Light microscopy morphological characteristics of the sperm flagellum may be related to axonemal abnormalities.

Although electron microscopy provides a detailed analysis of ultrastructural abnormalities, this technique is not available in all laboratories. We sought to determine whether certain characteristics of the flagellum as assessed by light microscopy were related to axonemal abnormalities. Forty-one patients with an absence of outer dynein arms (type I), a lack of a central complex (type III) and an absence of peripheral doublets (type IV) were studied. Sperm morphology was scored according to David's modified classification. Flagella with an irregular thickness were classified as being of normal length, short or broken. There were correlations between missing outer dynein arms and abnormal, short or coiled flagellum. Type III patients showed the highest flagellar defects (a short (P = 0.0027) or an absent flagellum (P = 0.011)). Just over 68% of the irregular flagella were short in Type III patients, whereas this value was only 34.5% in type I and 26.4% in type IV (P = 0.002). There was a negative correlation between misassembly and spermatozoa of irregular flagella (r = -0.79; P = 0.019). It is concluded that light microscopy analysis of flagellum abnormalities may help provide a correct diagnosis, identify sperm abnormalities with fertility potentials and outcomes in assisted reproduction technologies and assess the genetic risk.

[ICSI treatment in severe asthenozoospermia]. / Asthénozoospermie sévère à vitalité normale et ICSI.

In the management of asthenozoospermia, the spermogram-spermocytogram plays an important role during diagnosis. It is of major importance to distinguish between necrozoospermia and sperm vitality. An ultrastructural study of spermatozoa is processed in the case of primary infertility without female implication, severe, unexplained and irreversible asthenozoospermia, sperm vitality at least 50 % and normal concentration of spermatozoa. Ultrastructural flagellar abnormalities are numerous and involve most spermatozoa. ICSI provides a suitable solution for patients with sperm flagellar defects to conceive children with their own gametes but the rate of ICSI success may be influenced by the type of flagellar abnormality. Some fertilization and birth rate failures which are related to some flagellar abnormalities might occur.

Assessment of acrosome and nuclear abnormalities in human spermatozoa with large vacuoles.

BACKGROUND: Spermatozoa with large vacuoles (SLV) may have a negative impact on embryo development. The origin of these vacuoles is unknown. We evaluated acrosome and nucleus alterations in isolated SLV, versus unselected spermatozoa. METHODS: We studied 20 patients with teratozoospermia. Spermatozoa from the native semen sample and spermatozoa presenting a vacuole occupying >13.0% total head area, isolated under high magnification (×6600), were assessed. Confocal and transmission electron microscope evaluations were performed on SLV and native sperm, respectively. Acrosome morphology and DNA fragmentation were analysed using proacrosin immunolabelling (monoclonal antibody 4D4) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay. Chromatin condensation was evaluated with aniline blue staining. Sperm aneuploidy was assessed using fluorescence in situ hybridization. RESULTS: SLV represented 38.0 ± 5.10% of motile spermatozoa obtained after gradient density centrifugation. Vacuoles were mainly in the anterior and median sperm head (45.7 ± 2.90 and 46.1 ± 3.00%, respectively). Abnormal acrosomes were increased in SLV compared with unselected spermatozoa (77.8 ± 2.49 versus 70.6 ± 2.62%; P = 0.014). Microscopic observations showed an exclusively nuclear localization of large vacuoles. Complete DNA fragmentation was higher in native spermatozoa (P < 0.0001) than SLV, while chromatin condensation was altered in SLV (P < 0.0001). Aneuploidy and diploidy rates were increased in SLV (P < 0.0001). CONCLUSIONS: Sperm vacuoles were exclusively nuclear. In our selected teratozoospermic population, aneuploidy and chromatin condensation defects were the main alterations observed in SLV. Based on results from this small sample of spermatozoa, we propose a global impairment of the spermatogenesis process as a common origin of the morphological alterations.

Absence of annulus in human asthenozoospermia: case report.

The annulus is a septin-based ring structure located at the junction of the midpiece (MP) and the principal piece (PP) of spermatozoa flagellum. In the mouse, deletion of Septin 4, a structural component of the sperm annulus, prevents annulus formation and leads to MP-PP disjunction, flagellar bending, asthenozoospermia and male sterility. Testis anion transporter 1 (Tat1) is a germ cell-specific member of the SLC26 anion transporter family and is co-expressed with Septin 4 at the sperm annulus. Interestingly, Tat1 null sperm bear an atrophic annulus, causing a phenotype similar to that of Sept4 null sperm. We searched for Tat1 misexpression and/or mislocalization in spermatozoa from asthenozoospermic subjects (n = 75) and controls by performing an immunofluorescence detection assay on sperm smear preparations. We found one patient showing moderate asthenozoospermia, with 97% of sperm lacking Tat1, Septin 4 and Septin 7 proteins at the annulus. We confirmed the absence of the annulus structure by transmission electron microscopy and observed that spermatozoa from the patient displayed MP-PP disjunction and abnormal mitochondrial organization. We show that the structural defects in sperm are not caused by abnormal transcription or point mutations of the TAT1 and SEPT4 genes; however, although both proteins are expressed, they are not properly localized at sperm annulus. The case we studied, so far unreported in human, confirms the involvement of Tat1 and Septin proteins in the constitution of the annulus, but also raises questions about the function of this structure in human sperm motility.

[Knockout mice in the service of reproduction]. / Les souris génétiquement modifiées au service de la reproduction.

At least 600 infertile knockout mice have been produced and this review is limited to recent models involving unexpected genes in reproduction or genes involved in recently identified molecular biology pathways. They concern the female meiosis (Brca1), primordial follicles (Lhx8), granulosa cells (Lrh1), and, for both sexes, mitochondria (Immp2l) and meiosis (Ubb). Germ cells can be altered differently following the sex, as it is the case for Dicer, known to be involved in the formation of miRNA. Knockout mice can support data obtained in human, such as for HNRNPGT, whose role in the human spermatogenesis remained questionable. However, due to numerous factors involved, positive results obtained by the "candidate gene approach" remain limited (for example, SCP3 and CREM). Nevertheless, knockout mouse models bring considerable knowledge on genes possibly involved in men and women infertilities.

Ultrastructural nuclear defects and increased chromosome aneuploidies in spermatozoa with elongated heads.

BACKGROUND: Cellular and molecular mechanisms leading to elongated sperm heads are not known. We have analysed the nuclear status of spermatozoa with elongated heads. METHODS: Fourteen men with at least 30% of spermatozoa with an elongated nucleus were studied and compared with five fertile men as controls. Sperm morphology was analysed by a quantitative ultrastructural analysis. Sperm chromosomal content was assessed by three-colour fluorescence in-situ hybridization (chromosomes X, Y, 18). Y chromosome microdeletion and karyotype were analysed. RESULTS: Elongated sperm head rates of the patients were 46.9% (30-75 versus 0-2% in the control group) by light microscopy and 34.4% by electron microscopy. In all patients, the chromatin was poorly condensed in elongated sperm heads (50% of elongated nuclei). No anomalies of sperm biochemical markers were found. All the men showed normal karyotype (46,XY) and absence of Y chromosome microdeletion. Aneuploidy rates of gonosomes and chromosome 18 were significantly increased in patients (1.64- and 3.6-fold, P = 0.006 and 0.026, respectively). CONCLUSIONS: This study demonstrates that impaired chromatin compaction and slightly increased chromosome aneuploidies are found in spermatozoa with an elongated head, suggesting possible mechanisms such as meiotic non-disjunctions or spermiogenesis anomalies.

[Animal models: Candidate genes for human male infertility]. / Modèles animaux: gènes candidats pour l'infertilité masculine.

More than 300 genes necessary for the normal completion of the spermatogenesis have been identified by means of the production of knockout mice. The data cover the whole male reproduction apparatus and thus allow defining candidate genes that could be related to various dysfunctions of human male fertility. Data obtained from mouse models have allowed identifying genetic mutations with loss of function for men with: (i) early meiotic arrest, (ii) maturation arrest of the round spermatid and (iii) morphological anomalies of the spermatozoa. Also numerous Drosophila mutants are models for the knowledge of genes involved in the spermatogenesis. Finally, there are other important models sharing cilia and flagella, and thus, having a structure in common with the sperm flagellum, the axoneme. First, these organisms have allowed the identification of genes involved in human respiratory diseases. But interestingly, these last two years, a great number of human syndromes have been discovered to be related to cilia pathologies, and among them, complex phenotypes including an abnormal spermatogenesis.

Arrest of flagellum morphogenesis with fibrous sheath immaturity of human spermatozoa.

Morphogenesis of the mammalian sperm flagellum is characterized by the assembly of axonemal and peri-axonemal structures. The incorporation of mitochondria into the flagellum results from complex cellular events, including flagellum compartmentalization and membrane and organelle reorganization. These events are striking in the annulus, which progressively relocates from the neck to the principal piece of the flagellum. This study presents a human sperm phenotype with failure of the annulus relocation, absence of mitochondrial sheath and a fibrous sheath at intermediate step of assembly. The sperm nucleus was fully condensed but with deep invaginations engulfing the acrosome. The distal pole of some mitochondria exhibited an unusual dense substance. This rare human sperm phenotype was found in a consanguineous patient, suggesting a genetic origin. These anomalies raise the question of the mechanisms that lead to impairment of both the annulus relocation and the deposit of proteins on the fibrous sheath during spermiogenesis.

Impact of genetic engineering on the understanding of spermatogenesis.

To date, about 100 genes have been found, by genetic engineering, to be implicated in spermatogenesis. Primordial germ cells, spermatogonia, spermatocytes I and elongating spermatids are particularly sensitive. Transgenic and knockout mice permit an approach to be made to the question of genetic factors involved in DNA damage repair, thermal injury, sperm chromatin compaction and sex-specific recombination. Knockout mice reveal unexpected functional redundancies of testis-specific genes. This review considers how functional divergences can exist among homologous genes from different species, and to what extent the phenotypes of knockout mice can be similar to those from spontaneous mutations. Additional anomalies in reproductive function have frequently been found in these mice, as were found factors leading to tumour susceptibility and/or various diseases. Finally, knockout mice remind us that, in nearly all cases, hemizygous individuals retain a fertility and a wild-type sperm phenotype, although half of the spermatozoa share a genetic defect. The findings strongly emphasize the importance of understanding epidemiology in male infertility, to identify hereditary forms of impaired spermatogenesis, and to create DNA and pathological germ cell banks.

XMR is associated with the asynapsed segments of sex chromosomes in the XY body of mouse primary spermatocytes.

The XMR (Xlr-related, meiosis-regulated) protein is an M(r) 30,000 nuclear protein closely associated with the XY body in mouse primary spermatocytes. It shows sequence similarity with several other meiosis-specific proteins. In the present study, we investigated the fine immunolocalization of XMR in the XY body by laser confocal and electron microscopy. It was found that XMR was associated with the asynapsed segments of sex chromosomes, including their axes and the surrounding chromatin loops. In contrast, the pseudoautosomal region and the opposite free end of the X were unlabeled for XMR. In mice with the reciprocal T(X;16)16H translocation, XMR was also associated with the heterochromatic translocation product that emerges from the XY body. These findings at the subchromosomal level point to a role for XMR in chromatin condensation and transcriptional inactivation. XMR is unique among proteins in being capable of association with the XY body. It could play a specific role in a mechanism of male X-chromosome inactivation in mammals.

Mammalian spermatogenesis investigated by genetic engineering.

Genes involved in mammal spermatogenesis can now be identified through mutants created by genetic engineering. Information has been obtained on male meiosis, but also on the factors regulating the proliferation, maintenance and differentiation of male germ cells. Its has also increased our knowledge of the germ cell phenotype emerging from an altered germ cell genotype. This review is focused on data from genes expressed in male germ cells and on the question of how germ cells and Sertoli cells cope with the molecular lesions induced. The conservation of a wild-type phenotype of male germ cells in mutant mice is discussed, and how the mouse genetic background can lead to different germ cell phenotypes for a given gene mutation.

What are the germ cell phenotypes from infertile men telling us about spermatogenesis?

Drosophila mutants for known genes and those obtained following germline genetic engineering in mice have led to the identification of genes involved in the initiation and the maintenance of spermatogenesis and in the different steps of meiosis. Mutants allow the definition of meiosis-specific checkpoint controls that ensure the transmission of complete and undamaged genetic information. They reveal what spermatogenesis events are interdependent. In the light of these data, an attempt is made to define which events of spermatogenesis could be defective in some well-defined human spermatogenesis failures. They appear to be good models to study the decouplages of spermatogenesis events, the morphogenetic relationships between germ cell structures and the occurrence of pleiotropic sperm phenotypes. It is discussed whether a germ cell with a normal phenotype can transmit a non-functional gene involved in spermatogenesis and how homologous genes can lead to different germ cell phenotypes depending on the species.

High level expression of the Xlr nuclear protein in immature thymocytes and colocalization with the matrix-associated region-binding SATB1 protein.

The X-linked lymphocyte-regulated (Xlr) protein is a 30,000 Mr nuclear protein bearing homology with meiosis-specific proteins and expressed in late stage B lymphoid cell lines. In the present study we investigated its expression in the T lymphoid lineage. In adults, a high level of expression was detected in CD4-CD8- thymocytes. Most remarkably, the peak of Xlr expression occurred early during thymus cell ontogeny, precisely on days 14-15 of gestation, and was associated with the first wave of pre-T cell differentiation. Its onset preceded the rearrangement of TCR genes, as Xlr expression was conserved in thymus cells from RAG1(0/0) mice. The lower expression of Xlr on day 13 of fetal development, the bright Thy1+ phenotype of Xlr-positive cells, their large size, and their absence from subcapsular areas suggest that Xlr expression must be turned on within the thymus and not in prethymic precursors. From day 16 of gestation, Xlr expression decreased markedly. At birth and later, Xlr(high) cells were mostly large cells scattered throughout the cortical area. As shown by confocal microscopy, expression of Xlr closely overlapped that of SATB1, which binds special AT-rich DNA sequences associated with the nuclear matrix and plays an important regulatory role for many genes. The remarkably regulated expression of Xlr in the lymphoid cell lineage and of its homologue Xmr in the germ cell lineage suggests that they might play an important role in chromatin metabolism at critical stages of differentiation during which the genome undergoes irreversible rearrangements.

A 21-kDa polypeptide belonging to a new family of proteins is expressed in the Golgi apparatus of neural and germ cells.

We have isolated a full-length murine clone corresponding to the rat neuronal p1A75 partial cDNA (Sutcliffe, J. G., Milner, R. J., Shinnick, T. M., and Bloom, F. E. (1983) Cell 33, 671-682). It encodes a 185-residue polypeptide that displays 56% identity with p19, a protein selectively expressed in the Golgi apparatus of neural cells (Sabéran-Djoneidi, D., Marey-Semper, I., Picart, R., Studler, J.-M., Tougard, C., Glowinski, J., and Lévi-Strauss, M. (1995) J. Biol. Chem. 270, 1888-1893). An antibody directed against the recombinant polypeptide allowed us to demonstrate the existence of the natural 21-kDa protein (p21) in brain and its prominent juxtanuclear Golgi-like localization in cultured neurons. Ultrastructural observation of cultured neurons and analysis of transfected COS cells revealed a specific labeling of the Golgi apparatus, suggesting, as for p19, the presence of a Golgi targeting signal in its primary sequence. Surprisingly, p21, which is much more strongly expressed in the olfactory epithelium than p19, is also present in the Golgi complex of spermatocytes and in the flagellar middle piece of late spermatids.

Immunochemical characterization of a human sperm fibrous sheath protein, its developmental expression pattern, and morphogenetic relationships with actin.

Among the monoclonal antibodies (MAbs) prepared against human sperm extracts, MAb 4F7 was found to be specific to the human and Macaca fascicularis sperm cytoskeletal fibrous sheath (FS). In Western blotting, MAb 4F7 stains a doublet of polypeptides of about M(r) 95 x 10(3) in extracts of human sperm cells. These polypeptides are not recognized by the KL1 anti-cytokeratin MAb, nor by the MAbs known to bind to the carboxy terminal (IFA) and to the amino terminal (ME101) rod domain of intermediate filaments. Sequential extraction procedures shows that the FS polypeptides recognized by MAb 4F7 are exposed after treatment with 8 M urea 4F7 immunoreactivity is lost after treatment with high ionic solutions (NaCl; KCl, Kl). Immunogold electron microscopy reveals that this protein is present throughout the FS. This FS antigenic determinant first accumulates in an FS proximal body in late spermatids, then in granules extending distally along the flagellum. Staining of spermatozoa with flagellar dysgenesis reveals that this FS protein colocalizes with actin no matter what the location of their abnormal assembly. These data suggest that the transient microtubule-like spindle-shaped body of as yet unknown function could be involved in FS protein deposition and that the assembly of the FS and actin could be under the control of some common morphogenetical factor(s). MAb 4F7 should allow further investigations of this peri-axonemal structure in both normal and pathological conditions.

A putative human equivalent of the murine Xlr (X-linked, lymphocyte-regulated) protein.

The murine Xlr (X-linked, lymphocyte-regulated) gene family was originally identified by subtractive cDNA hybridization and cloning. It was found to encode two 30-kDa nuclear proteins expressed in lymphoid cells and in primary spermatocytes in a developmentally regulated manner. Our data show that, in contrast to most X-linked genes, the Xlr family is not conserved at the DNA level between mouse and human. However, using anti-Xlr antibodies, an Xlr-immunoreactive nuclear protein of M(r) 30,000 was characterized in human RAJI B-lymphoblastoid cells by flow cytofluorimetry, by immunoblotting, and by immunocytolabeling. An Xlr-like molecule was also found to be expressed in human activated lymphocytes and in human primary spermatocytes, with a stage specificity similar to that known in the mouse. In contrast, no Xlr-immunoreactive protein was detected in a series of human tissues including brain, skeletal muscle, colon, liver, and kidney, revealing a tissue-specific expression pattern similar to that of murine Xlr. These findings most likely identify a human equivalent of Xlr. The Xlr genes belong to a small category of X-linked genes, including STS, MIC2, CSF2RA, and KAL, that diverge at the DNA level in human and in mice. Characterization of the human XLR gene(s) should now be feasible with anti-Xlr antibodies and an expression cloning system. It should provide new insights into the evolution of mammalian X Chromosome (Chr).

Proacrosin as a marker of meiotic and post-meiotic germ cell differentiation: quantitative assessment of human spermatogenesis with a monoclonal antibody.

A quantitative immunohistochemical study of human spermatogenesis was performed using the 4D4 anti-proacrosin monoclonal antibody (mAb 4D4) as a marker of meiotic and post-meiotic germ cell differentiation. Cells from 15 testicular biopsies with normal spermatogenesis, 18 with slight and nine with marked hypospermatogenesis and six with maturation arrest were assigned to spermatogenic stages according to both nuclear maturation and proacrosin labelling patterns. The results showed that four spermatogenesis steps (mid- and late-pachytene primary spermatocytes, early and late spermatids) have to be separately considered for the classification of a given biopsy. Conversely, data from primary spermatocytes in the metaphase, anaphase and telophase stages and secondary spermatocytes did not show significant differences between biopsies. We conclude that: (1) slight hypospermatogenesis is due only to fewer cells entering meiosis, whereas in marked hypospermatogenesis there is also germ cell loss during the later meiotic steps and spermiogenesis; (2) the sloughing of germ cells from the epithelium could be of pathological significance; and (3) immunodetection with mAb 4D4 improves the assessment of spermatogenesis because it can label a protein expressed as early as meiotic prophase. In addition, mAb 4D4 labels a protein which is a marker of the Golgi complex allowing the detection of disturbances of cytoplasmic events during meiosis or spermiogenesis. Such an analysis is facilitated by mAb 4D4 labelling of paraffin-embedded sections.

The meiosis-specific Xmr gene product is homologous to the lymphocyte Xlr protein and is a component of the XY body.

The Xlr (X-chromosome linked, lymphocyte regulated) multigene family was previously found to determine, in the lymphoid cell lineage, the stage-specific expression of a nuclear protein with a primary sequence suggestive of a transcriptional activator function. We report here the characterization of a second functional member of the Xlr gene family that is abundantly transcribed in testis in a tissue-specific and developmentally regulated manner. The protein product of this newly identified gene, called Xmr (Xlr-related, meiosis regulated), is located in the nuclei of spermatocytes, early in the prophase of the first meiotic division, and later becomes concentrated in the XY nuclear subregion where it is in particular associated with the axes of sex chromosomes. The Xmr protein provides a new tool for the investigation of sex chromosome behaviour during meiosis in mammals.

Pregnancy after subzonal insemination with spermatozoa lacking outer dynein arms.

The absence of outer dynein arms in the sperm flagellum induces an abnormal movement pattern associated with male infertility. These spermatozoa can decondense in zona-free hamster oocytes but result in a very low fertilization rate in in vitro fertilization. We hypothesized that subzonal insemination could help achieve fertilization and pregnancy. A randomized prospective trial (five couples, five cycles) comparing subzonal insemination (n = 31 oocytes) and routine IVF (n = 23 oocytes) was carried out. Oocytes were microinjected with 8.5 +/- 3.6 spermatozoa. In a second series (nine cycles), all the oocytes were microinjected with 10.5 +/- 4.3 spermatozoa. In the randomized series, the fertilization rate was 16.1% without polyploidy, whereas no fertilization was obtained after control IVF insemination. In the second series involving nine couples, six of whom were included in the first series, the fertilization rate increased to 57.8% with a 27.8% polyspermic rate. Eighty-eight per cent of the zygotes cleaved normally (29 out of 33). A total of 11 embryo transfers resulted in three pregnancies, one of which terminated one month later, a second being ongoing and the third delivering a healthy girl. A 21.4% pregnancy rate per cycle, with a 37.5% pregnancy rate per couple, justifies the use of subzonal insemination to treat this particular flagellar dyskinesia.

[A new entity of sperm pathology: peri-axonemal flagellar dyskinesia]. / Une nouvelle entité pathologique du spermatozoïde: la dyskinésie flagellaire péri-axonémale.

The study of 17 infertile men has led to define a new entity of sperm pathology as part of the more general field of flagellar dyskinesias. Sperm parameters of the studied patients and a control series have been first estimated by routine analysis (concentration, motility, morphology). To precise their characteristics, kinetic and ultrastructural investigations, as the zona-free hamster oocyte penetration test, have been performed. Sperm parameters of the studied cases, as revealed by routine analysis, were close to the control group. However, a major kinetic anomaly was found which was characterized by an important decrease of the amplitude of lateral head displacement (1.6 microns vs 5.3 microns, p < 0.001), although the progressive velocity was only slightly impaired (20.3 microns vs 24.9 microns, p < 0.05). Electron microscopy revealed anomalies limited to the peri-axonemal structures such as the outer dense fibers and the fibrous sheath. Rates of sperm-oocyte attachment were normal but rates of oocyte penetration were low (27.7% of decondensed sperm heads vs 85.6%, p < 0.001). Attempts to assisted fertilization with the studied patients (51 cycles of insemination, 8 cycles of in vitro fertilization) were unsuccessful. All these data suggest that the infertility can be attributed to the movement disturbances which should impair sperm propulsion throughout the cervical mucus and the zona pellucida.