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An expression cassette carrying 426 basepairs of the rat probasin (PB) gene promoter and 28 basepairs of 5'-untranslated region is sufficient to target the expression of the bacterial chloramphenicol acetyltransferase (CAT) gene specifically to the prostate in transgenic mice. The PB-CAT transgene was expressed in three of five (60%) independent lines of mice, and this expression, as reported previously for the endogenous rat gene, was male specific, restricted primarily to the lateral, dorsal, and ventral lobes of the prostate, with only very low levels of CAT activity detected in the anterior prostate and seminal vesicles. The developmental and hormonal regulation of the transgene also paralleled that reported for the rat gene, with a 70-fold increase in CAT activity in the mouse prostate observed between 2-7 weeks of age, a time corresponding to sexual maturation. PB-CAT activity in the prostate declined after castration to 3.5% of the precastration level, and the CAT activity in castrated males approached precastration levels when mice were supplemented with testosterone. Transgene expression in castrated males was not induced by dexamethasone. Coinjection of PB-CAT with a chicken lysozyme gene matrix attachment region resulted in their cointegration and further restricted the pattern of PB-CAT to the dorsolateral prostate, with suppressed expression observed in the ventral prostate. These studies demonstrate that a minimal rat probasin promoter can target heterologous gene expression specifically to the prostate in a developmentally and hormonally regulated fashion.

Purpose To investigate the effects of male ageing on DNA fragmentation and chromatin packaging in the spermatozoa of oligoasthenoteratozoospermic (OAT) patients. Methods Sixty-one OAT patients and 49 men with proven fertility (controls) were included in the present study. DNA fragmentation was detected by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labelling (TUNEL) assay, while chromatin packaging was assessed by chromomycin A 3 (CMA 3 ) staining. Results In the patient group, semen volume, percentage of normally shaped spermatozoa and sperm motility decreased significantly (P<0.05) with age, while sperm concentration and the percentage of TUNEL and CMA 3 positive spermatozoa showed a statistically significant increase with age (P<0.05). In the control group, conventional semen parameters as well as DNA fragmentation and chromatin packaging did not show a statistically significant change with age (P>0.05). Conclusion Increased age in OAT patients is associated with an increase in sperm concentration, DNA fragmentation and poor chromatin packaging, as well as a decline in semen volume, sperm morphology and motility.

Sex determination is a complicated process involving large-scale modifications in gene expression affecting virtually every tissue in the body. Although the evolutionary origin of sex remains controversial, there is little doubt that it has developed as a process of optimizing metabolic control, as well as developmental and reproductive functions within a given setting of limited resources and environmental pressure. Evidence from various model organisms supports the view that sex determination may occur as a result of direct environmental induction or genetic regulation. The first process has been well documented in reptiles and fish, while the second is the classic case for avian species and mammals. Both of the latter have developed a variety of sex-specific/sex-related genes, which ultimately form a complete chromosome pair (sex chromosomes/gonosomes). Interestingly, combinations of environmental and genetic mechanisms have been described among different classes of animals, thus rendering the possibility of a unidirectional continuous evolutionary process from the one type of mechanism to the other unlikely. On the other hand, common elements appear throughout the animal kingdom, with regard to a) conserved key genes and b) a central role of sex steroid control as a prerequisite for ultimately normal sex differentiation. Studies in invertebrates also indicate a role of epigenetic chromatin modification, particularly with regard to alternative splicing options. This review summarizes current evidence from research in this hot field and signifies the need for further study of both normal hormonal regulators of sexual phenotype and patterns of environmental disruption.

Human sperm DNA damage may have adverse effects on reproductive outcome. Infertile men possess substantially more spermatozoa with damaged DNA compared to fertile donors. Although the extent of this abnormality is closely related to sperm function, the underlying etiology of ensuing male infertility is still largely controversial. Both intra-testicular and post-testicular events have been postulated and different mechanisms have been proposed to explain the presence of damaged DNA in human spermatozoa. Three among them, i.e. abnormal chromatin packaging, oxidative stress and apoptosis, are the most studied and discussed in the present review. Furthermore, results from numerous investigations are presented, including our own findings on these pathological conditions, as well as the techniques applied for their evaluation. The crucial points of each methodology on the successful detection of DNA damage and their validity on the appraisal of infertile patients are also discussed. Along with the conventional parameters examined in the standard semen analysis, evaluation of damaged sperm DNA seems to complement the investigation of factors affecting male fertility and may prove an efficient diagnostic tool in the prediction of pregnancy outcome.

Determining sexual fate is an integral part of reproduction, used as a means to enrich the genome. A variety of such regulatory mechanisms have been described so far and some of the more extensively studied ones are being discussed.For the insect order of Hymenoptera, the choice lies between uniparental haploid males and biparental diploid females, originating from unfertilized and fertilized eggs accordingly. This mechanism is also known as single-locus complementary sex determination (slCSD). On the other hand, for Dipterans and Drosophila melanogaster, sex is determined by the ratio of X chromosomes to autosomes and the sex switching gene, sxl. Another model organism whose sex depends on the X:A ratio, Caenorhabditis elegans, has furthermore to provide for the brief period of spermatogenesis in hermaphrodites (XX) without the benefit of the "male" genes of the sex determination pathway.Many reptiles have no discernible sex determining genes. Their sexual fate is determined by the temperature of the environment during the thermosensitive period (TSP) of incubation, which regulates aromatase activity. Variable patterns of sex determination apply in fish and amphibians. In birds, while sex chromosomes do exist, females are the heterogametic (ZW) and males the homogametic sex (ZZ). However, we have yet to decipher which of the two (Z or W) is responsible for the choice between males and females.In mammals, sex determination is based on the presence of two identical (XX) or distinct (XY) gonosomes. This is believed to be the result of a lengthy evolutionary process, emerging from a common ancestral autosomal pair. Indeed, X and Y present different levels of homology in various mammals, supporting the argument of a gradual structural differentiation starting around the SRY region. The latter initiates a gene cascade that results in the formation of a male. Regulation of sex steroid production is also a major result of these genetic interactions. Similar observations have been described not only in mammals, but also in other vertebrates, emphasizing the need for further study of both normal hormonal regulators of sexual phenotype and patterns of epigenetic/ environmental disruption.

Studies have documented the presence of herpes viruses in semen. The aim of our study was to determine whether they persist in semen samples following two-density gradient centrifugation for IVF purposes. Semen samples were collected from 109 men seeking fertility evaluation, prior to IVF treatment. Routine semen analysis was performed according to WHO guidelines. Each sample was treated in a two-density gradient centrifugation using PureSperm (PS). Both untreated and treated samples were screened for the presence of herpes viruses, using PCR. Kruskal-Wallis, chi-square and binomial statistical tests were used; P ≤ 0.05 was considered statistically significant. No statistically significant associations were observed between semen parameters and viral presence. Viral DNA was detected in 54% of semen samples: HSV1/2 in 32 samples, EBV in 49, CMV in 47, HHV6 in 9, HHV7 in 4 and VZV in none. PS gradient failed to remove CMV in 89.36%, HSV1/2 in 59.38% and EBV in 22.45% of samples, while HHV6 and 7 were completely removed. Especially HSV1/2 and CMV seem to persist even following PS treatment. These observations indicate the possible risk of oocyte becoming infected during insemination, by IVF or intracytoplasmic sperm injection, with unknown sequelae. Further studies are required to determine whether any correlation exists between their presence, implantation rate and the outcome of pregnancy.

The successful treatment of breast cancer is dependent upon a number of complex factors. Her-2/neu gene amplification is known to be one of the most common genetic alterations associated with breast cancer and its accurate determination has become necessary for the selection of patients for trastuzumab therapy. The aim of this study was to prove the consistency of chromogenic in situ hybridisation (CISH) technique after analyzing the overexpression of the Her-2/neu proto-oncogene in 100 invasive breast carcinomas and by comparing CISH results with immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH). Moreover, it was done to evaluate the possible correlation of estrogen (ERs) and progesterone receptors (PRs), the proliferation marker Ki67 and the tumour suppressor gene p53 with HER-2/neu status of these breast carcinomas. Of the 100 breast carcinomas that were analysed, 22 cases showed HER-2/neu amplification, 66 cases showed no amplification, whereas 12 cases were non-interpretable in both assays (FISH and CISH). Consequently, the overall concordance between FISH and CISH was 100%. Additionally, it was observed that when HER-2/neu gene was overexpressed, there was an association with negative PRs and ERs status, negative p53 protein expression and high Ki67 labelling index. It is concluded that patients with tumours scoring 2+ with the CBE356 antibody (borderline immunohistochemistry-tested cases) would also benefit from CISH as it is shown to be highly accurate, practical and can be easily integrated into routine testing in any histopathology laboratory. Finally, CISH represents an important addition to the HER2 testing algorithm.