Infertility affects an estimated 7% of men worldwide, nearly a quarter of whom are diagnosed as idiopathic. The genetic etiologies of idiopathic male infertility are unknown, partly due to lack of simple diagnostic techniques. Moreover, the transmission risk of such genetic defects to offspring born from assisted reproductive techniques is increasingly becoming a concern for physicians and infertile couples. We explored the feasibility of obtaining full-length mRNAs from transcriptionally inert human spermatozoa in semen as a non-invasive diagnostic tool for identifying germline mutations in candidate infertility-associated genes. The efficacy of reverse-transcription PCR on spermatozoal RNA from infertile patients with wide-ranging sperm concentrations varied between 91 and 99% for multiple haploid germ cell-expressed genes. Using this methodology, we identified seven oligozoospermic patients with missense and splicing mutations in the germ cell-specific gene, KLHL10. Three of 270 (1.1%) severely oligozoospermic patients (<10(6) sperm/ml) harbor KLHL10 alterations that were absent in 394 controls and exhibited significant association (P=0.02). Two KLHL10 missense mutations (A313T and Q216P) resulted in impaired homodimerization with the wild-type protein in yeast interaction assays, suggesting a functional deficiency. This study demonstrates the utility of this approach for analysis of haploid germ cell-expressed genes regulating post-meiotic events including sperm maturation, motility and fertilization. The development of non-invasive techniques to analyze genetic defects of human spermatogenesis, previously possible only with invasive testis biopsies, provides important diagnostic and therapeutic implications for reproductive medicine.

Nearly 7% of men are afflicted by male infertility worldwide, and genetic factors are suspected to play a significant role in the majority of these patients. Although sperm morphology is an important parameter measured in the semen analysis, only a few genetic causes of teratozoospermia are currently known. The objective of this study was to define the association between alterations in the genes encoding the Golgi-associated PDZ- and coiled-coil motif containing protein (GOPC), the protein interacting with C kinase 1 (PICK1) and the acrosomal protein zona pellucida binding protein 1 (ZPBP1/sp38) with abnormal sperm head morphology in infertile men. Previous reports demonstrated that mice lacking Gopc, Pick1 and Zpbp1 are infertile due to abnormal head morphology. Herein, using our validated RNA-based method, we studied spermatozoal cDNA encoding the human GOPC, PICK1 and ZPBP1 genes in 381 teratozoospermic and 240 controls patients via direct sequencing. Among these genes, we identified missense and splicing mutations in the sperm cDNA encoding ZPBP1 in 3.9% (15/381) of men with abnormal sperm head morphology. These mutations were not observed in 240 matched controls and the dbSNP database (χ(2) = 9.3, P = 0.002). In contrast, statistically significant and functionally relevant mutations were not discovered in the GOPC and PICK1 genes. In our study ZPBP1 mutations are associated with abnormal sperm head morphology, defined according to strict criteria, resembling the mouse Zpbp1 null phenotype. We hypothesize that missense mutations exert a dominant-negative effect due to altered ZPBP1 protein folding and protein:protein interactions in the acrosome.

We recently noted that immature rats failed to exhibit a normal uterine response to exogenously administered estradiol as assessed by both biochemical (induction of gene expression) and morphological (altered uterine and vaginal histology, and size) end points. An initial analysis suggested that this was due to a high degree ofestrogenization from a dietary source which was producing a near maximal uterotrophic response prior to hormone treatment. Subsequent chemical analysis indicated that the feed in question contained high amounts of two well-known phytoestrogens, genistein (210 mglkg) and daidzen (14 mg/kg), and the lot of feed in question produced a large uterotrophic ect when fed to immature ovariectomized rats. These findings illustrate that, despite increased awareness of phytoestrogens, some batches of animal feed contain very high amounts of estrogenic components which have marked efects on in vivo end points of hormone action. These observations have important implications for both basic research and screenin methods that utilize in uivo approaches.

We recently noted that immature rats failed to exhibit a normal uterine response to exogenously administered estradiol as assessed by both biochemical (induction of gene expression) and morphological (altered uterine and vaginal histology and size) end points. An initial analysis suggested that this was due to a high degree of estrogenization from a dietary source which was producing a near maximal uterotrophic response prior to hormone treatment. Subsequent chemical analysis indicated that the feed in question contained high amounts of two well-known phytoestrogens, genistein (210 mg/kg) and daidzen (14 mg/kg), and the lot of feed in question produced a large uterotrophic effect when fed to immature ovariectomized rats. These findings illustrate that, despite increased awareness of phytoestrogens, some batches of animal feed contain very high amounts of estrogenic components which have marked effects on in vivo end points of hormone action. These observations have important implications for both basic research and screening methods that utilize in vivo approaches.ImagesFigure 2Figure 3

The Sertoli cell is thought to play a significant role in the hormonal regulation of spermatogenesis within the rat testis. Little, however, is known about Sertoli cell function in man, largely because of the difficulties associated with the isolation of pure cell populations from human tissue. We have now developed a rapid and reproducible technique for establishing a human Sertoli cell monolayer culture. This has involved mechanical separation of the tissue, sequential trypsin and collagenase enzyme digestion, and final disruption of tubules by passage through a wire mesh grid. Using this technique, primary cultures can be maintained for up to 45 days. Ultrastructural studies of these cells have demonstrated the presence of the perinucleolar spheres, cell to cell junctional complexes, abundant lipid droplets, and smooth endoplasmic reticulum, all characteristic of Sertoli cells. Furthermore, biochemical markers of animal Sertoli cells, androgen-binding protein and gamma-glutamyl transpeptidase, have also been identified in these human cells. Concentrated media electrophoresed on nondenaturing gels containing 2 nM [3H]dihydrotestosterone produced a single peak of bound activity which coelectrophoresed with rat androgen-binding protein. This binding activity persisted despite media changes, thus ruling out contamination by serum binding proteins; fresh media lacked demonstrable binding activity. Using a colorimetric assay, these cells were also found to contain significant gamma-glutamyl transpeptidase activity compared to human foreskin fibroblasts and Leydig cells. Enzyme activity increased in a characteristic dose-response fashion in the presence of FSH (0.05-0.5 microgram/ml) and dibutyryl cAMP (0.1-1 microgram/ml), but not with LH or testosterone. These data offer the first demonstration of human Sertoli cells in monolayer culture and their production of a marker specifically regulated by FSH.