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Title: Sperm and Tissue-dependent differential gene expression in the reproductive tract of the Turkey (Meleagris Gallapovo) Hen

Author
item Toomer, Ondulla
item Long, Julie
item Bakst, Murray
item McMurtry, John
item BECKER, K - NIH
item WOOD, W - NIH

Submitted to: American Society of Cell Biology Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 11/30/2008
Publication Date: 12/5/2008
Citation: Foye Jackson, O.T., Long, J.A., Bakst, M.R., Mcmurtry, J.P., Becker, K.G., Wood, W.H. 2008. Sperm and Tissue-dependent differential gene expression in the reproductive tract of the Turkey (Meleagris Gallapovo) Hen. Molecular Biology of the Cell, 19 (suppl), Abstract 2802, p. 810 (CD-Rom).

Interpretive Summary: Due to decades of intensive selection for greater breast yield, the domestic turkey tom can no longer procreate naturally with the female. Consequently, artificial insemination has become a necessity for the turkey industry to ensure reproductive efficiency and species preservation. However, efficient methodology for long-term in vitro storage of turkey semen collected for artificial insemination is lacking. Sperm stored in vitro maintains fertility for approximately 6-8 hours under optimal conditions, while in vivo sperm are viably maintained in the sperm storage tubules (SST) up to 10 weeks. Therefore, we aim to determine the molecular and cellular mechanisms involved in regulating long-term sperm storage in vivo in the reproductive tract of turkey hens. We utilized subtractive cDNA library procedures (Suppression Subtractive Hybridization) to determine differential gene expression between the SST and vaginal epithelium (VE). Two distinct cDNA libraries were constructed from ten pooled samples of each treatment (SST or VE) of 38-week old turkey hens 48h post-artificial insemination. Genes unique to the SST were sequenced and gene identities were determined using NCBI Chicken Genome database. Validation of these results was conducted by quantitative real-time PCR (qrPCR) analysis. Approximately 9% of the total putative genes (480) were identified as unique to the SST of AI hens. Expression levels of seventeen genes were determined by qrPCR in the original 10 VE and SST samples, with only avidin demonstrating a 40-fold (p<0.05) increase in expression in the SST over the VE of AI hens. Additionally, there was 9-fold increase in gene expression of avidin-related protein 2 in the SST versus the VE of AI hens (p<0.05). In a separate set of experiments, there was a 9- and 4-fold increase in avidin and avidin-related protein 2 gene expression, respectively in the SST of AI versus sham inseminated turkey hens, (p<0.05). Avidin protein was found to be expressed in the surface epithelium of the uterovaginal (UVJ) region of both controls (non-inseminated) and AI hens, while protein expression was higher in the controls. Interestingly, the UVJ SST of control hens was avidin positive, while the SST of AI hens was avidin negative in immunohistochemical analysis. Greater insight of the regulatory mechanisms involved in prolonged sperm storage may improve in vitro semen storage conditions, reproductive efficiency and ultimately meat and egg production, which will be of great economic value.

Technical Abstract: Due to decades of intensive selection, the domestic turkey tom can no longer procreate naturally with the female. Consequently, artificial insemination (AI) has become a necessity for the turkey industry to ensure reproductive efficiency and species preservation. However, efficient methodology for long-term in vitro storage of turkey semen collected for AI is lacking. Sperm stored in vitro maintains fertility for approximately 6-8 hours under optimal conditions, while in vivo sperm are viably maintained in sperm storage tubules (SST) up to 10 weeks. This project aims to elucidate the molecular and cellular mechanisms involved in regulating long-term sperm storage in vivo in the reproductive tract of turkey hens. Suppression Subtractive Hybridization (SSH) was utilized to determine differential gene expression between the SST and vaginal epithelium (VE). Equimolar amounts of total RNA was pooled from the SST and VE tissues of ten 38-week old turkey hens 48h post-AI and used to construct distinct SSH cDNA libraries. SST unique transcripts were isolated and sequenced in their entirety. Gene annotations were determined utilizing Chicken Genome database NCBI. Confirmation of differential expression within the subtracted libraries was determined by quantitative real-time PCR (qrPCR) analysis. Approximately 9% of the total putative transcripts (480) were identified as differentially expressed to the SST of AI hens. Validation of SSH was determined by qrPCR analysis of the original 10 VE and SST samples. Expression levels of seventeen genes were determined by qrPCR, with only avidin demonstrating a 40-fold (p<0.05) increase in expression in the SST over the VE of AI hens. Additionally, there was 9-fold increase in mRNA expression of avidin-related protein 2 in the SST versus the VE of AI hens (p<0.05). In a separate set of experiments, there was a 9- and 4-fold increase in avidin and avidin-related protein 2 mRNA expression, respectively in the SST of AI versus sham inseminated turkey hens, (p<0.05). Avidin protein was found to be expressed in the surface epithelium of the uterovaginal (UVJ) region of both controls (non-inseminated) and AI hens, while protein expression was higher in the controls. Interestingly, the UVJ SST of control hens was avidin positive, while the SST of AI hens was avidin negative in immunohistochemical analysis. Greater insight of the regulatory mechanisms involved in prolonged sperm storage may improve in vitro semen storage conditions, reproductive efficiency and ultimately meat and egg production, which will be of great economic value.