This article reports the results of the most recent in a series of EHSRE workshops designed to synthesize the current state of the field in Andrology and provide recommendations for future work (for details see Appendix). Its focus is on methods for detecting sperm DNA damage and potential application of new knowledge about sperm chromatin organization, vulnerability and repair to improve the diagnosis and treatment of clinical infertility associated with that damage. Equally important is the use and reliability of these tests to identify the extent to which environmental contaminants or pharmaceutical agents may contribute to the incidence of sperm DNA damage and male fertility problems. A working group (for workshop details, see Appendix) under the auspices of ESHRE met in May 2009 to assess the current knowledgebase and suggest future basic and clinical research directions. This document presents a synthesis of the working group's understanding of the recent literature and collective discussions on the current state of knowledge of sperm chromatin structure and function during fertilization. It highlights the biological, assay and clinical uncertainties that require further research and ends with a series of 5 key recommendations.
The development of germ cells is a highly ordered process that begins during fetal growth and is completed in the adult. Epigenetic modifications that occur in germ cells are important for germ cell function and for post-fertilization embryonic development. We have previously shown that male germ cells in the adult mouse have a highly distinct epigenetic state, as revealed by a unique genome-wide pattern of DNA methylation. Although it is known that these patterns begin to be established during fetal life, it is not known to what extent DNA methylation is modified during spermatogenesis. We have used restriction landmark genomic scanning (RLGS) and other techniques to examine DNA methylation at multiple sites across the genome during postnatal germ cell development in the mouse. Although a significant proportion of the distinct germ cell pattern is acquired prior to the type A spermatogonial stage, we find that both de novo methylation and demethylation occur during spermatogenesis, mainly in spermatogonia and spermatocytes in early meiotic prophase I. Alterations include predominantly non-CpG island sequences from both unique loci and repetitive elements. These modifications are progressive and are almost exclusively completed by the end of the pachytene spermatocyte stage. These studies better define the developmental timing of genome-wide DNA methylation pattern acquisition during male germ cell development.
It is well established that the epididymis is the site where spermatozoa are matured and stored, but our understanding of the regulation of epididymal epithelium functions and their effects on spermatozoa is still fairly limited. The most active regulator of epididymal functions seems to be dihydrotestosterone, the 5 alpha-reduced metabolite of testosterone. Our laboratory has focused on the regulation of 5 alpha-reductase, with studies encompassing its messenger RNA, protein and enzyme activity. We have also investigated the hormonal regulation and distribution of other specific key proteins found in epididymal epithelial cells that play critical roles in the function of these cells. These proteins include clusterin or sulfated glycoprotein-2 and the glutathione S-transferases (GST). Using complementary experimental approaches, including orchidectomy and hormonal replacement, efferent duct ligation, and developmental studies, we have established that 5 alpha-reductase enzyme activity is present in both nuclear and microsomal fractions; the nuclear enzyme appears almost exclusively in the initial segment of the epididymis. In addition, 5 alpha-reductase activity and the mRNAs for both the type 1 and type 2 form of the enzyme are regulated differentially with respect to age and site within the epididymis. Immunolocalization of the protein has revealed that it is located in principal cells and that its subcellular location is dependent on the region of the epididymis. These results indicate that there is both transcriptional and post-transcriptional regulation of the expression of 5 alpha-reductase. Clusterin is a hydrophobic protein secreted by Sertoli cells and found in high concentration in the epididymis. This glycoprotein is expressed at its highest levels in the initial segment and caput epididymidis and at very low levels in the corpus and cauda epididymidis of the intact rat, and it exhibits a novel pattern of androgen regulation. In the areas of highest expression, there is no androgen dependence; however, orchidectomy causes a dramatic increase in the message for clusterin, which is suppressible by androgens in the segments where expression is normally lowest. The GSTs are a family of enzymes thought to play a key role in detoxification. Members of the GST family are expressed in a region-dependent manner along the rat epididymis. We have found that the localization of one member of this enzyme family, GST P, or subunit Yp, is selective for basal cells in the corpus and cauda epididymidis.(ABSTRACT TRUNCATED AT 400 WORDS)
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