Mary Ann Handel

Education

PhD Biology, Kansas State University, 1970

Research Interests

Genetic Analysis of Meiotic Chromosome Dynamics, Spermatogenesis and Male Fertility

The focus of our laboratory is on genetic regulation of spermatogenesis and male fertility. Appropriate dynamics and behavior of chromosomes during meiosis, a specialized cell division unique to germ cells, ensure genetic integrity and reproductive success. We study the mechanisms by which germ cells form condensed chromosomes as they enter the meiotic division phase. This process is of crucial importance for gametogenesis because it assures the haploid chromosome content of the future gamete. Work in our laboratory has shown that remodeling of chromatin into condensed division-phase chromosomes in mouse spermatocytes is prompted by events involving multiple kinases and inactivation of phosphatases. Our investigations focus on factors extrinsic and intrinsic to meiotic division phase I chromosome structure that establish mechanisms of meiotic division in both male and female germ cells and identify sexually dimorphic events. We anticipate that these studies will provide significant new information about assembly of mammalian meiotic chromosomes, a process that safeguards genomic integrity of gametes. This is important because errors in these meiotic mechanisms are a major cause of aneuploidy, or inappropriate chromosome number, in offspring. Additionally, we take an unbiased genetic approach to identify new mutations that affect meiotic processes, spermatogenic differentiation, and male fertility. Because the phenotypes we study—spermatogenic “maturation arrest” and fertilization failure—occur in many unexplained cases of human male infertility and reproductive toxicity, this approach can shed light on infertility, and possibly identify potential targets for contraception.

Meiotic Chromosome Assembly and Onset of Meiotic Cell Divisions

The complex events of the first meiotic prophase and division phase are only beginning to be understood. Previous work in our laboratory demonstrated the importance of metaphase promoting factor (MPF, comprised of the catalytic subunit CDC2A and the regulatory subunit CCNB1), but also implicated other kinases. Our studies of mitogen-activated protein kinases (MAPKs) suggest a novel pathway for MAPK activation in spermatocytes. We investigated the MAPKs that are commonly known as the extracellular signal-regulated protein kinases ERK1 (formally designated MAPK3) and ERK2 (formally designated MAPK1), their kinases, and the regulatory phosphatase CDC25C by both genetic and cellular analyses. ERK1 and ERK2, which are active when phosphorylated, are expressed in specific developmental patterns throughout spermatogenesis. Phosphorylated variants of these kinases are predominant in the mitotically dividing spermatogonia, but diminished in pachytene spermatocytes. Treatment of pachytene spermatocytes with okadaic acid, to induce the division phase, resulted in phosphorylation and enzymatic activation of ERK1 and ERK2. Analysis of spermatocytes lacking MOS, a mitogen-activated protein kinase kinase kinase responsible for phosphorylation of MAP kinase kinase and, ultimately, MAPK activation, revealed that MOS is not required for okadaic acid-induced activation of the MAPKs or for chromosome condensation. These findings suggest a role for MAPKs in spermatogenic cell divisions, but not in meiotic chromosome condensation. The induced activation of MAPKs was inhibited by butyrolactone I, an inhibitor of MPF. Thus MPF may regulate MAPK activity, which is a previously unsuspected relationship. Spermatocytes lacking CDC25C condensed division-phase chromosomes and activated both MPF and MAPKs in response to okadaic acid treatment; therefore in this model system there is a CDC25C-independent pathway for MPF and MAPK activation, also previously unanticipated.

Because our focus is on meiotic chromosome assembly, we study subcellular localization of proteins and the order of events in meiotic remodeling of chromatin to form condensed division-phase chromosomes. Surface-spread chromatin from spermatocytes is examined using immunofluorescent labeling with specific antibodies to determine subcellular localization of relevant kinase and chromosomal proteins. These include aurora kinases (AURKA and AURKB) that initiate the division phase, synaptonemal complex (SC) proteins SYCP1 and SYCP3 (also known as SCP1 and SCP3), chromosomal condensin subunits SMC2L1 and SMC4L1, and phosphorylated histone H3. These studies reveal that AURKA and AURKB are associated with chromatin and the SC in spermatocytes and show dramatic re-localization in the division phase, when AURKB, but not AURKA, re-localizes to centromeric regions of condensed chromosomes. This re-localization is accompanied by remodeling of chromatin, detected by disassembly of the SC and chromosome condensation. We find that degradation of SYCP1 occurs quite rapidly as spermatocytes enter the division phase, even before phosphorylation of histone H3, previously thought to be the earliest marker of chromosome condensation. Proteins involved in chromosome condensation, the SMC2L1 and SMC4L1 subunits of the condensin protein complex, are found throughout the chromatin in prophase and division-phase spermatocytes. Thus they appear in the right place and at the right time to play roles in resolution of sister chromatids and chromosome condensation during the meiotic division process.

An Unexpected Modification of Meiotic Sex Chromosomes

The XY body is a specialized chromatin territory that forms during meiotic prophase of spermatogenesis, and is comprised of the transcriptionally repressed sex chromosomes. Remodeling of the XY chromatin to form the XY body is brought about by recruitment of specific proteins to the X and Y chromosomes during meiosis and also by post-translational modifications of histones and other chromatin-associated proteins. We have demonstrated that SUMO, a small ubiquitin-related modifier protein that regulates a wide variety of nuclear functions in somatic cells, dramatically localizes to the XY body. Sumoylated substrates are first detected in the XY body of early pachytene spermatocytes and gradually accumulate, reaching maximal levels during the mid-prophase stages, and exiting from this chromatin domain in the division phase. Several known substrates for SUMO modification, including PML and DAXX, were also found to accumulate in the XY body of spermatocytes. These same proteins localize to PML nuclear bodies of somatic interphase nuclei. Together, these findings indicate a role for sumoylation in regulating the structure and function of the XY chromatin domain and suggest a functional similarity between the XY body and PML nuclear bodies.

Identification of New Genes Involved in Spermiogenesis and Male Fertility

Most of the genes that regulate spermatogenesis and sperm function in mammals are as yet unidentified. The ReproGenomics mutagenesis screen to generate and identify mutations that cause infertility is described in the ReproGenomics report in this volume. Many of the mapped mutations that affect only males result in abnormal sperm morphology and motility. These are being studied in more detail to provide models for human male infertility syndromes of sperm tail dysplasia. One genetic model identifies sperm function critical for fertilization of oocytes, and this is being studied in detail to identify events during sperm capacitation that are essential for recognition and penetration of the oocyte zona pellucida. Ultimately, dissection of gene function from these mutant phenotypes will enlarge our knowledge of pathways of spermiogenic differentiation and aspects of sperm function in fertilization.

Selected Publications