Geneticists measure time in generations and celebrate immortality with reproductive success. My lab is driven by a passion to understand the cell biological basis of gamete (sperm and egg) development. We study how germline stem cells balance self-renewal with differentiation. Stem cell self-renewal at the expense of differentiation can cause germ cell tumors while differentiation at the expense of self-renewal can cause sterility. Our long-term goal is to understand the mechanisms that regulate germline stem cell fate.
Other research interests include understanding the molecular function of the hormone testosterone in spermatogenesis. Our work has revealed that specialized tight junctions between Sertoli cells, which are integral to the blood/testis barrier, are regulated by testosterone. We are studying how germ cells pass through these tight junctions without compromising barrier function.
We are also investigating molecular mechanisms of translational regulation—a major form of gene regulation in both male and female germ cells—during spermatogenesis. We use both forward and reverse genetics to identify the genes involved. Phenotypic analysis includes microscopy, biochemistry and cell physiology.
Gametes (sperm and eggs) are highly specialized cells. Spermatozoa contain condensed chromatin, a flagellum, and an acrosomal vesicle whose contents are used for penetrating the outer layer of the egg. Oocytes, unfertilized eggs, have an exceptionally large cytoplasm containing RNA and protein that drives the early cleavage divisions and reprograms the male pronucleus to support early development. In mammals, inductive signaling early in embryogenesis creates a small number of primordial germ cells (PGCs). In both sexes, PGCs migrate to the presumptive gonads, the genital ridges, where in females they initiate meiosis and in males they undergo mitotic arrest. Spermatogenesis occurs postnatally within the seminiferous tubules of the testis. Seminiferous tubules contain a single type of somatic cell, the Sertoli cell, and germ cells at all stages of proliferation and differentiation: spermatogonial stem cells, proliferating and differentiating diploid spermatogonia, spermatocytes in different stages of meiosis, and haploid spermatids. Postnatal oogenesis involves the development of oocytes in growing ovarian follicles, where oocytes grow to be one of the largest cells in the body and communicate with proliferating somatic cells. In mammals, oocytes complete the first meiotic division during ovulation and the second meiotic division upon fertilization.
Germline stem cells
Spermatogonial (germline) stem cells (SSCs) in the testis maintain continuous gamete production postnatally. Cell intrinsic and extrinsic signaling maintains a balance between self-renewal and differentiation. We have shown that loss-of-function mutations in the gene encoding the promyelocytic leukemia zinc finger protein PLZF cause an age-dependent loss of germline stem cells leading to sterility. PLZF is expressed in germline stem cells and is required for their self-renewal. PLZF is a DNA binding protein that facilitates transcriptional repression through recruitment of chromatin modifying enzymes to specific DNA sequences. We are currently attempting to identify transcriptional targets of PLZF and determine how the somatic niche regulates PLZF. We have also shown that SSCs have unique epigenetic methyl-histone modifications that are distinct from their progeny. We suspect that these modifications are important determinants of cell fate and are currently testing this idea through SSC specific ablation of genes encoding key chromatin modification enzymes.
Testosterone is an androgenic steroid that binds to the androgen receptor (AR),a sequence-specific transcription factor. Whole body null mutations in the gene encoding AR result in complete androgen insensitivity syndrome. These individuals are externally female yet have undescended testes. Androgens also function postnatally. In the testis, AR is expressed in the steroid producing Leydig cells, where we have shown it functions in a short negative feedback loop to regulate cholesterol and steroid metabolism. It is also expressed in Sertoli cells, where it functions to support spermatogenesis, and in peritubular myoid cells, where its function is unknown. Using conditional genetic ablation of Ar in Sertoli cells, we have shown that AR signaling is required for three stages of spermatogenesis: progression through meiosis I, the differentiation of haploid round spermatids into elongating spermatids, and spermiogenesis, the release of fully differentiated elongated spermatids into the lumen of the seminiferous epithelium.
We have used gene expression profiling to identify several transcriptional targets of AR in Sertoli and in Leydig cells. These data suggest that androgens have both instructive and permissive roles in supporting spermatogenesis. One of the targets of AR in Sertoli cells, the Claudin 3 (Cldn3) gene, is expressed during the period when preleptotene spermatocytes move from the basal to the adluminal compartment of the seminiferous tubule. CLAUDIN 3 is a transient component of the Sertoli cell tight junctions. We have shown that the permeability of the blood/testis barrier is compromised in Ar mutants and are currently testing the role of the loss of Cldn3 in the phenotype. Experiments are also underway to determine the importance of the stage-specific expression of Cldn3 and its importance in maintaining immune privilege to differentiating germ cells in the adluminal compartment.
Translational control is a major form of gene regulation during gametogenesis. We are investigating the importance and mechanism of translational control during spermatogenesis. Genes encoding the basic proteins required for chromatin condensation--transition protein 1 and 2 and protamine 1 and 2--are all under translational control. The protamine 1 (Prm1) gene is transcribed in round spermatids and its mRNA stored as an RNA/protein particle (mRNP) for up to 7 days before it is eventually translated in elongating spermatids. We showed that sequence elements important for both translational repression and activation lie entirely in its 3' untranslated region (3' UTR). Temporal translation of Prm1 mRNA is essential for spermatogenesis. Deletion of its 3' UTR and replacement with a heterologous 3' UTR leads to premature translation of Prm1, early DNA condensation, an arrest in spermatid differentiation and sterility.
We have identified a 17-nucleotide sequence referred to as the translational control element (TCE) that confers both translational repression and activation on a heterologous mRNA in mice. We are currently studying the position-dependence of this element and the mechanism by which it confers translational regulation. A second sequence element in the 3' UTR, the Y box recognition sequence (YRS), appears to be important for mRNA stability during the period of translational repression. We have identified two Y box proteins, MSY2 and MSY4, that bind to the YRS in vitro and that are associated with the endogenous mRNP in vivo. To determine if MSY4 can mediate translational repression in vivo, we generated transgenic mice in which the temporal window of MSY4 expression was extended during spermatogenesis. Expression of MSY4 disrupted the normal completion of spermatogenesis and caused dominant sterility. Translation of several mRNAs, including the Prm1 and Prm2 mRNAs, was repressed. Loss-of-function mutations in either Msy2 or Msy4 causes sterility. We are currently investigating the reason for the sterility in Msy2 and Msy4 mutants and are creating Msy2 Msy4 double mutants to examine possible redundancy in function.
Little is known about the mechanism of translational activation of translationally repressed mRNAs. However, we have shown that a loss-of-function mutation in the gene encoding TAR RNA binding protein 2 (TARBP2) leads to delayed activation of Prm1 translation and sterility. The timing of TARBP2 expression suggests that it may function as a chaperone in the assembly of specific translationally regulated RNPs. Several groups have recently shown that the human orthologue of TARBP2 interacts with DICER1, the enzyme that catalyzes the cleavage of precursor microRNAs into their mature products. We are currently examining the processing of small noncoding RNAs in Tarbp2 mutants and investigating the role of microRNAs in translational repression and activation. We have also created a germline specific mutation in the gene encoding Dicer1 and are characterizing the sterility in the mutant.
Professor: Robert Braun, Ph.D.
Research Scientist: Anne Greenlee, Ph.D.
Associate Research Scientist: Bill Buaas, Ph.D., Manju Sharma, Ph.D.
Postdoctoral Fellows: Papia Chakraborty, Ph.D., Tongjun Gu, Ph.D., Elizabeth Snyder, Ph.D.
Master of Science Teacher Intern: Zachary Batz, B.S.
Research Assistant I: Nichelle Gray, B.S.
Research Administrative Assistant: Melissa Rockwood, M.S.
Gallagher SJ, Kofman AE, Huszar JM, Dannenberg JH, DePinho RA, Braun RE, Payne CJ. 2013. Distinct requirements for Sin3a in perinatal male gonocytes and differentiating spermatogonia. Dev Biol 373: 83-94.
Greenlee AR, Shiao MS, Snyder E, Buaas FW, Gu T, Stearns TM, Sharma M, Murchison EP, Puente GC, Braun RE. 2012. Deregulated sex chromosome gene expression with male germ cell-specific loss of Dicer1. PLoS One 7: e46359.
Smith BE, Braun RE. 2012. Germ cell migration across Sertoli cell tight junctions. Science 338: 798-802.
Gu T, Buaas FW, Simons AK, Ackert-Bicknell CL, Braun RE, Hibbs MA. 2011. Canonical A-to-I and C-to-U RNA editing is enriched at 3’ UTRs and microRNA target sites in multiple mouse tissues. PLoS One 7: e33720. PMCID: PMC3308996.
Navarro VM, Gottsch ML, Wu M, Garcia-Galiano D, Hobbs SJ, Bosch MA, Pinilla L, Clifton DK, Dearth A, Ronnekleiv OK, Braun RE, Palmiter RD, Tena-Sempere M, Alreja M, Steiner RA. 2011. Regulation of NKB pathways and their roles in the control of Kiss1 neurons in the arcuate nucleus of the male mouse. Endocrinology 152: 4265-4275. PMCID: PMC3198996.
Meng J, Greenlee AR, Taub CJ, Braun RE. 2011. Sertoli cell-specific deletion of the androgen receptor compromises testicular immune privilege in mice. Biol Reprod 85: 254-260. PMCID: PMC3142254.
Kaneko H, Dridi S, Tarallo V, Gelfand BD, Fowler BJ, Cho WG, Kleinman ME, Ponicsan SL, Hauswirth WW, Chiodo VA, Kariko K, Yoo JW, Lee DK, Hadziahmetovic M, Song Y, Misra S, Chaudhuri G, Buaas FW, Braun RE, Hinton DR, Zhang Q, Grossniklaus HE, Provis JM, Madigan MC, Milam AH, Justice NL, ALbuquerque RJ, Blandford AD, Bogdanovich S, Hirano Y, WItta J, Fuchs E, Littman DR, Ambati BK, RUdin CM, Chong MM, Provost P, Kugel JF, Goodrich JA, Dunaief JL, Baffi JZ, Ambati J. 2011. DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration. Nature 471: 325-330.
Spradling A, Fuller MT, Braun RE, Yoshida S. 2011. Germline stem cells. Cold Spring Harbor Perspect Biol. 3(11): a002642.
Payne CJ, Gallagher SJ, Foreman O, Dannenberg JH, DePinho RA, Braun RE. 2010. Sin3a is required by Sertoli cells to establish a niche for undifferentiated spermatogonia, germ cell tumors, and spermatid elongation. Stem Cells 28: 1424-1434.
Nakagawa T, Sharma M, Nabeshima Y, Braun RE, Yoshida S. 2010. Functional hierarchy and reversibility within the murine spermatogenic stem cell compartment. Science 328: 62-67.
Wang G, Weng CC, Shao SH, Zhou W, De Gendt K, Braun RE, Verhoeven G, Meistrich ML. 2009. Androgen receptor in Sertoli cells is not required for testosterone-induced suppression of spermatogenesis, but contributes to Sertoli cell organization in Utp14bjsd mice. J Androl 2009 Jan 8 [Epub ahead of print].
Nalam RL, Andreu-Vieyra C, Braun RE, Akiyama H, Matzuk MM. 2009. Retinoblastoma protein plays multiple essential roles in the terminal differentiation of Sertoli cells. Mol Endocrinol 23: 1900-1913.
Daher A, Laraki G, Singh M, Melendez-Pe–a CE, Bannwarth S, Peters AHFM, Meurs EF, Braun RE, Patel RC, Gatignol A. 2009. TRBP control of PACT-induced phosphorylation of protein kinase R is reversed by stress. Mol Cell Biol 29: 254-265.
Sadate-Ngatchou PI, Payne CJ, Dearth AT, Braun RE. 2008. Cre recombinase activity specific to postnatal, premeiotic male germ cells in transgenic mice. Genesis 46:738-742.
Payne CJ, Braun RE. 2008. Human adult testis-derived pluripotent stem cells: revealing plasticity from the germline. Cell Stem Cell 3:471-472.
Eacker SM, Agrawal N, Qian K, Dichek HL, Gong EY, Lee K, Braun RE. 2008. Hormonal regulation of testicular steroid and cholesterol homeostasis. Mol Endocrinol 22:623-635.
Zhao M, Rohozinski J, Sharma M, Ju J, Braun RE, Bishop CE, Meistrich ML. 2007. Utp14b: a unique retrogene within a gene that has acquired multiple promoters and a specific function in spermatogenesis. Dev Biol 304:848-859.
Eacker SM, Shima JE, Connolly CM, Sharma M, Holdcraft RW, Griswold MD, Braun RE. 2007. Transcriptional profiling of androgen receptor (AR) mutants suggests instructive and permissive AR signaling in germ cell development. Mol Endocrinol 21:895-907.
DiNardo S, Braun RE. 2007. Developmental biology. Home for the precious few. Science 217:1696-1697.
Seydoux G, Braun RE. 2006. Pathways to totipotency--lessons from germ cells. Cell 127:891-904.
Payne C, Braun RE. 2006. GDNF maintains a POZ-itive influence on stem cells. Proc Natl Acad Sci USA 103:9751-9752.
Payne C, Braun RE. 2006. Histone lysine trimethylation exhibits a distinct perinuclear distribution in Plzf-expressing spermatogonia. Dev Biol 293:461-472.
Greenbaum MP, Yan W, Wu MH, Lin YN, Agno J, Sharma M, Braun RE, Rajkovic A, Matzuk MM. 2006. TEX14 is essential for intercellular bridge development and fertility in male mice. Proc Natl Acad Sci USA 103:4982-4987.
Smith JT, Dungan HM, Stoll EA, Gottsch ML, Braun RE, Eacker SM, Clifton DK, Steiner RA. 2005. Differential regulation of Kiss1 gene expression by sex steroids in the brain of the male mouse. Endocrinology 146:2976-2984.
Nie Y, Ding L, Kao PN, Braun RE, Yang J-H. 2005. ADAR1 interacts with NF90 through double-stranded RNA and regulates NF90-mediated gene expression independently of RNA editing. Mol Cell Biol 25:6956-6963.
Meng J, Holdcraft RW, Shima J, Griswold MD, Braun RE. 2005. Androgens regulate the permeability of the blood-testis barrier. Proc Natl Acad Sci USA 102:16696-16700.
Connolly CM, Dearth AT, Braun RE. 2005. Disruption of murine Tenr results in teratospermia and male infertility. Dev Biol 278:13-21.
Holdcraft RH, Braun RE. 2004. Hormonal regulation of spermatogenesis. Int J Andrology 27:335-342.
Holdcraft RH, Braun RE. 2004. Androgen receptor function is required in Sertoli cells for terminal differentiation of haploid spermatids. Development 131:459-467.
Buaas FW, Kirsh AL, Sharma M, McLean DJ, Morris JL, Griswold MD, de Rooij DG, Braun RE. 2004. PLZF is required in adult male germ cells for stem cell self-renewal. Nature Genetics 36:647-652.
Giorgini F, Davies HG, Braun RE. 2002. Translational repression by MSY4 inhibits spermatid differentiation in mice. Development 129:3669-3679.
Zhong J, Peters AHFM, Kafer K, Braun RE. 2001. A highly conserved sequence that is essential for translational repression of protamine mRNAs in murine spermatids. Biol of Reprod 64:1784-1789.
Peters AHFM, Drumm J, Ferrell C, Roth DA, Roth DM, McCaman M, Novak PL, Friedman J, Engler R, Braun RE. 2001. Absence of germ line infection in male mice following intraventricular injection of adenovirus. Mol Therapy 4:603-613.
Meagher MJ, Braun RE. 2001. Requirement for the murine zinc finger protein ZFR in perigastrulation growth and survival. Mol Cell Biol 21:2880-2890.
Giorgini F, Davies HG, Braun RE. 2001. MSY2 and MSY4 bind a conserved sequence in the 3' untranslated region of Protamine 1 mRNA in vitro and in vivo. Mol Cell Biol 21:7010-7019.
Braun RE. 2001. Packaging paternal chromosomes in protamine. Nature Genetics 28:10-12.
Nadler JJ, Braun RE. 2000. Fanconi anemia complementation group C is required for proliferation of murine primordial germ cells. Genesis 27:117-123.
DePaolo LV, Hinton BT, Braun RE. 2000. Male contraception: Views to the 21st century. Trends Endocrinol Metab 11:66-69.
Davies HG, Giorgini F, Fajardo MA, Braun RE. 2000. A sequence-specific RNA binding complex expressed in murine germ cells contains MSY2 and MSY4. Dev Biol 221:87-100.
Zhong J, Peters AHFM, Lee K, Braun RE. 1999. A double-stranded RNA binding protein required for activation of repressed messages in mammalian germ cells. Nature Genetics 22:175-178.
Meagher MJ, Schumacher JM, Lee K, Holdcraft RW, Edelhoff S, Disteche C, Braun RE. 1999. Identification of ZFR, an ancient and highly conserved murine chromosome-associated zinc finger protein. Gene 228:197-211.
Buaas FW, Lee K, Edelhoff S, Disteche C, Braun RE. 1999. Cloning and characterization of the mouse interleukin enhancer binding factor 3 (Ilf3) homolog in a screen for RNA binding proteins. Mamm Genome 10:451-456.
Zhong J, Edelhoff S, Disteche C, Braun RE. 1998. The gene encoding PRBP, the mosue homolog of human TRBP, maps to distal chromosome 15. Mamm Genome 9:413-414.
Schumacher JM, Artzt K, Braun RE. 1998. Spermatid perinuclear ribonucleic acid-binding protein binds microtubules in vitro and associates with abnormal Manchettes in vivo in mice. Biol of Reprod 59:69-76.
Braun RE. 1998. Every sperm is sacred--or is it? Nature Genetics 18:202-204.
Whitney MA, Royle G, Low MJ, Kelly MA, Axthelm MK, Reifsteck C, Olson S, Braun RE, Heinrich MC, Rathbun RK, Bagby GC, Grompe M. 1996. Germ cell defects and hematopoietic hypersensitivity to g-interferon in mice with a targeted disruption of the Fanconi anemia C gene. Blood 88:49-58.
Lee KS, Fajardo MA, Braun RE. 1996. A testis cytoplasmic RNA binding protein that has the properties of a translational repressor. Mol Cell Biol 16:3023-3034.
Schumacher JM, Lee KS, Edelhoff S, Braun RE. 1995. Distribution of Tenr, an RNA-binding protein, in a lattice-like network within the spermatid nucleus in the mouse. Biol of Reprod 52:1274-1283.
Lee K, Haugen HS, Clegg CH, Braun RE. 1995. Premature translation of protamine 1 mRNA causes precocious nuclear condensation and arrests spermatid differentiation in mice. Proc Natl Acad Sci USA 92:12451-12455.
Schumacher JM, Lee KS, Edelhoff S, Braun RE. 1994. Spnr, a murine RNA-binding protein that is localized to cytoplasmic microtubules. J Cell Biol 129:1023-1032.
Fajardo MA, Butner KA, Lee K, Braun RE. 1994. Germ cell-specific proteins interact with the 3' untranslated regions of the Prm-1 and Prm-2 mRNA. Dev Biol 166:643-653.
Wilkie TM, Braun RE, Ehrman WJ, Palmiter RD, Hammer RE. 1991. Germline intrachromosomal recombination restores fertility in transgenic MyK-103 male mice. Genes & Devel 5:38-48.
Braun RE, Peschon JJ, Behringer RR, Brinster RL, Palmiter RD. 1989. Protamine 3' untranslated sequences regulate temporal translational control and subcellular localization of growth hormone in spermatids of transgenic mice. Genes & Devel 3:793-802.
Braun RE, Behringer RR, Peschon JJ, Brinster RL, Palmiter RD. 1989. Genetically haploid spermatids are phenotypically diploid. Nature 337:373-376.