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, and the signals that propel the germ cells to enter the 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 how germ cells program the meiotic division phase, and ultimately this will help us understand how errors in meiotic mechanisms cause aneuploidy, or inappropriate chromosome number, in offspring. Additionally, we exploit an unbiased genetic approach that has identified new mutations affecting meiotic processes 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.
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, are essential to genetic integrity and reproductive success. We study the genetic mechanisms by which germ cells regulate meiotic division, the process that ensures 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. Because the spermatogenic "maturation arrest" phenotype we study occurs 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 prophase exit 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 CDK1 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. We tested a role for the aurora kinases (AURKA and AURKB) in transition from prophase to the meiotic division phase. Both AURKA and AURKB associated with chromatin and the synaptonemal complex (SC) in pachynema. Inhibition of AURKs in cultured spermatocytes inhibits the MPF histone H3 kinase activity characteristic of division phase and also chromosome individualization, but not desynapsis or the initial stage of chromatin condensation, providing evidence that these processes are subject to differential regulation. Recently we have discovered a role for polo-like kinase 1 (PLK1) in promoting desynapsis of homologous chromosomes and entry into the meiotic division phase. Moreover, in vitro, PLK1 exhibits kinase activity toward several proteins of the SC, providing new insight into mechanisms of desynapsis. 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.
Recent genetic evidence reveals unexpected complexity in the regulation of the onset of the meiotic division phase. Few of the genes that control this process in mammals are known, but we have shown that a novel ENU-induced meiotic arrest phenotype is due to a mutation in the Eif4g3 gene, encoding eukaryotic translation initiation factor 4, gamma 3. Mutant germ cells appear to execute events of meiotic prophase normally. Although many proteins characteristic of the prophase to metaphase transition were not obviously depleted in mutant spermatocytes, the activity of CDK1 kinase was dramatically reduced in mutant spermatocytes, and mutant spermatocytes did not enter metaphase I in vivo, or when induced to do so in vitro. Strikingly, HSPA2, a chaperone protein for CDK1 kinase, was absent in the mutant spermatocytes in spite of the presence of Hspa2 transcript, and the phenotype of the Eif4g3repro8 mutation is similar to the phenotype of the knockout mutation of Hspa2. Therefore, EIF4G3 is required for HSPA2 translation in spermatocytes, and consequent activation of cyclin-dependent kinase and exit from meiosis. These results provide the first evidence for acute and selective translational control of meiotic exit in mammalian spermatocytes.
Identification of new genes involved in fertility
Most of the genes that regulate mammalian fertility are as yet unidentified. The Reproductive Genomics Program mutagenesis screen has been successful in generating and identifying mutations that cause infertility. Interestingly, the majority of these mutations affect male fertility only. Many of these result in abnormal sperm morphology and motility. Several 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, and 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, poor sperm morphology and reduced or absent sperm 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 late stages of spermatid differentiation. Thus distinctly different single gene mutations can cause similar, complex, and non-specific sperm pathologies, a point with important implications for managing cases of human male infertility and understanding the action of reproductive toxins.
The repro4 mutation causes failure of spermatocytes to exit meiotic prophase I into the division phase. Nonetheless, major events of meiotic prophase I occurred normally in affected spermatocytes and known regulators of the meiotic G2/MI transition were present and functional. Deep sequencing of mutant DNA revealed a mutation located in an intron of Mtap2 gene, encoding microtubule-associated protein 2, and levels of Mtap2 transcript were reduced in mutant testes. This evidence implicates MTAP2 as required directly or indirectly for completion of meiosis and normal spermatogenesis in mammals.
Another ENU-induced mutation, repro42, causes both male and female infertility, with no other apparent phenotypes. Positional cloning led to the discovery of a nonsense mutation in Spata22, a hitherto uncharacterized gene conserved among bony vertebrates. Expression of both transcript and protein is restricted predominantly to germ cells of both sexes. Germ cells of repro42 mutant mice express Spata22 transcript, but not SPATA22 protein. Gametogenesis is profoundly affected by the mutation, and germ cells in repro42 mutant mice do not progress beyond early meiotic prophase, with subsequent germ cell loss in both males and females. The Spata22 gene is essential for one or more key events of early meiotic prophase, as homologous chromosomes of mutant germ cells do not achieve normal synapsis or repair meiotic DNA double-strand breaks. The repro42 mutation thus identifies a novel mammalian germ cell-specific gene required for meiotic progression.
Another genetic model of infertility, ferf1 (fertilization failure 1), identifies sperm function critical for fertilization of oocytes. Mutant sperm are unable to recognize and penetrate the oocyte zona pellucida, although they are capable of fertilizing zona-free oocytes. Sperm from homozygous ferf1 mutant males are morphologically normal, although they clump and exhibit abnormalities of both acrosomal exocytosis and motility, suggesting that the product of the ferf1 gene affects the processes of epididymal maturation and/or capacitation.
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.
Meiotic sex chromosomes and fertility consequences of unpaired chromosomesThe 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, and accumulates proteins and protein modifications that are usually restricted to the XY body, whether or not that chromosome is associated with the XY pair. This suggests that unpaired chromosomes may together establish a unique chromatin territory within meiotic nuclei.
Associate Research Scientist: Fengyun Sun, Ph.D.
Postdoctoral Fellow: Philip Jordan, Ph.D.
Research Assistants: Kristina Palmer, M.S., Marilyn O'Brien
Research Administrative Assistant: Maxine Friend
Su YQ, Sun F, Handel MA, Schimenti JC, Eppig JJ. 1012. Meiosis arrest female 1 (MARF 1) has nuage-like function in mammalina oocytes. Proc Natl Acad Sci. 109:18653-18660. PMC3503166
Guan Y, Gorenshteyn D, Burmeister M, Wong AK, Schimenti JC, Handel MA, Bult CJ, Hibbs MA, Troyanskaya OG. 2012. Tissue-specific funtional networks for prioritizing phenotype and disease genes. PLoS Comput Biol. 8:e1002694. PMC3459891
Jordan PW, Karppinen J, Handel MA. 2012. Polo-like kinase is required for synaptonemal complex disassembly and phosphorylation in mouse spermatocytes. J Cell Sci. 125:5061-5072.
Fritsche M, Reinholdt L, Lessard M, Handel MA, Bewersdorf J, Heermann DW. 2012. Entropy-driven spatial organization of synaptonemal complexes within the cell nucleus. PLoS Biol. 7:e36282. PMC3344857
Su YQ, Sugiura K, Sun F, Pendola JK, Cox GA, Handel MA, Schimenti JC, Eppig JJ. 2012. MARF1 regulates essential oogenic processes in mice. Science 335:1496-1499.
La Salle S, Palmer K, O'Brien M, Schimenti JC, Eppig JJ, Handel MA. 2012. Spata22, a novel vertebrate-specific gene, is required for meiotic progress in mouse germ cells. Bio Reprod. 86:1-12. PMC3290669
Bolcun-Filas E, Bannister LA, Barash A, Schimenti KJ, Hartford SA, Eppig JJ, Handel MA, Shen L, Schimenti JC. 2011. A-MTB (MYBL1) transcription factor is a master regulator of male meiosis. Development 138:3319-3330. PMC3133921
Sun F, Handel MA. 2011. A mutation in Mtap2 is assocated with arrest of mammalian spermatocytes before the first meiotic division. Genes 2(1):21-25.
Sun F, Palmer K, Handel MA. 2010. Mutation of Eif4g3, encoding a eukaryotic translation initiation factor, causes male infertility and meiotic arrest of mouse spermatocytes. Development. 137:1699-1707. PMC286051
Handel MA, Schimenti JC. 2010. Genetics of mammalian meiosis: regulation, dynamics and impact on fertility. Nat Rev Genet. 11:124-136.
Reinholdt LG, Czechanski A, Kamdar S, King BL, Sun F, Handel MA. 2009. Meiotic behavior of aneuploid chromatin in mouse models of Down syndrome. Chromosoma. 118:723-736. PMC2848991
La Salle S, Sun F, Handel MA. 2009. Isolation and short-term culture of mouse spermatocytes for analysis of meiosis. Methods Mol Biol. 558:279-297.
Geyer CB, Inselman AL, Sunman JA, Bornstein S, Handel MA, Eddy EM. 2009. A missense mutation in the Capza3 gene and disruption of F-actin organization in spermatids of repro32 infertile male mice. Dev Biol. 330:142-152. PMC26888473
La Salle S, Sun F, Zhang XD, Matunis MJ, Handel MA. 2008. Developmental control of sumoylation pathway proteins in mouse male germ cells. Dev Biol. 32: 227-237. PMC2599952
Sun F, Handel MA. 2008. Regulation of the meiotic prophase 1 to metaphase 1 transition in mouse spermatocytes. Chromosoma. 117:471-485. PMC2737826
Eppig JJ, Handel MA. 2008. Editorial from the BOR Editors-in-Chief. The dreaded "d" word. Biol Reprod. 78:566.
Ryu KY, Sinnar SA, Reinholdt LG, Vaccari S, Hall S, Garcia MA, Zaitseva TS, Bouley DM, Boekelheide K, Handel MA, Conti M, Kopito RR. 2008. The mouse polyubiquitin gene Ubb is essential for meiotic progression. Mol Cell Biol. 28:1136-1146. PMC2223379
Good JM, Handel MA, Nachman MW. 2008. Asymmetry and polymorphism of hybrid male sterility during the early stages of speciation in house mice. Evolution. 62:50-65. PMC2907743
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.