Sperm with fibrous sheath dysplasia and anomalies in head-neck junction: focus on centriole and centrin 1.

Spermatozoa with a rare combination of two monomorphic sperm defects, dysplasia of the fibrous sheath (DFS) and alterations in head-mid-piece junction were analysed. The main focus was to explore the status of the centriole, a key organisation during fertilisation, using the centrin 1, a calcium-binding protein linked to this structure. The sperm quality was examined by light, scanning and transmission electron microscopy (SEM, TEM); immunocytochemistry was performed for tubulin, A-kinase anchor protein 4 (AKAP4) and centrin 1. Spermatozoa showed DFS defect associated with anomalies in head-tail attachment detected by SEM and TEM. Immunolocalisation of tubulin, AKAP4 and centrin 1 confirmed these alterations. Centrin 1 was visible in 67% of spermatozoa (in only 13% centrin localised in a normal position); in the majority of sperm centrin 1's location was altered, sometimes bent; often four spots, indicating the presence of two implantation fossae, were detected. At the centriolar level, immunoreactive fragments, frequently invading the entire short and thick tail, were observed. Centrin 1 is an essential component of the spermatozoa connecting piece and plays a role in centrosome dynamics during sperm morphogenesis and in zygotes and early embryos during spindle assembly. It is important to shed light on these rare conditions in order to better manage the patients during assisted reproductive technology.

In vitro effect of gold and silver nanoparticles on human spermatozoa.

The cytotoxicity of Au/Ag nanoparticles (NPs) on human spermatozoa was investigated in vitro. Semen from donors were incubated (37 °C, 60'-120') with 30, 60, 125, 250 and 500 µM Au/Ag-NPs. Sperm motility was evaluated following WHO guidelines; sperm viability was assessed with eosin Y test. Au-NPs were characterised and localised with field emission gun-based scanning transmission electron microscope/energy dispersive spectroscopy and transmission electron microscopy. Both tested NPs exerted a significant dose-dependent effect on motility and viability of human spermatozoa (P < 0.001). Ag-NPs seem to show a slightly elevated toxicity although not significant (P > 0.05). Au-NPs were localised in spermatozoa, whereas Ag-NPs were undetectable. In conclusion, Au-NPs and Ag-NPs do not appear to be harmful for human spermatozoa up to high concentrations (250-500 µM) that are probably difficult to reach in vivo. It is mandatory to explore the genotoxic effect of NPs in germ cells.

Effects of diadenosine polyphosphates and seminal fluid vesicles on rabbit sperm cells.

Membrane vesicles were isolated from rabbit seminal plasma. Electron microscopy analyses showed the presence of numerous small, round vesicles with a diameter of about 70 nm. Determination of enzyme activities was carried out by high performance liquid chromatography and showed that the vesicles can degrade the diadenosine polyphosphates (ApnA), Ap3A and Ap4A and ATP and ADP, but not AMP. Studies of the degradation of diadenosine compounds by the vesicles present in seminal fluid showed an increasing production of AMP as the by-product and a time-dependent generation of dephosphorylated products consistent with the presence of ecto-ATP diphosphophosphatase (ecto-apyrase). In the presence of rabbit spermatozoa, AMP did not accumulate because 5'nucleotidase and adenosine deaminase, present at the surface of sperm cells, transformed AMP into adenosine and inosine. The effects of seminal fluid vesicles and diadenosine compounds on the acquisition of fertilizing capacity by rabbit spermatozoa were evaluated by Pisum sativum agglutinin fluorescein isothiocyanate conjugated staining. The results obtained with uncapacitated spermatozoa showed that the capacitating effector BSA could be substituted efficiently by the addition of diadenosine compounds and vesicles previously incubated for 2 h to the capacitative medium. Under these experimental conditions, the spontaneous acrosome reaction rate was not increased. Capacitated rabbit spermatozoa did not undergo acrosome reaction when l-alpha-lysophosphatidylcholine was substituted by diadenosine compounds previously incubated with vesicles. In conclusion, this study has shown that rabbit seminal fluid vesicles can degrade diadenosine compounds to AMP and that the addition of the vesicles and diadenosine compounds to uncapacitated rabbit spermatozoa favours the acquisition of the fertilizing capacity.

Culture and co-culture of DU145 prostate carcinoma, osteoblasts and HT-29 colon carcinoma cells on a fabricated type III collagen matrix.

DU145 prostate carcinoma cells cultured on type III collagen possessed a highly migratory potential which was twice as much as HT-29 colon carcinoma cells. Prior to attachment to collagen, DU145 cells were highly reactive for fibronectin and after attachment clear zones between cells and collagen suggested protease activity. HT-29 cells attached to type III collagen forming dome-like polyps, however, tight and/or gap junctions were not observed. hFob osteoblasts were co-cultured with DU145 to establish a prostate cancer-collagen matrix barrier-bone cell metastasis model. Osteoblasts maintained their differentiated osteoblastic characteristics on one side of the collagen barrier, demonstrating high alkaline phosphatase, osteocalcin and insulin growth factor (IGF) activities. hFob cell growth was prominent adjacent to demineralized bone matrix particles (BMPs) embedded in type III collagen. The collagen matrix was deteriorated on the DU145 side of the collagen barrier. The DU145-collagen III-hFob model will allow an evaluation of the influence of the matrix on prostate cancer-bone cell interaction and regulation by growth factors.

Helicobacter pylori infection and infertility.

OBJECTIVE: To determine (1) the prevalence of Helicobacter pylori infection in male and female patients with reproductive disorders and controls; (2) the presence of anti-H. pylori antibodies in samples of follicular fluid, vaginal secretions and sperm; and (3) the existence of a structural homology between a major spermatozoa protein, tubulin, and H. pylori proteins. PATIENTS AND METHODS: Serum samples from 167 patients with infertility and 837 age- and gender-matched controls (blood donors) were examined by enzyme-linked immunosorbent assay (ELISA) and Western blotting to determine the seropositivity for H. pylori infection. The presence of anti-H. pylori antibodies in samples of follicular fluid, vaginal secretions and sperm was determined using the same techniques. The possible cross-reactivity with spermatozoa of anti-H. pylori hyperimmune sera and human antibodies was studied by immunofluorescence. The N-acid homology of human tubulin with the principal H. pylori proteins was assayed by the WU-blastp program available on the Internet. RESULTS: The prevalence of infection was significantly higher in patients than controls (49.1% v. 33.5%, P < 0.001). Follicular fluids from infected patients contained specific antibodies in all cases, sperm samples in about 50% of cases, and vaginal secretions in a minority of cases. Sera to H. pylori whole antigens and VacA reacted with the tails and the pericentriolar area of human spermatozoa (which are rich in tubulin); sera to urease and heat-shock protein (Hsp) did not. Follicular fluids with anti-H. pylori antibodies immune reacted with spermatozoa. A linear homology was found between beta-tubulin and three H. pylori proteins, flagellin, VacA and CagA. CONCLUSIONS: H. pylori infection may increase the risk of developing reproductive disorders or worsen the clinical expression of this syndrome.

Notulae seminologicae. 4. Mathematical evaluation of submicroscopical alterations in spermatozoa of sterile men with varicocele.

In this paper the mathematical formula recently proposed by Baccetti & Mirolli in this journal is used for the quantitative electron microscopical evaluation of six submicroscopical defects frequently present in the spermatozoa ejaculated by patients affected by various degrees of varicocele. All these characteristics are independent from each other and are in some way related to imperfect sperm maturation. They concern the shape and position of the acrosome, the status of the chromatin, of the mitochondria, of the axoneme, the individuality of the cell. The incidence in the sperm population of these submicroscopical characters is clearly related to the degree of the affection as estimated by usual clinical parameters. This demonstrates that the electron microscopical sperm analysis evaluated by the present formula is a very sensitive tool in revealing fine prognostical parameters.

Notulae seminologicae. 1. New combinations of Kartagener's syndrome.

In this note new consequences of the Kartagener's syndrome are described. In males the syndrome involved a diffused sperm immaturity; in one female severe skeletal defects were present probably resulting from bad organization of the renal apparatus.

Notulae seminologicae. 2. The 'short tail' and 'stump' defect in human spermatozoa.

In this note several cases of stunted tails involving the total sperm population in sterile humans are described. Half of the cases are classified as 'short tailed' spermatozoa, the other half as 'stump defect' previously described in bulls. Both defects are referred in details at electron microscopical level.

Studies on varicocele. 1. Submicroscopical and endocrinological features.

In this study we have selected a group of patients affected by a more or less severe condition of varicocele. After the evaluation of spermatogenesis and sperm function by electron microscopy we have demonstrated that the sperm malformations are mostly due to immaturity. Subsequently we have observed low FSH levels in the blood, concomitant with inhibin high contents, and we have studied Sertoli cells at submicroscopical level. In conclusion we suggest the following mode of action of varicocele in endocrinologically and spermatologically altered patients: varicocele----Sertoli cells----increased inhibin----hypophysis----decreased FSH----decreased testosterone----aberrant spermatogenesis----immature spermatozoa. The research will continue.

HIV particles detected in spermatozoa of patients with AIDS.

In this paper the Authors describe the presence of HIV particles on and in mature spermatozoa either ejaculated by AIDS patients or incubated in vitro with HIV. Both kinds of spermatozoa have particles localized around the sperm organelles. In the first case, the nucleoid of the virus can be enveloped by a membrane-like coat or be devoid of it and form buddings in the plasma membrane. In the in vitro infected spermatozoa, only membrane enveloped nucleoids are present, and no process of budding can be found. The Authors conclude considering that the spermatozoa of the AIDS patients can be penetrated by the virus particles in different moments of their life, and show the HIV particles in different stages of their cycle: some of them have freshly penetrated the sperm, and are still contained in a membrane-like coat, others are replicated and are budding through the sperm plasma membrane. On the contrary, in vitro infected spermatozoa have only freshly penetrated virus particles, and lack buddings and membrane-free nucleoids. The presence of the HIV virus in spermatozoa is substantiated by labelling with monoclonal or polyclonal anti-HIV antibodies.

A "miniacrosome" sperm defect causing infertility in two brothers.

A sperm defect causing infertility in two brothers is described. The malformation, named the "miniacrosome sperm defect," occurs in the total sperm population. It involves both the nuclei, which have a roundish apex, and the acrosomes, which are small, located in an apical position on the top of the nucleus, and sometimes ring-shaped. The midpiece and tail segments are normal, and the sperm are perfectly motile but fail to fertilize zona-free hamster ova in vitro. In spermatids, the absence of a microtubular manchette and reduction of the Golgi complex are evident.

Morphogenesis of the decapitated and decaudated sperm defect in two brothers.

The "decapitated sperm" defect, found in both of two sterile brothers, may be assumed to have a genetic origin. The present material suggests that the term "decapitated spermatozoa" is not exact, because detached heads and tails were found in the brothers' ejaculate that could be regarded as "decapitated tails" and "decaudated heads." The present report describes frequent, more or less advanced stages of detachment. Both heads and tails showed a normal structure in which only the postnuclear region was deficient, lacking basal plate and implantation fossa. A break at a different level of the midpiece, and therefore three kinds of separation, were observed. The defect, according to the present research, must originate in the testicular region, whereas the detachment occurs in the epididymis.

Localization of acrosomal enzymes in Arthropoda, Echinodermata and Vertebrata.

The presence and localization of acrosin and hyaluronidase have been detected by immunocytochemistry in animals belonging to different phyla and characterized by the different structure of the acrosomal complex. Acrosin is widely distributed from Insecta to Echinodermata and Vertebrata; hyaluronidase has a similar diffusion, but seems to be absent from an Insect and from Echinodermata. The presence of extraacrosomal layer and perforatorium seems not to be related to that of the two enzymes, that are absent from Teleost species devoid of acrosome or having only a pseudoacrosome.

Crater defect in human spermatozoa.

This report describes the "crater defect" in human spermatozoa, a malformation that consists of a nuclear and acrosomal invagination present in 100% of the cells, whereas tail structure and motility are fairly normal. The defect occurs during spermiogenesis. A possible concomitance with abnormalities in the microtubular apparatus involved in the sperm molding is discussed.

Polarized site of sperm entrance in the egg of a freshwater bivalve, Unio elongatulus.

We studied the organization of the egg of a freshwater bivalve, Unio elongatulus. This egg is markedly polarized. At the vegetal pole there is a crater which constitutes the point of attachment of the growing oocyte to the ovarian wall. This has previously been interpreted as a micropyle. We show that the sperm does not enter the egg through the crater but in a differentiated region around it, mostly at its base. This region is characterized by a wrinkled surface and is the only site of the vitelline coat which specifically binds the lectin from Lotus tetragonolobus. The egg reacts explosively upon fertilization, ejecting vacuolar material from the crater. The role of this "egg reaction" in relation to the prevention of polyspermy is discussed.

Immunocytochemistry and sperm pathology.

In this paper the Authors describe the localization of some significative proteins (acrosin, actin, tubulin, vimentin) detected by immunocytochemical methods in human infertile spermatozoa and correlate their distribution with the ultrastructural characteristics of the same spermatozoa, in order to clarify the structural bases of infertility.

Action of gossypol on rat germinal cells. II. The acrosome.

The action of gossypol on the acrosomal complex in rats has been investigated by microscopical and submicroscopical methods. The drug displays its morphological action only on spermatozoa during the transit through the epididymis, causing malformations and vesiculations. It appears to exert a primary action on the S-S groups formation, disturbing morphological molding occurring in this period and inhibiting capacitation.

Sperm morphogenesis, structure and function in humans over 70 years old.

Semen and testicular biopsy samples from different aged human donors have been examined for any age related changes in sperm morphogenesis, structure and function. Various abnormalities and defects, some being unique to spermatozoon of elderly men, were observed at the light and electron microscope levels. In spite of this, the best fractions of ejaculated spermatozoa population maintains his function, as demonstrated by some complete egg penetrations observed with the 'zona free' hamster egg test system.

Scanning electron microscopy and human sperm pathology.

This paper has been carried out in order to inquire whether some of the best known human malformations can be recognized by scanning electron microscopy. The SEM appearance of "straight tailed", "empty tailed", "short tailed", "round headed", "double", "old" human spermatozoa is described, and related to the inner structure. Straight tails and empty tails are quite evident at SEM. These defects are due to severe axonemal defects, related to dynein and tubulin deficiencies: "9+0", "arm less" and "axoneme less" spermatozoa are included in this category. Short tails and round heads are still more evident defects, due to complete absence of tail structures or acrosome and easily recognizable at SEM. Double spermatozoa are consistently in patients having abnormally high prolactin level and SEM is obviously sufficient for the diagnosis. Finally, spermatozoa in aged individuals show in SEM a peculiar shape due to absence of immature stages, reduced cytoplasm, disordered axoneme. It is concluded that SEM examination is a good tool for the identification of most of the best known human sperm abnormalities.

Human dynein and sperm pathology.

Human spermatozoa with normal structure and with different axonemal deficiencies (absence of axoneme, of arms, or of central structures) were studied by electron microscopy, SDS-polyacrylamide gel electrophoresis, and ATPase activity measurements. Normal human sperm possess a complement of high molecular weight polypeptides with an electrophoretic migration similar to that of sea urchin and other mammalian sperm dyneins. Human high molecular weight bands are numbered one to four in order of increasing of electrophoretic mobility; all of them are absent in spermatozoa that lack axoneme. The absence of doublet arms, coincides with the absence of bands 2, 3, and 4; the absence of central structures coincides with a reduction in intensity of band 2. In the latter two abnormal conditions, band 1 has an increased intensity. The data are tentatively interpreted by attributing the polypeptides forming bands 3 and 4 to the arm structure, whereas band 2 is supposed to contain a mixture of polypeptides localized in the arms and in the central structures; these abnormal sperm contain modified polypeptides which gather in band 1. Histochemical ATPase stainings indicate that this enzyme is localized mainly in the doublet arms and, to a minor extent, in the central structures.