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In recent years, investigators have noted a trend toward a declining proportion of male births in many industrialized nations. While men bear the sex-determining chromosome, the role of the female partner as it pertains to fertilization or miscarriage may also alter the gender ratio. We attempted to determine a man's secondary sex ratio (F1 generation) by directly examining the sex chromosomes of his sperm. We examined our male infertility clinic database for all men who had undergone a semen fluorescence in situ hybridization (FISH). Patient demographic and semen parameters were recorded. Chi-squared analysis was used to compare gender ratios (Y chromosomes/total chromosomes). Multivariable logistic regression was used to predict the odds of possessing a Y-bearing sperm after accounting for demographic and semen parameters. A total of 185 men underwent sperm FISH. For the entire cohort, the proportion of Y chromosome-bearing sperm was 51.5%. Men with less than five million motile sperm had a significantly lower proportion of Y chromosome-bearing sperm (50.8%) compared to men with higher sperm counts (51.6%; P=0.02). After multivariable adjustment, a higher sperm concentration, total motile sperm count and semen volume significantly increased the odds of having a Y chromosome-bearing sperm (P

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.

The primary objective of this study was to correlate simultaneous measures of prostate-specific antigen (PSA) and serum testosterone among large samples of eugonadal, untreated hypogonadal and hypogonadal men treated with testosterone replacement therapy (TRT). From 2001 to 2007, laboratory records were reviewed to identify men who underwent simultaneous measurement of PSA and serum testosterone levels. The data were stratified based on three groups of men: group 1 consisted of eugonadal men (T>300 ng per 100 ml) evaluated for BPH, reproductive failure or sexual dysfunction; group 2 consisted of untreated hypogonadal men (T

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 noninvasive 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 (

Protein kinase C (PKC) is a key regulatory enzyme in the control of growth and differentiated function in many cell types. Recently it has become clear that cross talk occurs between PKC and steroid hormone-activated signaling pathways. In this work we have thus used the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to investigate the relationship between PKC activation and estrogen-induced proliferation in an in vivo model of hormone action, the immature rat uterus. A single injection of estradiol (E2) to immature female animals increases DNA synthesis in all major uterine cell types. Administration of TPA alone, simultaneous administration of TPA and E2, or administration of TPA 12 h after E2 did not alter uterine DNA synthesis. However, administration of TPA 6 h after E2 markedly decreased [3H]thymidine incorporation and the labeling indices in uterine epithelial, stromal, and myometrial cells. This inhibition represents a decrease in DNA synthesis per se rather than a change in the time course of the tissue response to the hormone. The inhibitory effect of TPA was reversible within 72 h and did not appear to be due to a decrease in the level or degree of occupancy of uterine estrogen receptors. These results suggest that a discrete regulatory event(s) in the pathway of estradiol-induced proliferation is inhibited by PKC activation approximately 6 h after hormonal stimulation.

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. Images Figure 2 Figure 3

Oligozoospermia (low sperm count) is a common semen deficiency. However, to date, few genetic defects have been identified to cause this condition. Moreover, even fewer molecular genetic diagnostic tests are available for patients with oligozoospermia in the andrology clinic. Based on animal and gene expression studies of oligozoospermia, several molecular pathways may be disrupted in post-meiotic spermatozoa. One of the disrupted pathways is protein ubiquitination and cell apoptosis. A critical protein involved in this pathway is the ubiquitin-conjugating enzyme 2B, UBE2B. Absence of Ube2b in male mice causes spermatogenic meiotic disruption with increased apoptosis, leading to infertility. To examine the association between messenger RNA defects in UBE2B and severe oligozoospermia (0.1-10 × 10(6) cells/ml), sequencing of sperm cDNA in 326 oligozoospermic patients and 421 normozoospermic men was performed. mRNA alterations in UBE2B were identified in sperm in 4.6% (15 out of 326) of the oligozoospermic patients, but not found in control men, suggesting strong association between mRNA defects and oligozoospermia (χ(2) = 19, P = 0.0001). Identified UBE2B alterations include nine splicing, four missense and two nonsense alterations. The follow-up screen of corresponding DNA regions did not reveal causative DNA mutations, suggesting a post-transcriptional nature of identified defects. None of these variants were reported in the dbSNP database, although other splicing abnormalities with low level of expression were present in 11 out of 421 (2.6%) controls. Our findings suggest that two distinct molecular mechanisms, mRNA editing and splicing processing, are disrupted in oligozoospermia. We speculate that the contribution of post-transcriptional mRNA defects to oligozoospermia could be greater than previously anticipated.

Treatment of immature female rats with estradiol increases uterine levels of c-jun and jun-B mRNAs approx. 10-fold. This effect is specific for estrogenic steroids. The induction of jun transcripts is blocked by actinomycin D but not puromycin, suggesting that the hormonal effect is due at least in part to transcriptional activation. The hormone effect is rapid and peak levels of jun mRNAs are seen within 3 h after treatment. Inductions of jun and fos transcripts in the uterus by estradiol exhibit similar dose response curves (maximum responses at 4 micrograms/kg). Estradiol also elevates uterine levels of jun-D, and this induction is insensitive to puromycin. In vivo treatment with the phorbol ester TPA rapidly elevates uterine levels of fos, jun, and myc transcripts, indicating that expression of these protooncogenes is under non-estrogenic as well as estrogenic regulation in this target tissue. These results suggest that multiple members of the jun and fos protooncogene families may play a role in amplifying the uterine response to estrogens.

Estrogen-stimulated endometrial proliferation is an important preparatory event for implantation and is thought to be initiated by estrogen receptor (E-R)-mediated changes in gene expression. Many laboratories have thus focused on the biochemical nature of E-R interactions and how this complex interacts with the consensus estrogen response element (ERE) originally identified in the vitellogenin genes of birds and amphibians.

Publisher Summary Numerous agents, including growth factors, protooncogenes, and transcription factors have been implicated in the regulation of uterine growth and function by estrogenic hormones. Another major signaling pathway involved in the cellular responses to many hormones, neurotransmitters, and growth factors involves protein phosphorylations catalyzed by protein kinase C. This chapter describes a study that used TPA to assess the effects of PKC activation on estradiol (E2)-induced proliferation of uterine cells in the immature rat model. TPA treatment decreased estrogen stimulated uterine DNA synthesis when administered 6 hr after the hormone. This suggests that a discrete event in the E2-induced growth program, which occured 6 hr after the process is initiated, was altered as the result of PKC activation. This effect of the phorbol ester is reversible and occurs in all major uterine cell types of the immature rat uterus. In addition to inhibiting DNA synthesis, TPA also affects apoptosis. The inhibitory effect of PKC occurs distal to receptor occupancy.

Publisher Summary Progesterone decreases the induction of c-fos mRNA in the rodent uterus by estrogen. This inhibition raises the possibility that progesterone antagonism of estrogen-induced uterine growth is mediated in part by suppression of fos transcript levels. This effect of progesterone occurs in both the rat and mouse and it will be interesting to determine in future work if it occurs in other species. The dosage of progesterone that elicits a decrease in estrogen induced fos mRNA is in the range that produces other tissue responses to progesterone. These studies with other classes of steroids indicate that the effect of progesterone exhibits some specificity because neither an androgen nor a mineralocorticoid inhibits estrogen induction of c-fos mRNA. The results suggest that the most likely mechanism of the observed effect is a direct inhibition of transcription at the level of the c-fos gene. This inhibition could occur by one of several mechanisms. First, the progesterone receptor could bind to a regulatory region of the fos gene and prevent or alter the binding of estrogen receptor and or other transcription factors. Alternatively, the antagonistic effect of progesterone could be mediated by way of protein-protein interactions between its receptor and transcription factors required for estrogen induction of fos transcription.

Our basic approach to the problem of hormonal carcinogenesis has been to investigate the mechanisms by which physiologically relevant levels of estrogens regulate the growth and function of normal target tissues. This information is required to understand the mechanisms of carcinogenesis in hormone target tissues, the role of the hormone in these processes, and rational approaches to the prevent,ion and treatment of major human diseases such as prostate, endometrial, and breast cancer. The experimental system we have used for our studies is the immature rat uterus.

supplementation in men post-RP. METHODS: Men with an early morning total T level in the hypogonadal (HG) range (

Many naturally occurring and man-made chemicals present in the environment possess estrogenic activity. Examples include plant and fungal products, pesticides, plasticizers, and other agricultural and industrial chemicals. These environmental estrogens as well as endogenous ovarian estrogens are thought to initiate their physiological actions in target tissues largely via interactions with a nuclear receptor system. The resultant estrogen-receptor complex in turn affects transcription via its interactions with nucleotide sequences known as estrogen response elements (EREs) present in the regulatory regions of hormone responsive genes. A "consensus" ERE sequence GGTCAnnnTGACC was originally identified in the vitellogenin genes of birds and amphibians, but it is now clear that most naturally occurring EREs differ from this sequence in one or more bases. We and others have obtained both in vivo and in vitro data suggesting a differential interaction of receptor complexes containing different ligands with the multiple EREs present in mammalian systems. This raises the possibility that the toxicity of environmental estrogens may arise in part from a differential pattern of ERE activation by environmental compounds relative to endogenous ovarian estrogens. The experimental basis for such a paradigm and its toxicological implications are discussed in this paper.

This study describes the use of adult rats as a model to determine whether chronic estrogen treatment irreversibly alters the capacity of Sertoli cells to secrete androgen binding protein (ABP). Twenty-five adult rats were implanted with silastic tubing containing 17 beta-estradiol. After 1 month of estradiol the epididymides had regressed to 47 per cent of the weight of the epididymides in a control group. The amount of ABP in control epididymides was 9.9 fmol./mg. protein, whereas no ABP was detectable in those from rats treated with estradiol for 1 month. Serum testosterone levels had been depressed by 90 per cent. Estradiol treatment for 8 months resulted in a 60 per cent lower body weight and a 91 per cent decrease in testicular weight compared to control animals. During this period the epididymides regressed significantly. When Sertoli cells were cultured from the testes of the estradiol implanted rats, in spite of the dramatic changes which had occurred in the testes, within 4 days the cultured cells synthesized ABP in response to FSH and testosterone. Furthermore, the direct addition of estrogen at a concentration of 200 ng./ml. to rat Sertoli cell cultures failed to depress ABP below control levels. Thus, although estradiol depresses ABP synthesis in vivo, probably through its depression of both gonadotrophin secretion and testosterone biosynthesis, it has no direct effect on the Sertoli cells, and its long-term inhibition of ABP synthesis by an indirect mechanism is reversible.

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.