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Heat-shock protein 110 (HSP110) family members act as nucleotide exchange factors (NEF) of mammalian and yeast HSP70 chaperones during the ATP hydrolysis cycle. In this study, we describe the expression pattern of murine HSPA4, a member of the HSP110 family, during testis development and the consequence of HSPA4 deficiency on male fertility. HSPA4 is ubiquitously expressed in all the examined tissues. During prenatal and postnatal development of gonad, HSPA4 is expressed in both somatic and germ cells; however, expression was much higher in germ cells of prenatal gonads. Analyses of Hspa4-deficient mice revealed that all homozygous mice on the hybrid C57BL/6J×129/Sv genetic background were apparently healthy. Although HSPA4 is expressed as early as E13.5 in male gonad, a lack of histological differences between Hspa4(-/-) and control littermates suggests that Hspa4 deficiency does not impair the gonocytes or their development to spermatogonia. Remarkably, an increased number of the Hspa4-deficient males displayed impaired fertility, whereas females were fertile. The total number of spermatozoa and their motility were drastically reduced in infertile Hspa4-deficient mice compared with wild-type littermates. The majority of pachytene spermatocytes in the juvenile Hspa4(-/-) mice failed to complete the first meiotic prophase and became apoptotic. Furthermore, down-regulation of transcription levels of genes known to be expressed in spermatocytes at late stages of prophase I and post-meiotic spermatids leads to suggest that the development of most spermatogenic cells is arrested at late stages of meiotic prophase I. These results provide evidence that HSPA4 is required for normal spermatogenesis.

Background: Multipotent adult germ-line stem cells (maGSCs) represent a new pluripotent cell type that can be derived without genetic manipulation from spermatogonial stem cells (SSCs) present in adult testis. Similarly to induced pluripotent stem cells (iPSCs), they could provide a source of cellular grafts for new transplantation therapies of a broad variety of diseases. To test whether these stem cells can be rejected by the recipients, we have analyzed whether maGSCs and iPSCs can become targets for cytotoxic T lymphocytes (CTL) or whether they are protected, as previously proposed for embryonic stem cells (ESCs)., Results: We have observed that maGSCs can be maintained in prolonged culture with or without leukemia inhibitory factor and/or feeder cells and still retain the capacity to form teratomas in immunodeficient recipients. They were, however, rejected in immunocompetent allogeneic recipients, and the immune response controlled teratoma growth. We analyzed the susceptibility of three maGSC lines to CTL in comparison to ESCs, iPSCs, and F9 teratocarcinoma cells. Major histocompatibility complex (MHC) class I molecules were not detectable by flow cytometry on these stem cell lines, apart from low levels on one maGSC line (maGSC Stra8 SSC5). However, using a quantitative real time PCR analysis H2K and B2m transcripts were detected in all pluripotent stem cell lines. All pluripotent stem cell lines were killed in a peptide-dependent manner by activated CTLs derived from T cell receptor transgenic OT-I mice after pulsing of the targets with the SIINFEKL peptide., Conclusion: Pluripotent stem cells, including maGSCs, ESCs, and iPSCs can become targets for CTLs, even if the expression level of MHC class I molecules is below the detection limit of flow cytometry. Thus they are not protected against CTL-mediated cytotoxicity. Therefore, pluripotent cells might be rejected after transplantation by this mechanism if specific antigens are presented and if specific activated CTLs are present. Our results show that the adaptive immune system has in principle the capacity to kill pluripotent and teratoma forming stem cells. This finding might help to develop new strategies to increase the safety of future transplantations of in vitro differentiated cells by exploiting a selective immune response against contaminating undifferentiated cells., Reviewers: This article was reviewed by Bhagirath Singh, Etienne Joly and Lutz Walter.

The primary goal of this study was to determine the 5'region of the Insl3 gene that specifically targets the expression of human insulin to Leydig cells, and to explore whether the testicular proinsulin is efficiently processed to insulin that is able to rescue the diabetes in different mouse models of diabetes. We show here that the sequence between nucleotides -690 and +4 of mouse Insl3 promoter is sufficient to direct the Leydig cell-specific expression of the human insulin transgene (Insl3-hIns). We also found that the 3'untranslated region (3'UTR) of Insl3 was effective in enhancing transgene expression of the insulin in vivo. Expression analysis revealed that the temporal expression pattern of the hIns transgene in Leydig cells of transgenic testes is roughly the same as that of the endogenous Insl3. Despite the Leydig cells translate human proinsulin and secrete a significant level of free C-peptide into the serum, the Leydig cell-derived insulin is not able to overcome the diabetes in different mouse models of diabetes, suggesting a lack of glucose sensing mechanisms in the Leydig cells. A consequence of overexpression of the human proinsulin in Leydig cells was the decrease of fertility of transgenic males at older ages. Germ cells in transgenic males were able to initiate and complete spermatogenesis. However, there was a progressive and age-dependent degeneration of the germ cells that lead to male infertility with increasing age.

During mammalian spermiogenesis somatic histones are replaced at first by transition proteins, which are in turn replaced by the protamines, forming the sperm nucleoprotamines. It is believed that transition protein 2 (Tnp2) is necessary for maintaining the normal processing of protamines and, consequently, the completion of chromatin condensation. The transition protein mRNAs are stored in translationally inert messenger ribonucleoprotein particles for up to 7 days until translational activation in elongated spermatids. Substantial evidence suggests an involvement of 3'untranslated region (UTR) in the translational regulation of the Tnp2 mRNAs. In order to determine the role of Tnp2 3'UTR in translational regulation and to study whether the translational repression of Tnp2 mRNA is necessary for normal spermatid differentiation in mice, we generated transgenic mice that carry a Tnp2-hGH transgene. In this transgene, 3'UTR of Tnp2 gene was replaced by 3' 3'UTR of human growth hormone gene. In these transgenic animals, transcription and translation of Tnp2 occur simultaneously in round spermatids which is an evidence for involvement of Tnp2 3'UTR in its translation repression. Premature translation of Tnp2 mRNA caused abnormal head morphogenesis, reduced sperm motility and male infertility. These results show clearly that a strict temporal and stage-specific Tnp2 translation is necessary for the correct differentiation of round spermatids into mature spermatozoa and for male fertility., ((c) 2006 Wiley-Liss, Inc.)

Testis expressed gene 18 (Tex18) is a small gene with one exon of 240 bp, which is specifically expressed in male germ cells. The gene encodes for a protein of 80 amino acids with unknown domain. To investigate the function of (Tex18) gene, we generated mice with targeted disruption of the (Tex18) gene by homologous recombination. Homozygous mutant males on a mixed genetic background (C57BL/6J x 129/Sv) are fertile, while they are subfertile on the 129/Sv background, although mating is normal. We showed that Tex18(-/-) males are subfertile because of abnormal sperm morphology and reduced motility, which is called asthenoteratozoospermia, suggesting that (Tex18) affects sperm characteristics. Maturation of spermatids is unsynchronized and partially impaired in seminiferous tubules of Tex18(-/-) mice. Electron microscopical examination demonstrated abnormal structures of sperm head. In vivo experiments with sperm of Tex18(-/-) 129/Sv mice revealed that the migration of spermatozoa from the uterus into the oviduct is reduced. This result is supported by the observation that sperm motility, as determined by the computer-assisted semen analysis system, is significantly affected, compared to wild-type spermatozoa. Generation of transgenic mice containing Tex18-EGFP fusion construct revealed a high transcriptional activity of (Tex18) during spermiogenesis, a process with morphological changes of haploid germ cells and development to mature spermatozoa. These results indicate that (Tex18) is expressed predominantly during spermatid differentiation and subfertility of the male Tex18(-/-) mice on the 129/Sv background is due to the differentiation arrest, abnormal sperm morphology and reduced sperm motility.

Germ cells must develop along distinct male or female paths to produce the spermatozoa or oocyte required for sexual reproduction. Male germline stem cells maintain spermatogenesis in the postnatal human testis. Here we show that a small population of bone marrow cells is able to transdifferentiate to male germ cell-like cells. We show expression of early germ cell markers (Oct4, Fragilis, Stella and Vasa) and male germ cell specific markers (Dazl, TSPY, Piwil2 and Stra8) in these cells. Our preliminary findings provide direct evidence that human bone marrow cells can differentiate to putative male germ cells and identify bone marrow as a potential source of male germ cells that could sustain sperm production.

Recent studies have demonstrated that somatic stem cells have a more flexible potential than expected, whether put into tissue or cultured under different conditions. Bone marrow (BM)-derived stem cells can transdifferentiate into multilineage cells, such as muscle of mesoderm, lung and liver of endoderm, and brain and skin of ectoderm origin. Here we show that BM stem cells are able to transdifferentiate into male germ cells. For derivation of male germ cells from adult BM stem (BMS) cells, we used the Stra8-enhanced green fluoresence protein (EGFP) transgenic mouse line expressing EGFP specifically in male germ cells. BMS cell-derived germ cells expressed the known molecular markers of primordial germ cells, such as fragilis, stella, Rnf17, Mvh and Oct4; as well as molecular markers of spermatogonial stem cells and spermatogonia including Rbm, c-Kit, Tex18, Stra8, Piwil2, Dazl, Hsp90alpha, beta1- and alpha6-integrins. Our ability to derive male germ cells from BMS cells reveals novel aspects of germ cell development and opens the possibilities for use of these cells in reproductive medicine.

Triple knockout mice were used to investigate the interactions of five genes that were expressed in meiotic and haploid spermatogenic cells in mice, transition protein 2 (Tnp2), proacrosin (Acr), histone H1.1 (H1.1), histone H1t (H1t), and sperm mitochondria-associated cysteine-rich protein (Smcp). TNP2 functions in the replacement of histones and the initial condensation of the spermatid nucleus. The linker histone subtypes H1.1 and H1t are expressed at high levels in meiotic and early haploid cells. ACR, a protease that is stored as a proenzyme in the acrosome, is activated during the acrosome reaction and functions in binding of sperm to the zona pellucida. SMCP is a structural protein in the outer membranes of sperm mitochondria that functions in motility. Previous work demonstrates that homozygous knockout mice lacking each of these proteins individually exhibit no defect in fertility on mixed genetic backgrounds. In contrast, the present study demonstrates that five triple knockout lines, Acr/H1.1/Smcp, Acr/Tnp2/Smcp, Tnp2/H1.1/Smcp, Acr/H1t/Smcp, Tnp2/H1t/Smcp, exhibit drastic reductions in fertility on mixed genetic backgrounds. Analysis of fertility parameters reveal that the decreased fertility is due to line-dependent defects in sperm motility in vitro correlated with reduced migration in the female reproductive tract, and decreased fertilization due to defects in adhesion of sperm to the zona pellucida, the membrane surrounding the egg. It was also found that triple knockout males, that are hemizygous for one locus and homozygous for two other loci, are as subfertile as homozygous triple knockout males, a phenomenon known as haploinsufficiency. These findings demonstrate that male fertility involves synergistic interactions of genes that function in sperm motility and sperm-egg adhesion during fertilization., (2005 Wiley-Liss, Inc.)

Infertility affects 13-18% of couples and growing evidence from clinical and epidemiological studies suggests an increasing incidence of male reproductive problems. There is a male factor involved in up to half of all infertile couples. The pathogenesis of male infertility can be reflected by defective spermatogenesis due to failure in germ cell proliferation and differentiation. We report here in vitro generation of a germ cell line (SSC1) from the pluripotent teratocarcinoma cells by a novel promoter-based sequential selection strategy and show that the SSC1 cell line form mature seminiferous tubule structures, and support spermatogenesis after transplantation into recipient testes. To select differentiated germ cell population, we generated a fusion construct (Stra8-EGFP) harbouring the 1.4 kb promoter region of germ line specific gene Stra8 and coding region of enhanced green fluorescence protein. This region was sufficient to direct gene expression to the germinal stem cells in testis of transgenic mice. The purified cells expressed the known molecular markers of spermatogonia Rbm, cyclin A2, Tex18, Stra8 and Dazl and the beta1- and alpha6-integrins characteristic of the stem cell fraction. This cell line undergoes meiosis and can develop into sperm when transplanted into germ cell depleted testicular tubules. Sperm were viable and functional, as shown by fertilization after intra-cytoplasmic injection into mouse oocytes. This approach provides the basis that is essential for studying the development and differentiation of male germ line stem cell, as well as for developing new approaches to reproductive engineering and infertility treatment.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein belonging to the family of glutathione peroxidases and has been implicated in antioxidative defense and spermatogenesis. PHGPx accounts for almost the entire selenium content of mammalian testis. In an attempt to verify the expression pattern of PHGPx, testes of mouse mutants with arrest at different stages of germ cell development and testes of mice at different ages were subjected to immunostaining with a monoclonal anti-PHGPx antibody. PHGPx was detected in Leydig cells of testes in all developmental stages. In the seminiferous tubuli, the PHGPx staining was first observed in testes of 21-day-old mice which is correlated with the appearance of the first spermatids. This result was confirmed when the testes of mutant mice with defined arrest of germ cell development were used. An immunostaining was observed in the seminiferous tubuli of olt/olt and qk/qk mice which show an arrest at spermatid differentiation. In Western blot analysis of proteins extracted from testes of mutant mice and from developing testes, two signals at 19- and 22-kDa were observed which confirm the existence of two PHGPx forms in testicular cells. In mouse spermatozoa, a subcellular localization of PHGPx and sperm mitochondria-associated cysteine-rich protein (SMCP) was demonstrated, indicating the localization of PHGPx in mitochondria of spermatozoa midpiece. For verifying the midpiece localization of PHGPx in other species, spermatozoa of Drosophila melanogaster, frog, fish, cock, mouse, rat, pig, bull, and human were used in immunostaining using anti-PHGPx antibody. A localization of PHGPx was found in the midpiece of spermatozoa in all species examined. In electronmicroscopical analysis, PHGPx signals were found in the mitochondria of midpiece. These results indicate a conserved crucial role of PHGPx during sperm function and male fertility., (Copyright 2004 Wiley-Liss, Inc.)

The ADAM (a disintegrin and a metalloprotease) family includes the best-characterized proteins involved in gamete interaction and membrane fusion in mammals. Previous studies have shown that the murine testase 2 (ADAM 25) gene is expressed specifically in testis. We found two different restriction patterns of subcloned fragments of the gene, indicating the presence of two different testase 2 transcripts. Further experiments and Celera database searches demonstrated that the two transcripts are the products of two testase 2 genes, which are located on mouse chromosome 8 in close distance of 24 kb. They show high sequence similarity to the published testase 2 gene (87.8 and 95.5%, respectively). The genomic structure of both testase 2 genes (alpha and beta) is different from other ADAM family genes like that for cyritestin and fertilin. While these genes are composed of about 20 exons, the testase 2alpha and 2beta genes contain only two exons. The first exon is very short approximately 85 bp while the second exon is approximately 2.4 kb long. Both testase 2 genes are specifically expressed in testis and they exhibit the same temporal and spatial expression pattern during male germ cell differentiation with the onset of expression in haploid stages., (Copyright 2003 Wiley-Liss, Inc.)

Over 200 genes have been shown to be associated with infertility in mouse models. However, knockout mice reveal unexpected functional redundancy of some germ cell expressed genes. Single null mutations in mouse genes encoding four male germ cell proteins, transition protein 2 (Tnp2), proacrosin (Acr), histone H1.1 (H1.1), histone H1t (H1t) and sperm mitochondria-associated cysteine-rich protein (Smcp) have been generated and analysed. Tnp2 is believed to participate in the removal of the nuclear histones and initial condensation of the spermatid nucleus. Proacrosin is an acrosomal protease synthesized as a proenzyme and activated into acrosin during the acrosome reaction. The linker histone subtype H1.1 belongs to the group of main-type histones and is synthesized in somatic tissues as well as in germ cells during the S-phase of the cell cycle. The histone gene Hist1h1t is expressed exclusively in spermatocytes and may have a function in establishing an open chromatin structure for the replacement of histones by transition proteins and protamines. Sperm mitochondria-associated cysteine-rich protein (Smcp) is a major structural element of the mitochondria in the midpiece of the sperm tail. Male mutant mice lacking any of these proteins show no apparent defects in spermatogenesis or fertility. To examine the synergistic effects of these proteins in spermatogenesis and during fertilization four lines of double knockout mice Hist1h1a/Mcsp, Hist1h1t/Mcsp, Tnp2/Mcsp and Acr/Mcsp were established. It was found that even when knockout mice are heterozygous for one allele (-/+) and homozygous for the other allele (-/-), mice were subfertile. Homozygous double knockout mice of all four lines are nearly infertile. However, in the four homozygous double knockout mouse lines, different characteristic abnormalities are prominently manifested: In Hist1h1a-/-/Mcsp-/- the migration of spermatozoa is disturbed in female genital tract, in Hist1h1t-/-/Mcsp-/- spermatozoa show morphological head abnormalities, in Tnp2-/-/Mcsp-/- the motility of sperm is affected, and in Acr-/-/Mcsp-/- the sperm-oocyte interaction is impaired. These findings indicate strongly that male germ cell expressed genes have synergistic effects on male fertility., (Copyright 2003 S. Karger AG, Basel)

The sperm mitochondria-associated cysteine-rich protein (SMCP) is a cysteine- and proline-rich structural protein that is closely associated with the keratinous capsules of sperm mitochondria in the mitochondrial sheath surrounding the outer dense fibers and axoneme. To investigate the function of SMCP, we generated mice with a targeted disruption of the gene Smcp by homologous recombination. Homozygous mutant males on a mixed genetic background (C57BL/6J x 129/Sv) are fully fertile, while they are infertile on the 129/Sv background, although spermatogenesis and mating are normal. Homozygous Smcp(-/-) female mice are fertile on both genetic backgrounds. Electron microscopical examination demonstrated normal structures of sperm head, mitochondria, and tail. In vivo experiments with sperm of Smcp(-/-) 129/Sv mice revealed that the migration of spermatozoa from the uterus into the oviduct is reduced. This result is supported by the observation that sperm motility as determined by the computer-assisted semen analysis system (CASA) is significantly affected as compared to wild-type spermatozoa. In vitro fertilization assays showed that Smcp-deficient spermatozoa are able to bind to the oocyte but that the number of fertilized eggs is reduced by more than threefold relative to the wild-type control. However, removal of the zona pellucida resulted in an unaffected sperm-egg fusion which was monitored by the presence of pronuclei and generation of blastocyts. These results indicate that the infertility of the male Smcp(-/-) mice on the 129/Sv background is due to reduced motility of the spermatozoa and decreased capability of the spermatozoa to penetrate oocytes.

The mouse zona pellucida glycoprotein, mZP2, is thought to be the secondary receptor on eggs for retention of acrosome-reacted sperm during fertilization. Here, we present evidence that one of its complementary binding proteins on sperm is proacrosin/acrosin. mZP2 binds to proacrosin null sperm considerably less effectively than to wild-type sperm. Binding is mediated by a strong ionic interaction between polysulphate groups on mZP2 and basic residues on an internal proacrosin peptide. The stereochemistry of both sulphate groups and basic amino acids determines the specificity of binding. Structurally relevant sulphated polymers and suramin, a polysulphonated anticancer drug, compete with mZP2 for complementary binding sites on proacrosin/acrosin in solid-phase binding assays. The same competitors also displace attached sperm from the zona pellucida of eggs in an in vitro fertilization system. This combination of genetic, biochemical and functional data supports the hypothesis that mZP2-proacrosin interactions are important for retention of acrosome-reacted sperm on the egg surface during fertilization. Safe mimetics of suramin have potential as non-steroidal antifertility agents.

Impaired ciliary and flagellar functions resulting in male infertility and recurrent respiratory tract infections are found in patients suffering from primary ciliary dyskinesia (PCD). In most cases, axonemal defects are present, i.e. PCD patients often lack inner and/or outer dynein arms in their sperm tails and cilia, supporting the hypothesis that mutations in dynein genes may cause PCD. However, to date it is unclear whether mutations in dynein heavy chain genes are responsible for impaired flagellar and ciliary motility in mammals. To elucidate the role of the mouse dynein heavy chain 7 (MDHC7) gene, which encodes a component of the inner dynein arm, we have generated mice lacking this dynein heavy chain isoform. Both MDHC7(+/-) and MDHC7(-/-) mice are viable and show no malformations; however, homozygous males produce no offspring. In comparison to MDHC7(+/-) and wild-type mice the spermatozoa of MDHC7(-/-) mice revealed a dramatic reduced straight line velocity and progressive movement, resulting in the inability of MDHC7-deficient sperm to move from the uterus into the oviduct. Additionally, we measured the beat frequency of tracheal cilia and observed a decrease in the beat frequency of approximately 50% in MDHC7(-/-) mice. The reduction in both ciliary and flagellar motility is not correlated with any gross defects in the axonemal structure. The phenotype of MDHC7(-/-) mice is similar to that observed in some patients suffering from PCD, and our data strongly suggest that in some patients this disease could be due to mutations in the homologous human gene DNAH1 (HDHC7).

Germ cell cyritestin is a membrane-anchored protein belonging to the ADAM family of proteins. Sequencing of eight human cyritestin cDNA clones revealed that they are identical at their 5' and 3' ends but differ from each other in the length of an internal deletion, suggesting that the human cyritestin mRNA is alternatively spliced. Internal deletions that are present in some cDNA isoforms do not cause a frameshift in the C-terminal coding region. Analysis of the predicted amino acid sequences demonstrated that the human cyritestin is a polymorphic protein that could include membrane-anchored and soluble forms. Southern blot analysis and characterization of human cyritestin genomic fragments revealed that the human genome contains two copies of the cyritestin gene instead of one as in the mouse. The human CYRN1 and CYRN2 genes were assigned to the region p12-21 of chromosome 8 and q12 of chromosome 16, respectively. Northern blot and RT-PCR analyses revealed that both human genes are expressed in the testis. Amino acid sequence comparisons between cyritestin and other members of the metalloprotease-disintegrin family of proteins suggested that human and mouse cyritestin and monkey tMDCI are homologous molecules.

Cell-type specific tumorigenesis can be induced in transgenic mice by the directed expression of simian virus 40 (SV 40) large tumour antigen (TAg). In an attempt to determine the susceptibility of haploid male germ cells to neoplastic transformation by this oncogene, transgenic mice were generated that harboured a chimeric gene composed of the SV40 T antigen genes fused to the 2.3-kb 5' flanking sequences of the rat proacrosin gene. It was previously shown that this regulatory sequence is able specifically to direct the expression of CAT reporter gene in male germ cells with the onset of translation in early haploid male germ cells. The transgene showed regulated expression in male germ cells. Although T antigen immunostaining was detected specifically in spermatids, no testicular pathology was observed. This indicates that spermatids show no susceptibility to transformation by oncogene TAg. However, in about 10% of animals of two independent transgenic lines, we could find non-testicular tumours in abdomen with a sarcoma-like structure in advanced age which showed SV40 TAg expression., (Copyright 1998 Academic Press.)

Objective: To report the initiation of a pregnancy that was achieved by intracytoplasmic sperm injection (ICSI) with sperm from a patient with Klinefelter's syndrome., Design: Case report., Setting: University women's hospital IVF center., Patient(s): A couple with primary infertility and nonmosaic 47,XXY karyotype of the male partner., Intervention(s): Intracytoplasmic sperm injection after ovarian stimulation and transvaginal ultrasound-guided oocyte pick-up with sperm from a hypergonadotropic man with a nonmosaic 47,XXY karyotype., Main Outcome Measure(s): Clinical pregnancy., Result(s): Despite a 47,XXY karyotype in all 50 analyzed lymphocyte metaphases, the sperm of the patient led to a clinical pregnancy with the first attempt of ICSI and intrauterine transfer of three embryos. The pregnancy stopped developing in the ninth week. Cytogenetic investigation of the abortion material revealed a numerical normal 46,XXY karyotype., Conclusion(s): Sperm from a patient with hypergonadotropic nonmosaic Klinefelter's syndrome, when used for ICSI, can lead to a pregnancy.

Sperm chromosome analysis offers the opportunity to gather information about the origin of chromosome aberrations in human germ cells. Over the last 20 years more than 20,000 sperm chromosome complements from normal donors and almost 6000 spermatozoa from men with constitutional chromosome aberrations (inversions, translocations) have been analyzed for structural and numerical chromosome abnormalities, as well as for segregation of the constitutional chromosome aberrations after the sperm had penetrated hamster oocytes. On the other hand, it took only 6 years to screen more than 3 million mature spermatozoa from healthy probands for disomy rates of 20 autosomes (chromosomes 19 and 22 not evaluated) and the sex chromosomes, and for diploidy rates by in situ hybridization techniques. In the present paper the results arising from both methods are compiled and compared.

Meiotic segregation of the sex chromosomes was analysed in sperm nuclei from a man with Klinefelter's karyotype by three-colour FISH. The X- and Y-specific DNA probes were co-hybridized with a probe specific for chromosome 1, thus allowing diploid and hyperhaploid spermatozoa to be distinguished. A total of 2206 sperm nuclei was examined; 958 cells contained an X chromosome, 1077 a Y chromosome. The ratio of X:Y bearing sperm differed significantly from the expected 1:1 ratio (chi2 = 6.96; 0.001 < P < 0.01). Sex-chromosomal hyperhaploidy was detected in 2.67% of the cells (1.22% XX, 1.36% XY, 0.09% YY) and a diploid constitution in 0.23%. Although the frequency of 24,YY sperm was similar to that detected in fertile males, the frequencies of 24,XX, 24,XY and diploid cells were significantly increased. A sex-chromosomal signal was missing in 4.26% of the spermatozoa. This percentage appeared to be too high to be attributed merely to nullisomy for the sex chromosomes and was considered, at least partially, to be the result of superposition of sex-chromosomal hybridization signals by autosomal signals in a number of sperm nuclei. The results contribute additional evidence that 47,XXY cells are able to complete meiosis and produce mature sperm nuclei.

Acrosin (ACR), a serine proteinase located in the acrosome of the sperm, has been presumed to be involved in the recognition and binding of the sperm to the zona pellucida of the ovum and the sperm penetration through the zona pellucida. To examine the function of acrosin in vivo, we have generated mice carrying a mutation at the acrosin locus (Acr) through targeted disruption in embryonic stem (ES) cells. One chimeric male and female transmitted the targeted gene through their germ line. Homozygous Acr-/- mice are fertile and yield litters comparable in number and size to those of Acr+/+ mice. These data show that sperm of the homozygous Acr-/- mice are able to penetrate the zona pellucida, fertilize the ovum, and produce viable offspring. However, spermatozoa lacking acrosin protein show a delayed fertilization. One chimeric male which contained the targeted gene in 20% of its sperm transmitted only the Acr+ allele to its progeny. Furthermore, in vitro fertilization with equally mixed sperm cells of Acr+/+ and Acr-/- mice resulted in fertilization only with the Acr+ sperm cells. Incubation of oocytes with Acr+ or Acr- sperm show that the Acr+ sperm are faster to fertilize the oocytes than the Acr- sperm cells. These results suggest that Acr- sperm have a selective disadvantage when they are in competition with Acr+ sperm.

Sperm cells from 45 infertile patients were investigated for disomy rates of chromosomes 1, 7, 10, 17, X and Y as well as for diploidy by single- and double-target in-situ hybridization. The patients who attended the infertility clinic were aged 23-46 years. Semenograms showed that the patients had oligo-, astheno-, oligoastheno-, oligoterato-, oligoasthenoterato-, or asthenoteratozoospermia. The average disomy rates determined in the patients were similar for all chromosomes, ranging from 0.10% (chromosome Y) to 0.14% (chromosomes 10 and X). Diploidy was detected with a mean incidence of 0.1%. With the exception of two patients who exhibited significantly increased diploidy rates of 0.35 and 1.6%, neither disomy nor diploidy was increased in the group of infertile patients as compared to healthy, fertile males.

We have cloned and sequenced the cDNA of a novel gene from the rat protamine gene cluster. This gene, preliminarily referred to as Prm3, is intronless and resides between the genes for protamine 2 and transition protein 2. Prm3 is transcribed from the same strand as these genes and is expressed in haploid stages of spermatogenesis. The 410-bp-long cDNA possesses an ORF, coding for a putative 104-amino acid polypeptide with a high content of glutamic acid.

During spermiogenesis, the successive replacement of the somatic histones by basic proteins, the transition proteins and protamines, allows normal sperm nuclear condensation. It was suggested that disturbances in nuclear condensation may result in male infertility. Here we report the first molecular analysis of the structure of three genes which code for germ cell-specific nuclear proteins, namely protamine 1 (PRM1), protamine 2 (PRM2) and transition protein 1 (TNP1) in infertile men with disturbed sperm chromatin condensation. In 36 infertile men whose spermatozoa showed a positive reaction with aniline blue, which is an indication for the presence of histones in the nuclei, the complete nucleotide sequences of the coding regions and 5' and 3' untranslated regions of the three genes were evaluated. In addition, 10 infertile patients with oligoasthenoteratozoospermia were studied in the same way, as well as nine infertile patients whose spermatozoa showed a reduction of the protamine 2 content. We did not detect any mutation in the three genes in any of the patients. We assume that the disturbances in the sperm chromatin condensation of our patients, and those described in the literature, are not primarily due to mutations in the genes for PRM1, PRM2 and TNP1.

Seventy-one semen samples of 60 infertile patients with a suggested high number of immature germ cells were assigned for chromosomal analysis. The sperm probes of nine patients did not contain immature germ cell stages apart from spermatids. In 22 probands we could only find early prophase stages or not readable metaphase structures. Only 25 patients could be analysed cytogenetically. The semen sample of each patient contained degenerated germ cell structures associated with desynaptic and fragmented/pulverized metaphase cells. It follows that in each analysed probe we could detect anomalies which can be attributed to errors in the gametic development process and which are possibly connected with the existing teratozoospermia and oligozoospermia. The easy practicality for cytogenetic analysis represents the most important advantage of the described method. The analysis of immature germ cell stages in semen samples makes it possible to bypass or to reduce the incidence of tests biopsies for meiotic chromosome investigation.

Acrosin is a serine proteinase located in a zymogen form, proacrosin in the acrosome of the sperm. It is released as a consequence of the acrosome reaction and is believed to be the most important enzyme in the fertilization process. In the mouse, the proacrosin gene is transcribed premeiotically in spermatocytes, but protein biosynthesis starts in haploid spermatids and is restricted to the emerging acrosome. Four lines of transgenic mice harboring 2.3 kb of 5' untranslated region of the rat proacrosin gene fused to the CAT-reporter gene were generated by microinjection of fertilized eggs. The chimeric gene was found to be present in 10-100 copies per genome in the different strains. The 5' untranslated region of rat proacrosin gene could properly direct CAT-gene expression to spermatocytes and CAT-mRNA translation to round spermatids as it is known for mouse proacrosin gene. However, CAT protein is not restricted to the acrosome; rather, it is distributed in the spermatid cytoplasm. This could be due to the lack of DNA sequences for a hydrophobic leader peptide that have been found in all mammalian proacrosins studied until now but that was not present in transgene. It can be concluded from our results that cis-acting sequences required for tissue specific proacrosin expression reside on a 2.3-kb restriction fragment and are conserved in the proacrosin genes of mouse and rat.

The spontaneous acrosomal reaction of mouse spermatozoa during their passage through the male and female genital tract was studied by indirect immunofluorescence staining techniques using antibodies against the outer acrosomal membrane (OAM). Low rates of spontaneously acrosome-reacted spermatozoa could be observed during the passage through the male reproductive tract, namely 4% in the testis (SD +/- 0.8%) and in the caput epididymidis (SD +/- 1.5%), and up to 8% in the cauda epididymidis (SD +/- 0.5%) and in the ductus deferens (SD +/- 0.5%), whereas a higher value of 13% (SD +/- 1%) of reacted spermatozoa was obtained from the corpus epididymidis. Spermatozoa removed from the uterus 20 min up to 14 h post copulation (p.c.) exhibited a constant degree of 8% (SD +/- 6%) acrosomal reaction, whereas 43% (SD +/- 6%) of the oviductal sperms were acrosome-reacted, independent of the time of removal p.c. Rates of spontaneously acrosome-reacted spermatozoa from the male and female reproductive tract could be increased by 2-fold and up to 7-fold by an in vitro incubation in modified Tyrode's medium for 210 min. On the basis of our results wer postulate a basic level of spontaneously acrosome-reacted spermatozoa in the oviduct at ovulation time, which might be relevant to ensure the environmental conditions for the fertilization process. Additionally, we offer a possibility of optimizing in vitro fertilization techniques.

Rat preproacrosin primary structure as predicted from a 1431 nucleotide (nt) cDNA indicates that the molecule is synthesized as a preproenzym consisting of a putative 19 amino acid signal sequence, a 23 amino acid light chain and finally a 395 amino acid heavy chain. Functional domains like the catalytic triad (His-70, Asp-124, Ser-222) are highly conserved not only between the available acrosin primary structures of different mammals but also in comparison with other serine proteinases. Number of amino acid exchanges and the degree in amino acid identity between the different serine proteinases and rat acrosin leads to the assumption that acrosin is one of the early descendants within the phylogenetic tree of the serine proteinase superfamily.

The sperm enzyme acrosin has long been known as one of the key enzymes in the mammalian fertilization process. Elucidation of primary structures of preproacrosin from various species have allowed a deeper insight into the structural organization and the complex evolution of the sperm proteinase acrosin. In addition to the typical elements of serine proteases, the acrosin molecule possesses one novel domain that might convey DNA-binding properties.

Acrosome-reacted CB6F1 mouse spermatozoa with slight flagellar motility were microinjected under the zona pellucida of CB6F1 mouse oocytes. Electron microscopy revealed the presence of swollen and decondensed sperm heads in the oocyte cytoplasm. Sixty-one percent of the microinjected oocytes reached a morphologically apparent two-cell stage, but chromosomal analysis demonstrated only haploid chromosomal complements in all cases. The exposure of microinjected oocytes to suspensions of spermatozoa of mice homozygous for a 2,4 reciprocal translocation resulted in normal fertilization and embryonic development with a maternally as well as a paternally derived haploid genome. Identical results were obtained with oocytes microinjected with medium and subjected to in vitro fertilization thereafter. Thus it can be suggested that the microinjected spermatozoa with insufficient flagellar motility are incorporated into the oocyte cytoplasm by phagocytosis. These spermatozoa do not induce a polyspermy block but induce the oocyte to parthenogenetic development.

The nuclei of spermatozoa in all mammals examined so far contain P1 protamine. A second protamine variant, protamine P2, has to date been isolated only from human and murine spermatozoa where it represents the major fraction of basic nuclear protein. In order to elucidate the reason for this unusual distribution of the protamine variants among mammals we have investigated the expression of protamine P2 in boar and bull. It can be shown that also in these species protamine 2 is transcribed and translated on low levels. Various mutational events though have altered the primary structure of the protein: In boar, a deletion of 8 aminoacids has removed a sequence motif from the amino-terminus of the molecule, which highly probable is of functional relevance. The bovine sequence, as a consequence of numerous point mutations has accumulated neutral and hydrophobic aminoacids which reduce the affinity of the protamine 2 to DNA.

Using the indirect immunofluorescence staining technique, the occurrence and localization of proacrosin, the zymogen form of acrosin, was studied during spermatogenesis in the bull, ram, boar and rabbit. Proacrosin staining was demonstrable for the first time in the early haploid spermatid and increased with the differentiation of the spermatid to spermatozoon. The spermatozoon is covered by a cap-like structure of uniform fluorescence corresponding to the acrosomal compartment of the male gamete. No fluorescence could be found in diploid spermatogenic cells, i.e., in spermatogonia and spermatocytes. An identical developmental pattern of proacrosin was observed with the indirect immunoperoxidase staining technique. However, with this staining technique a distinct distribution of proacrosin staining was observed in the acrosome of epididymal and ejaculated spermatozoa of the bull, ram, boar, rabbit and man. Proacrosin seems to be distributed in the acrosome in granules rather than in the homogeneous form, as was indicated by the results of indirect immunofluorescence staining.

Acrosin and its zymogen form, proacrosin, were extracted from early and late spermatids, from ejaculated and epididymal spermatozoa (caput, corpus, and cauda) of the bull. Activity of proacrosin/acrosin and the time course of proacrosin activation were studied. It turned out that proacrosin/acrosin activity is first demonstrable in haploid spermatids, increases during spermiohistogenesis in the testis, and remains nearly constant in epididymal and ejaculated spermatozoa.

Chromosomal analysis was performed on 109 males with teratozoospermia. Four cases of anomaly were found, including three cases of mosaic Klinefelter's syndrome and one balanced autosomal translocation. Among chromosomal heteromorphisms, 9qh+ was found in 25% of the patients. The fraction of malformed spermatozoa in the semen of the 9qh+ group is significantly higher than in the chromosomally normal group. It is speculated that heterochromatic variants like 9qh+ could be one of several unknown factors disturbing normal gametogenesis.

Using the indirect immunofluorescent staining technique, the developmental patterns of (pro) acrosin and the outer acrosomal membrane were studied in human spermatogenesis. Specific antibodies against purified acrosin and outer acrosomal membranes from boar spermatozoa were raised in the rabbit and were found to crossreact with (pro)acrosin and outer acrosomal membrane from human spermatogenic cells. It was concluded that (pro)acrosin as well as the molecules building up the outer acrosomal membrane have been highly conserved during mammalian evolution. In the course of human spermatogenesis (pro)acrosin as well as the outer acrosomal membrane first appear in the haploid spermatids; the fluorescent areas of the individual cells steadily increase during spermiogenesis. Staining for acrosin and the outer acrosomal membrane, respectively, was found in identical compartments of the spermatogenic cells in juxtaposition to the nucleus. Round-headed spermatozoa from an infertile patient did not stain for (pro)acrosin or outer acrosomal membrane. The lack of the acrosin system was further substantiated by the gelatin substrate film technique demonstrating the absence of a gelatinolytic protease in round-headed spermatozoa. Hence, round-headed spermatozoa lack the acrosome with its constituent membrane proteins and the acrosin system housed by the acrosome of normal spermatozoa.

Acrosin and the outer acrosomal membrane (OAM) were studied in the spermatozoa of 9 infertile patients who differed in the number of round-headed spermatozoa between 14 and 71% in their ejaculates. These sperm components were also investigated in two infertile brothers who exhibited exclusively round-headed spermatozoa in their ejaculates, and in their fertile father. It turned out that round-headed spermatozoa lack both acrosin and the OAM as studied by indirect immunofluorescent and immunoperoxidase staining technique, gelatinolysis tests and by acrosin activity measurements. The normally shaped spermatozoa of 6 of the 9 infertile patients were found to be positive for acrosin and the OAM as expected, but in the remaining three patients even these spermatozoa were abnormal; in one patient they were unstainable for acrosin and in two patients they were unstainable both for acrosin and the OAM. These results have been confirmed by studies with the gelatinolysis test. The father of the two brothers with exclusively acrosomeless spermatozoa had more than 94% of normally shaped spermatozoa. However, only 10% of these spermatozoa were acrosin positive and only 30% were positive for the OAM. On the basis of these results we postulate that the mode of inheritance of the round-headed spermatozoa syndrome is polygenic rather than monogenic as suggested by previous authors.

An antiserum to the purified porcine outer acrosomal membrane (OAM) was raised in rabbits and the IgG fraction isolated by ammonium sulphate precipitation and ion exchange chromatography. The antibodies reacted exclusively with the acrosomal cap of the sperm head as revealed by indirect immunofluorescence. In addition they cross-reacted not only with the acrosomal part of the spermatozoa of all mammalian species tested (bull, horse, rabbit, rat, mouse, hamster, mole, antelope, monkey, man) but also with the spermatozoa of the cock (Class: birds) and the rainbow trout (Class: fish). All the species exhibited similar development of the acrosomal cap during spermatogenesis, with the appearance of the immunofluorescent stain in early round spermatids. In the mole the localization of the acrosome in elongated testicular spermatids differed from that in all other species: Instead of prominent fluorescence over the apical part of the sperm an equatorial belt was formed. The cross-reactivity of the anti-boar OAM antibody with the acrosomes of different vertebrate species at the morphological level was supported by the results of Western blotting experiments with purified boar OAM proteins and the SDS-extractable proteins of bull and human spermatozoa, respectively. Using anti-OAM antibodies and antibodies against the acrosin inhibitors I and II described recently by Tschesche et al. (1982), in absorption and Western blotting experiments, it was demonstrated that the acrosin inhibitor proteins are integrated in the outer acrosomal membrane.

Acrosin, the outer acrosomal membrane (OAM) and an acrosin inhibitor were studied in testicular cells and ejaculated spermatozoa of fertile men and in those of an infertile patient with exclusively round-headed spermatozoa in his ejaculates. The investigations were performed with the aid of immunohistochemical techniques using specific antibodies against the three acrosomal markers isolated from boar spermatozoa. The spermatozoa of fertile men exhibit staining for acrosin, the OAM and the acrosin inhibitor in the cap region while the round-headed spermatozoa of the patient are totally negative for the three markers, clearly supporting the conclusions of other authors that round-headed spermatozoa lack acrosomes. The lack of the acrosin system was further substantiated by the gelatin substrate film technique. In the course of normal human spermatogenesis acrosin, the OAM and the acrosin inhibitor we first demonstrable in early round spermatids, namely in identical compartments adjacent to the cell nucleus. During spermatid differentiation the staining for the three markers becomes flattened over the nucleus, resulting in a cap-like structure in testicular spermatozoa. In contrast to the ejaculated round-headed spermatozoa, the early round spermatids in the testis of the infertile patient exhibit fluorescent staining for the three markers in the region adjacent to the nuclear membrane. In the course of further spermiogenesis, the staining did not extend over the nuclear membrane, as was observed during normal spermiogenesis, but became separated from the nuclear membrane, as was observed during normal spermiogenesis, but became separated from the nuclear membrane, was translocated at various locations in the cytoplasm and was finally eliminated with the loss of the cytoplasm. These results are in accordance with the results of electron microscopically investigations on the teratogenesis of round-headed spermatozoa. Furthermore, the developmental pattern of the acrosin inhibitor during normal and abnormal spermiogenesis supports the intraacrosomal location of the acrosin inhibitor recently described by Tschesche et al. (1982).

Using the indirect immunofluorescence staining technique, the developmental pattern of acrosin during spermatogenesis of boar, ram, rabbit, mouse, rat, and Russian hamster (Phodopus sungorus) was studied. Specific antibodies against purified boar acrosin raised in rabbits cross-reacted with the acrosin of all species investigated thus suggesting that the antigenic determinants of the acrosin molecule cross-reacting with anti-boar acrosin antiserum have been highly conserved in mammalian evolution. During spermatogenesis acrosin was first demonstrable in haploid spermatids and increased in the course of the differentiation of the spermatids to spermatozoa. During the entire period of spermatid differentiation acrosin appeared in juxtaposition to the nucleus. In boar and ram the results obtained with the indirect immunofluorescence staining procedure were confirmed with the indirect immunoperoxidase staining method.

A patient with nekrozoospermia is presented. His clinical, andrological and endocrinological data are described. Cytogenetically a complex chromosomal mosaic of 46,XX; 46,XY; 47,XXY and 48,XXXY was found.

Acrosin was prepared from boar and bull spermatozoa and its lytic effect in vitro on the zona pellucida of mouse, golden hamster, rabbit, pig and cow was investigated. Depending upon the species studied, ovarian oocytes, ovulated oocytes and preimplantation embryos were obtained for the experiments. While in golden hamster and rabbit the zona pellucida was removed by both acrosins, this effect was absent for bull acrosin in cow and mouse eggs and for boar acrosin in pig and mouse eggs. In those species in which the zona pellucida was not removed by the acrosins after an incubation period of 2 hours even a prolongation up to 24 hours with higher amounts of acrosin, the addition of acrosomal extracts to the incubation buffer (Tyrode solution pH 7.2) or an increase of the pH value up to 8.6 of the acrosin solution had no effect upon the zona pellucida. Our results indicate that at least in vitro, acrosin does not possess the capacity to lyse the zona pellucida in a species specific fashion. Since the lytic effect of boar and bull acrosin on the zona pellucida of ovarian oocytes and preimplantation embryos is not different from that on ovulated oocytes it can be assumed that neither the maturation of the zona pellucida during oogenesis nor its modification after fertilization, change the susceptibility of the zona pellucida to acrosin digestion.