Source: CORNELL UNIVERSITY submitted to
SPERM STORAGE PROTEINS AND THEIR RELATIONSHIP TO BULL FERTILITY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0214096
Grant No.
2008-35203-19031
Project No.
NYCV-435552
Proposal No.
2008-00544
Multistate No.
(N/A)
Program Code
41.0
Project Start Date
Aug 1, 2008
Project End Date
Jul 31, 2012
Grant Year
2008
Project Director
Suarez, S.
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
BIOMEDICAL SCIENCES
Non Technical Summary
Our overall goal is to improve success of bovine artificial insemination. In the US, most dairy cows are made pregnant by artificial insemination with frozen semen. Companies that sell semen to farmers have succeeded in greatly improving the milk output of the daughters of inseminated cows, but this improvement has come at the expense of fertility. Normally, inseminated sperm that reach the oviduct in the cow are held in a storage reservoir by sticking to the wall of the oviduct. We have identified three proteins on sperm, called BSP proteins, and four proteins on the wall of the oviduct, called annexins, that interact with each other. We have already demonstrated that the BSP proteins keep sperm alive longer when they are incubated with membranes from the lining of the oviductal wall. Aim 1 is to verify that annexins are the oviductal partners for the BSP proteins and to determine whether they serve to maintain sperm viability during oviductal storage. Aims 2 and 3 are to determine how the BSP proteins and annexins act to release sperm from storage and assist them in proceeding to the site of fertilization. Aim 4 is to determine whether testing for the levels of BSP proteins remaining on sperm after they have been frozen for artificial insemination can be used to predict bull fertility. Altogether this information should be useful for improving preparation and storage of sperm for artificial insemination and for developing assays for predicting bull fertility.
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3013399100020%
3013399102020%
3013499100030%
3013499102030%
Goals / Objectives
After insemination, a few thousand bull sperm reach the oviduct where they are kept fertile in a storage reservoir until ovulation. The reservoir forms because three bovine seminal plasma proteins (BSPs) on sperm bind to four annexins (ANXAs) on oviductal epithelium. Our long-term goal is to elucidate the mechanisms of sperm storage in order to improve success of artificial insemination. Aim 1 is to test the hypothesis that ANXAs play a role in preserving sperm viability during storage by suppressing capacitation of sperm. Aim 2 is to test the hypothesis that ANXAs play a key role in guiding sperm movement out of storage and toward the site of fertilization. Aim 3 is to test the hypothesis that BSPs regulate sperm movement out of storage by detaching gradually from the sperm surface. Aim 3 is to test the hypothesis that the amounts of BSPs on sperm that survive freezing and thawing can be used to predict bull fertility. This project addresses the FY 2008 priority of gonadal function, particularly preservation of gametes.
Project Methods
Our approaches for meeting each of the project aims are as follows. Aim 1 is to test the hypothesis that bovine oviductal annexins (ANXAs) play a role in sperm storage. ANXAs will be purified from oviductal epithelium and used to test whether they prolong sperm viability and whether they do so by suppressing capacitation of sperm. Suppression of capacitation will be assayed by determining whether incubation of sperm with ANXAs prevents capacitation-associated increases in protein tyrosine phosphorylation and in intracellular calcium. Aim 2 is to test the hypothesis that ANXAs play a key role in sperm movement. Differential distribution of the four ANXA proteins could guide sperm toward the oocyte as the sperm intermittently stick to and detach from epithelium during release from the reservoir. Distributions of each ANXA will be analyzed by immunohistochemistry of sections of oviduct and by immunoblotting of proteins extracted from oviductal epithelium from different regions of the oviduct. Aim 3 is to test the hypothesis that BSPs regulate sperm movement. Sperm shed BSPs as they undergo capacitation. Immunoblot analysis will be used to test whether each BSP is shed at a different rate. This could account for gradual release of sperm from the reservoir, which serves to reduce incidence of polyspermy, and also for guiding released sperm towards the oocyte. Aim 3 is to test the hypothesis that the amounts of BSPs on live sperm after freezing and thawing can be used as predictors of bull fertility. We will separate live sperm from dead sperm and seminal plasma in frozen/thawed semen samples, extract membrane proteins from sperm, quantify the amount of BSPs in the extracts, and test for correlation of BSP quantities with the field fertility data for the bulls.

Progress 08/01/08 to 07/31/12

Outputs
OUTPUTS: Dr. Susan Suarez, Project Director, and her research group conducted the experiments described in the USDA proposal, which were designed to test the hypotheses that (1) annexin proteins play a key role in bovine sperm storage, (2) annexin proteins play a key role in bovine sperm movement, (3) BSP proteins regulate sperm movement, and (3) the amounts of BSP proteins on sperm predict bull fertility. Dr. Suarez was invited to share the information gained from this project by presenting seminars at Harvard Medical School, Aug 2009; Society for the Study of Reproduction (SSR), Pittsburgh, Aug 2009; University of Monash, Australia, Feb 2009; University of Melbourne, Australia, Feb 2009; University of Newcastle, Australia, April 2009; Institute for Conservation Research of the San Diego Zoo, Jan 2010; University of California at San Diego, Jan 2010; Locomotion Workshop, Institute for Mathematics and Its Applications, University of Minnesota, June 2010; International Symposium on Spermatology, Okinawa, Japan, June 2010; Joint Annual Meeting of the American Dairy Science Association/American Society of Animal Sciences, Denver July 2010; International Meeting on Ruminant Reproduction, Anchorage, AK, Sept 2010; Greenwald Symposium on Reproduction, Kansas City, MO, Oct 2010; Gordon Research Conference on Cilia, Mucus, and Mucociliary Interactions, Feb 2011; Hannover University, Hannover Germany, Oct 2011; International CAESAR Conference on Sperm Signaling and Motility, Bonn, Germany, Oct 2011; Penn State University, Nov 2011; Universidad de Antofagasta, Chile, Jan 2012; Universidad de Santiago, Chile, Jan 2012; Huazhong University of Science ND Technology, Wuhan, China, May 2012; Johns Hopkins Bloomberg School of Public Health, Oct 2012. Dr. Suarez also presented her results and participated in the USDA Project Directors meetings in 2009-2011. Postdoctoral Associate Pei-hsuan Hung presented results from the project at the Molecular Reproduction and Development Conference in Philadelphia, June 2011; Gordon Conference on Fertilization and the Activation of Development, Holderness, NH, July 2011; Annual Meeting of the SSR, Portland, OR, Aug 2011; Reproductive and Developmental Genomics Retreat, June 2012, Ithaca, NY; Center for Vertebrate Genomics 8th Symposium, July 2012, Ithaca, NY; and the Annual Meeting of the SSR, State College, PA, July 2012. Postdoctoral Associate Florencia Ardon presented results from the project at the Annual Meeting of the SSR, State College, PA, Aug 2012. Lastly, Dr. Suarez disseminated this information to ~100 veterinary students at Cornell each year in the class VETMED 5100 "The Animal Body", twice to graduate students in the course BIOAP 7570 "Current Concepts in Reproductive Biology", and twice to undergraduate students in the courses Animal Science 4250 "Gamete Physiology and Fertilization in Mammals" and BIOAP 4130 "Histology", and also provided three undergraduate students with research experience by guiding them in research projects that earned them graduation with honors. PARTICIPANTS: Project Director, Dr. Susan Suarez, planned the research, supervised all other participants, was the senior author on publications, presented the work at national and international meetings and in invited talks at other research institutions, presented the to graduate students, veterinary students, and undergraduate students at Cornell University. Postdoctoral Associate Pei-hsuan Hung oversaw day-to-day operations in the laboratory, assisted in training 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 Florencia Ardon joined the team in 2011 and completed a project on the effect of cryopreservation on the BSP coating of sperm. Four undergraduate students, Lisa Opdycke, Thomas Yaros, Zarah Deutsch, and Ross Markello were paid as student laboratory assistants to perform laboratory maintenance chores associated with the project. They worked on these chores altogether about 6 hours per week. In addition, undergraduate students Lisa Opdycke, Thomas Yaros, and Lindsay Knable carried out supervised research projects under the auspices of the USDA grant. Dr. Suarez collaborated with Dr. Sutovsky of the University of Missouri at Columbia by sharing the anti-BSP5 anti-polypeptide antibodies with him to examine the relationship between the BSP coating on sperm and sperm morphology as well as bull fertility. 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 USDA research. Bovine oviducts were obtained at Cargill Taylor Beef in Wyalusing, PA. 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 included publication in peer-reviewed scientific journals (listed under PUBLICATIONS), invited seminars at other research and educational institutions (detailed under OUTPUT), papers presented at national and international meetings (detailed under OUTPUT), and a lecture and laboratory exercise in a veterinary course, as well as providing undergraduates with research experiences in the laboratory (detailed under OUTPUT). PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
When bull sperm enter the oviduct, most bind to the oviductal epithelium to form a storage reservoir. The Suarez lab found that three proteins in the BSP family (BSP1, BSP3, BSP5) enhance binding of bull sperm to the oviductal epithelium. BSPs are secreted by bovine seminal vesicles and adsorbed onto sperm when they come in contact with vesicular secretions. Loss of BSPs from sperm during capacitation was proposed to play a role in releasing sperm from the oviductal reservoir to move up the oviduct; therefore, the effect of capacitation on the BSP coat of sperm was examined. Fresh bull sperm were incubated in medium with or without addition of capacitation enhancers. Proteins were extracted from sperm and western blot assays were performed using antibodies made against unique polypeptides of each BSP. At 5 hours, little BSP1 had been shed from the sperm under any condition; however, BSP5 was shed quickly, even in minimally capacitating conditions. On blots probed with anti-BSP3 antibody, a band of lower molecular mass appeared during incubation. Analysis by mass spectrometry confirmed that the lower molecular mass contained BSP3. It was determined that the smaller size of the protein was not due to deglycosylation; however, appearance of the smaller BSP3 could be inhibited by serine protease inhibitors. Because serine proteases have been identified on the surface of sperm, proteolytic cleavage is a plausible explanation for the modification of BSP3. Altogether, the different responses of BSPs to capacitation of sperm could modulate the release of sperm from the reservoir and even guide sperm toward the site of fertilization. In a second project, the Suarez lab examined changes in the BSP coating on frozen/thawed sperm. Results indicated that capacitation and modification of the BSP proteins varied considerably among samples from different bulls. Greater amounts of BSPs were detected on freshly-thawed live sperm than on live fresh sperm. More BSP1 was detected when fresh semen was diluted in milk extender rather than in TALP sperm medium indicating a role for milk. Two BSP3 isoforms were detected at 13 and 15 kDa in frozen-thawed samples, but only the 15 kDa isoform was detected in fresh samples. Abnormally high levels of BSPs on sperm could alter the timing of storage and release of sperm, thus reducing the chances of sperm reaching oocytes at the optimal time after ovulation. This may explain field observations that high amounts of BSPs on sperm are correlated with low fertility. In another project, the distributions of 4 annexin proteins, the putative receptors for BSPs on the oviductal epithelium, were examined using immunohistochemistry and western blot of extracts of oviductal epithelium. The distributions were found to vary along the length of the oviduct. This may provide a mechanism for directing sperm out of the reservoir and up the oviduct as the coat on the sperm surface is modified and interactions between BSPs and annexins change. Altogether, this work is elucidating how the storage and movement of sperm is regulated in the oviduct and how cryopreservation may reduce fertility.

Publications

  • Suarez SS (2010) How do sperm get to the egg Bioengineers wanted! Exp Mech 50: 1267-1274, DOI: 10.1007/s11340-009-9312-z
  • Hung PH and Suarez SS (2010) Regulation of sperm storage and movement in the ruminant oviduct. Soc Reprod Fertil Suppl. 2010;67:257-266.
  • Suarez SS (2012) Unsolved problems in the locomotion of mammalian sperm. in Childress S, Hosoi A, Schultz WW, Wang ZJ (eds) Natural Locomotion in Fluids. Springer, NY.
  • Hung PH, Suarez SS (2012) Alterations to the bull sperm surface proteins that bind sperm to oviductal epithelium. Biol Reprod, Epub Jul 25. 87:1-11, PMID: 22837481.


Progress 08/01/10 to 07/31/11

Outputs
OUTPUTS: Dr. Susan Suarez, Project Director, was invited to share the information gained from this project by presenting seminars at the International Meeting on Ruminant Reproduction, Anchorage, AK, Sept 2010; Greenwald Symposium on Reproduction, Kansas City, MO, Oct 2010; Gordon Research Conference on Cilia, Mucus, & Mucociliary Interactions, Feb 2011. She also presented her results and participated in the USDA Project Directors meeting in April 2011. Postdoctoral Associate Pei-hsuan Hung presented results from the project at the MRD Conference, Philadelphia, PA, June 2011; Gordon Conference on Fertilization and the Activation of Development, Holderness, NH, July 2011; and the Annual Meeting of the Society for the Study of Reproduction, Portland, OR, Aug 2011. Lastly, Dr. Suarez disseminated this information to veterinary students at Cornell in the class VETMED 5100 "The Animal Body" and provided three undergraduate students with research experience by guiding them in research projects. PARTICIPANTS: Project Director, Dr. Susan Suarez, planned the research, supervised all other participants, was the senior author on publications, presented the work at national and international meetings and in invited talks at other research institutions, presented the to graduate students, veterinary students, and undergraduate students at Cornell University. Postdoctoral Associate Pei-hsuan Hung oversaw day-to-day operations in the laboratory, assisted in training 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. Two undergraduate students, Lisa Opdycke and Thomas Yaros, were paid as student laboratory assistants to perform laboratory maintenance chores associated with the project. They worked on these chores altogether about 6 hours per week. In addition, both students, as well as an additional student, Lindsay Knable, carried out supervised research projects under the auspices of the USDA 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. 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 included publication in peer-reviewed scientific journals (listed under PUBLICATIONS), invited seminars at other research and educational institutions (detailed under OUTPUT), papers presented at national and international meetings (detailed under OUTPUT), and a lecture and laboratory exercise in a veterinary course, as well as providing undergraduates with research experiences in the laboratory (detailed under OUTPUT). PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
When bull sperm enter the oviduct, most bind to the oviductal epithelium to form a sperm reservoir. The Suarez lab had found that three proteins in the Binder of Sperm Protein family (BSP1, BSP3, BSP5) enhance binding of bull sperm to the oviductal epithelium. BSPs are secreted by bovine seminal vesicles and adsorbed onto sperm when they come in contact with vesicular secretions. Loss of BSPs from sperm during capacitation was proposed to play a role in releasing sperm from the oviductal reservoir to move up the oviduct; therefore, the effect of capacitation on the BSP coat of sperm was examined. Fresh bull sperm were incubated in medium with or without addition of capacitation enhancers. Proteins were extracted from sperm and western blot assays were performed using antibodies made against unique polypeptides of each BSP and anti-phosphotyrosine antibodies to assay capacitation. At 5 hours, little BSP1 had been shed from the sperm under any condition; however, BSP5 was shed quickly, even in minimally capacitating conditions. On blots probed with anti-BSP3 antibody, a band of lower molecular mass appeared during incubation. Mass spectrometry was used to confirm that the lower molecular mass contained BSP3. It was determined that the smaller size of the protein was not due to deglycosylation; however, the appearance of the modified form of BSP3 could be reduced by serine protease inhibitors. Because serine proteases have been identified on the surface of sperm, this is a plausible explanation for the modification of BSP3. Altogether, the different responses of BSPs could modulate the release of sperm from the reservoir and even guide sperm toward the site of fertilization. In a second project, the Suarez lab examined changes in the BSP coating on frozen/thawed sperm. Results indicated that capacitation and modification of the BSP proteins varied considerably among samples from different bulls, but that much less BSP1 was present on frozen/thawed sperm than fresh sperm. Anti-BSP3 antibodies detected two lower molecular mass fragments on frozen-thawed sperm capacitated for only 2 hours; whereas, only one had been detected in fresh sperm incubated for 5 hours. The reduction of BSP proteins on frozen/thawed sperm and the increased fragmentation of BSP3 could contribute to lowering the success rate of artificial insemination. In a third project, the distributions of annexins, the putative receptors for BSPs on the oviductal epithelium, were examined, using immunohistochemistry and western blot of extracts of oviductal epithelium. The distributions of the four annexin proteins that the Suarez lab had identified as putative receptors were found to vary along the entire length of the oviduct. This may provide a mechanism for directing sperm out of the reservoir and up the oviduct as the coat on the sperm surface is modified. That is, as the BSP coating on sperm in modified, the interactions of sperm with each of the four annexins would also change and sperm could advance up the oviduct via a series of different interactions with annexins. Altogether, this work is elucidating how the storage and movement of sperm is regulated in the oviduct.

Publications

  • PUBLICATIONS: Hung PH and Suarez SS (2010) Regulation of sperm storage and movement in the ruminant oviduct. Soc Reprod Fertil Suppl. 2010;67:257-266.


Progress 08/01/09 to 07/31/10

Outputs
OUTPUTS: Dr. Susan Suarez, Project Director, was invited to share the information gained from this project by presenting seminars at the Institute for Conservation Research of the San Diego Zoo (01/20/2010) and the University of California at San Diego (01/21/2010). Also, she was invited to speak at the Locomotion Workshop at the Institute for Mathematics and Its Applications (IMA) at the University of Minnesota (6/04/2010), the International Symposium on Spermatology in Okinawa Japan (06/26/2010), and the Joint Annual Meeting of the American Dairy Science Association/American Society of Animal Sciences meetings in Denver (07/13/2010). Lastly, she disseminated this information to veterinary students at Cornell in the class VETMED 5100 The Animal Body, to graduate students in the course BIOAP 7570 Current Concepts in Reproductive Biology, and to undergraduate students in the courses Animal Science 4250 Gamete Physiology and Fertilization in Mammals and BIOAP 4130 Histology. PARTICIPANTS: Project Director, Dr. Susan Suarez, planned the research, supervised all other participants, was the senior author on publications, presented the work at national and international meetings and in invited talks at other research institutions, presented the work in various courses to graduate students, veterinary students, and undergraduate students at Cornell University. Postdoctoral Associate Pei-hsuan Hung oversaw day-to-day operations in the laboratory, assisted in training 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. Two undergraduate students, Lisa Opdycke and Thomas Yaros, were paid as student laboratory assistants to perform laboratory maintenance chores associated with the project. They worked on these chores altogether about 6 hours per week. In addition, both students, as well as an additional student, Lindsay Knable, are in the process of carrying out supervised research projects under the auspices of the USDA grant in hopes of completing an honors thesis. 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. 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 included publications in peer-reviewed scientific journals (which are listed under PUBLICATIONS), invited seminars at other research and educational institutions (detailed under OUTPUT), papers presented at national and international meetings (detailed under OUTPUT), and a lecture and laboratory exercise in a veterinary course and an undergraduate course, as well as lectures in a graduate and an undergraduate course (detailed under OUTPUT). PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
When bull sperm enter the oviduct, most bind to the epithelium to form a sperm reservoir. Sperm become capacitated before they leave the reservoir. The Suarez lab had found that that proteins in the Binder of Sperm Proteins (BSP) family enhance binding of bull sperm to the oviductal epithelium. BSPs are secreted by bovine seminal vesicles and adsorbed by sperm when they come in contact with vesicular secretions. There are three BSPs in the secretions: PDC109, BSPA3, and BSP30K. It had been shown that PDC109 is shed from sperm during capacitation, thereby releasing sperm from oviductal epithelium; however, it was not known whether BSPA3 or BSP30K are also shed. If each BSP is shed differently in response to capacitating conditions, it could serve to regulate movement of sperm out of the reservoir. Fresh bull sperm were incubated in TALP medium with or without heparin or 3-isobutyl-1-methylxanthine (IBMX) and dbcAMP added to enhance capacitation. Proteins were extracted from sperm and western blot assays were performed, using antibodies made against unique polypeptides from the N-terminal domain of each BSP. Sperm capacitation was assayed by western blot analysis of protein tyrosine phosphorylation. At 5 hours, little PDC109 had been shed from the sperm under all of the enhanced capacitation conditions. BSP30K was shed quickly and was even shed in minimally capacitating conditions, when little tyrosine phosphorylation was detected. On blots probed with anti-BSPA3 antibody, a band of lower molecular mass appeared during incubation, indicating that BSPA3 is modified by the sperm. This was confirmed by identifying fragments of BSPA3 in both bands, using mass spectrometry. It was concluded that each BSP responds differently to various capacitating conditions. Surprisingly, the response was not always directly related to the extent of tyrosine phosphorylation. Altogether, the different responses of BSPs could modulate the release of sperm from the reservoir, perhaps causing a more gradual release or even guiding sperm toward the site of fertilization. In a second project, the Suarez lab tested whether the amount of BSPs on sperm from extended/frozen/thawed semen could be correlated to field fertility of bulls. Variation among bulls and ejaculates was very high and no difference was detected between high fertility and low fertility bulls. The most significant finding was how little PDC109 and BSPA3 proteins remain adsorbed onto sperm after they are extended, frozen, and thawed. Whereas, sperm from fresh semen samples had adsorbed 10-25 ng/million sperm of PDC109 from seminal plasma, sperm from frozen/thawed samples retained only 1-5 ng/million sperm. Sperm from fresh semen samples had adsorbed 6-18 ng/million of BSPA3; whereas, sperm from frozen/thawed samples retained less than 1 ng/million. Interestingly, there was little difference for BSP30K: sperm from fresh and frozen/thawed semen samples contained about 6-11 ng/million. Success of artificial insemination may be improved by adjusting extension and freezing protocols to optimize adsorption and retention of BSP proteins.

Publications

  • Suarez SS (2010) How do sperm get to the egg Bioengineers wanted! Exp Mech published online 20 Nov 2009.
  • Hung PH and Suarez SS (2010) Regulation of sperm storage and movement in the ruminant oviduct. Reproduction in Domestic Ruminants VII, M Lucy et al., (eds), Nottingham University Press, in press.


Progress 08/01/08 to 07/31/09

Outputs
OUTPUTS: I was invited to share the information gained from this project by presenting a minisymposium at the 2009 Annual Meeting of the Society for the Study of Reproduction in Pittsburgh, PA (07/20/09) and invited talks at the Monash University (02/26/09), Melbourne University (02/27/09), and Newcastle University (04/03/09) in Australia. I also disseminated this information to veterinary students at Cornell in the class VETMED 5100, The Animal Body. Finally, I presented this information in a workshop for high school biology teachers (07/09/08), a workshop for middle school biology teachers (07/08/09) and to three high school biology classes in Groton NY (05/12/09). 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 a veterinary course 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. Two undergraduate students, first Shailly Prasad and later Lisa Opdycke, were paid as student laboratory assistants to perform laboratory maintenance chores associated with the project. Their work amounted to 4 hours per week. Juliana Chang completed a research project that partially fulfilled one aim of the grant and wrote a thesis enabled her to graduate with high honors. 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 with the help of USDA inspectors. 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 included 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 a lecture and laboratory exercise in a veterinary course. In addition, the Project Director has presented this information in a local high school and at a Cornell workshop for New York State middle school science teachers. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
After insemination, a few thousand bull sperm reach the oviduct, where they are kept fertile in a storage reservoir until ovulation. The reservoir forms because three bovine seminal plasma proteins (BSPs) on sperm bind to four annexin proteins (ANXAs) on oviductal epithelium. Our long-term goal is to elucidate the mechanisms of sperm storage in order to improve success of artificial insemination. As of 06/15/09, we obtained the following outcomes on the four specific aims for the first 10.5 months of our three-year project. Aim 1 is to test the hypothesis that ANXAs on the surface of oviductal epithelium play a role in bovine sperm storage. ANXA extracts made from the apical plasma membranes of oviductal epithelium were found to extend the motile life spans of bull sperm in vitro and to suppress rises in intracellular calcium ions that usually occur during incubation of sperm. Aim 2 is to test the hypothesis that ANXAs play a key role in sperm movement. At this point, we found that ANXA distribution is patchy on the oviductal epithelium; however, additional samples are required to determine whether the four species of ANXA are differentially distributed along the oviduct. Aim 3 is to test the hypothesis that BSPs regulate sperm movement. Sperm shed BSPs as they capacitate and this could account for gradual release of sperm from the oviductal reservoir. The gradual release plays a role in reducing polyspermic fertilization. To quantify BSPs, a sensitive ELISA was developed using specific, non-cross-reacting rabbit polyclonal antibodies made against nonoverlapping peptide segments of each of the three species of BSP. All capacitation enhancers promoted the loss of BSP-A1/A2 (aka PDC109) from sperm, and HDL was more effective than heparin. Addition of IBMX and dbcAMP to heparin, but not to HDL, enhanced BSP loss. Likewise, capacitation with both heparin and HDL promoted the loss of BSPA3, although to a lesser extent than with BSPA1/A2. In contrast, capacitation with either heparin or HDL (with or without IBMX and dbcAMP) did not enhance loss of BSP30K. Aim 4 is to test the hypothesis that the amounts of BSPs on sperm predict bull fertility by comparing the amounts of BSPs on frozen-thawed sperm to relative conception rates of bulls. We are currently adapting the ELISA test for this analysis.

Publications

  • Suarez SS (2008) Regulation of sperm storage and movement in the mammalian oviduct. Int J Dev Biol 52:455-462.
  • Pitnick S, Wolfner M, Suarez SS (2009) Ejaculate-female and sperm-female interactions. Pp 247-304 in Birkhead TR, Hosken DJ, Pitnick S (eds) Sperm Biology: An Evolutionary Perspective. Elsevier LTD, Oxford.