Our long-term objective is to understand better the regulation of the initial steps of spermatogenesis, namely spermatogonial proliferation and differentiation In the current granting period, we demonstrated that proliferation of type A spermatogonia is stimulated by stem cell factor (SCF) Other studies demonstrated that Sertoli cell-spermatogonial co-cultures allow for spermatogonial differentiation, i.e., the isolated type A (presumably stem cells) divided a number of times to yield rows of interconnected differentiated type A. We also established that the PI-3 kinase/AKT/p70 S6 kinase signaling pathway is involved in SCF-induced proliferation of type A spermatogonia. In addition, we demonstrated that differentiation of type A into sperm is associated with changes in telomere length and telomerase activity. In this competing renewal application, in aim 1, we will further examine the biology of the type A spermatogonia. We hypothesize that in addition to SCF, other ligands such as leukemia inhibitory factor (LIF) and glial cell line-derived neurotrophic factor (GDNF) may be important for spermatogonial development. Our preliminary results indicate that receptors (GFRalpha1 and LIFr) for these two ligands are expressed on the surface of type A spermatogonia. We will use whole mount immunocytochemistry to examine whether all type A spermatogonia possess the GFRalpha1, c-kit, and LIF receptors. Using the immunomagnetic bead procedure, we will separate a GFRalpha1 positive group of cells as well as a c-kit and LIFr positive group of cells and stimulate each group with the respective ligands. To determine whether these subsets of type A spermatogonia retain their ability to differentiate, we will transplant them into sterile recipient mice and follow spermatogenesis in vivo.
In aim 2, we will continue to investigate the molecular signals responsible for spermatogonial proliferation. We have generated exciting preliminary data to suggest that SCF and LIF may initiate distinct intracellular signaling pathways. Through these signaling pathways, specific candidate genes are induced by ligands leading to spermatogonial proliferation. In the third aim, we will examine gene expression in isolated type A spermatogonia and in seminiferous tubules in culture using serial analysis of gene expression (SAGE) and DNA array procedures in response to ligands. The experiments outlined in this proposal should provide new data on the basic biology of the spermatogonia. Furthermore, these studies will help in developing techniques of in-vitro spermatogenesis and contribute to the use of healthy germ cells in assisted reproduction.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD033728-06
Application #
6738131
Study Section
Reproductive Biology Study Section (REB)
Program Officer
Rankin, Tracy L
Project Start
1997-04-01
Project End
2008-06-30
Budget Start
2004-07-01
Budget End
2005-06-30
Support Year
6
Fiscal Year
2004
Total Cost
$524,611
Indirect Cost
Name
Georgetown University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
049515844
City
Washington
State
DC
Country
United States
Zip Code
20057
He, Zuping; Jiang, Jiji; Kokkinaki, Maria et al. (2013) MiRNA-20 and mirna-106a regulate spermatogonial stem cell renewal at the post-transcriptional level via targeting STAT3 and Ccnd1. Stem Cells 31:2205-17
Dwyer, Andrew A; Sykiotis, Gerasimos P; Hayes, Frances J et al. (2013) Trial of recombinant follicle-stimulating hormone pretreatment for GnRH-induced fertility in patients with congenital hypogonadotropic hypogonadism. J Clin Endocrinol Metab 98:E1790-5
Kokkinaki, Maria; Lee, Tin-Lap; He, Zuping et al. (2010) Age affects gene expression in mouse spermatogonial stem/progenitor cells. Reproduction 139:1011-20
He, Zuping; Kokkinaki, Maria; Jiang, Jiji et al. (2010) Isolation, characterization, and culture of human spermatogonia. Biol Reprod 82:363-72
Balasinor, Nafisa H; D'Souza, Ryan; Nanaware, Padma et al. (2010) Effect of high intratesticular estrogen on global gene expression and testicular cell number in rats. Reprod Biol Endocrinol 8:72
Kokkinaki, Maria; Lee, Tin-Lap; He, Zuping et al. (2009) The molecular signature of spermatogonial stem/progenitor cells in the 6-day-old mouse testis. Biol Reprod 80:707-17
He, Zuping; Jiang, Jiji; Kokkinaki, Maria et al. (2009) Nodal signaling via an autocrine pathway promotes proliferation of mouse spermatogonial stem/progenitor cells through Smad2/3 and Oct-4 activation. Stem Cells 27:2580-90
Golestaneh, Nady; Kokkinaki, Maria; Pant, Disha et al. (2009) Pluripotent stem cells derived from adult human testes. Stem Cells Dev 18:1115-26
He, Zuping; Kokkinaki, Maria; Dym, Martin (2009) Signaling molecules and pathways regulating the fate of spermatogonial stem cells. Microsc Res Tech 72:586-95
He, Zuping; Kokkinaki, Maria; Pant, Disha et al. (2009) Small RNA molecules in the regulation of spermatogenesis. Reproduction 137:901-11

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