缺失类型, 其中全部4个拷贝缺失仅见于严重寡精症患者, 频率为11.7%; DAZ1/DAZ2缺失的频率在严重寡精症患者中显著高于正常男性(9.4% vs 2.8%, P = 0.004); 在严重寡精症患者中DAZ基因拷贝完全缺失与DAZ1/DAZ2缺失的总发生率为21.1%。DAZ3/DAZ4缺失的频率在两组人群中无显著差异(7.0% vs 3.8%, P > 0.05)。这些结果提示, DAZ基因全部拷贝缺失是严重寡精症患者生精障碍的常见遗传病因, 而DAZ1/DAZ2缺失则可能是一种高风险因素。鉴于上述DAZ基因缺失在严重生精障碍精子供体中较高的发生率, 在应用ICSI进行辅助生育前, 建议对严重寡精症的精子供体进行DAZ基因全缺失与DAZ1/DAZ2共缺失筛查, 以评估其男性后代患病的风险。     

A successful second delivery outcome using refrozen thawed testicular sperm from an infertile male true hermaphrodite with a 46, XX/46, XY karyotype: case report

We report a successful second delivery of a healthy infant fathered using refrozen thawed testicular sperm from an infertile male chimera. We also examined sex chromosome distribution of the seminiferous tubule. Intracytoplasmic sperm injection (ICSI) was performed using the remaining refrozen testicular sperm, which had been stored during the first treatment. Biopsied testicular cells were examined by fluorescence in situ hybridization (FISH) and the peripheral lymphocyte karyotype was tested using a G-band. Following ICSI, a second pregnancy was established, and a healthy girl was successfully delivered at 40 gestational weeks without complications. Although the husband’s lymphocyte chromosomal analysis revealed a 46, XX [28]/46, XY [2] karyotype, the seminiferous tubule cells on histological examination by FISH were chimeric sex chromosome type XX [18]/XY [82]. In conclusion, this is a very rare case report of a successful subsequent delivery of a healthy infant (46, XX) from an infertile true hermaphrodite (46, XX/46, XY) using refrozen thawed testicular sperm. The seminiferous tubule cells’ karyotype ratio differed from that of the lymphocytes.     

Intracytoplasmic sperm injection effects in infertile azh mutant mice

Several reports in the literature describe men with infertility resulting from abnormal sperm head shape or decapitation defects of their spermatozoa. These defects are similar to those shown for the spermatozoa from azh (abnormal spermatozoon head shape) mice. The present study examines the efficiency and effects of intracytoplasmic sperm injection (ICSI) in successive generations of azh mice generated with this method. Three successive generations of azh mice were produced with ICSI. In all three ICSI series, more than 80% of 2-cell embryos were obtained, and more than 35% of embryos transferred gave rise to normal live offspring. In addition, ICSI was used to cross homozygous azh/azh males with homozygous azh/azh females, and live offspring were obtained. The ICSI-derived males were tested for their fecundity and abnormalities of sperm morphology. Spermatozoa from ICSI-derived azh/+ males did not show any impairment of fecundity in in vitro fertilization. These spermatozoa successfully fertilized oocytes from both C57BL/6 and B6D2F1 females, with fertilization rates ranging from 70%- 92%. The proportion of morphologically normal spermatozoa was similar in azh/+ males from three successive generations of ICSI (57.8%, 54.8%, and 49.0%, respectively), and no differences were noted when comparing ICSI-derived males with males derived by mating (57.6%) and with wild-type controls (61.6%). Detailed analysis differentiating between specific types of anomalies of sperm morphology did not reveal significant differences among the examined groups. The results of the present study demonstrate that ICSI does not enhance the azh mutation phenotype in the offspring and brings no risks when applied continuously. Moreover, serial (successive generations) ICSI is highly efficient in maintaining valuable mice with fertility problems.     

Use of mouse oocytes to evaluate the ability of human sperm to activate oocytes after failure of activation by intracytoplasmic sperm injection

The objective of the present study was to investigate the nuclei of human sperms that failed to fertilize human oocytes after intracytoplasmic sperm injection (ICSI). The sperms were injected into mouse oocytes by a piezo-micromanipulator, and some of these oocytes were artificially activated with strontium chloride (SrCl2) after ICSI. The oocytes were fixed, stained, and subjected to chromosomal analysis. The survival rate of mouse oocytes injected with infertile human sperms was 92.0% (46/50), while that of the control mouse oocytes injected with fertile human sperms was 73.6% (81/110). The rate of two pronuclei (2PN) formation was 0 (0/46) by the infertile sperms and 81.5% (66/81) by the fertile ones, a significant difference (p < 0.01). Sperm chromosomes in non-activated oocytes were present as premature chromosome condensation (PCC). Artificial activation after ICSI increased the 2PN formation rate in the infertile group to 90.3% (28/31). The results of the present study suggest that infertile sperms have a low potential to spontaneously activate oocytes and to form pronuclei. Thus, artificial activation after ICSI may rescue oocytes fertilized with infertile human sperms that do not produce 2PN. The present study proved the usefulness of mouse oocytes as specimens in evaluating the oocyte-activating capacity of objective human sperms prior to ICSI treatment.