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.

Publications

• Mailhes JB, MA Handel. 2008. Animal models for investigating the causes and mechanisms of mammalian germ cell aneuploidy. In: Sourcebook of Models for Biomedical Research (Ed. M. Conn), pp. 527-537. Humana Press.
• Good JM, MA Handel, MW Nachman. 2008. Asymmetry and polymorphism of hybrid male sterility during the early stages of speciation in house mice. Evolution 62:50-65.
• Ryu KW, SA Sinnar, LG Reinholdt, S Vaccari, S Hall, MA Garcia, TS Zaitseva, DM Bouley, K Boekelheide, MA Handel, M Conti, RR Kopito. 2008. The mouse polyubiquitin gene Ubb is essential for meiotic progression. Mol Cell Biol 28:1136-1146.
• La Salle S, F Sun, MA Handel. 2008. Isolation and short-tem culture of mouse spermatocytes for analysis of meiosis. In: Methods in Molecular Biology, Molecular Medicine and Biotechnology (Series Editor: J. N. Walker), Meiosis Protocols (Ed. S. Keeney). Humana Press. In press.
• Philipps, D., Wigglesworth, K., Hartford, S., Sun, F., Pattabiraman, S., Schimenti, K., Handel, M.A., Eppig, J.J. and Schimenti, J. 2008. The dual bromodomain and WD repeat-containing mouse protein BRWD1 is required for normal spermiogenesis and the oocyte-embryo transition. Devel. Biol. 317:72-82.
• Sun F, MA Handel. 2008. Regulation of the meiotic prophase I to metaphase I transition in mouse spermatocytes. Chromosoma: In press.
• La Salle S, F Sun, XD Zhang, MJ Matunis, MA Handel. 2008. Developmental control of sumoylation pathway proteins in mouse male germ cells. Dev Biol 321:227-237.
• Davisson MT, MA Handel. 2007. Cytogenetics. In: Mouse in Biomedical Research, 2nd Ed. (Eds., Fox J, Barthold S, Davisson MT, Newcomer C, Quimby F, Smith A), Elsevier Press. Vol. 1 Ch. 9 pp 145-164.
• Lessard C, H Lothrop, JC Schimenti, MA Handel. 2007. Mutagenesis-generated mouse models of human infertility with abnormal sperm. Hum Reprod 22:159-166.
• Davisson M, E Akeson, C Schmidt, B Harris, J Farley, MA Handel. 2007. Impact of trisomy on fertility and meiosis in male mice. Hum Reprod 22:468-476.
• Handel, MA, C Lessard, L Reinholdt, J Schimenti, JJ Eppig. 2006. Mutagenesis as an unbiased approach to identify novel contraceptive targets. Mol Cell Endo 250:201-205.
• Matulis S, MA Handel. 2006. Spermatocyte responses in vitro to induced DNA damage. Molec Reprod Devel 73:1061-1072.
• Handel MA, Sun F. 2005. Regulation of meiotic cell divisions and determination of gamete quality:Impact of reproductive toxins. Sem Reprod Med. 23:213-221.
• Sharan SK, A Pyle, V Coppola, J Babus, S Swaminathan, J Benedict, D Swing, BK Martin, L Tessarollo, JP Evans, JA Flaws, MA Handel. 2004. BRCA2 deficiency in mice leads to meiotic impairment and infertility. Development 131:131-142.
• Handel MA. 2004. News and Views: Marking Xs, together and separately. Nature Genetics 36:12-13.
• Qin J, LL Richardson, M Jasin, MA Handel, N Arnheim. 2004. Mouse strains with an active H2-Ea meiotic recombination hot spot exhibit increased levels of H2-Ea -specific DNA breaks in testicular germ cells. Molec Cell Biol 24:1655-1666.
• Lessard C, JK Pendola, SA Hartford, JC Schimenti, MA Handel, JJ Eppig. 2004. New mouse genetic models for human contraceptive development. Cytogenet Genome Res 105:222-227.
• Handel MA. 2004. The XY body: A specialized meiotic chromatin domain. Exptl Cell Res 296:57-63.
• Lin Q, A Inselman, X Han, H Xu, W Zhang, MA Handel, AI Skoultchi. 2004. Reductions in linker histone levels are tolerated in developing spermatocytes but cause changes in specific gene expression. J Biol Chem 279:23525-35.
• Inselman A, MA Handel. 2004. Mitogen-activated protein kinase dynamics during the meiotic G2/MI transition of mouse spermatocytes. Biol Reprod 71:570-578.
• Cho YS, Chennathukuzhi VM, MA Handel, Eppig JJ, Hecht NB. 2004. The relative levels of translin-associated factor X (TRAX) and testis brain RNA-binding protein determine their nucleocytoplasmic distribution in male germ cells. J Biol Chem 279:31514-31523.
• Rogers RS, Inselman A, MA Handel, Matunis MJ. 2004. SUMO modified proteins localize to the XY body of pachytene spermatocytes. Chromosoma 113:233-243.
• Pyle P, MA Handel. 2003. Meiosis in male PL/J mice: A genetic model for gametic aneuploidy. Molec Reprod Devel 64: 471-481.
• Inselman A, S Eaker, MA Handel. 2003. Temporal expression of cell cycle related proteins during spermatogenesis: Establishing a timeline for onset of the meiotic divisions. Cytogenet Genome Res 103:277-284.
• Libby BJ, R De La Fuente, MJ O’Brien, K Wigglesworth, J Cobb, A Inselman, S Eaker, MA Handel, JJ Eppig, JC Schimenti. 2002. The mouse meiotic mutation mei1 disrupts chromosome synapsis with sexually dimorphic consequences for meiotic progression. Dev Biol 242:174-187.
• Eaker S, J Cobb, A Pyle, MA Handel. 2002. Meiotic prophase abnormalities and metaphase cell death in MLH1-deficient mouse spermatocytes: Insights into regulation of spermatogenic progress. Dev Biol 249:85-95.
• Eaker S, A Pyle, J Cobb, MA Handel. 2001. Evidence for meiotic spindle checkpoint from analysis of spermatocytes from Robertsonian-chromosome-heterozygous mice. J Cell Sci 114:2953-2965.

Grants

• 1996 to 2011 — — Cell Cycle Regulation during Spermatogenesis from NIH
• 2002 to 2008 — — Reproductive Genetics: Mutant Models for Infertility from NIH
• 2005 to 2010 — — Chromatin Structure during Spermatogenesis from NIH