Male infertility is a prevalent disease (7% of men are infertile) that affects the quality-of-life of many reproductive-age couples in the US. Most cases of male infertility which result from a man's complete inability to produce sperm (azoospermia) or arise from low sperm production (oligospermia) are idiopathic. Therefore, research to uncover novel causes of male infertility are highly significant to reproductive medicine and may lead to innovative diagnostics or therapeutics. Spermatogonial stem cells (SSCs) are adult stem cells in mammalian testes that maintain spermatogenesis and are essential for male fertility, yet the mechanisms that control their activity remain elusive. Undifferentiated embryonic cell transcription factor 1 (UTF1) is a transcription factor expressed in undifferentiated spermatogonia of the testis (including SSCs) which has an established role in proliferation and differentiation of pluripotent stem cells. Preliminary studies demonstrated that UTF1 expression responds to differentiation signals in cultured SSCs, suggesting it plays a prominent role in SSC differentiation. Thus, I hypothesized that UTF1 promotes SSC proliferation and is required for SSC differentiation. I will test this hypothesis using innovative methods in mice to demonstrate whether UTF1 plays a role in SSC proliferation and differentiation.
In Specific Aim 1, I will manipulate UTF1 protein levels in cultured Thy1+ spermatogonia (which contain SSCs) using lentiviral-delivered, inducible, over-expression and knockdown constructs. I will examine SSC proliferation using Thy1+ spermatogonial cultures from Id4-GFP mice, which express GFP specifically in SSCs, and evaluate proliferation using three separate assays. These studies will determine the proliferative index among Id4-GFP+ SSCs when UTF1 levels are altered. The results of Aim 1 will determine whether 1) excess UTF1 promotes SSC proliferation and 2) UTF1 deficiency blocks SSC proliferation.
In Specific Aim 2, I will determine the degree of SSC differentiation when UTF1 is over-expressed or knocked-down in GFP+ Thy1+ spermatogonial cultures. For these studies, I will force SSC differentiation by retinoic acid treatment using cultures harboring inducible over-expression or knockdown constructs and I will assess differentiation by measuring expression of differentiation marker genes and SSC numbers by transplantation. The results of Aim 2 will determine whether 1) excess UTF1 promotes SSC differentiation and 2) UTF1 deficiency blocks SSC differentiation. The studies proposed in this application will examine the fundamental mechanisms through which UTF1 contributes to the biological activity of SSCs. Overall, the results of these studies clarify the role of UTF1 in spermatogenesis and will determine whether UTF1 (and, by extension, its target genes) may play a role in the pathogenesis of male infertility. Moreover, the proposed studies will provide ample opportunities for my career development, including mastery of cutting-edge techniques in stem cell, developmental, and molecular biology, scholarly contributions to the field of spermatogenesis, and help me to develop my research niche.
Infertility affects 1 in 10 couples in the United States and many couples are unable to conceive because the man fails to produce sperm. Thus research proposed in this fellowship application will address key questions about how men become infertile and the role of a specific protein, UTF1, in normal male fertility. The proposed research will also help to train a postdoctoral fellow, Kazadi Mutoji, in the field of male infertility by promoting her career development and providing opportunities for her to develop her scientific ideas related to male infertility.
|Lovelace, Dawn L; Gao, Zhen; Mutoji, Kazadi et al. (2016) The regulatory repertoire of PLZF and SALL4 in undifferentiated spermatogonia. Development 143:1893-906|
|Mutoji, Kazadi; Singh, Anukriti; Nguyen, Thu et al. (2016) TSPAN8 Expression Distinguishes Spermatogonial Stem Cells in the Prepubertal Mouse Testis. Biol Reprod :|
|Hermann, Brian P; Mutoji, Kazadi N; Velte, Ellen K et al. (2015) Transcriptional and translational heterogeneity among neonatal mouse spermatogonia. Biol Reprod 92:54|
|Benavides-Garcia, Roberto; Joachim, Rose; Pina, Nancy A et al. (2015) Granulocyte colony-stimulating factor prevents loss of spermatogenesis after sterilizing busulfan chemotherapy. Fertil Steril 103:270-80.e8|