Martin H. Brinkworth

Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences.

During the haploid phase of mammalian spermatogenesis, nucleosomal chromatin is ultimately repackaged by small, highly basic protamines to generate an extremely compact, toroidal chromatin architecture that is critical to normal spermatozoal function. In common with several species, however, the human spermatozoon retains a small proportion of its chromatin packaged in nucleosomes. As nucleosomal chromatin in spermatozoa is structurally more open than protamine-packaged chromatin, we considered it likely to be more accessible to exogenously applied endonucleases. Accordingly, we have used this premise to identify a population of endonuclease-sensitive DNA sequences in human and murine spermatozoa. Our results show unequivocally that, in contrast to the endonuclease-resistant sperm chromatin packaged by protamines, regions of increased endonuclease sensitivity are closely associated with gene regulatory regions, including many promoter sequences and sequences recognized by CCCTC-binding factor (CTCF). Similar differential packaging of promoters is observed in the spermatozoal chromatin of both mouse and man. These observations imply the existence of epigenetic marks that distinguish gene regulatory regions in male germ cells and prevent their repackaging by protamines during spermiogenesis. The ontology of genes under the control of endonuclease-sensitive regulatory regions implies a role for this phenomenon in subsequent embryonic development.

Association of cyclophosphamide-induced male-mediated, foetal abnormalities with reduced paternal germ-cell apoptosis

To investigate the mechanism by which malformed offspring can result from the exposure of males to mutagens, we treated adult male rats with 0, 1.4, 3.4 or 5.1 mg/kg cyclophosphamide, 6 days per week for 9 weeks, a treatment regimen known to induce heritable abnormalities. Testis samples from some of the animals were then collected for fixation in Carnoys fluid and subsequent analysis of germ-cell apoptosis and proliferation. The remainder were mated, resulting in a greater than 11-fold increase in the proportion of abnormal offspring produced in the 5.1 mg/kg group. The number of apoptotic cells per stage XII/XIII tubular cross-section decreased with increasing dose, significantly so at 5.1 mg/kg (P<0.05). No statistically significant effect was found on spermatocyte numbers at this dose, indicating that a reduction in the amount of cells available to undergo apoptosis cannot explain the decrease. The inappropriate survival of damaged germ-cells caused by a lowering of the incidence of apoptosis may, therefore, account for the rise in the proportion of foetal malformations.

Intrauterine bisphenol A exposure leads to stimulatory effects on Sertoli cell number in rats

Using the optical disector for quantifying cell numbers, we investigated whether oral treatment of rats on days 6-21 of gestation with the weakly estrogenic bisphenol A (BPA, 0.1 or 50 mg/kg) or the highly estrogenic ethinyl estradiol (EE, 0.02 mg/kg) alters testicular histology, in those offspring 9-12 month of age. Since production of male germ cells depends on Sertoli cell number, possible changes in that parameter were investigated using unbiased stereology. Spermatogenesis was qualitatively normal in all groups. BPA increases Sertoli cell number per organ but not when expressed as per gram testis. EE did not affect cell number per organ but did affect numbers on a per gram testis basis due to a lowered testis weight. In contrast to the lowering of Sertoli cell numbers that might have been expected according to the estrogen hypothesis, intrauterine administration of these xenoestrogens in fact resulted in minor increases in Sertoli cell numbers and had no qualitative effect on spermatogenesis.

Genetic effects of 1,3-butadiene on the mouse testis

1,3-Butadiene is a known male mouse germ-cell mutagen, to which humans may either be occupationally or environmentally exposed. Prolonged exposure to moderate or high doses in male mice can cause dominant lethal mutations and one report has indicated that 10 week inhalation administration of low doses can result in the production of malformed foetuses. The present study had dual purposes: (a) to attempt to clarify the suspected ability of sub-chronic (6 h/day, 5 days/wk, 10 weeks) low-dose exposure to 1,3-butadiene to induce heritable mutations in mouse male germ cells: (b) investigation of the relationships between testicular DNA damage, testicular DNA repair and foetal outcome. Adult male mice were exposed to low or moderate doses of 1,3-butadiene by inhalation sub-chronically or for a single 6 h period and either used for mating (sub-chronic exposure only) or for studies of DNA damage and repair. Litter size, dominant lethality and numbers of abnormal foetuses were determined the day preceding the normal day of parturition. Testicular DNA damage and repair were assessed by the Comet assay (for DNA damage) and the unscheduled DNA synthesis assay (for DNA repair). 1,3-Butadiene caused a statistically significant increase in dominant lethality at 125 ppm but not 12.5 ppm. No significant increase in DNA repair was found with either dose level or exposure period while only 6 h exposure to 125 ppm caused a small but significant increase in DNA damage as detected by the Comet assay. These effects demonstrate the reproductive genotoxicity of (125 ppm) 1,3-butadiene but do not confirm its ability to cause abnormalities in the offspring via the sperm. It is suggested that the relationship between 1,3-butadiene-induced DNA damage, DNA repair and heritable defects in the offspring may depend on the pattern of metabolites produced.

Key gene regulatory sequences with distinctive ontological signatures associate with differentially endonuclease-accessible mouse sperm chromatin

Using a well-established endonuclease-based chromatin dissection procedure in conjunction with both experimental comparative genome hybridisation (CGH) array profiling and in silico data mining, we show that mouse spermatozoa contain chromatin that is sensitive and resistant to digestion with micrococcal nuclease (MNase). Sequences represented in the micrococcal nuclease digestion solubilised (MNDS) but not the MND insoluble (MNDI) chromatin are strongly enriched in chromosomal regions of high gene density. Furthermore, by fluorescence in situ hybridisation (FISH) analysis, we show that MNDS and MNDI DNAs occupy distinct domains of decondensed mouse sperm nuclei that may also retain abundant histones. More detailed in silico analysis of CGH probe location in relation to known promoters and sequences recognised by CCCTC binding factor (CTCF) shows a significant excess of both in MNDS chromatin. A functional analysis of gene promoters reveals strong ontological signatures for ion transport on methylated promoters associated with CTCF binding sequences in MNDS chromatin. Sensory perception is the only strong ontological signature present in MNDI chromatin, driven by promoters that are not associated with CTCF regardless of their methylation status.

Development of an in vitro test system for assessment of male, reproductive toxicity

There is a need for improved reproductive toxicology assays that do not require large numbers of animals but are sensitive and informative. Therefore, Staput velocity-sedimentation separation followed by culture of specific mouse testicular cells was used as such a system. The specificity of separation was assessed using immunocytochemistry to identify spermatids, spermatocytes and spermatogonia. The efficacy of the system to detect toxicity was then evaluated by analysing the effects of hydrogen peroxide (H2O2) by the terminal uridine-deoxynucleotide end-labelling (TUNEL) assay to show the rate of apoptosis induced among the different types of germ cells. We found that 2 h of treatment at both 1 and 10 μM induced increases of over ∼10-fold in the percentage of apoptotic cells (p≤0.001), confirming that testicular germ cells are prone to apoptosis at very low concentrations of H2O2. It was also demonstrated for the first time for this compound that spermatogonia are significantly more susceptible than spermatocytes, which are more affected than spermatids. This reflects the proportion of actively dividing cells in these cell types, suggesting a mechanism for the differential sensitivity. The approach should thus form the basis of a useful test system for reproductive and genetic toxicology in the future.

An investigation of male-mediated F1 effects in mice treated acutely and sub-chronically with urethane

In order to investigate the alleged potential of paternally administered urethane to cause foetal abnormalities and heritable tumours, male CD-1 mice were treated with urethane, either acutely by intraperitoneal injection at doses of 1.25 and 1.75 g/kg bodyweight (bwt) or sub-chronically in the drinking water at 1.25 for 10 weeks, and 3.75 mg/ml for 9 weeks or vehicle for the control groups. They were mated to untreated females 1 week later. Uterine contents of half the pregnant females were examined just before full term, while the remaining females were allowed to deliver their litters. The resulting F1 offspring were observed for approximately 18 months and 12 months for acute and sub-chronic exposures respectively and subjected to necropsy examination. Some of the mice treated acutely with 1.75 g/kg bwt exhibited partial infertility but none of those treated with 1.25 g/kg bwt had an adverse effect on their reproductive ability. There was no genetic effect of acute urethane treatment on male germ cells as indicated by dominant lethality. After birth, there was an increase (P < 0.05) in post-implantation deaths possibly due to perinatal mortality. There was an increased incidence and earlier onset of liver tumours induced in F1 male offspring from F0 males treated with 1.75 g/kg bwt, (20.7% vs. 10.1%, P = 0.026) but not in the female offspring. F1 males from both treatment groups had mean bodyweights significantly higher than controls (P < 0.01). Some males from each dose group of the acute study were examined using the restriction site mutation assay involving analysis of exon sequences. No mutations were identified in testes, liver or spleen of DNA isolated from the urethane-treated animals. No reproductive or genetic effects were seen with sub-chronic treatment at either 1.25 or 3.75 mg/ml urethane nor was there any predisposition of F1 animals to tumours although observation times were shorter.

Germ Cell Responses to Doxorubicin Exposure in Vitro

Anthracyclines such as doxorubicin (Dox), widely used to treat various types of tumours, may result in induced testicular toxicity and oxidative stress. The present investigation was designed to determine whether exposure of isolated and purified mouse germ cells to Dox induces DNA damage in the form of strand breaks (presumably) resulting in apoptosis and to investigate the relative sensitivity of specific cell types. DNA damage was assessed using the Comet assay and the presence of apoptosis was determined by TUNEL assay. Isolated mouse germ cells were treated with different concentrations (0.05, 0.5 and 1mM, respectively) of Dox, and fixed 1h after treatment. The incidences of both DNA damage shown by single cell gel-electrophoresis and of apoptosis increased significantly in each specific cell type in a concentration-dependent manner. The DNA damage and apoptosis incidences gradually increased with concentration from 0.05 to 1mM with Dox. Our results indicate that apoptosis plays a vital role in the induction of germ cell phase-specific toxicity caused by Dox with pre-meiotically and meiotically dividing spermatogonia and spermatocytes respectively as highly susceptible target cells.

Diethylstilbestrol induces oxidative DNA damage, resulting in apoptosis of spermatogonial stem cells in vitro

The spermatogonial stem cells (SSCs) are the only germline stem cells in adults that are responsible for the transmission of genetic information from mammals to the next generation. SSCs play a very important role in the maintenance of progression of spermatogenesis and help provide an understanding of the reproductive biology of future gametes and a strategy for diagnosis and treatment of infertility and male reproductive toxicity. Androgens/oestrogens are very important for the suitable maintenance of male germ cells. There is also evidence confirming the damaging effects of oestrogen-like compounds on male reproductive health. We investigated the effects in vitro, of diethylstilbestrol (DES) on mouse spermatogonial stem cells separated using Staput unit-gravity velocity sedimentation, evaluating any DNA damage using the Comet assay and apoptotic cells in the TUNEL assay. Immunocytochemistry assays showed that the purity of isolated mouse spermatogonial cells was 90%, and the viability of these isolated cells was over 96%. Intracellular superoxide anion production (O2-) in SSCs was detected using p-Nitro Blue Tetrazolium (NBT) assay. The viability of cells after DES treatment was examined in the CCK8 (cell counting kit-8) cytotoxicity assay. The results showed that DES-induced DNA damage causes an increase in intracellular superoxide anions which are reduced by the flavonoid, quercetin. Investigating the molecular mechanisms and biology of SSCs provides a better understanding of spermatogonial stem cell regulation in the testis.

Environmental Influences on Male Reproductive Health

Humans are exposed to a vast array of chemicals through a wide range of portals. Innumerable environmental, toxic hazards arising from nature have always existed but the additional exposures to man-made chemicals have increased progressively since the Industrial Revolution. Regulatory agencies, themselves the product of public demand for drug regulation following thalidomide and other catastrophes, have developed licensing guidelines for new chemicals in order to protect human populations from damaging environmental exposures, while permitting the production and use of important new chemicals, which are an integral part of our built environment. Over the last 3 decades the scope and sophistication of testing requirements has increased greatly as the quantity and diversity of chemical entities to which humans may be exposed continues to increase. The earliest safety screening mainly concerned acute toxicity, carcinogenesis and teratogene-sis, however, reproductive and genetic toxicity are now being evaluated to an ever increasing extent. Although historically, reproductive effects have been most studied in females, it has been recognized for some years that adverse effects on male reproductive function are also important. Indeed, a recent, comprehensive review has listed published data on over 600 chemicals to which humans can be exposed that affect the male reproductive system (Krause 2008).