Role of the oviduct environment in sperm storage
There is emerging evidence that creatine may be relevant to pre-fertilization events. In this study we sought to identify and localize the creatine transport channel SLC6A8. We performed SDS-PAGE and immunoblotting to detect SLC6A8 in sperm protein lysates and determined that it is present. To further characterize the distribution of SLC6A8 in sperm, we used immunofluorescence. Our findings demonstrated that the creatine transport channel co-localizes in the acrosomal region of the head and is present in the principle piece. Supplementation of capacitating medium with 5 mM creatine doubled (26% motile vs 12% motile) the percentage of sperm that remained motile after 24 hrs of incubation at 39 C. However, creatine supplementation did not increase sperm motility parameters during shorter incubations (0, 4, 8 hrs). Although the creatine transport channel localized to the acrosomal region, 5 mM creatine supplementation had no effect on acrosome stability or the ability of sperm to undergo the acrosome reaction when induced by calcium ionophore. In conclusion, while creatine has no immediate effect on sperm motility, it did increase the percentage of motile porcine sperm during extended time periods. After insemination, either by natural mating or artificial insemination, a select number of sperm populate the porcine oviduct. These sperm are stored until the time of ovulation. Sperm storage in the oviduct extends sperm lifespan, maintains their motility, and extends their fertility. This allows for a modest desynchronization of insemination and ovulation. However, the support the oviduct provides the pre-fertilization environment is not well defined. To investigate the oviduct’s genetic regulation of sperm storage we evaluated the transcriptome of the oviduct in two different conditions. Eighteen pre-pubertal gilts were estrus induced and synchronized. The oviducts of 9 animals were collected 12 hrs after displaying signs of estrus and artificially inseminated. The oviducts of the remaining 9 animals were collected seven days after displaying signs of estrus and were not inseminated. Epithelial cells were collected from both oviducts of each animal and RNA was extracted. RNA was used to generate libraries and over 30 million reads were generated per sample. After alignment to the Sus scrofa genome and filtering of low expression genes, the two treatment groups were compared. In pigs, 2741 genes were upregulated and 2391 genes were down regulated in oviducts storing sperm; expression of 10,714 genes did not differ from the control group. A similar experiment was carried out using cows. The oviducts of five animals were collected 12 hours after displaying signs of estrus and being artificially inseminated. The oviducts of 5 other animals were collected ten days after displaying signs of estrus and were not inseminated. Epithelial cells were collected from the ipsilateral oviduct (side where the ovary that ovulated most recently) of each animal and RNA was extracted. In cattle, 955 genes were upregulated and 931 genes were down regulated in oviducts storing sperm; 13,686 genes were not significantly different from the control group. Gene ontology analysis revealed upregulated pathways involved in cellular regulation, ciliary structure, and secretory functions. These were common to both species. Additionally, exploration of individual genes identified upregulation of genes encoding several antioxidants, specifically members of the glutathione peroxidase family. There was also a significant upregulation of SNARE genes, highlighting the secretory role of the oviduct. Overall, this transcriptomic analysis revealed several interesting pathways and specific genes that are important to sperm storage in the oviduct and pre-fertilization events.
University of Illinois - Urbana-Champaign
Networked Digital Library of Theses and Dissertations
University of Illinois at Urbana-Champaign