Human sperm tail fibrous sheath undergoes phosphorylation during its development.

The phosphorylation of the human sperm tail fibrous sheath as a maturational step during its development is reported for the first time. This was demonstrated using GDA-J/F3 and RT97 monoclonal antibodies (MoAb) which recognize the fibrous sheath. In indirect immunofluorescence microscopy of frozen sections of human adult testes, the two antibodies reacted with the assembled fibrous sheath only, but the numbers of sperm tails stained with RT97 were consistently lower than those treated with GDA-J/F3. Furthermore, by using double indirect immunofluorescence, although the majority of spermatozoa were doubly stained with the two MoAbs, some GDA-J/F3-positive sperm tails were negative with RT97. In epididymal and ejaculated spermatozoa, the two antibodies stained all the tails. This indicated that the ontogenic appearance of the GDA-J/F3 epitope precedes that of RT97. In Western blotting and/or indirect immunofluorescence of spermatozoa, treatment of samples with alkaline phosphatase abolished the reactivity of RT97 while that of GDA-J/F3 MoAb was not affected. This finding indicated that the RT97 but not the GDA-J/F3 epitope was phosphorylated. Together, these results therefore reveal that during tail morphogenesis, the fibrous sheath undergoes phosphorylation as part of its structural maturation. Screening of sperm cell precursors recovered from oligozoospermic donors showed reaction of some abnormal germ cells with GDA-J/F3 MoAb but not with RT97, suggesting the possible failure of phosphorylation of the fibrous sheath protein in these cells. The significance of these findings is discussed together with the biological importance of phosphorylation to the fibrous sheath.     

Isolation and characterization of human and rabbit sperm tail fibrous sheath

Using mechanical and chemical dissection methods, fibrous sheath was isolated both from normal ejaculated human spermatozoa and from rabbit cauda epididymal spermatozoa. The same techniques did not produce a pure preparation of fibrous sheath from ejaculated rabbit spermatozoa, suggesting that further cross-linking and stabilization of sperm structures occurs in response to components of the seminal plasma. The isolation procedures were monitored by phase contrast microscopy and the purity of the fibrous sheath was verified by electron microscopy. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human fibrous sheath revealed at least 14 protein bands of which the most intensely stained were of molecular weight 84, 72, 66.2, 57, 32 and 28.5 kDa. The rabbit fibrous sheath revealed at least 10 protein bands, of which the most intensely stained were 35.2, 32.7 and 28.5 kDa. The amino acid composition of the purified fibrous sheath from human and rabbit spermatozoa was similar, being high in aspartic acid and/or asparagine and glutamic acid and/or glutamine, serine, alanine, leucine, lysine and glycine, but low in histidine, tyrosine and isoleucine. This composition is similar to that reported for the rat and suggests that mammalian sperm tail fibrous sheaths are composed of similar types of proteins, although there are apparent differences in protein components between species.      

GDA-J/F3 monoclonal antibody as a novel probe for the human sperm tail fibrous sheath and its anomalies

GDA-J/F3 monoclonal antibody (MoAb) recognized an antigen in the fibrous sheath (FS) of human spermatozoa. This was based on: (i) intracellular localization of the antigen which was limited to the principal piece of the sperm tail; (ii) its absence from the cilia of trachea and nasal mucosa which lack FS; (iii) immunogold electron microscopy (IEM) which confirmed its ultrastructural localization to the FS. The antibody was used for the detection of abnormal germ cells in human semen. Nucleated cells other than spermatozoa (NCOS) obtained from oligozoospermic donors were screened with GDA-J/F3 MoAb using the indirect immunofluorescence test. The antibody which stains only the tails in normal cells, produced diffuse cytoplasmic immunofluorescence inside some spermatids. Using phase contrast microscopy, the tails in these spermatids were either present or undetectable. Electron microscopy studies of the NCOS showed the lack of the FS in those which had the tails (afibrous tail), or the presence of disorganized tails (tail dysgenesis) in the others. This antibody therefore provides a useful analytical tool for probing the FS as well as for the easy identification of certain abnormal germ cells in human semen.     

AJ-FSI monoclonal antibody detects a novel group of non-glycostlated antigens within the human sperm tail fibrous sheath

AJ-FS1 is one of a new series of monoclonal antibodies (MoAbs)raised by immunizing mice with isolated human sperm tail fibroussheath. Usinjg indirect immunofluorescence (IIF), the AJ-FS1MoAb didnot react with the surface antigens of viable sperm,but didstain the falgellar principal piece of sperm dried ontoslides or those demenbranated with 1% Triton X-100. The specificityof the antibody for the fibrous sheatht was confirmed by immunogoldelectron microscopy which showed the distrobution of gold particleson the outer fibrous sheath surface, and its reaction with theWestern blots of purified fibrous sheath preparations wheremultiple protein bands with mol. wt ranging between 97 and 28kDa were identified. The same peptides were alsod detected inurea-dithiothreitol (DTT) fraction of sequentially extractedspermatozoa but not in Triton or Triton-DTT sperm lysates. Thefailure of sodium metaperiodate to abrogate the antobody reactionin both Western blotting and IIf indicated on the non-glycosylatednature of the antigens. IIF screening of human testicular cryosatatsections with AJ-FS1 MoAb showed its reactivity with the assembledfibrous sheath of maturing sperm tails only; thus indicatingthe late apperance of the antigens during spermatogensis. Theanti-body did not react with skin, oesophagus, tongue, liver,kidney, placenta, uterus, cervix or their blood vessels. Thesignificance of these results is discussedtogether with theimportnce of AJ-FS1 MoAb as a specific probe for the characterizationof the fibrous sheath antigens in both normal and abnormal falgella.     

The target antigen for GDA-J/F3 monoclonal antibody in the human sperm tail fibrous sheath is a non-collagenous asialo-glycoprotein: implications and significance

Enzymes and chemicals were used to analyse the biochemical structureof the antigenic epitope recognized by GDA-J/3 monoclonal antibody(MoAb) in the human sperm tail fibrous sheath. Treatment ofsperm dried onto slides with trypsin or dispase enzymes abolishedtheir immunofluorescence staining with GDA-J%3 MoAb, thus indicatingthe proteinaceous nature of the antigen. The proteolytic cleavageof GDA-J%F3 protein by trypsin, which also caused sperm decapitation,indicated the presence of peptide bonds involving the carboxylgroups of the basic amino acids, arginine and%or lysine. Theepitope was also glycosylated as demonstrated by its sensitivityto sodium metaperiodate treatment which was dose–dependent.The GDA-J%F3 antigenic epitope lacked sialic acid since pre–treatmentof spermatozoa with sialidase enzyme (neur–aminidase)had no effect on their reactivity with the antibody. The lackof collagenous domains in the GDA-J%F3 antigen was demonstratedby the failure of collagenase to abrogate sperm immunostainingwith the MoAb. Furthermore, type VII collagen of the skin basementmembrane (BM) was previously thought of as a potential targetantigen for GDA-J%F3 MoAb. This was ruled out since severalmonoclonal and polyclonal antibodies failed to detect the antigenin the spermatozoa using immunofluorescence and Western blotting.These data, therefore, show that the target antigen for GDA-J%F3MoAb is a non-collagenous asialo–glycoprotein, and byinference provide the first evidence for the glycosylation ofthe sheath proteins as another step of post–translationalmodification occurring during sperm tail development.     

Gene deletions in an infertile man with sperm fibrous sheath dysplasia

BACKGROUND: Asthenozoospermia may sometimes be related to geneticstructural defects of the sperm tail detectable by transmissionelectron microscopy. Dysplasia of the fibrous sheath (DFS) isa genetic sperm defect, characterized by dysplastic developmentof the axonemal and periaxonemal cytoskeleton. We report thecase of an infertile man with normal sperm count and total spermimmotility in which dysplasia of the fibrous sheath, Akap3,Akap4 gene deletions, meiotic segregation of chromosomes 18,X and Y and Y microdeletions were investigated. METHODS: A 32-year-oldman with a 3-year history of primary infertility presented atour Regional Referral Center for Male Infertility. Family medicalhistory, lymphocyte karyotype, PCR analysis, physical examination,hormone assays and semen analysis were performed. RESULTS: Ultrastructuralsperm evaluation showed dysplasia of the fibrous sheath. Immunostainingof AKAP4 protein was negative in sperm tails. PCR analysis revealedintragenic deletions of the Akap3 and Akap4 genes. Fluorescencein situ hybridization on sperm showed a high frequency of XYdisomy. CONCLUSION: In this infertile patient, our results suggesta possible relationship between dysplasia of the fibrous sheath,partial deletions in the Akap3 and Akap4 genes and absence ofAKAP4 protein in the fibrous sheath. These findings, however,were not detected in another four patients with dysplasia ofthe fibrous sheath. Our results require future confirmatorymolecular analyses.     

Sperm ubiquitination in patients with dysplasia of the fibrous sheath

BACKGROUND: Human sperm with structural abnormalities display an increased content of the cellular proteolytic marker peptide, ubiquitin. We investigated whether dysplasia of the fibrous sheath (DFS), a severe structural anomaly found in the sperm of some asthenozoospermic patients, is accompanied by (i) increased ubiquitination of the sperm surface and (ii) by increased ubiquitination of the sperm mitochondria. METHODS AND RESULTS: Five DFS patients and eight fertile donors were studied by immunocytochemistry with anti-ubiquitin antibodies. Increased cross-reactivity of the ubiquitinated mitochondrial epitopes was seen in 32-50% of DFS sperm, but only 2-4.1% of sperm from fertile donors. Sperm surface ubiquitination assessed by sperm-ubiquitin tag immunoassay (SUTI) and immunofluorescence demonstrated an increased sperm ubiquitination in all DFS patients. The average median value of ubiquitin-induced fluorescence in DFS patients was 25.8 counts (range 19.8-37.9), as opposed to 13.4 counts range (9.3-16.6) in fertile men. Sperm with 'stump tails', coiled tails, twin and triplet sperm, and clusters of immature spermatogenic cells were common. CONCLUSIONS: DFS sperm have increased cross-reactivity to anti-ubiquitin antibodies, a finding consistent with the ubiquitination of defective sperm shown in animal models. These results justify the use of ubiquitin-based assays for objective semen analysis in infertile men with heritable defects.     

Ultrastructural pathology of the sperm flagellum: association between flagellar pathology and fertility prognosis in severely asthenozoospermic men

An ultrastructural study of spermatozoa in a series of 247 severely asthenozoospermic patients disclosed two kinds of anomalies. The first was dysplasia of the fibrous sheath, a primary defect of spermatozoa with hypertrophy and hyperplasia of the fibrous sheath, associated axonemal anomalies, familial incidence and chronic respiratory disease. The patients could be divided into two subgroups: the complete form (all spermatozoa affected) and the incomplete form (alterations in 70- 80% spermatozoa). There were no spontaneous or in-vitro fertilization (IVF) pregnancies. Intracytoplasmic sperm injection (ICSI) in six patients resulted in successful fertilizations, but only two pregnancies were obtained. These features configure a phenotype that suggests a genetic origin. The second anomaly was non-specific flagellar anomaly (NSFA), random secondary flagellar alterations affecting variable numbers of spermatozoa, without respiratory disease or familial incidence. 54 men with NSFA were followed for 2-6 years. Of these, 18 achieved conception, either spontaneous or by means of assisted fertilization, followed by 14 pregnancies and 12 live births. Their sperm motility significantly increased during the follow-up period. In the remaining 36 men motility did not change during the follow-up period and there were no fertilizations or pregnancies. We conclude that in severe asthenozoospermia, ultrastructural examination of spermatozoa has an effective prognostic value, identifying two syndromes with very different flagellar alterations and fertility potentials.      

Isolation and biochemical characterization of the human sperm tail fibrous sheath.

The isolation and biochemical characterization of the human sperm tail fibrous sheath (FS) is described for the first time. Initially, the solubilization properties of the FS were assessed immunocytochemically using GDA-J/F3 and RT97 monoclonal antibodies (MoAbs) and morphologically by electron microscopy. Following extensive investigations to optimize the conditions for the FS isolation, a simple method was developed which involved sequential extraction of the flageller components with Triton-dithiothreitol (DTT) and urea-DTT. The procedure was monitored by phase contrast microscopy and the purity of the FS preparations was confirmed by electron microscopy. SDS-PAGE of the isolated FS revealed seven major protein bands with mol. wt of 97, 76, 62, 55, 33, 28 and 25 kDa. In Western blotting, the reaction of RT97 MoAb with supernatants from the various extraction steps and the isolated FS indicated that its target antigen (AJ-p97) was an integral FS product and that disulphide bonding was probably involved in its stabilization. The reactivity of normal and aprotruded sperm tails with GDA-J/F3 and RT97 MoAbs was not affected by Triton while the GDA-J/F3 staining of the cytoplasmic matrix of other abnormal spermatids was abolished, thus suggesting variation in the biochemical properties of GDA-J/F3 in normal and abnormal germ cells. These and other data indicate that the FS could be a modified form of intermediate filament.     

Morphogenesis of the fibrous sheath in the marsupial spermatozoon

The spermatozoon fibrous sheath contains longitudinal columns and circumferential ribs. It surrounds the axoneme of the principal piece of the mammalian sperm tail, and may be important in sperm stability and motility. Here we describe its assembly during spermiogenesis in a marsupial, the brush-tail possum, and compare its structural organization with that of eutherian mammals, birds and reptiles. Transmission electron microscopy showed that possum fibrous sheath assembly is a multistep process extending in a distal-to-proximal direction along the axoneme from steps 4 to 14 of spermiogenesis. For the most part, assembly of the longitudinal columns occurs before that of the circumferential ribs. Immunohistochemical and immunogold labelling showed that fibrous sheath proteins are first present in the spermatid cytoplasm; at least some of the proteins of the sheath precursors differ from those in the mature fibrous sheath. That immunoreactivity develops after initiation of chromatin condensation suggests that fibrous sheath proteins, or their mRNAs, are stored within the spermatid cytoplasmic lobule prior to their assembly along the axoneme. These findings are similar to those in laboratory rats, and thus suggests that the mode of fibrous sheath assembly evolved in a common ancestor over 125 million years ago, prior to the divergence of marsupial and eutherian lineages.     

A pathology of the sperm centriole responsible for defective sperm aster formation,syngamy and cleavage

BACKGROUND: In the present report we analyse the structural and functional features of sperm from a patient with severe asthenoteratozoospermia and failure of cleavage after ICSI. METHODS: Sperm were studied by phase contrast and transmission electron microscopy and microinjected into bovine oocytes to examine aster formation using antibodies against acetylated alpha- and beta-tubulins. RESULTS: Acephalic sperm, headless tails and abnormal alignments of the head-tail junction were observed. Flagella evidenced the features of dysplasia of the fibrous sheath. Bovine oocytes injected with patient's sperm showed male and female pronuclei but a faulty development of microtubules from the sperm-derived centrosome. The first ICSI attempt using conventional sperm selection methods resulted in fertilized two pronuclei zygotes, but no syngamy or cleavage. Three more ICSI attempts were performed, carefully avoiding sperm with obvious anomalies of the connecting piece. Fertilization and cleavage took place in all cycles, and in two of them positive betahCG plasma levels were detected but preclinical abortions ensued. CONCLUSIONS: We propose that the alterations in the head-tail junction and attachment, responsible for the observed sperm phenotype, result from centriolar dysfunctions that cause insufficient sperm aster formation, lack of syngamy and cleavage or defective embryos leading to early abortions.     

Three cases of genetic defects affecting sperm tail: a FISH study

Submicroscopic alterations in the cytoskeletal structure of sperm flagellum are associated with severely reduced or completely absent motility in subfertile or infertile men. Sometimes these alterations can be related to well known genotypic defects when the same anomaly affects the whole sperm population. Transmission electron microscopy (TEM) is the only tool able to specifically characterize the morphological features of genetic sperm defects. In this study, the frequencies of aneuploid and diploid spermatozoa were identified in three patients showing specific flagellar anomalies, each of them affecting the whole sperm population: dysplasia of the fibrous sheath, primary ciliary dyskinesia and absence of fibrous sheath. All these defects were highlighted by TEM. Fluorescence in situ hybridization (FISH) analysis was performed on decondensed sperm nuclei for chromosomes 18, X and Y, highlighting higher diploidies and sex chromosome disomies in cases of dysplasia of the fibrous sheath and primary ciliary dyskinesia, in agreement with other reports. We have also described FISH results in spermatozoa with absence of fibrous sheath. In this case, the only one reported due to the rarity of this defect, the aneuploidies and diploidies were within normal range. These data contribute to the growing evidence that genetic sperm defects of sperm flagella are generally correlated with meiotic segregation derangement. For this reason, genetic counseling is advisable, although all the genes involved and the possible mechanisms of these mutations have not yet been fully characterized.     

Andrology: The use of GDA-J/F3 monoclonal antibody for the detection of dead spermatozoa (necrosperm) during semen analysis

The distinction between immotile necrosperm (dead spermatozoa)and those with immotility due to other causes is of the utmostclinical importance. The supravital dyes currently used forthe identification of necrosperm are not highly reliable oraccurate. In this study, GDA-J/F3 monoclonal antibody (MoAb)which reacts with the fibrous sheath (FS) was used as a specificprobe for the detection of necrosperm using indirect immunofluorescence(IIF). Previously, several lines of evidence indicated the reactionof the antibody with necrosperm. This was confirmed in the currentstudy where GDA-J/F3 MoAb failed to react with viable swim-upseparated spermatozoa; such cells were only stained followingsperm demembranation with 1% Triton X-100. Furthermore, by usingimmunogold electron microscopy of a normozoospermic sperm sample,all the spermatozoa which reacted with GDA-J/F3 MoAb showeddamaged cytoplasmic membranes. Following these initial studies,sperm samples were obtained from 42 men attending infertilityclinics and assessed by conventional semen analysis and GDA-J/F3MoAb screening using IIF. The results showed a wide variationin sperm immotility and GDA-J/F3 reactivity; the ranges were19–99 and 0–50% respectively. This novel immunologicalapproach provides a simple and specific method of necrospermenumeration for the investigation of male infertility.     

Genetic sperm defects and consanguinity.

BACKGROUND: The existence of a genetic component to human infertility has been suggested, although neither the specific abnormalities involved, nor their genetic mechanism of transmission, are currently defined. We have examined, by transmission electron microscopy (TEM), ejaculate from 1600 males with fertility problems. Among the subjects studied, we focused on a group of patients whose family histories revealed different degrees of consanguinity, in order to evaluate the relationship between consanguinity and particular sperm alterations. METHODS AND RESULTS: A total of 64 consanguineous individuals were identified. In this group, excluding two azoospermic patients, 17 patients (27%) were found to have well recognized genetic ultrastructural defects affecting their entire sperm population: eight subjects had spermatozoa with "stunted tails", four "detached tail" spermatozoa, two "Kartagener's syndrome", two "miniacrosome" and one "round headed" spermatozoa. Since these alterations affect the total sperm population and do not respond to medical treatment, they are suspected of having a genetic origin. The remaining group of 1506 non-consanguineous patients suffered from the same genetic defects in only 15 cases (<1%). CONCLUSIONS: From the data presented, it appears that some very peculiar and rare sperm defects may have a genetic basis since they occur more frequently in consanguineous patients, and are related to different degrees of consanguinity. Since the ejaculate of the remaining patients, both consanguineous and not, showed diverse types of ultrastructural sperm anomalies that did not affect the entire sperm population, they might represent pathologies lacking a genetic basis.     

Origin, development and ultrastructure of boar spermatozoa with folded tails and with two tails.

Spermatozoa from the three epididymal regions (head, body and tail) of healthy and sexually mature boars have been examined by light microscopy, and scanning and transmission electron microscopy. The origin, development and external and internal morphologies of aberrant spermatozoa with folded tails and spermatozoa with one or two heads and two fused tails have been established. A count carried out in each region of the epididymis indicated that significant differences (P less than 0.01) exist in the frequencies of each type of malformation and the epididymal region from which the spermatozoa come. Spermatozoa with folded tails at Jensen's ring originate in the cauda of the epididymis from immature spermatozoa that have not ejected the distal cytoplasmic droplet. The plasma membrane which covers the main piece is fused with the membranes of the midpiece, the connecting piece and the head. The fibrous sheath deforms the mitochondrial sheath and is placed between the plasma membrane and the postacrosomal dense lamina. Spermatozoa with one head and two fused tails originate in the epididymal body from spermatozoa with one head and two unfused tails coming from the cephalic region of the epididymis. Spermatozoa with two heads and two fused tails originate in the cephalic region of the epididymis by head-to-head agglutination of two spermatozoa and later fusion of their tails. The frequency of spermatozoa with two fused tails increases as they progress through the epididymal duct. Their tails, parallel in monocephalic spermatozoa and helicoid in bicephalic spermatozoa, have two complete axonemal axes. In their midpiece, the mitochondrial sheaths of the two axes are fused, producing an 8-shaped sheath.     

Morphometric assessment of mature and diminished-maturity human spermatozoa: sperm regions that reflect differences in maturity.

As part of our studies on sperm maturity and function, we examined the head, midpiece and tail of human spermatozoa using computerized morphometry in order to determine which regions reflect the differences between mature spermatozoa and spermatozoa of diminished cellular maturity. We studied 20 men, who were divided into two groups based on their lower (LCKM: 14.6 +/- 7.0%, n = 8) and higher sperm creatine kinase (CK-M) isoform ratios (HCKM: 48.0 +/- 4.3%, n = 12) in the initial semen. Using a sequential centrifugation method which relies on the lower density of immature spermatozoa with retained extra cytoplasm, we prepared three sperm fractions with progressively declining maturity, as confirmed with CK-M isoform ratio measurements. Following the sequential fractionation, we affixed the spermatozoa to glass slides, stained the midpiece and the sperm contour, and photographed 25 spermatozoa in each of the 60 fractions (1509 spermatozoa in all). The spermatozoa were then individually digitized on the Image-1 system, and the dimensions of the head, midpiece, and tail were determined. While the data showed significant differences in the midpiece and tail dimensions between the mature and diminished-maturity sperm fractions, the head dimensions were similar and did not reflect sperm maturity. We postulated that the relationship between the biochemical markers of sperm maturity and sperm morphology is based on common spermiogenic events. The data support this idea. In immature spermatozoa in which cytoplasmic extrusion, CK-M isoform expression, and tail sprouting are all diminished, the retained extra cytoplasm in the midpiece and shorter tail length contribute to the morphological variations that we identified by morphometry and considered in sperm morphology. These morphometric features, in association with fluorochrome-coupled biochemical probes, can facilitate the identification of mature spermatozoa in computer-assisted semen analysis.     

Sperm autoantigens and fertilization. III. Ultrastructural localization of guinea pig autoantigens

Guinea pig sperm autoantigens have been localized by direct and indirect immunoferritin techniques in (1) plasma membrane over the entire sperm head, (2) acrosomal contents, (3) fibrous sheath and outer dense fibers of the tail filament, and (4) the inner acrosomal membrane of 50% of acrosome reacted spermatozoa. These cellular structures are known to be involved in guinea pig sperm rouleaux formation, acrosome reaction, interaction of acrosome-reacted sperm with zona pellucida and with the vitellus of guinea pig ova. Since IgG and Fab of autoantiserum to guinea pig spermatozoa have been shown to interfere with these cellular reactions, this study provides further evidence, albeit indirect, that sperm autoantigens are involved in these cellular events.     

Immunology: AJ-FS9 monoclonal antibody detects masked antigens within the human sperm tail fibrous sheath

AJ-FS9 is one of a new series of monoclonal antibodies (mAbs)raised by immunizing mice with isolated human sperm tail fibroussheath (FS). Using indirect immunofluorescence (IIF) of humanspermatozoa dried onto slides, the AJ-FS9 mAb reacted with theprincipal piece of occasional spermatozoa. Following their enzymatictreatment with trypsin, dispase or collagenase, but not sulphatase,all the spermatozoa were stained at their principal piece. Theultrastructural localization of the antigens to the FS was establishedby immunogold electron microscopy, which showed the distributionof gold particles on the FS outer surface of spermatozoa sequentiallytreated with 1% Triton and dispase; spermatozoa demembranatedby Triton alone showed no reaction. For biochemical characterization,spermatozoa were lysed with 1% Triton, and the sperm pelietwas run through a reducing sodium dodecyl sulphate-polyacrylamidegel electrophoreesis, Western blotted and immunostained withAJ-FS9. The results showed the reaction of the antibody withthree protein bands with molecular masses ranging between 46and 56 kDa. IIF screening of human testicular cryostat sectionswith AJ-FS9 mAb showed its reactivity with occasional spermtails; but following their dispase treatment, all spermatozoawere stained. The restricted staining of the assembled FS ofmaturing sperm tails indicated the late appearance of the antigensduring spermatogenesis. The antibody did not react with spermcell precursors or other cell types within/without the seminiferoustubules. Untreated and dispase-treated frozen sections of skin,oesophagus, tongue, liver, kidney, stomach, ileum or their bloodvessels showed no reaction. These data provide the first evidencefor the presence of masked antigens within the human sperm tailFS, and their significance is discussed.     

Easily decapitated spermatozoa defect: a possible cause of unexplained infertility.

We report the first 16 cases of a new sperm abnormality which we call 'easily decapitated spermatozoa defect'. This was discovered during intracytoplasmic sperm injection (ICSI) in couples with unexplained infertility. Semen analysis was normal, but minimal micromanipulation for ICSI resulted in decapitation of the spermatozoon during immobilization. For some oocytes the head and tail were injected separately, in others the intact sperm was injected after minimal immobilization. A fertilization rate of 47.5% was obtained using ICSI. Conventional in-vitro fertilization (IVF) on sibling oocytes (three cases) or in a previous cycle (three cases) resulted in total failure of fertilization. All patients reached the embryo transfer stage and three pregnancies resulted. Findings on electron microscopy in four cases included spermatozoa with degeneration or absence of the basal plate, abnormalities of the proximal centriole and degeneration of the midpiece with a large cytoplasmic droplet. We conclude that an occult sperm abnormality presenting as easily decapitated spermatozoa during ICSI could be a cause of unexplained infertility, as it resulted in total failure of fertilization in conventional IVF. Further research is necessary to investigate this sperm abnormality.     

Sperm selection for ICSI: shape properties do not predict the absence or presence of numerical chromosomal aberrations

BACKGROUND: We hypothesize that the potential relationship between abnormal sperm morphology and increased frequency of numerical chromosomal aberrations is based on two attributes of diminished sperm maturity: (i) cytoplasmic retention and consequential sperm shape abnormalities; and (ii) meiotic errors caused by low levels of the HspA2 chaperone, a component of the synaptonemal complex. Because sperm morphology and aneuploidies were assessed in semen, but not in the same spermatozoa, previous studies addressing this relationship were inconclusive. We recently demonstrated that sperm shape is preserved following fluorescence in situ hybridization (FISH). Thus, we examined the shape and chromosomal aberrations in the same sperm. METHODS: We performed phase contrast microscopy and FISH, using centromeric probes for chromosomes X, Y, 10, 11 and 17 in 15 men. The fluorescence and respective phase contrast images were digitized using the Metamorph program. We studied 1286 sperm (256 disomic, 130 diploid and 900 haploid sperm) by three criteria: head and tail dimensions, head shape and Kruger strict morphology. Furthermore, in each analysis, we considered whether disomic or diploid sperm may be distinguished from haploid sperm. RESULTS: There was an overall, but not discriminative, relationship between abnormal sperm dimensions or shape and increased frequencies of numerical chromosomal aberrations. However, approximately 68 of the 256 disomic, and four of 130 diploid sperm showed head and tail dimensions comparable with the most normal, lowest tertile of the 900 haploid spermatozoa. Considering all 1286 sperm, among those with the most regular, symmetrical shape (n = 367), there were 63 and five with disomic and diploid nuclei, respectively. In line with these findings, among the 256 disomic sperm, 10% were Kruger normal. CONCLUSIONS: Sperm dimensions or shape are not reliable attributes in selection of haploid sperm for ICSI.