Male gametes originate from a small population of spermatogonial stem cells (SSCs). These cells are believed to divide infinitely and to support spermatogenesis throughout life in the male. Here, we developed a strategy for the establishment of SSC lines from embryonic stem (ES) cells. These cells are able to undergo meiosis, are able to generate haploid male gametes in vitro, and are functional, as shown by fertilization after intracytoplasmic injection into mouse oocytes. Resulting two-cell embryos were transferred into oviducts, and live mice were born. Six of seven animals developed to adult mice. This is a clear indication that male gametes derived in vitro from ES cells by this strategy are able to induce normal fertilization and development. Our approach provides an accessible in vitro model system for studies of mammalian gametogenesis, as well as for the development of new strategies for the generation of transgenic mice and treatment of infertility.

The influence of E. coli on human sperm motility was studied in vitro. Semen samples were prepared by a swim-up technique and adjusted to 22 x 10(6) spermatozoa/ml. Samples were then inoculated with different concentrations of a uropathogenic strain of E. coli, serotype 06, with initial sperm/bacteria ratios varying between 10:1 and 10000:1. Motion parameters were analysed by computer-aided motility analysis directly, and 2, 4 and 6 h after inoculation. In a second series of experiments, bacterial replication was inhibited by addition of chloramphenicol. In a third series, the effect of E. coli culture filtrates on sperm motility was investigated. The direct inhibitory effect of E. coli on progressive motility of spermatozoa was found to depend upon the bacterial concentration. A distinct inhibitory effect was observed only at a sperm/bacteria ratio of approximately 1, achieved by growth of E. coli during the experiments. For modality of motion, no distinct changes were observed. When growth of bacteria was prevented by chloramphenicol, no inhibitory effect on sperm motility was detected. Sperm motility was not inhibited by E. coli culture filtrates. Analysis by electron microscopy revealed multiple adhesions of E. coli to spermatozoa, causing variable ultrastructural damage as probable morphological correlates of immobilization.

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.

This study evaluated if the negative influence of Escherichia coli on the motility of human spermatozoa is a consequence of E. coli-induced ultrastructural alterations. Suspensions of spermatozoa were artificially infected with E. coli from a serotyped, pathogenic strain and incubated at 37 degrees C for 6 h. After incubation, spermatozoa were fixed in glutaraldehyde, stained with osmium tetroxide and ruthenium red and embedded in Spurr(R)-resin followed by ultramicrotomy. The sections were analysed subsequently by use of transmission electron microscopy. Uninfected suspensions of spermatozoa in medium and bacterial suspensions served as controls. Negative contrast technique was performed to facilitate visualization of ultrastructural details of the bacterial capsule after experimental exposure to spermatozoa. Electron microscopic evaluation revealed multiple and profound alterations in the ultrastructure of spermatozoa such as membrane defects and cytoplasmic vacuoles exclusively in spermatozoa of infected samples (> 90%). Morphological alterations involved all superficial structures of spermatozoa, in particular the plasma membrane of the mid-piece and neck as well as the inner and outer acrosomal membrane of the acrosome, indicating that morphological defects account for the immobilization of spermatozoa by E. coli. The results suggest that E. coli infection of ejaculates results in immobilization and impaired acrosomal function in human spermatozoa, findings that support the indication for antimicrobial chemotherapy in symptomatic and silent infections that affect the ejaculate.

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.

Background Patients increasingly use health portals and Web-based expert forums (ask-the-doctor services), but little is known about the specific needs of Internet users visiting such websites, the nature of their requests, or how satisfied they are with Internet health experts.Objective The aim of this study was to analyze the information requests of (mostly female) patients visiting an Internet expert forum on involuntary childlessness and their satisfaction with the experts' feedback.Methods We posted an electronic questionnaire on a website hosting an expert forum on involuntary childlessness. The questionnaire was “activated” whenever a visitor sent a question or request to the expert forum. The survey focused on the reasons for visiting the expert forum and whether the visitors were satisfied with the experts' answers to previously posted questions. The free-text questions of visitors who answered the survey were analyzed using Atlas-ti, a software program for qualitative data analysis.ResultsOver a period of 6 months, 513 out of 610 visitors (84%) answered the questionnaire. The majority of respondents (65.5%) expected general information about involuntary childlessness, conception, or an evaluation of drugs. Others were concerned about their actual treatment (40.6%) and therapeutic options (28.8%). Out of 225 respondents who had previously contacted the forum, 223 had received an answer, and 123 (55.2%) were satisfied with the experts' answers. About half (105/223) of those users who had previously received an answer from the expert forum stated that they had discussed it with their own doctor. More of these users were satisfied with their subsequent care in fertility clinics than users who did not talk to their doctor about their Internet activities (93.9% vs 76.1%; P = .015 ). According to the qualitative analysis, many requests (n = 194) were more or less trivial, especially those for information on basic aspects of reproduction. More than one-third of visitors (n = 199) sent detailed results of diagnostic tests and asked for a first or second opinion. Requests to the expert forum were also sent in order to obtain emotional support (17%) or to complain about a doctor (15%).Conclusions Visitors who sent their laboratory findings to receive a thorough evaluation or a second opinion had a good command of the opportunities that an expert forum offers. One important expectation of the forum was emotional support, indicating psychological needs that were not met by medical providers. Future websites must find a compromise in order to protect experts from being overwhelmed by general, nonspecific requests while supporting patients with individualized answers.

Urogenital infections are considered important factors in male infertility. In this in vitro study we have evaluated the impact of leucocytes in association with an artificial infection with Escherichia coli on the motility of human spermatozoa. Ejaculates and blood samples were obtained from healthy donors with normal semen parameters. Ejaculates were prepared by swim-up technique and five fractions were isolated for incubation. Leucocyte subtypes were separated from blood samples by gradient centrifugation. Purified sperm suspensions were adjusted to a concentration of 20 x 106 ml-1 and incubated with lymphocytes/ monocytes, polymorphonuclear granulocytes (PMN), and E. coli. Samples were incubated for up to 6 h at 37 degrees C. Motility analysis was performed using a computer-assisted sperm analyzer (CASA). Spermatozoa incubated with 3 x 106 PMN ml-1 revealed a significant (P=0.003) decrease in progressive motility after 2 h. This decrease remained weakly significant (P=0.024) after 4 and 6 h. Lymphocytes and monocytes had no effect on sperm motility. Spermatozoa incubated with granulocytes and E. coli demonstrated highly significant alterations in motility after 4 and 6 h of incubation (P < 0.001). The PMN indicate an effect on motility of spermatozoa under experimental conditions. However, the results suggest that bacteria are the primary agents that interfere with sperm motility.

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.