Progress 09/01/04 to 08/31/08
Outputs
OUTPUTS: The long-term goal of this research is to elucidate the mechanisms of bovine sperm storage in order to improve success of artificial insemination and prediction of male fertility. After insemination, a few thousand bull sperm pass from the uterus into the oviduct, where they are held in a storage reservoir until ovulation by binding to the wall of the oviduct. The overall objective of this project was to determine whether Bovine Seminal Plasma (BSP) proteins play a role in storing sperm in the reservoir. Each of the three BSP proteins were isolated from seminal plasma and used to coat epididymal bull sperm, which lack these proteins. Sperm coated with each protein alone gained the ability to bind to oviductal epithelium. Next, the oviductal receptors for each BSP protein were captured by passing protein extracts of apical membranes of oviductal epithelium through BSP affinity purification columns. The columns trapped four oviductal proteins, which were identified by mass spectrometry as annexins. To confirm the role of annexins as oviductal receptors, antibodies against each of the four annexins were shown to block sperm binding to oviductal epithelium. The same antibodies were used to show that annexins are located on the apical surface of oviductal epithelium, including the cilia to which sperm bind. The role of BSP proteins in maintaining sperm viability during storage was confirmed by coating epididymal sperm with each BSP and then incubating them with vesicles isolated from apical plasma membranes of oviductal epithelium. Each BSP protein alone prolonged the motile life spans of sperm. Further experiments revealed that the BSP proteins prolonged sperm survival by suppressing escalations in intracellular calcium and in sperm protein phosphorylation. These escalations prepare sperm for fertilization but also destabilize them; therefore, by suppressing the escalations, BSP proteins act to preserve sperm during storage. Overall, these findings demonstrate a significant role for the interaction of BSP proteins on sperm and annexin proteins on oviductal epithelium in prolonging the lives of sperm during storage and thus these proteins may be useful as indicators of fertility and also to improve success of sperm storage and artificial insemination. This information was imparted to undergraduate and graduate students enrolled in two courses in reproductive biology. Veterinary students were also taught the basics of sperm transport and storage in the female reproductive tract. Six undergraduate students took part in the experiments and wrote up their results for research credit. Two of them wrote theses and graduated with honors. The above results were presented at annual USDA Program Directors meetings, annual meetings of the Society for the Study of Reproduction, Univ of Warwick (UK), Univ of Montreal, Univ of Pennsylvania, Swedish Univ of Agricultural Science, International Embryo Transfer Society, Tzu Chi Univ (Taiwan), National Taiwan Univ, Gordon Research Conference on Fertilization, Wells College, National Autonomous Univ of Mexico, Univ of Massachusetts at Amherst, Population Council (NYC), and Univ of California at San Diego. PARTICIPANTS: Project Director, Dr. Susan Suarez, planned the research, supervised all other participants, was the senior author on all publications, presented the work at national and international meetings and in invited talks at other research institutions, presented the work in undergraduate and graduate courses at Cornell University, presented the work to local high schools and New York State teachers, and participated in various experiments. Research Associate, Dr. George Ignotz, oversaw day-to-day operations in the laboratory, trained students in the lab who participated in the research for class credit and for graduation with honors, and obtained the major part of the data. Postdoctoral Associate Osamu Kawase worked for one year on the first aim of the project, which was to determine whether the bovine seminal plasma proteins form complexes on the surface of sperm. Two undergraduate students, first Margaret Cho and later Aaron Mendelson, were paid as student laboratory assistants to perform laboratory maintenance chores associated with the project. Margaret Cho completed a research project on an aim of the USDA grant and her thesis enabled her to graduate with honors. Aaron Mendelson also graduated with honors, but his research project was based on another grant. Genex Cooperative, Inc, in Ithaca, NY (a subsidiary of CRI International, based in Shawano WI) generously provided all of the bull sperm used for the research. Bovine oviducts were obtained at Cargill Taylor Beef in Wyalusing, PA. The company was not very cooperative until USDA inspectors stepped in and very kindly obtained the oviducts for us. Training and professional development was provided to the two undergraduate students mentioned above and also for undergraduates Natalie Pozzi and Neda Simaika, who each worked on a research project in the laboratory for a semester. TARGET AUDIENCES: The primary target audience for this research is the scientific community, including animal scientists and basic scientists whose interests lie in the realm of reproductive biology. Efforts to deliver the knowledge to the target audience include publications in peer-reviewed scientific journals (which are listed under the section on publications), invited seminars at other research and educational institutions (listed under OUTPUT) papers presented at national and international meetings (listed under OUTPUT), and lectures in three upper level undergraduate/graduate courses. In addition, the Project Director has presented this information in two local high schools annually and at a Cornell workshop for New York State science teachers in August 2008. PROJECT MODIFICATIONS: There were no major changes in this research project. Some challenges were encountered in getting appropriate fresh material from the only regional slaughterhouse available; however, USDA inspectors stepped in and greatly facilitated the process.
Impacts
This was a basic research project with the goal of elucidating the natural mechanisms used by the female bovine to store sperm. After insemination, a few thousand bull sperm pass from the uterus into the oviduct, where they are held in a storage reservoir until ovulation by binding to the wall of the oviduct. The overall objective of this project was to determine whether Bovine Seminal Plasma (BSP) proteins play a role in storing sperm in the reservoir. Each of the three BSP proteins were isolated from seminal plasma and used to coat epididymal bull sperm, which lack these proteins. Sperm coated with each protein alone gained the ability to bind to oviductal epithelium. Next, the oviductal receptors for each BSP protein were captured by passing protein extracts of apical membranes of oviductal epithelium through BSP affinity purification columns. The columns trapped four oviductal proteins, which were identified by mass spectrometry as annexins. To confirm the role of annexins as oviductal receptors, antibodies against each of the four annexins were shown to block sperm binding to oviductal epithelium. The same antibodies were used to show that annexins are located on the apical surface of oviductal epithelium, including the cilia to which sperm bind. The role of BSP proteins in maintaining sperm viability during storage was confirmed by coating epididymal sperm with each BSP and then incubating them with vesicles isolated from apical plasma membranes of oviductal epithelium. Each BSP protein alone prolonged the motile life spans of sperm. Further experiments revealed that the BSP proteins prolonged sperm survival by suppressing escalations in intracellular calcium and in sperm protein phosphorylation. These escalations prepare sperm for fertilization but also destabilize them; therefore, by suppressing the escalations, BSP proteins act to preserve sperm during storage. Overall, these findings demonstrate a significant role for the interaction of BSP proteins on sperm and annexin proteins on oviductal epithelium in prolonging the lives of sperm during storage and thus these proteins may be useful as indicators of fertility and also to improve success of sperm storage and artificial insemination. This new basic information has been disseminated via national and international talks and publications with the intent of impacting the way artificial insemination could be modified to improve pregnancy rates of cows and to improve prediction of bull fertility. This laboratory is currently developing a test for quantifying BSP proteins on sperm after sperm have been diluted and frozen for artificial insemination in order to determine whether the amount of retention of BSP proteins on sperm after thawing can be used to predict bull fertility. If so, the laboratory will systematically modify the process of semen extension and freezing in attempt to optimize retention of BSP proteins on sperm.
Publications
- Suarez SS, Pacey AA (2006) Sperm transport in the female reproductive tract. Hum Reprod Update 12:23-37.
- Suarez SS (2006) Gamete and zygote transport. In: Neill JD (ed.) Knobil and Neill's Physiology of Reproduction, 3rd ed., vol 1. Oxford: Elsevier, pp 113-146.
- Gwathmey TM, Ignotz GG, Mueller JL, Manjunath P, Suarez SS (2006) Bovine seminal plasma proteins PDC-109, BSP-A3, and BSP-30-kDa share functional roles in storing sperm in the oviduct. Biol Reprod 75:501-507.
- Suarez SS (2007) Interactions of spermatozoa with the female reproductive tract: inspiration for assisted reproduction. Reprod Fertil Devel 19:1-8.
- Ignotz GG, Cho MY, Suarez SS (2007) Annexins are candidate oviductal receptors for bovine sperm surface proteins and thus may serve to hold bovine sperm in the oviductal reservoir. Biol Reprod 77:906-913.
- Suarez SS (2008) Regulation of sperm storage and movement in the mammalian oviduct. Int J Dev Biol 52:455-462.
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Progress 01/01/07 to 12/31/07
Outputs
The information gained from this project has been shared by speaking at the following venues in 2007: International Embryo Transfer Society, Kyoto, Japan, January; Tzu Chi University, Taiwan, January; National Taiwan University, Taiwan, January; Gordon Research Conference on Fertilization, Plymouth, NH, July; annual meeting of the Society for the Study of Reproduction, San Antonio, Texas, July; USDA Investigator Meeting, San Antonio, Texas, July; Wells College, Aurora, NY, September; Autonomous University of Mexico, Cuernavaca Mexico, October; and the University of Massachusetts at Amherst, December. In addition, I have disseminated this information in lectures in various courses at Cornell: ANSC 425, Gamete Physiology and Fertilization; BIOAP 757, Current Concepts in Reproductive Biology; and BIOG 101-106, Explorations in the Biological Sciences; VETMED 510, The Animal Body.
Impacts
Aim 1: To test the hypothesis that all Bovine Seminal Plasma (BSP) proteins enhance sperm binding to oviductal epithelium and to determine whether BSP proteins form functional complexes in sperm plasma membranes. Epididymal sperm were coated with purified PDC109, BSPA3, or BSP30K, then added to oviductal epithelium. Each BSP significantly and specifically increased sperm binding. Pre-incubation of oviductal epithelium with excess purified BSPs competitively inhibited ejaculated sperm binding. To determine whether BSPs form complexes on sperm, we used crosslinkers to see whether the BSPs on the surface of live sperm were interacting closely enough to allow covalent crosslinking. Sperm were treated with Bis[sulfosuccinimidyl] suberate and then membrane extracts were probed on immunoblots with antibodies to each BSP. No bands were detected by anti-BSP30K above 30 kDa, indicating that BSP30K does not form complexes with the other BSPs. More than one band was identified by
anti-PDC109 and anti-BSPA3 at twice the size of each protein, indicating formation of heterodimers in addition to homodimers, which were expected for PDC109. Aim 2: To test the hypothesis that the BSP proteins bind to different ligands in the oviduct. We developed an immunoaffinity purification procedure to identify oviductal ligands for BSPs by binding purified BSPs to their corresponding antibodies conjugated to agarose beads. Proteins extracted from apical plasma membranes of oviductal epithelium were passed through the columns and protein bands of 34 and 36 kDa bound to each column. Mass spectroscopy identified them as annexins (ANXAs), which are known as calcium-regulated membrane-binding proteins whose functions include cell adhesion. Anti-ANXA antibodies blocked sperm binding to epithelium and detected ANXAs on apical surfaces of oviductal epithelium. We concluded that the BSPs bind to ANXAs. Aim 3: To test the hypothesis that prolongation of sperm survival depends upon the
binding of sperm to oviductal membranes via the BSP proteins. BSP-treated epididymal sperm were incubated with vesicles of apical plasma membranes of oviductal epithelium. Each BSP alone prolonged the motile lives of sperm, although PDC109 was more effective than the others. Aim 4: To test the hypothesis that binding of sperm to oviductal epithelial membranes prolongs their survival by inhibiting signaling events of capacitation. Because calcium rises in bull sperm as they capacitate, sperm were loaded with the fluorescent calcium indicator fluo3-AM, then incubated alone or with apical plasma membrane vesicles of oviductal epithelium. Flow cytometry was used to show that more sperm incubated with vesicles maintained low intracellular calcium than sperm incubated without vesicles. Because proteins in capacitating sperm undergo tyrosine phosphorylation, we also tested whether incubation with oviductal plasma membrane vesicles would inhibit this process. Immunoblots of sperm proteins
probed with anti-phosphotyrosine antibodies showed that incubation with vesicles inhibits phosphorylation. Thus capacitation is suppressed in sperm bound to oviductal epithelium.
Publications
- Suarez SS, Pacey AA (2006) Sperm transport in the female reproductive tract. Hum Reprod Update 12: 23-37.
- Suarez SS (2006) Gamete and zygote transport. In: Neill JD (ed.) Knobil and Neill's Physiology of Reproduction, edition 3, volume 1. Oxford: Elsevier, pp 113-146.
- Gwathmey TM, Ignotz GG, Mueller JL, Manjunath P, Suarez SS (2006) Bovine seminal plasma proteins PDC-109, BSP-A3, and BSP-30-kDa share functional roles in storing sperm in the oviduct. Biol Reprod 75: 501-507.
- Suarez SS (2007) Interactions of spermatozoa with the female reproductive tract: inspiration for assisted reproduction. Reprod Fertil Devel 19: 1-8.
- Ignotz GG, Cho MY, Suarez SS (2007) Annexins are candidate oviductal receptors for bovine sperm surface proteins and thus may serve to hold bovine sperm in the oviductal reservoir. Biol Reprod 77: 906-913.
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