Contents

CHAPTER 1: INTRODUCTION3

CELL CYCLE PATHWAY DURING SPERMATOGENESIS -4

DNA REPAIR AND CELL DEATH PATHWAY DURING SPERMATOGENESIS-5

REGULATION OF GENE EXPRESSION DURING SPERMATOGENESIS-6

CHAPTER 2: Microarray Based Analysis of Cell Cycle Gene Expression During Spermatogenesis in the Mouse10

MATERIALS AND METHODS11

Microarray Data Mining11

Validation of microarray results15

CHAPTER 3: RESULTS18

Chapter 4: DISCUSSION25

Classification of annotated genes27

Analysis of chromatin remodeling and epigenetic programming genes as a function of age -28

Standard RT-PCR -33

Results33

Description of the microarray(s)33

Global Analysis of Epigenetic programming and chromatin remodeling gene expression during spermatogenesis:34

Comparison of epigenetic programming and chromatin remodeling gene expression in spermatogonia and spermatocytes and spermatids35

Comparison of Epigenetic programming and chromatin remodeling gene expression in pachytene spermatocytes as a function of age38

Comparison of Epigenetic programming and chromatin remodeling gene expression in round spermatids as a function of age40

CHAPTER 5: CONCLUSION50

REFERENCES53

CHAPTER 1: INTRODUCTION

Spermatogenesis is the process by which male germ cells develop and differentiate to give rise to the mature sperm. In the adult male mammal, spermatogenesis is thus a continuum of differentiation of a cell lineage in which three principal stages can be discerned - 1) a mitotic stage in spermatogonia including cellular renewal and proliferation, 2) a meiotic stage in spermatocytes including meiosis and genetic recombination, and 3) a post-replicative stage in spermatids when haploid male germ cells undergo the differentiative process of spermiogenesis in the absence of any further replication (Zirkin, 1993,McCarrey JR 1993, Zhao and Garbers, 2002, Eddy, 2002).

The process of spermatogenesis takes approximately 85 days in humans and 36 days in the mouse. The spermatogonial stem cell population replicates mitotically to both maintain itself and give rise to mitotically active differentiating spermatogonia. These differentiating cells then enter meiosis as primary spermatocytes and proceed through the first meiotic division to yield secondary spermatocytes. During meiosis each cell progresses through a series of cytologically identifiable stages known as leptotene (chromosomes begin to condense), zygotene (pairing of homologous chromosomes is initiated), pachytene (complete synopsis of homologous chromosomes which undergo recombination), diplotene (homologous chromosomes begin to separate), diakinesis (chromosomes condense), metaphase I (separation of homologous chromosomes) and metaphase II (separation of sister chromatids). These secondary spermatocytes then enter the second meiotic division to produce postmeiotic spermatids. Spermatids then undergo extensive differentiation via the process of spermiogenesis to form spermatozoa. In mice, the proliferation of spermatogonia occurs soon after birth, at around 5 days post-partum (dpp), and this is followed by the first wave of spermatogenic differentiation that gives rise to primary spermatocytes at around 10 dpp, haploid round spermatids at 20 dpp, and mature spermatozoa at 35 dpp (Bellve et al., 1977 and Zhao and Garbers, 2002).

Spermatogenesis is thus an excellent system in which to study a developmental process, as it is very well characterized morphologically, and because it is possible to isolate relatively pure populations of cells at specific stages (McCarrey JR., 1993). Over the years, researchers have found that mouse spermatogenic cell lineage provides a unique utility for elucidating the developmental order of regulatory events associated with gene expression. Spermatogenesis features the germ cell-specific events associated with meiosis, as well as certain unique ...