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.

Apoptosis plays a major role in the development of the central nervous system. Previous studies of apoptosis induction during retinal development are difficult to interpret, however, because they explored different mouse strains, different developmental periods, and used different assays. Here, we first established a comprehensive sequential pattern of cell death during the whole development of the C57BL/6J mouse retina, from E10.5 to postnatal day (P) 21 by using the terminal deoxynucleotidyl transferase (TdT) -mediated deoxyuridine triphosphate (dUTP)-biotinylated nick end labeling (TUNEL) assay. We confirmed the existence of three previously described apoptotic peaks and identified another, later peak at P15, in both the outer nuclear layer, in which the photoreceptors differentiate, and the ganglion cell layer. Comparison of wild-type C57BL/6 mice, gld mice, defective in the death ligand fasL, and bax-/-mice, defective in the pro-apoptotic BAX protein, revealed a minor role for FAS ligand but a crucial role for BAX in both apoptosis and normal retinal development. The lack of BAX resulted in thicker than normal inner neuroblastic and ganglion cell layers in adults, with larger numbers of cells and an impaired electroretinogram response related to a decreased number of responsive cells. Our findings indicate that cell death during normal retinal development is important for the modeling of a functional vision organ and showed that the pro-apoptotic BAX protein plays a crucial role in this process. Developmental Dynamics 228:231-238, 2003.

Understanding meiosis is facilitated by in vitro experimental approaches, but this has not been easily applicable to mammalian meiocytes. Available methods for in vitro analysis of mammalian oocytes are generally limited to experimental analysis of the late prophase period. Short-term cultures of male germ cells have been useful for analysis of earlier meiotic prophase pathways, as well as onset of the meiotic division phase, but no studies have achieved reliable spermatogenesis in vitro. Here we describe a method for preparing highly enriched pachytene spermatocytes from mouse testicular cell suspensions using cell-size fractionation by sedimentation through a bovine serum albumin gradient at unit gravity. We also provide a procedure for short-term culture of spermatocytes and the pharmacological induction of the prophase-to-division phase transition.

Protein sumoylation regulates a variety of nuclear functions and has been postulated to be involved in meiotic chromosome dynamics as well as other processes of spermatogenesis. Here, the expression and distribution of sumoylation pathway genes and proteins were determined in mouse male germ cells, with a particular emphasis on prophase I of meiosis. Immunofluorescence microscopy revealed that SUMO1, SUMO2/3 and UBE2I (also known as UBC9) were localized to the XY body in pachytene and diplotene spermatocytes, while only SUMO2/3 and UBE2I were detected near centromeres in metaphase I spermatocytes. Quantitative RT-PCR and Western blotting were used to examine the expression of sumoylation pathway genes and proteins in enriched preparations of leptotene/zygotene spermatocytes, prepubertal and adult pachytene spermatocytes, as well as round spermatids. Two general expression profiles emerged from these data. The first profile, where expression was more prominent during meiosis, identified sumoylation pathway participants that could be involved in meiotic chromosome dynamics. The second profile, elevated expression in post-meiotic spermatids, suggested proteins that could be involved in spermiogenesis-related sumoylation events. In addition to revealing differential expression of protein sumoylation mediators, which suggests differential functioning, these data demonstrate the dynamic nature of SUMO metabolism during spermatogenesis.