Browsing by Subject "testis"
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Item Changes in Gene Expression of Goat Developing Testes and Sperm During Breeding and Non-breeding Season(2012-07-16) Faucette, AzureTesticular function is fundamental to male fertility, since testicular cells act in collaboration with each other to signal sex differentiation, the initiation of puberty and spermatogenesis. Complications that can be influenced by many factors will affect sperm number, morphology, motility, chromatin quality and acrosomal integrity. The purpose of these studies was to analyze the changes in gene expression in the developing testes and analyze the seasonal changes in gene products in sperm of mature bucks. In the first experiment, testes were harvested from five Alpine bucks at 0, 2, 4, 6, and 8 months of age. Northern and in situ hybridization indicated that the largest change in gene expression occurred during the first 4 months of goat testes development. Sex determining region Y-box 9 (SOX9) and Heat Shock protein A8 (HSPA8) peaked at 2 months of age, and were expressed in Sertoli cells and spermatogonium, respectively. At 4 months, expression of Stimulated by Retinoic Acid gene 8 (STRA8), Protamine1 (PRM1) and Outer Dense Fiber protein 2 (ODF2) was strongly up-regulated in early and maturing germ cells, respectively. In the second experiment, RNA from ejaculated spermatozoa collected from mature Alpine bucks in peak (October) and non-peak (April) breeding season were analyzed on a 4 x 44K Agilent bovine microarray. One thousand three hundred and eighteen gene products were differentially expressed 2-fold or more (p ? 0.05 ) was expressed in mature goat sperm collected October and April. To eliminate the likelihood of false positives, the cut off was set to fold change of 3 or more at p ? 0.01 which narrowed the list of genes to 50 transcripts. Real time PCR results confirmed the expression of Sperm Adhesion Molecule 1 (SPAM1) in April, and the expression of Glycerol kinase 2(GK2) and Myc Binding Protein 2 (MYCBP2) in October. Based on the results from both experiments, it can be concluded that: SOX9 and HSPA8 expression play an important role in tubular formation and germ cell maintenance; two months after SOX9 and HSPA8 expression, genes that are associated with spermatogenesis initiation and completion are upregulated; and validation of the seasonal changes in sperm mRNA levels may provide additional insight to testicular events as they relate to breeding and non-breeding season.Item Characterization of a Gene Abundantly Expressed in Stallion Testis(2012-02-14) Shields, Jordan ElizabethNMES1 is a gene of unknown function first characterized in 2002. Reduction of the expression of this gene has been implicated in skin tumorigenesis in mice. Expression of NMES1 is observed in epithelial tissue but expression in the testis is significantly higher than in epidermis. Because stallion fertility is an economically important trait, we decided to characterize the NMES1 gene in stallions. We screened the CHORI241 library and obtained the full length equine NMES1 genomic sequence by direct sequencing off of clone CH241-11J8. In order to experimentally determine the 5? and 3? untranslated regions (UTRs) we conducted RLM-RACE experiments using stallion testis RNA. The equine NMES1 mRNA is 534 nt long and contains 5 exons. Fluorescence in situ hybridization mapped NMES1 to chromosome Eca1q23. In situ experiments to testis tissue sections were inconclusive and yielded no data confirming the physical expression pattern of NMES1 in stallion testis tissue. In order to determine the expression pattern of NMES1 mRNA we conducted qRT-PCR assays on a panel of stallion testis samples from horses with normal and abnormal fertility. We found that expression was variable among both groups, with significantly less expression in some individuals. We also conducted the qRT-PCR assay on a panel of five equine tissues and found that the expression of NMES1 was more than 100-fold greater in testis than in other tissues examined. miR-147b is a miRNA of unknown target found within the 3? UTR of NMES1. We conducted a miRNA qRT-PCR assay to determine the expression levels in stallion testis samples from fertile and sub-fertile stallions. We observed similar expression among both groups and the ratio of mRNA to miRNA did not appear constant. We also investigated miR-147b expression in a panel of five equine tissues and found that equine spleen had more than 8-fold greater expression than testis.