Browsing by Author "William L. Flowers, Committee Member"

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Effect of Season on Sperm Membrane Protein 22 and Selected mRNAs in Fresh and Cryopreserved Stallion Sperm
(2008-12-07) Wrench, Nicola; Carlos R.F. Pinto, Committee Co-Chair; William L. Flowers, Committee Member; Charlotte E. Farin, Committee Chair; David J. Dix, Committee Member; Gary R. Klinefelter, Committee Member
Abstract Wrench, Nicola. Effect of season on SP22 protein and selected mRNAs in fresh and cryopreserved stallion sperm. (Under the direction of Drs. Charlotte E. Farin and Carlos R.F. Pinto). The objective of this study was to determine if season or semen cryopreservation had an effect on the expression of fertility-related protein SP22 and selected mRNA transcripts in stallion sperm. Six stallions were collected in June 2005, September 2005, December 2005 and March 2006. Each ejaculate was partitioned for evaluation of sperm parameters in fresh and cryopreserved samples. The percentages of normal morphology, primary abnormalities, secondary abnormalities, membrane integrity, viability, total motility, progressive motility and acrosome integrity were recorded. In addition, aliquots of fresh and cryopreserved ejaculates were analyzed for SP22 protein expression and expression of mRNA transcripts. For SP22 immunocytochemistry, samples were stained using a sheep anti-rat recombinant SP22 primary antibody and a FITC-conjugated secondary antibody. At least 200 stained sperm were counted using a fluorescent microscope and categorized into one of two patterns: Pattern 1, overlying the equatorial region (ER) only; Pattern 2, overlying the acrosomal and equatorial region (AER), neck (N) and tail (T). For mRNA transcript expression, sperm were washed with a hypoosmotic solution to induce somatic cell lysis. RNA was extracted from sperm samples using Tri-Reagent and cDNA was synthesized. PCR was performed using the cDNA to assess mRNA expression. Data were analyzed for all six stallions as well as for the subset of four stallions whose collections were repeated every season (subset stallions). Data were analyzed using general linear model procedures (SAS Institute Inc., Cary, NC, USA). The process of cryopreservation significantly (P<0.05) affected all sperm parameters. In general, freezing decreased the proportion of sperm exhibiting normal morphology as well as the proportions of viable and motile sperm. In contrast, freezing was associated with an increased incidence of primary and secondary morphology abnormalities. A significant (P<0.05) effect of season was noted for normal morphology, primary abnormalities, total motility and progressive motility for all 6 stallions. In general, the same seasonal effects were present for the 4 subset stallions. However, there was no effect of season on total motility or progressive motility. Significant (P<0.05) season by freezing treatment interactions were found for progressive motility and intact acrosomes for all 6 stallions. For the 4 subset stallions a significant (P<0.05) season by treatment interaction was found for total motility, progressive motility and secondary abnormalities. There were no significant stallion by treatment interactions. The proportion of sperm stained for SP22 was significantly (P<0.05) affected by season, stallion and freezing treatment. A significant (P<0.05) season by freezing treatment interaction was also present. A tendency (P<0.08) for the intensity of staining for SP22 to differ among stallions was noted for the 4 subset stallions. For cryopreserved sperm, the proportion of sperm staining for SP22 on the equatorial segment was affected differently by stallion or season depending on the SP22 antibody used. RNA yield from sperm was not affected by season, stallion, cryopreservation or their interactions. There was no effect of season or freezing treatment on relative quantity of mRNAs for PGK2, TPX1, TIMP3 or ACTB. Also, no season by freezing treatment or stallion by freezing treatment interactions were found. However, differences between stallions (n=6) were apparent for PGK2 (P=0.08) and ACTB (P=0.01) content. For the 4 subset stallions there was a tendency (P=0.1) for a stallion effect on ACTB mRNA content. Sperm parameters and proportion of sperm staining for SP22 were affected by season, stallion and freezing treatment. Certain mRNA transcripts (PGK2 and ACTB) were also affected by stallion but season and freezing treatment do not appear to have an effect on any mRNA transcripts. Understanding these differences that exist in sperm from different seasons and stallions may prove to be beneficial to determine the best stallions and the best time to collect semen for cryopreservation.
Expression and localization of mRNA for the VEGF system and FP receptor in porcine corpora lutea.
(2002-04-30) Boonyaprakob, Ukadej; William L. Flowers, Committee Member; John E. Gadsby, Committee Co-Chair; Glen W. Almond, Committee Co-Chair; Neil C. Olson, Committee Member
The purpose of this research was to establish a complete cDNA sequence for porcine prostaglandin F2a receptor (FPr), and to examine the expression and localization of FPr and components of the VEGF system in the porcine CL throughout the estrous cycle. FPr cDNA was isolated and cloned from porcine CL. This isolate (Genbank accession number U91520) contains an open reading frame of 1086 bases coding for a protein of 362 amino acids with seven potential transmembrane domains. The predicted amino acid sequence of the porcine FPr had 83% homology with the FPr amino acid sequence for sheep, cattle and humans. The phylogenetic tree of prostanoid receptor family members clustered porcine FPr into the same group with other mammalian FPr, with the closest relationship to ovine and bovine FPr. Northern blot analysis revealed a single transcript of ˜5 kilobases for FPr message. The signals of FPr mRNA expression were observed throughout the estrous cycle. Relatively weak signals of FPr mRNA expression were detected on day 4 and day 7 of the estrous cycle. The signal was greater (p<0.05) on day 10, day 13 and day 15 than day 4 or day 7. In situ hybridization analysis revealed that mRNA for ovarian FPr was expressed predominantly in the CL. Cellular localization of the FPr signals was primarily in steroidogenic large luteal cells. The FPr signals were also present in small cells, morphologically similar to (steroidogenic) cells which expressed LH receptor message. Expression mRNAs for vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGFR-1) and VEGF receptor 2 (VEGFR-2) were observed in porcine CL throughout the estrous cycle. Northern blot analysis revealed single transcripts of approximately 3.9-kb, 7-kb and 5-kb for VEGF, VEGFR-1 and VEGFR-2 respectively. Signals for VEGF mRNA expression were consistent throughout the cycle. Relatively weak signals of VEGFR-1 mRNA expression were observed on day 4. Signal intensity increased on day 7, and obtained higher levels (P<0.05) on days 10, 13 and 15. In contrast, relative levels of VEGFR-2 mRNA were lower on day 4 than on day 7, peaked on day 10, remained unchanged on day 13, and decreased (P<0.05) on day 15. In situ hybridization revealed that VEGF mRNA was localized predominantly in large luteal cells, whereas VEGFR-1 and VEGFR-2 were limited to small endothelial-like cells. Collectively, the results provide information on: 1) a complete cDNA sequence for porcine FP receptor; 2) the expression and localization of mRNA for luteal FPr, which indicated that the number of luteal FPr contributes to the acquisition of luteolytic capacity in the pig; 3) the expression and localization of mRNA for VEGF, VEGFR-1 and VEGFR-2, which suggested that the VEGF system plays a role in angiogenesis and maintenance of vasculature in porcine CL.
Reproductive Gene Expression in Male Sus scrofa: An examination of the differential gene expression of Divergent Testosterone selection and development of a Ribonucleic Acid extraction protocol from whole Porcine Spermatozoa
(2009-12-04) Sisk, Dana Stanley; Melissa S. Ashwell, Committee Chair; William L. Flowers, Committee Member; Alison A. Motsinger-Reif, Committee Member
The ability to characterize and enhance market traits in livestock has facilitated a greater interest in determining the genetic tool kit available for manipulation. In swine, using new approaches in genomics, such as microarray analysis and biological pathway analysis, we can show genes up and down regulated in a variety of processes and conditions. To this end, we identify the genes, pathways, and disease biomarkers affected by divergent selection of testosterone in boars. Testicular samples were taken from boars at 0, 30, 120, 150, and 180 days of age for lines of high (HT) and low testosterone (LT). Evidence that many of the differences in gene expression were at the pubertal period of 150 days led to a subsequent microarray study of the 150 day HT and LT animals. Microarray studies were followed by validation with real-time RT-PCR of 11 genes and extensive GeneGo pathway analysis (Metacore) of differentially expressed genes. While increased testosterone has long been associated with increased growth rates, we now have supporting genomic evidence of the genes and pathways up-regulated and down-regulated in these lines. To this end, this study has identified several disease biomarkers that may require further investigation and biological pathways associated with growth and metabolism that allow the recommendation of selective breeding for high testosterone to increase lean growth traits. The genetic blueprint contained in the spermatozoan transcriptome can also illuminate key issues in swine reproduction. By developing a procedure for effective RNA extraction of boar spermatozoa we are one step closer to elucidating the porcine sperm transcriptome and the genes implicated in growth and fertility. A viable protocol was developed to handle the complexities of large scale extraction of RNA from porcine semen utilizing an RNA carrier and Dnase treatment. This protocol was validated with PCR amplification of Sus scrofa prm1 in order to provide evidence of a successful RNA extraction from sperm.