Animal Science Department

 

Date of this Version

4-20-2012

Comments

A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Animal Science, Under the Supervision of Professor Andrea S. Cupp. Lincoln, NE: May 2012

Copyright (c) 2012 Kevin M. Sargent

Abstract

Vascular Endothelial Growth Factor A (VEGFA) and its receptors are important for vascular development in the testis. Roles of VEGFA have been proposed in undifferentiated spermatogonia where proangiogenic isoforms are suggested to promote their self-renewal while antiangiogenic isoforms stimulate their differentiation. Thus, we treated male rats in vivo (perinatally with IP injections from P0-P2) with different VEGFA isoforms or an antibody to VEGFAxxxb (antiangiogenic isoforms) to determine if this would affect testis morphogenesis, expression of genes regulating the spermatgonial stem cell (SSC) niche and genes that regulate cell survival. Testis morphogenesis with VEGFA164 (1mg) isoform treatment increased cord area, reduced interstitium and tended to increase germ cell numbers while VEGFA165b (0.5 mg) decreased cord area and increased interstitium. The antiVEGFAxxxb treatment reduced expression of mRNA for Nanos2 and Ret- two genes important in SSC renewal. Furthermore, mRNA for Bax, a pro-apoptotic gene, was reduced, and the ratio of Bcl2:Bax tended to be increased. Serum testosterone tended to be increased with the antiVEGFAxxxb treatment while it was reduced with perinatal VEGFA165b (0.5 mg) treatment. A second study was performed with Amhr2-cre;Nrp-1-/- mice where Nrp-1, the co-receptor that binds proangiogenic isoforms of VEGFA, was knocked out in Sertoli cells. Mice at 78.6 days had reduced Bcl2 (pro-survival), Sin3a (niche establishment), Gdnf (SSC renewal), Neurog3 (differentiation) with a tendency for Ret and Kitl (survival and differentiation) to be reduced. Both the mRNA and the protein for PLZF (renewal) were elevated in knockout testes. Knockout males were mated to control females and allowed to breed until cessation. Compared to control matings, the knockout male matings to controls resulted in fewer pups per litter, a tendency for increased days between parturitions and fewer pups surviving to weaning. These data further confirm the balance of VEGFA isoforms is necessary for appropriate expression of genes regulating the SSC niche, and cell survival and they are also critical for male fertility.

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