Overview
Our laboratory investigates the genetic regulation of spermatogenesis and male fertility. We focus on meiosis, a specialized cell division unique to germ cells that reduces the number of chromosome sets from two (diploid) to one (haploid), producing the gametes, the eggs and sperm that come together during sexual reproduction. We study the mechanisms by which germ cells form condensed chromosomes as they enter the meiotic division phase. Appropriate dynamics and behavior of chromosomes during meiosis is of crucial importance for the formation of gametes, ensuring the haploid chromosome content of the future gamete, as well as genetic integrity and reproductive success. Our studies are providing significant new information about assembly of mammalian meiotic chromosomes, and ultimately will help us understand how errors in these meiotic mechanisms cause 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 traits 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.
Scientific report
Genetic Analyses of Meiosis, 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. 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. These studies are providing significant new information about assembly of mammalian meiotic chromosomes, and ultimately will help us understand how errors in these meiotic mechanisms cause 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 cell-cycle-active kinases, including metaphase promoting factor (MPF), comprised of the catalytic subunit CDC2A and the regulatory subunit CCNB1, and mitogen-activated protein kinases (MAPKs). In order to better understand how these molecules act at the cellular level, subcellular localization of proteins and order of events in meiotic remodeling of chromatin to form condensed metaphase chromosomes has been studied, primarily by immunofluorescence. By inhibitor analysis, we tested a role for the aurora kinases (AURKA and AURKB) in the late prophase to metaphase transition (G2/MI). Both AURKA and AURKB associated with chromatin and the synaptonemal complex (SC) in pachynema. Inhibition of AURKs in cultured spermatocytes inhibits the G2/MI histone H3 kinase activity and chromosome individualization, but not desynapsis or the initial stage of chromatin condensation, providing evidence that these processes are subject to differential regulation. In fact, neither CDKs nor AURKs are involved in disassembly of synaptonemal complex protein 1 (SYCP1) and de-synapsis during the G2/MI transition, while inhibition of either CDKs or AURKs affects synaptonemal complex protein 3 (SYCP3) disassembly. Thus, different mechanisms are responsible for degradation of different components of the SC. Together, these observations provide information about both the temporal order and regulatory relationships of the earliest events in the transition from meiotic prophase to metaphase.
These relationships were bolstered by the discovery of a new mutation affecting these processes. The repro4 mutation was identified through an ENU mutagenesis and phenotype screen for infertility. The repro4/repro4 males are characterized by small testes, with spermatogenesis arrested at the pachytene stage of meiotic prophase. Localization of SYCP1 and SYCP3 revealed that homologous chromosomes paired and the SC was apparently normally assembled. Moreover, mutant repro4 spermatocytes did not arrest until mid- to late-pachynema, as revealed by presence of the mid-pachynema marker, histone HIST1H1T. Although spermatocytes did not enter the division phase, many proteins characteristic of the late prophase-metaphase G2/MI transition were not obviously different in mutant spermatocytes. Meiotic competence of repro4/repro4 spermatocytes was tested by exposing them to okadaic acid (OA), which induces the G2/MI transition in vitro. Mutant spermatocytes were not competent to undergo the induced G2/MI transition. Chromatin condensation in response to cell cycle regulators was abnormal. After OA treatment, most repro4/repro4 spermatocytes exhibited chromosome condensation only in regions of heterochromatin, and metaphase chromosomes failed to become individualized. Genetic fine mapping has localized the gene identified by the repro4 mutation to a region of 0.57 Mb on Chromosome 1 containing 1 gene, parathyroid receptor 2, which has not yet been demonstrated to be involved in fertility. Thus, ENU mutagenesis has induced a novel meiotic phenotype, which reveals a previously undescribed terminal stage of meiotic chromosome condensation.
Meiotic sex chromosomes and fertility consequences of unpaired 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, previously studied in our laboratory. Among the post-translational modifications of proteins associated with the XY body, sumoylation is quite prominent, and in the spermatocyte nucleus is highly specific to the XY body. Protein sumoylation regulates a variety of functions, including transcriptional silencing and formation of specific nuclear heterochromatin domains. Expression of the mediators of sumoylation in the male germ line has been analyzed by quantitative RT-PCR. General expression profiles emerging from these data identify sumoylation pathway genes that could be involved in meiosis-related sumoylation functions and spermiogenesis-related sumoylation events. In collaboration with Dr. Muriel Davisson, we have used models for trisomy to study similar modifications on unpaired autosomal chromatin and the impact of unpaired chromatin on fertility. Impairment to spermatogenesis in trisomic males could be due to the presence of extra genomic material (i.e., triplicated genes) or to the chromosomal abnormality and presence of an unpaired chromosome in meiosis. We found that it is the presence of an intact, extra chromosome, rather than trisomy per se, that is associated with male sterility. Moreover, in sterile males, the unpaired chromosome is frequently associated with the XY body, which contains the largely unpaired X and Y chromosomes. This suggests that unpaired chromosomes may together establish a unique chromatin territory within meiotic nuclei.
We have also found that an extra intact chromosome accumulates proteins and protein modifications that are usually restricted to the XY body, whether or not that chromosome is associated with the XY pair. However, an unpaired chromosomal segment with the same gene content is not similarly modified when attached to normally paired autosomes. These data suggest that the proteins and modifications that are unique to the sex body during male meiosis are a response to the presence of unpaired chromosomes and not restricted to the XY chromatin.
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, which is used to generate and identify mutations that cause infertility, is described elsewhere in this volume. Many of the mapped mutations that affect only males result in abnormal sperm morphology and motility. These were studied in more detail to provide models for human male infertility syndromes of oligoasthenoteratozoospermia. Genes identified by three independent mutations were designated swm2, repro2, repro3. Although the mutant males were infertile, they were apparently normal in all other respects and the gross morphology of their reproductive organs was also normal. All mutant males were characterized by low sperm concentration and poor sperm morphology and motility. Sperm from mutant males failed to fertilize oocytes in vitro. Histological assessment of testes of mutant males revealed that the abnormalities were first apparent in step VIII-IX spermatids. Thus single gene mutations can cause complex and non-specific sperm pathologies, a point with important implications for managing cases of human male infertility.
Another genetic model of infertility, ferf1 (fertilization failure 1), identifies sperm function critical for fertilization of oocytes, and this is being studied in detail to identify events that are essential for the sperm to recognize and penetrate the oocyte zona pellucida. Sperm from ferf1/ferf1 mutant males show abnormalities of both acrosomal exocytosis and motility, suggesting that the product of the ferf1 gene affects the processes of epididymal maturation and/or capacitation. The ferf1 gene has been mapped to a small interval on Chromosome 14. The ENU-induced mutation repro7 affects germ-cell survival and the autosomal recessive mutation causes infertility in both male and female mice. Although the timing of germ cell loss in male and female repro7/repro7 mice correlates with meiotic prophase, our data suggest that repro7/repro7 animals do not exhibit meiotic defects at the chromosomal level. Therefore, the repro7 mutation may affect a gene(s) required for the progression and survival of germ cells through meiosis. The repro7 mutation has been mapped to a 1 Mb region on Chromosome 17. This region does not contain any genes now known required for the progression/survival of germ cells through meiosis. Ultimately, dissection of gene function from these mutant phenotypes will enlarge our knowledge of pathways of gametogenic differentiation and aspects of sperm function in fertilization.
Lab staff
Principal Investigator: Mary Ann Handel, Ph.D.Postdoctoral Fellows: Fengyun Sun, Ph.D., Sophie La Salle, Ph.D.
Research Assistant I: Kristina Palmer, M.S.
Laboratory Technician III: Heather Lothrop
Research Administrative Assistant: Maxine Friend
Publication listings
Lessard C, Lothrop H, Schimenti JC, MA Handel. 2007. Mutagenesis-generated mouse models of human infertility with abnormal sperm. Hum Reprod. 22:159-166.
Davison M, Akeson E, Schmidt C, Harris B, Farley J, MA Handel. 2007. Impact of trisomy on fertility and meiosis in male mice. Hum Reprod. 22:468-476.
Matulis S, MA Handel. 2006. Spermatocyte responses in vitro to induce DNA damage. Mol Reprod Dev. 73:1061-1072.
MA Handel, Lessard C, Reinholdt L, Schimenti JC, Eppig JJ. 2006. Mutagenesis as an unbiased approach to identify novel contraceptive targes. Mol Cell Endo. 250:201-205.
Cho YS, Iguchi N, Yang J, MA Handel, Hecht NB. 2005. Meiotic mRNA and Non-Coding RNA Targets of the RNA-Binding Protein Translin (TSN) in Mouse Testis. Biol Reprod. 73:840-847.
MA Handel, Sun F. 2005. Regulation of meiotic cell divisions and determination of gamete quality:Impact of reproductive toxins. Sem Reprod Med. 23:213-221.
Rogers RS, Inselman A, MA Handel, Matunis MJ. 2004. SUMO modified proteins localize to the XY body of pachytene spermatocytes. Chromosoma 113:233-243.
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.
Inselman A, MA Handel. 2004. Mitogen-activated protein kinase dynamics during the meiotic G2/MI transition of mouse spermatocytes. Biol Reprod 71:570-578.
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.
Handel MA. 2004. The XY body: A specialized meiotic chromatin domain. Exptl Cell Res 296:57-63.
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.
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.
Handel MA. 2004. News and Views: Marking Xs, together and separately. Nature Genetics 36:12-13.
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.
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.
Pyle P, MA Handel. 2003. Meiosis in male PL/J mice: A genetic model for gametic aneuploidy. Molec Reprod Devel 64: 471-481.
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.
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, 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.
