Progress 04/01/15 to 03/31/20
Outputs
Target Audience:Scientists, graduate students, veterinarians, livestock producers, general audiences Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Trainees 2015-2020: 1) Graduate Stundents: Dalen Zuidema, 2017-present, MS/PhD in progress Karl Kerns, PhD may 2019 Wonhee Song, PhD Dec. 2017 2) Postdocs: Lauren Hamilton 2020-present Karl Kerns 2019-present Michal Zigo 2014-2019 3) Visitig Scholars: 2019: Clio Maicas, University College of Dublin, Ireland, 3 month fellowship 2019: Filip Tirpak, Slovak Ag University, Nitra, SK, 5 month fellowship 2018: Lucie Tumova, Czech University of Life Sciences, Prague, 2 month fellowship 2018: Miriam Stiavnicka, Charles University, Second Medical faculty, Pilsen, Czech Republic, 8 month fellowship 2018-2019: Seda Ocakli, Turkey, 1 year fellowship 2017-2018: Eriklis Nogueira, DVM, PhD, EMBRAPA, Brazil, 1 year fellowship 2017: Lauren Hamilton, Queen's University, Kingston, ON, visiting scholar, 1 month stay 2016: Lauren Hamilton, Queen's University, Kingston, ON, visiting scholar, 1 month stay 2015: Lina Baron Lastra, University of Antofagasta, Chile, visiting scholar, 2 month stay 2015: Marketa Sedmikova, Vice-Chancellor, Czech Life Science University in Prague 2014-15-Lin Yan (12 months), Szechuan University, China (visiting scholar) 4) Undergraduate Research Assistants: Meghan Lawlor 2019- Betsy Pascoe ANSCI 2019- Grace Wiley ANSCI & BIOCHEM 2017-2020 Jennifer Jankovitz ANSCI 2017 Sheridan Tompkins, ANSCI 2015 Awards to trainees 2015-2020: ?2020: University of Missouri Distinguished Dissertation Award to Karl Kerns. 2020: Selection of undergraduate researcher Grace Wiley for presentation at the 2020 Undergraduate Research Dayat the Capitol, Missouri State Capitol, Jefferson City MO, April 2, 2020 2019: USDA NIFA Postdoctoral Fellowship to past PhD student, current postdoc Dr. Karl Kerns 2019: University of Missouri MU Postdoctoral Association Travel Award to Dr. Michal Zigo, a postdoctoral fellow. 2019: University of Missouri Office of Undergraduate Research Travel Award to Grace Wiley, undergraduate research assistant, for presentation at the 52nd Annual SSR Meeting, San Jose, CA, July 18 - 21, 2019. 2019: Society for The Study of Reproduction Trainee Travel Award to Grace Wiley, undergraduate research assistant, for presentation at the 52nd Annual SSR Meeting, San Jose, CA, July 18 - 21, 2019. 2019: Society for The Study of Reproduction Trainee Travel Award to Karl Kerns, a PhD student, for presentation at the 52nd Annual SSR Meeting, San Jose, CA, July 18 - 21, 2019. 2019: Grace Wiley selected for Undergraduate Research Ambassador by University of Missouri Undergraduate Research Office. 2019: University of Missouri Lifesciences Week poster competition, first place to grad student Karl Kerns. 2019: IowaSTATEment Maker award to Karl Kerns, a PhD student, selected by Iowa State University Alumni Association and the Young Alumni Council 2019: Lawrence and Louise Stark Educational Internship in Animal Science, University of Missouri, awarded to Grace Willey. 2018: Student oral presentation competition prize (one of four awarded) to Dalen Zuidema, a PhD student, for oral presentation at the biannual meeting of the Association for Applied Animal Andrology, New Orleans, July 14-16, 2018. 2018: Larry Ewing Memorial Trainee Travel Fund Award to Dalen Zuidema, a PhD student, for presentation at the 51st Annual Meeting of the Society for The Study of Reproduction, New Orleans, LA, July 10 - 14, 2018 2018: Larry Ewing Memorial Trainee Travel Fund Award to Karl Kerns, a PhD student, for presentation at the 51st Annual Meeting of the Society for The Study of Reproduction, New Orleans, LA, July 10 - 14, 2018 2018: Larry Ewing Memorial Trainee Travel Fund Award to Michal Zigo, a postdoctoral fellow, for presentation at the 51st Annual Meeting of the Society for The Study of Reproduction, New Orleans, LA, July 10 - 14, 2018 2018: Lawrence and Louise Stark Educational Internship in Animal Science, University of Missouri, awarded to Grace Willey. 2018: University of Missouri Animal Science Graduate Student Forum PhD oral competition, first place to grad student Karl Kerns. 2018: University of Missouri Animal Science Graduate Student Forum MS oral competition, first place to collaborating grad student Evan Grusenmeyer. 2018: University of Missouri Lifesciences Week poster competition, second place to grad student Karl Kerns. 2017: National Swine Improvement Federation (NSIF) Graduate Student Award to Karl Kerns, PhD student. 2017: Paper by former PhD student Shawn Zimmerman rated top 10% most frequently cited papers published in PLoS One. Zimmerman et al., 2011, PLoS ONE, 6(2):e17256 2017: USDA-NIFA-AFRI Merit Award (SSR meeting 2017) to Dr. Michal Zigo, a postdoctoral fellow 2017: University of Missouri MU Postdoctoral Association Travel Award to Dr. Michal Zigo, a postdoctoral fellow. 2017: USDA-NIFA-AFRI Merit Award (SSR meeting 2017) to Karl Kerns, also a finalist in SSR 2017 trainee platform competition 2017: ASAS-SSR 2017 Triennial Reproduction Symposia, best poster award to Karl Kerns (Sutovsky lab, coauthor) and Momal Sharif (Miller lab, U. of Illinois, lead author). 2017: Outstanding Poster Award, Missouri Life Sciences Week graduate student poster competition, to PhD student Karl Kerns. 2017: Douglas D. Randall Young Scientists Development Fund award, University of Missouri, to PhD student Karl Kerns. 2017: USDA NIFA Graduate Fellowship to PhD student Karl Kerns. 2016: Larry Ewing Memorial Trainee Travel Fund Award to Michal Zigo, a postdoctoral fellow, for short oral talk presentation at the 49th Annual Meeting of the Society for The Study of Reproduction, San Diego, CA. 2016: Article published in PNAS by Wonhee Song et al., was Top 2% most downloaded/highest web impact research papers published in Fall 2016, out of >160,000 papers. 2016: Larry Ewing Memorial Trainee Travel Fund Award to Karl Kerns, a PhD student, for poster talk presentation at the 49th Annual Meeting of the Society for The Study of Reproduction, San Diego, CA. 2016: Larry Ewing Memorial Trainee Travel Fund Award to Wonhee Song, a PhD student, for poster presentation at the 49th Annual Meeting of the Society for The Study of Reproduction, San Diego, CA. 2015: Nadani - Josef, Marie & Zdenek Hlavka Family Foundation post-doctoral travel award to Michal Zigo. 2015: Douglas D. Randall Young Scientists Development Fund, University of Missouri travel award to Karl Kerns, a PhD student, for poster presentation at the 48th Annual Meeting of the Society for The Study of Reproduction, San Juan, Puerto Rico. 2015: Larry Ewing Memorial Trainee Travel Fund Award to Karl Kerns, a PhD student, for poster presentation at the 48th Annual Meeting of the Society for The Study of Reproduction, San Juan, Puerto Rico. 2015: Larry Ewing Memorial Trainee Travel Fund Award to Wonhee Song, a PhD student, for poster presentation at the 48th Annual Meeting of the Society for The Study of Reproduction, San Juan, Puerto Rico. How have the results been disseminated to communities of interest?Invited conference presentations 2015-2020: Upcoming 2020: International Meeting of Animal Reproduction, Viçosa, Brazil, July 30-August 2, 2020 2019: Symposium Repronutri - Reproduction, Production and Nutrition of Bovines, September 26&27, 2019, Campo Grande, MS, Brazil. 2019: Investment Forum Minneapolis, presented by Karl Kerns. 2019: IXth International Conference on Boar Semen Preservation, August ­ 11-14, 2019, Hunter Valley, Australia 2019: Scientific Days 2019, Czech University of Life Sciences, April 24-26, 2019, Prague, Czech Republic. 2018: International Bull Fertility Conference - Theory to Practice, May 27-30 2018, Westport, North Ireland 2018: 44th International Embryo Technology Society (IETS) Annual Conference, January 14-16, 2018, Bangkok, Thailand 2017: International Conference on Analytical Cytometry, Prague, Czech Republic, October 15-17, 2017 2017: XVI Reunion Cientifica ICBAR - Latin American science congress organized by Universidad Nacional San Marcos, August 9-11, 2017, Lima, Peru 2017: Collaborate for Cure, University of Kansas School of Medicine, Kansa City, July 24, 2017 2017: 50th annual meeting of the Society for the Study of Reproduction, July 14-17, Washington DC 2017: American Society of Animal Science, annual meeting, July 8-12, 2017, Baltimore, MD 2017: XlI International Veterinary Technical Meeting of MAGAPOR, 26-27 April 2016, Zaragoza, SPAIN 2017: XII Congreso Internacional de Reproducción Porcina Dr. Santiago Martín Rillo, México - León, Guanajuato March 30-31, 2017 2017: 43rd International Embryo Technology Society (IETS) Annual Conference, Morulae Workshop, Austin TX, January 13-17, 2017 2016: Ag Innovation Showcase, Donald Danforth Plant Sciences Center, September 12-14, St. Louis MO 2016: Gateway to Parenthood, St. Louis Infertility Awareness 2016, April 30 (presented by associate, Dr. Michal Zigo). 2016. Boar Stud Managers' Conference V, August 3-4, St. Louis MO (presented by grad student Karl Kerns). 2016: 11th International Scientific Conference on Biotechnology and Quality of Raw Materials and Foodstuffs, January 27-29, 2016, Stará Lesná, Slovak Republic. 2015: National Swine Improvement Federation Annual Meeting 2015, December 3&4, Nashville, TN. 2015: Warnick Lecturer, D.H. Barron Reproductive and Perinatal Biology Research Program, University of Florida. November 3, 2015 2015: Annual Meeting of the Chilean Society of Reproduction and Development, September 4-7, 2015, Antofagasta, Chile 2015: VIIIth International Conference on Boar Semen Preservation, August 9th_12th in Champaign, Illinois 2015: Gordon Research Conference on "Nanoscale Science and Engineering for Agriculture and Food Systems", June 7-12, 2015, Bentley University, Waltham, MA 2015: XIX Curso "Novos Enfoques na Producao e Reproducao de Bovinos", XIX Course on New Advanced in Bovine Production & Reproduction, Uberlandia, Brazil, March 19 & 20, 2015 What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In the last five years, we made advances in understanding how the UPS regulates in vitro capacitation (IVC) in livestock species' spermatozoa, including our discovery of new zinc-dependent pathway that intersects with sperm UPS and appears to be a master-regulator of the capacitation process. This work was published in Nature Communications and featured in the USDA-NIFA Annual Report 2018, released 6/28/2019. Other major published findings from previous period include the proteasome dependent remodeling of sperm surface glycoproteins and sperm proteasomal subunits during capacitation, and the essential role of ubiquitin tail fusion proteins (UBA52 [8]), glutathione transferases, and ubiquitin-regulated histone deacetylases (SIRT1) in pre-embryo development. In the final year of the project, we focused on zygotic/embryonic ubiquitin ligase NEDL2, a WW-domain protein required for fertilization and paternal pronucleus development ( manuscript in final stages of preparation). Preliminary studies have been completed on sperm zincoproteome, sperm-proteasomal interactome, and sperm-oviductal sperm reservoir binding, with manuscripts in preparation and submitted.Altogether, previous funding period resulted in 20+ publications in esteemed journals including Nature Communications (IF=11.8), Antioxidants (IF=4.5), Intl. Journal of Cell & Molecular Science (IF=4.2), Scientific Reports (IF=4.0), Cell Tissue Research (IF=3.4), Reproduction (IF=3.1) and Biology of Reproduction (IF=3.0), as well as two US patents and one pending patent application. Importantly, this project period afforded training opportunities to three PhD-graduate students (two graduated, one in progress), eight fellowship-funded long-term visiting scholars from seven different countries, and four MU undergraduate researchers. Collectively, these trainees earned a number of national and international awards and honors, including one predoctoral and one postdoctoral fellowship from USDA-NIFA, and multiple NIFA and SSR awards for presentations at the SSR meetings. To promote networking between NIFA Animal Reproduction awardees, we collaborated with three currently funded investigators, Drs. David Miller (Illinois), Wansheng Liu (U. Penn) and Tom Geary (USDA-ARS Fort Keogh MT). Translating our research to practice, research from previous report was instrumental in obtaining an ongoing USDA Phase I SBIR award with International Boar Semen, Eldora, IA, and founding a new semen extender/andrology startup, AndroLabb LLC in Columbia, MO, in addition to fostering ongoing collaborations with two major AI companies, The Maschhoffs and Fast Genetics/Sexing Technologies.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2020
Citation:
Zigo M, Man�skov�-Postlerov� P, Zuidema D, Kerns K, Jon�kov� V, Tumov� L, Buben�ckov� F, Sutovsky P (2020) Porcine model for the study of sperm capacitation, fertilization and male fertility. Cell Tissue Res., In press.
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2020
Citation:
Hamilton LEH, Oko R, Sutovsky P (2020) Cellular and molecular events after ICSI in clinically relevant animal models. In: Manual of Intracytoplasmic Sperm Injection in Human Assisted Reproduction. Palermo G and Nagy P, Editors. Cambridge University Press, In press.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Kelsey KM, Zigo M, Thompson WE, Kerns K, Gaurishankar Manandhar G, Sutovsky M, Sutovsky P (2020) Reciprocal surface expression of arylsulfatase A and ubiquitin in normal and defective mammalian spermatozoa. Cell Tissue Res. 379:561-576.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Hamilton LE, Zigo M, Mao J, Xu W3, Sutovsky P, OFlaherty C, Oko R (2019) GSTO2 isoforms participate in the oxidative regulation of the plasmalemma in eutherian spermatozoa during capacitation. Antioxidants 8: 601.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Zigo M, Manaskova-Postlerova P, Jonakova V, Kerns K, Sutovsky P (2019) Compartmentalization of the proteasome-interacting proteins during sperm capacitation. Sci. Rep. 9:12583
- Type:
Websites
Status:
Published
Year Published:
2019
Citation:
St John JC, Sutovsky P (2019) Mitochondrial DNA: Fate of the paternal mitochondrial genome. eLS - Wiley Online Library.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Kerns K, Sharif M, Zigo M, Xu W, Hamilton L, Sutovsky M, Ellersieck M, Drobnis EZ, Oko R, Miller D, Sutovsky P (2020) Sperm cohort-specific zinc signature acquisition and capacitation-induced zinc flux regulate sperm-oviduct and sperm-zona pellucida interactions. Int. J. Mol. Sci., 21:2121 Special Issue on Advances in Molecular Regulation of Spermatozoa Function.
|
Progress 04/01/18 to 03/31/19
Outputs
Target Audience:Scientists, graduate students, veterinarians, livestock producers, general audiences Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One graduate student and one postdoc participated in this research in the Sutovsky laboratory during this reporting period. One undergraduate student, taking an undergraduate research for three credit hours also had an opportunity to participate in the project. Work on ubiquitin-dependent fertilization and zygotic development has been advanced through international collaborations with scientists in Brazil, Canada, Czech Republic with scholars from all three countries visiting Sutovsky laboratory for multi-month research stays. Recent awards to trainees involved in this project: 2019: USDA NIFA Postdoctoral Fellowship to past PhD student, current postdoc Dr. Karl Kerns 2019: Society for The Study of Reproduction Trainee Travel Award to Grace Wiley, undergraduate research assistant, for presentation at the 52nd Annual SSR Meeting, San Jose, CA, July 18 - 21, 2019. 2019: Society for The Study of Reproduction Trainee Travel Award to Karl Kerns, a PhD student, for presentation at the 52nd Annual SSR Meeting, San Jose, CA, July 18 - 21, 2019. 2019: Grace Wiley selected for Undergraduate Research Ambassador by University of Missouri Undergraduate Research Office. 2019: University of Missouri Lifesciences Week poster competition, first place to grad student Karl Kerns. 2019: IowaSTATEment Maker award to Karl Kerns, a PhD student, selected by Iowa State University Alumni Association and the Young Alumni Council 2019: Lawrence and Louise Stark Educational Internship in Animal Science, University of Missouri, awarded to Grace Willey. 2018: Larry Ewing Memorial Trainee Travel Fund Award to Karl Kerns, a PhD student, for presentation at the 51st Annual Meeting of the Society for The Study of Reproduction, New Orleans, LA, July 10 - 14, 2018 2018: Larry Ewing Memorial Trainee Travel Fund Award to Michal Zigo, a postdoctoral fellow, for presentation at the 51st Annual Meeting of the Society for The Study of Reproduction, New Orleans, LA, July 10 - 14, 2018 2018: Lawrence and Louise Stark Educational Internship in Animal Science, University of Missouri, awarded to Grace Willey. 2018: University of Missouri Animal Science Graduate Student Forum PhD oral competition, first place to grad student Karl Kerns. How have the results been disseminated to communities of interest?Beside a number of research papers and book chapters, data from this project has been reported as part of invited lectures at the following major venues: 2018: International Bull Fertility Conference - Theory to Practice, May 27-30 2018, Westport, North Ireland 2018: 44th International Embryo Technology Society (IETS) Annual Conference, January 14-16, 2018, Bangkok, Thailand What do you plan to do during the next reporting period to accomplish the goals?Major goal of OBJECTIVE #1 will be to gain better understanding of how UPS and zinc ion fluxes co-regulate boar sperm capacitation. We will also address the mechanism by which UPS regulates the shedding of acrosomal surface-adsorbed seminal plasma proteins during capacitation. Ultimately, the knowledge gained from this work will allow us to develop optimal conditions for semen processing, extension, storage and distribution that will mitigate premature sperm capacitation and demise. Under OBJECTIVE #2, we will focus on the role of UPS in early zygotic development and particularly on the involvement of specific ubiquitin ligases, such as NEDL2/HECW2. Our preliminary data show NEDL2 infiltrating both maternal and paternal pronuclei shortly after fertilization and the interference with NEDL2 function having a detrimental effect on blastocyst quality. Such observations will give us better understanding of embryo development and possibly of early pregnancy loss which dramatically reduces reproductive performance in livestock species.
Impacts
What was accomplished under these goals?
OBJECTIVE #1: Zinc ion fluxes during mammalian sperm capacitation are regulated by ubiquitin-proteasome system. Sperm capacitation, the ultimate maturation event preparing mammalian spermatozoa for fertilization, was first described in 1951, yet its regulatory mechanisms remain poorly understood. The capacitation process encompasses an influx of bicarbonate and calcium ions, removal of decapacitating factors, changes of pH and sperm proteasomal activities, and the increased protein tyrosine phosphorylation. We discovered a novel biological phenomenon of a unique zinc (Zn2+) ion redistribution associated with mammalian sperm in vitro capacitation (IVC). Using image-based flow cytometry (IBFC), we identified four distinct types of sperm zinc ion distribution patterns (further zinc signature) and their changes during IVC. The zinc signature was altered after sperm capacitation, reduced by proteasomal inhibitors, removed by zinc chelators, and maintained with addition of external ZnCl2. Importantly, we were able to restore the pre-capacitation zinc signature by zinc reloading of extended, stored boar semen. IMPACT: Our findings represent a fundamental shift in the understanding of mammalian fertilization, paving the way for improved semen analysis, in vitro fertilization (IVF) and artificial insemination (AI). Relevant to livestock industry quest for better fertility management, zinc ion supplementation through nutrition and direct addition to extended semen will benefit reproductive performance and profitability of animal production. Published in Nature Communications, Theriogenology and International Journal of Molecular Science. OBJECTIVE #1: Proteasome-dependent deaggregation of sperm surface proteins occurs during boar sperm capacitation. We studied the participation of ubiquitin proteasome system (UPS) in spermadhesin release during in vitro capacitation (IVC) of domestic boar spermatozoa. At ejaculation, boar spermatozoa acquire low molecular weight (8-16 kDa) seminal plasma proteins, predominantly spermadhesins, aggregated on the sperm surface. Due to their arrangement, such aggregates are relatively inaccessible to antibody labeling. As a result of de-aggregation and release of the outer layers of spermadhesins from the sperm surface during IVC, antibody labeling becomes feasible in the capacitated spermatozoa. In vivo, the capacitation-induced shedding of spermadhesins from the sperm surface is associated with the release of spermatozoa from the oviductal sperm reservoir. We took advantage of this property to perform image-based flow cytometry to study de-aggregation and shedding of boar spermadhesins (AQN, AWN, PSP protein families) and boar DQH (BSP1) sperm surface protein which induces higher fluorescent intensity in capacitated vs. ejaculated spermatozoa. Addition of a proteasomal inhibitor (100 µM MG132) during IVC significantly reduced fluorescence intensity of all studied proteins (P<0.05) compared to vehicle control IVC. Western blot detection of spermadhesins did not support their retention during IVC with proteasomal inhibition (P>0.99) but showed the accumulation of DQH (P=0.03) during IVC, compared to vehicle control IVC. Our results thus demonstrate that UPS participates in the de-aggregation of spermadhesins and DQH protein from the sperm surface during capacitation, with a possible involvement in sperm detachment from the oviductal sperm reservoir and/or sperm-zona pellucida interactions. Published in Reproduction. OBJECTIVE #2: NEDD4-like ubiquitin ligase 2 (NEDL2) co-regulates porcine sperm function and fertilization. The ubiquitin-proteasome system (UPS) plays diverse regulatory and homeostatic roles in mammalian reproduction. Ubiquitin ligases are the substrate-specific mediators of ubiquitin binding to its subject proteins. The NEDD4-like ubiquitin ligase 2 (NEDL2) is a HECT-type ubiquitin ligase that contains one C2, one HECT, and two WW domains/protein-protein interaction modules. Beyond its predicted ubiquitin-ligase activity, its cellular functions are largely unknown. Our studies were designed to investigate the content and distribution of NEDL2 in porcine spermatozoa, oocytes, zygotes and early preimplantation embryos, and in cumulus cells before and after in vitro maturation with oocytes, and fibroblast cells as positive control by western blot and immunocytochemistry, and to examine its rules during oocyte fertilization. Two isoforms of NEDL2 were identified by western blot. One at approximately 52 kDa was detected only in the germinal vesicle stage and metaphase II oocytes, and in the early preimplantation embryos. Second isoform was a high mass band at 176 kDa, which was only detected in somatic cells. Interestingly, ejaculated spermatozoa prominently displayed the same 52 kDa band as oocytes; they also had two minor bands of 74 and 129 kDa (by densitometry), which were not detected in somatic cells or oocytes. By immunofluorescence, NEDL2 showed a diffused cytoplasmic localization in all cell types and accumulated in distinct foci in the germinal vesicles of immature oocytes, in maternal and paternal pronuclei of zygotes and nuclei of embryo blastomeres and somatic cells. In blastocysts, the labelling intensity of NEDL2 was stronger in the inner cell mass than in trophoblast, indicating higher NEDL2 content in the ICM cells than in trophectoderm. NEDL2 protein was ten times higher in post maturation oocyte-surrounding cumulus cells than that of cumulus cells before in vitro maturation with hormones, indicating that NEDL2 may have a unique role in cumulus cells after ovulation. Microinjection of anti-NEDL2 antibody into oocyte before IVF did not affect percent oocytes fertilized, percent oocytes cleaved, or blastocyst formation. However, anti-NEDL2 antibody decreased the number of pronuclei, accelerated the formation of nuclear precursor bodies at 6h post fertilization, inhibited sperm DNA decondensation, and resulted in more fertilized oocytes without male pronuclear formation. IMPACT: These studies, while still in progress hint at an essential role NEDL2 may play during fertilization, particularly during sperm DNA decondition and formation of the paternal pronucleus. Manucript in prepration, will be completed by adding new data generated by ongoing work.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Mao J, OGorman C, Sutovsky M, Zigo M, Wells KD, Sutovsky P (2018) Ubiquitin A-52 residue ribosomal protein fusion product 1 (Uba52) is essential for preimplantation embryo development. Biol. Open, 7(10)
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Kerns K, Zigo M, Sutovsky P (2018) Zinc: A necessary ion for mammalian sperm fertilization competency. Int. J. Mol Sci., 19(12)
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Zigo M, Jonakova V, Manaskova-Postlerova P, Kerns K, Sutovsky P (2019) Ubiquitin-proteasome system participates in the de-aggregation of spermadhesin and DQH protein during boar sperm capacitation. Reproduction, In press. doi: 10.1530/REP-18-0413. [Epub ahead of print]
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2019
Citation:
Geisert RD, Sutovsky P, Lucy M, Bartol FF, Meyer AE (2019) Reproductive Physiology of Swine. In: Animal Agriculture: Challenges, Innovations, and Sustainability. Edited by Fuller W. Bazer, G. Cliff Lamb, and Guoyao Wu, Elsevier, In press.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
Protopapas N, Hamilton L, Warkentin R, Xu W, Sutovsky P, and Oko R (2019) The post-acrosomal sheath and perforatorial regions of the perinuclear theca of rat spermatozoa share common developmental origins and protein constituents. Biol. Reprod., In press
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
Hamilton LE, Suzuki J, Aguila L, Meinsohn M-C, Smith O, Protopapas N, Wei Xu3, Sutovsky P, Oko R (2019) Sperm-borne glutathione-s-transferase omega 2 accelerates the nuclear decondensation of spermatozoa during fertilization in mice. Biol. Reprod., In press.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
Sutovsky P, Kerns K, Zigo M, Zuidema D (2019) Boar semen improvement through sperm capacitation management, with emphasis on zinc ion homeostasis. Theriogenology, In press.
|
Progress 04/01/17 to 03/31/18
Outputs
Target Audience:Scientists, graduate students, veterinarians, livestock producers, general audiences. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One graduate student, one postdoc and one research professor participated in this research in the Sutovsky laboratory. Two undergraduate students, both taking an undergraduate research for three credit hours also had an opportunity to participate in the project. As was the case during previous two reporting periods, work on ubiquitin-dependent zygotic histone modifications has been advanced through an international collaboration with a junior investigator in Czech Republic who previously trained in Sutovsky laboratory that resulted in a joint publication. How have the results been disseminated to communities of interest?Beside a number of research papers and book chapters, data from this project has been reported as part of invited lectures at the following major venues: 2018: 44th International Embryo Technology Society (IETS) Annual Conference, January 14-16, 2018, Bangkok, Thailand 2017: International Conference on Analytical Cytometry, Prague, Czech Republic, October 15-17, 2017 2017: XVI Reunion Cientifica ICBAR - Latin American science congress organized by Universidad Nacional San Marcos, August 9-11, 2017, Lima, Peru 2017: Collaborate for Cure, University of Kansas School of Medicine, Kansa City, July 24, 2017 2017: 50th Annual meeting of the Society for the Study of Reproduction, July 14-17, Washington DC 2017: American Society of Animal Science, annual meeting, July 8-12, 2017, Baltimore, MD 2017: XlI International Veterinary Technical Meeting of MAGAPOR, 26-27 April 2016, Zaragoza, SPAIN 2017: XII Congreso Internacional de Reproducción Porcina Dr. Santiago Martín Rillo, León, Guanajuato MEXICO March 30-31, 2017 What do you plan to do during the next reporting period to accomplish the goals?Major goal of OBJECTIVE #1 will be to gain better understanding of how UPS and zinc ion fluxes co-regulate boar sperm capacitation. We will also address the mechanism by which UPS regulates the shedding of acrosomal surface-adsorbed seminal plasma proteins during capacitation. Ultimately, the knowledge gained from this work will allow us to develop optimal conditions for semen processing, extension, storage and distribution that will mitigate premature sperm capacitation and demise. Under OBJECTIVE #2, we will focus on the role of UPS in early zygotic development and particularly on the involvement of specific ubiquitin ligases, such as NEDL2/HECW2. Our preliminary data show NEDL2 infiltrating both maternal and paternal pronuclei shortly after fertilization and the interference with NEDL2 function having a detrimental effect on blastocyst quality. Such observations will give us better understanding of embryo development and possibly of early pregnancy loss which dramatically reduces reproductive performance in livestock species.
Impacts
What was accomplished under these goals?
OBJECTIVE #1: Zinc ion fluxes during mammalian sperm capacitation are regulated by ubiquitin-proteasome system. Sperm capacitation, the ultimate maturation event preparing mammalian spermatozoa for fertilization, was first described in 1951, yet its regulatory mechanisms remain poorly understood. The capacitation process encompasses an influx of bicarbonate and calcium ions, removal of decapacitating factors, changes of pH and sperm proteasomal activities, and the increased protein tyrosine phosphorylation. We discovered a a novel biological phenomenon of a unique zinc (Zn2+) ion redistribution associated with mammalian sperm in vitro capacitation (IVC). Using image-based flow cytometry (IBFC), we identified four distinct types of sperm zinc ion distribution patterns (further zinc signature) and their changes during IVC. The zinc signature was altered after sperm capacitation, reduced by proteasomal inhibitors, removed by zinc chelators, and maintained with addition of external ZnCl2. IMPACT: These findings represent a fundamental shift in the understanding of mammalian fertilization, paving the way for improved semen analysis, in vitro fertilization (IVF) and artificial insemination (AI). Published in Nature Communications. OBJECTIVE #1: Modifications of the 26S proteasome occur during boar sperm capacitation. Protein ubiquitination is a stable, reversible posttranslational modification, targeting proteins for degradation/recycling by 26S proteasome in a well characterized enzymatic cascade. Studies revealed the role of UPS in the regulation of fertilization, including sperm-zona pellucida (ZP) interactions and the early event of sperm capacitation. We investigated the changes in proteasome compartmentalization, subunit composition and posttranslational modifications during in vitro capacitation of fresh boar spermatozoa.We observed capacitation dependent shedding of both 20S core and 19S regulatory particle from acrosome that was associated with decreased plasma membrane integrity, independent of proteasomal inhibition. Subunits PSMA1-7 of the 20S core did not appear to undergo post-translational modifications during capacitation, based on invariant molecular masses before and after capacitation; however, we observed multiple PSMD4 forms of 19S regulatory particle (50, 53, 70, 115-140, 160, and >176 kDa) sequentially released from spermatozoa. PSMD4 subunit was found to be post-translationally modified during the course of capacitation, resulting in change of apparent molecular mass, some of which were dependent on proteasomal inhibition. IMPACT: Our results show that the sperm proteasomes are being modified during sperm capacitation. Additional studies of individual 26S proteasome subunits will be required to elucidate these modifications and to understand how UPS modulates the sperm capacitation. Published in Cell & Tissue Research. OBJECTIVE #2: Ubiquitin-regulated histone deacetylase SIRT1 modulates the methylation and acetylation of histone H3 on lysine 9 (H3K9) in the zygotic pronuclei improves porcine embryo development. The histone code is an established epigenetic regulator of early embryonic development in mammals. The lysine residue K9 of histone H3 (H3K9) is a prime target of SIRT1, a member of NAD+-dependent histone deacetylase family of enzymes targeting both histone and non-histone substrates. At present, little is known about SIRT1-modulation of H3K9 in zygotic pronuclei and its association with the success of preimplantation embryo development. Therefore, we evaluated the effect of SIRT1 activity on H3K9 methylation and acetylation in porcine zygotes and the significance of H3K9 modifications for early embryonic development. Our results show that SIRT1 activators resveratrol and BML-278 increased H3K9 methylation and suppressed H3K9 acetylation in both the paternal and maternal pronucleus. Inversely, SIRT1 inhibitors nicotinamide and sirtinol suppressed methylation and increased acetylation of pronuclear H3K9. Evaluation of early embryonic development confirmed positive effect of selective SIRT1 activation on blastocyst formation rate (5.2 ± 2.9 versus 32.9 ± 8.1 % in vehicle control and BML-278 group, respectively; P≤0.05). Stimulation of SIRT1 activity coincided with fluorometric signal intensity of ooplasmic ubiquitin ligase MDM2, a known substrate of SIRT1 and known limiting factor of epigenome remodeling. IMPACT: We conclude that SIRT1 modulates zygotic histone code, obviously through direct deacetylation and via non-histone targets resulting in increased H3K9me3. These changes in zygotes lead to more successful pre-implantation embryonic development and, indeed, the specific SIRT1 activation due to BML-278 is beneficial for in vitro embryo production and blastocyst achievement. Published in Journal of Animal Science & Biotechnology. OBJECTIVE #2: Ubiquitin A-52 residue ribosomal protein fusion product 1 (Uba52) is essential for preimplantation embryo development. Ubiquitin A-52 residue ribosomal protein fusion product 1 (Uba52) is a major source of ubiquitin protein for covalent modification of proteinaceous substrates recycled by ubiquitin-proteasome system (UPS), but its role in early embryo development has not been studied until now. Using CRISPR/cas9 gene editing tool, our objective was to determine if UBA52 protein is required for mammalian embryogenesis. Matured metaphase II porcine oocytes were injected with CRISPR Cas9+guide RNAs (Uba52 gRNA) or cas9 without gRNAs as control, followed by in vitro fertilization (IVF) and embryo culture to day 7. Injection of cas9+gRNAs affected embryo development. On day 4 of embryo culture, the proportion of 2-, 4- and 8-cell stage embryos was significantly different between the Uba52 gRNA and control group (P<0.05), with more 8-cell stage embryos in the control and more 4- and 2-cell stage embryos in the Uba52g RNA group. This delay in the development of Uba52 gRNA embryos occurred at the transition from the 4- to 8-cell stages, around the time of major zygotic genomic activation. The percentage of blastocyst formation on day 7 and the cell number per blastocyst were significantly lower in the Uba52 gRNA group than in the control (P<0.05). Genotyping by PCR and DNA gel electrophoresis analysis showed that 91.8% of embryos that failed to develop to blastocyst had either a monoallelic or a biallelic modification of the Uba52 gene. In comparison, only 24.4% of embryos that reached blastocyst had a monoallelic modification and biallelic editing was not found in any of the blastocysts. Based on immuno-labeling intensity, both UBA52 and proteasome protein levels on days 4 and 7 of culture were significantly lower in the Uba52 gRNA group than in the control (P<0.05), in agreement with UBA52 Western blotting-densitometry of day 4 embryos. Morphological examination of blastomere nuclei revealed abnormal nuclear structure in the Uba52 gRNA group, such as reduced size, irregular shapes, nucleus fragmentation and uneven DNA distribution at all stages of embryo development. Nuclear morphology studies of embryos injected with cas9+gRNAs and co-injected with plasmid DNA encoding nuclear localized GFP further supported these observations. IMPACT: Our data indicate that Uba52 gene is essential in early embryogenesis. Under review in Biology Open.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Sutovsky P (2018) Review: Sperm-oocyte interactions and their implications for bull fertility, with emphasis on ubiquitin-proteasome system. Animal, 12: 1-12 doi: 10.1017/S1751731118000253
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Zigo M, Kerns K, Sutovsky M, Sutovsky P (2018). Modifications of the 26S proteasome during boar sperm capacitation. Cell Tissue Res., 372(3):591-601
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Hamilton LE, Acteau G, Xu W, Sutovsky P, Oko RJ (2017) The developmental origin and compartmentalization of glutathione-S-transferase Omega 2 isoforms in the perinuclear theca of Eutherian spermatozoa. Biol. Reprod., 97(4):612-621
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Adamkova K, Yi Y-J, Petr J, Zalmanova T, Hoskova K, Jelinkova P, Moravec J, Kralickova M, Sutovsky M, Sutovsky P, Nevoral J (2017) SIRT1-dependent modulation of methylation and acetylation of histone H3 on lysine 9 (H3K9) in the zygotic pronuclei improves porcine embryo development. J. Anim. Sci. Biotech., 8: 83.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Taylor JF, Schnabel RD, Sutovsky P (2018) Review: Genomics of bull fertility. Animal, 5:1-12 doi: 10.1016/j.anireprosci.2018.02.007
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2018
Citation:
Taylor JF, Schnabel RD, Sutovsky P (2018) Identification of Genomic Variants Causing Sperm Abnormalities and Reduced Male Fertility, Anim Reprod Sci, In press. doi: 10.1016/j.anireprosci.2018.02.007.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Kerns K, Zigo M, Drobnis EZ, Sutovsky M, Sutovsky P (2018) Zinc ion flux during mammalian sperm capacitation. Nature Communications, 9(1):2061. doi: 10.1038/s41467-018-04523-y
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2018
Citation:
Sutovsky P (2018). Pig overview (male reproduction) In: Encyclopedia of Reproduction, Second edition, Vol. 1., B. Jegou & MK Skinner, Ed., Elsevier.
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Progress 04/01/16 to 03/31/17
Outputs
Target Audience:Scientists, graduate students, veterinarians, livestock producers, general audiences. Changes/Problems:No major changes to the project, no major problems encountered. What opportunities for training and professional development has the project provided?Trainees participating in this project: Michal Zigo, postdoc, Sutovsky lab Karl Kerns, grad student, Sutovsky lab Momal Sharif, grad student, Miller lab How have the results been disseminated to communities of interest?In addition to submitted and published articles, results were presented at thefollowing conferences: XlI International Veterinary Technical Meeting of MAGAPOR, 26-27 April 2016, Zaragoza, SPAIN XII Congreso Internacional de Reproducción Porcina Dr. Santiago Martín Rillo, México - León, Guanajuato,March 30-31, 2017 Ag Innovation Showcase, Donald Danforth Plant Sciences Center, September 12-14, 2016, St. Louis MO Gateway to Parenthood, St. Louis Infertility Awareness 2016, April 30 Boar Stud Managers' Conference V, August 3-4, 2016, St. Louis MO What do you plan to do during the next reporting period to accomplish the goals?AIM 1: Examine the protesome-regulated mechanisms that control zinc ion fluctuation during boar sperm capacitation Evaluate the relationship of boar sperm zinc signature types with boar fertility in AI service Identify proteasomal subunits and proteasome-associated proteins that are modified during sperm capacitation AIM 2: Provide definitive evidence of NEDL2 role in zygotic development Examine sperm NEDL2 localization and changes during sperm capacitation Examine the role of ubiquitin-tail fusion ribonucleoprotein UBA52 in zygotic development up to and including blastocyst stage Characterize the relationship betwen ubiquitin-proteoasome system and sirtuins, and their influence on zygotic histone code. Develop a mammalian cell free system (permeabilized boar spermatozoa coincubated with porcine oocyte extract), to study the role of UPS in zygotic pronuclear development
Impacts
What was accomplished under these goals?
Previously unknown phenomenon of proteasome-modulated sperm zinc signature: Sperm capacitation, the ultimate maturation event preparing mammalian spermatozoa for fertilization, was first described in 1951, yet its regulatory mechanisms remain poorly understood. We discovereda never before described biological phenomenon of a unique zinc (Zn2+) ion signature in mammalian spermatozoa, and its changes during in vitro capacitation (IVC) of domestic boar spermatozoa under proteasomal proteolysis permissive and inhibitingconditions. Using image-based flow cytometry (IBFC), we identified four distinct types of sperm zinc signatures, and determined that the 26S proteasome is likely involved much earlier in sperm capacitation than previously realized. The zinc signature was altered after sperm capacitation, inhibited under proteasome inhibiting IVC conditions, altered by zinc chelators, and maintained with addition of external ZnCl2. Furthermore, zinc signature differs between different sperm fractions of boar ejaculate, lending support to new paradigms and refuting older dogmas on sperm function during fertilization. The 'zinc shield' established by the oocyte following fertilization could derail sperm zinc signaling as an added barrier to pathological polyspermic fertilization. Such findings represent a fundamental shift in the understanding of mammalian fertilization, paving the way for a more accurate semen analysis to ameliorate the methodology of in vitro fertilization (IVF) and artificial insemination (AI). Dynamics of the sperm proteasome during boar sperm capacitation: Parallel and sequential treatments of ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting conditions were used to isolate putative sperm proteasome-associated sperm proteins in a compartment-specific manner. Treated spermatozoa were screened with state of the art imaged based flow cytometer (FlowSight®, Amnis-Millipore) to assess changes in acrosome integrity (PNA lectin), and the quantity and electrophoretic migration patterns of candidate UPS substrate-proteins and proteasomal subunits. Resultant protein fractions were screened by Western blotting (WB) for posttranslational modifications of i) proteasomal subunits, and ii) candidate proteasomal substrate/interactor proteins that co-purify with sperm proteasomes. Employing differential sperm compartment fractionation and protein isolation approaches and conditions, proteins were isolated from the ejaculated and in vitro capacitated spermatozoa under various UPS-modulating conditions. Differential proteomic approach employing 1D PAGE revealed differences in accumulated proteins at the molar masses of 65, 49, and 35 kDa, as well as differential accumulation of spermadhesin proteins at 12-15 kDa. MS analysis revealed accumulation of proteins previously reported as proteasome co-purifying proteins as well as some novel proteins, such as acrosin, cathepsin F, alpha subunit of ATP synthase, and enzymes of citric acid cycle. Image-based flow cytometry revealed differential protein localization and accumulation patterns, under various UPS conditions, for lactadherin MFGE-8, ADAM-5, and acrosin inhibitor, a;l of which were previously found to co-purify with sperm proteasomes. Lectin PNA labeling revealed the capacitation-induced reorganization of outer acrosomal membrane, hindered in the presence of proteasomal inhibitors. Furthermore, proteasomal 19S regulatory particle subunit PSMD4 appeared to be posttranslationally modified during capacitation and unique bands, other than the expected band of 58 kD, were detected in capacitated spermatozoa regardless of UPS-altering treatment. Proteasomal 20S core particle did not appear to be modified during capacitation. Proteasomal inhibitors also prevented the detachment of acrosomal shrouds in non-fixed spermatozoa that is typically observed in a subpopulation of in vitro capacitating spermatozoa. These results further support the proposed role of UPS in the sperm capacitation. Role of sperm proteasomes in the sperm release from oviductal sperm reservoir (collaboration with project co-investigator & subcontractor, Dr. David Miller, U. of Illinois): We hypothesized that the UPS is involved in spermatozoa release from the reservoir in the oviduct by degrading putative spermatozoa glycan receptors. As an initial test of this hypothesis, porcine spermatozoa were allowed to bind beads to which either of two oviduct glycans were attached, a 6-sialylated branched glycan (bi-SiaLN) and a sulfated Lewis X trisaccharide (suLeX). Free spermatozoa were removed. The release of spermatozoa from glycan bound beads was induced by 80 and 800 nM progesterone. The number of spermatozoa released by either concentration was not different. Addition of progesterone induced the release of a similar number (58% and 65%) of spermatozoa from the bi-SiaLN and suLeX beads within 30 min. Combinations of three proteasomal inhibitors were tested for their effect on progesterone-induced spermatozoa release, one reversible and cell permeant inhibitor, MG132, and two irreversible inhibitors, clasto-Lactacystin β-Lactone (CLBL) and Epoxomicin (EPOX). MG132 (100 mM) inhibited 71% spermatozoa release (mean) from either immobilized oviduct glycans. Using both MG132 and CLBL (100 mM each) also inhibited 89% of spermatozoa release. Addition of all three inhibitors (10 mM each) inhibited 98% of spermatozoa detachment from both immobilized oviduct glycans. The mechanism by which release is blocked by proteasomal inhibition is under investigation. In summary, inhibiting spermatozoa proteasomes blocked the release of spermatozoa from immobilized oviduct glycans, suggesting that the UPS is a vital part of the mechanism by which spermatozoa move from storage in the isthmus to the site of fertilization. Role of EDD4-like Ubiquitin Ligase 2 (NEDL2) in Porcine Oocyte Fertilization and Early Embryo Development: The objectives of thisstudy were 1) to determine if the NEDL2 protein was imported into pronuclei after in vitro fertilization (IVF) and after sperm co-incubation with a cell-free porcine oocyte extract culture system; and 2) to test the function of NEDL during fertilization and early embryo development by micro-injection of anti-NEDL2 antibody into mature metaphase (MII) oocytes, followed by in vitro fertilization and embryo culture. Co-incubation of demembranated boar spermatozoa with porcine oocyte extract for 24 h triggered sperm decondensation and paternal pronucleus formation characterized by the presence of de novo constituted nuclear envelope with nuclear pore complex. NEDL2 protein was present both inside of pronucleus, a pattern similar to that observed in the IVF zygotes. NEDL2 was also present on the nuclear envelope of the oocyte extract-exposed sperm nuclei, which was different from the buffer-exposed control group and also different from NEDL2 localization in the post-acrosomal sheath of spermatozoa before oocyte extract treatment. Microinjection of anti-NEDL2 antibody into mature MII oocytes before IVF did not affect fertilization rate at 21 h post-fertilization , embryo cleavage at 48 h , or day 7 blastocyst formation. However, anti-NEDL2 antibody decreased the number of supernumerary pronuclei formed in polyspermic-fertilized oocytes at 21 h after IVF, increased the number of oocytes fertilized but lacking paternal pronucleus, and accelerated the formation of nuclear precursor bodies at 6h post fertilization. Taken together, NEDL2 appears to play a role in zygotic development, particularly during sperm nucleus decondition and paternal pronucleus formation.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2017
Citation:
Oko R., Aarabi M., Mao J, Balakier H, Sutovsky P (2017) Sperm specific WW-domain binding proteins. In: The Sperm Cell: Production, Maturation, Fertilization, Regeneration. Second Edition, DeJonge C, Barratt C, Eds., Cambridge University Press, Cambridge, UK, pages 157-176.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Kerns K, Morales P, Sutovsky P (2016) Regulation of sperm capacitation by the 26S proteasome: An emerging new paradigm in spermatology. Biol. Reprod., 94:1-9
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Yin Y, Liu L, Lin C, Veith GM, Wang C, Sutovsky P, Zhou P, Ma L (2016) Cullin-Family Ubiquitin Ligase CUL4B is Required for Mouse Spermatogenesis. J. Bol. Chem., 291(13):6923-35
- Type:
Journal Articles
Status:
Under Review
Year Published:
2017
Citation:
Karl Kerns, Michal Zigo, Miriam Sutovsky, Peter Sutovsky (2017) Unique Zinc Signature of Mammalian Spermatozoa. Nature Communications, under review.
- Type:
Book Chapters
Status:
Published
Year Published:
2017
Citation:
Nevoral J. Sutovsky P, (2017) Epigenome modification and ubiquitin-dependent proteolysis during pronuclear development of the mammalian zygote: Animal models to study pronuclear development. In: Animal Models and Human Reproduction. Schatten H, Constantinescu GM, Editors, Wiley-Blackwell, pages 435-466.
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Progress 04/01/15 to 03/31/16
Outputs
Target Audience:Scientists, graduate students, veterinarians, livestock producers, general audiences. Changes/Problems:No major problems were encountered and no major changes have been made to the experimental plan or timeline. What opportunities for training and professional development has the project provided?One graduatestudent and one postdoc participated in this research in the Sutovsky laboratory. Furthermore, a research assistant professor in Sutovsky laboratory, who is not funded from this project made significant contributions to itand benefited for his training and progress towards promotion to research associate professor. Several undergraduate stundents on of them taking an undergrdauate research for three credit hours also had an opportunity to participate in experiemnts described above.Finally, work on ubiquitin-dependent zygotic histone modifications has been advanced through an international collaboration with a junior investigator in Czech republic who previously trained in Sutovsky laboratory. No USDA funds were used outside of Sutovsky laboratory. How have the results been disseminated to communities of interest?So far, one book chapters and three papers have been published with ealry data collected in this project. Four abstracts lisitng this project funding have been submitted for the upcoming annual meeting of the Society of the Study of Reproduction. PI used data from this project in his lectures in USA, Chile,Brazil, Czech republic and Slovakia. What do you plan to do during the next reporting period to accomplish the goals?Objective #1 will continue focusing on the isolation, validation and characterization of sperm proteins that interact with 26S proteasome during capacitation. In addition to completing theexperiments described above, Objective #2 will focus on the functional studies of zygotic ubiquitin tail fusion protein UBA52, as outlined in the original proposal for this project. Recently, we were able to use CRISPR/Cas9 system to obliterate UBA52 in the zygote, a genomic modification that efficiently suppressed blastocyst formation,indicating that UBA52 has an a esential role in ealry development.
Impacts
What was accomplished under these goals?
OBJECTIVE #1 - Pilot studies on the involvement of ubiquitin-proteasome system (UPS) in mammalian sperm capacitation.The UPS has been implicated in theevent of sperm capacitation, responsible for the remodeling of sperm plasma membrane and acrosome, necessary for sperm fertilizing ability. Present trialinvestigates the changes in proteasome compartmentalization, subunit composition and activity during in vitro capacitation of fresh boar spermatozoa. Parallel and sequential treatments of ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting conditions were used to isolate putative sperm proteasome-associated sperm proteins in a compartment-specific manner.Treated spermatozoa were screened with state of the art imaged based flow cytometer, (FlowSight®, Amnis-Millipore) to asses changes in acrosome integrity and proteasomal 20S core subunit re-distribution and labeling intensity changes. Resultant protein fractions were screened by Western blotting for posttranslational modifications of i) 19S regulatory and 20S core proteasomal subunits, and ii) proteasomal substrate/interactor candidates known to co-purify with sperm proteasomes.Lactadherin MFGE8 co-purified with sperm proteasomes,and was processed during the course of capacitation, resulting in two post-capacitation isoformsincluding acapacitation-specific 35 kDa isoform. Furthermore, proteasomal 19S regulatory particle subunit PSMD4 of 58 kDa was less abundant in capacitated spermatozoa, and a unique PSMD4 band of 74 kDa was detected in both ejaculated and capacitated spermatozoa extracted with TrX-100. Image-based flow cytometry studies of sperm capacitation under proteasome permissive vs. inhibiting conditions revealed an increase of the fluorescence intensity of 20S core subunits in the capacitated spermatozoa. These preliminary results support the proposed role of of UPS in the sperm capacitation. OBJECTIVE #1 - Capacitation-induced Changes of Candidate Proteasomal Interactors ADAM5 and NEDL2, Measured by Image-based Flow Cytometry in Porcine Spermatozoa.Our goalis to establish therole of UPS in the acquisition of the spermatozoa's ability to fertilize oocytes, a property conveyed by sperm capacitation within the female reproductive system. The 26S proteasome, a multi-subunit ubiquitin-specific protease is highly enriched in thesperm acrosome, but present in all parts of the sperm head and flagellum. We investigated two candidate proteasome-interacting sperm proteins,implicated in sperm capacitation. The WW-domain containing NEDD4-like ubiquitin ligase 2 (NEDL2/HECW2) catalyzes covalent ligation of ubiquitin to internal Lys-residues of substrate proteins and may interact with WW-domain binding sperm proteins such as PAWP. A-disintegrin-and-metalloproteinase domain proteins ADAM5 and ADAM20-like are expressed in testis and may form multi- protein complexes with each other and with other ADAM-family members in spermatozoa. Mouse knockout studies have shown the deletion of Adam1a, Adam2, and Adam3 genes resulted in considerably decreased fertility.Sperm proteasomes co-purified with ADAM5 and ADAM20-like, which plays a role in one of the aforementioned ADAM complexes. We detected both ADAM5 and NEDL2 proteins by Western blotting and localized them in boar spermatids and spermatozoa by immunofluorescence. Using FlowSight image-based flow cytometer, we observed a decrease in fluorescence intensity of both ADAM5 and NEDL2 in the sperm head post-acrosomal sheath after in vitro capacitation in. The number of spermatozoa with pre-capacitation pattern of ADAM5 labeling decreased by 32% and the number of spermatozoa with pre-capacitation pattern of NEDL2 labeling decreased by 31%. The pre-capacitation labeling patterns and fluorescence intensities of ADAM5 and NEDL2 wereretained when pharmaceutical inhibitors of the 26S proteasome and/or the proteasome-presenting Valosin-containing Protein (VCP) were administered during capacitation.During sperm capacitation, the UPS may facilitatethe remodeling of the post-acrosomal sperm head region in preparation for sperm-oolemma fusion and oocyte activation/sperm factor release during fertilization. OBJECTIVE #2 - Examination of the NEDD4-like ubiquitin ligase 2 (NEDL2) in porcine spermatozoa, oocytes and preimplantation embryos. The UPS plays diverse regulatory and homeostatic roles in mammalian reproduction. We investigated the content and distribution of NEDL2, a HECT-type ubiquitin ligase that contains two WW domains/protein-protein interaction modules in porcine spermatozoa, oocytes zygotes and early preimplantation embryos, as well as in somatic cell controls. NEDL2 protein was detected in the cumulus-oocyte complexes (COCs) and oocytes were used for in vitro fertilization to produce embryos.NEDL2 labeling intensity was 10x higher in post maturation cumulus cells than before IVM.The NEDL2 protein was alsopresent in the post-acrosomal sheath of spermatozoa, in distinct foci in maternal and paternal pronuclei of zygotes, in the blastomere nuclei of all stage embryos and in the nuclei of cumulus and fibroblast cells. In blastocysts, the labelling intensity of NEDL2 was stronger in the inner cell mass than in trophoblast. A single band of 52 kDa was detected by WB in MII oocytes, and early preimplantation embryos from zygote to blastocyst.Somatic cells, displayed a major high mass band of ~142 kDa, but they lacked the 52 kDa band detected in gametes. To determine if NEDL2 protein may be (auto)ubiquitinated, the recombinant ubiquitin-binding UBA domain protein SQSTM1/p62, was used to pull down ubiquitinated proteins from ejaculated sperm extracts. The SQSTM1 bound protein fraction was resolved by 1-D PAGE and screened for NEDL2. Presence of the 129 kDa isoform in this fraction might result from the ubiquitination of lower mass NEDL2 species. In summary, there were multiple isoforms of NEDL2 in porcine gametes which could havearisen from alternative splicing, self-ubiquitination and/or processing by a protease. OBJECTIVE #2 - Modulation of histone H3 in porcine zygotes by BML-278, a specific SIRT1 activator. The emerging role of UPS in the regulation of epigenetic inheritanceimplies the participation ofthe sirtuin family of histone deacetylases, which are present in porcine oocytes and zygotes. The histone methylation benefits early embryos by limiting DNA fragmentation.Resveratrol, a polyphenols from red grape skinsimproves embryonic development in vitro and isthought to activate sirtuins all seven know sirtuinsSIRT1 - 7. Among them, SIRT1 is a potent factor improving early embryonic development, affecting both histones and non-histone targets. Lysine residue K9 of histone H3 (H3K9) is one of the nuclear substrates of SIRT1, which therefore modulates post-translational modifications of H3 such as acetylation and methylation. Activation of SIRT1 thus offers the means to modify the histone code in zygotic pronuclei, favoring H3K9 methylation, potentially improving embryonic development in vitro.We hypothesized that BML-278, a narrowly specific activator of SIRT1, will have such an effect and will surpass the benfit of resveratrol, which targets multiple sirtuins simultaneously. Hence, in vitro matured porcine oocytes were fertilized in mTBM, followed by 22hr cultivation of presumed zygotes in PZM3with/without resveratrol or BML-278. Epitope/modification specific anti-histone antibodies showed decreased H3K9ac accompanied by increased methylation of the same residue after resveratrol. This phenomenon was mirrored and amplified by SIRT1-specific BML-278 treatment which showed significantly higher level of methylation on H3K9 compared with resveratrol. Methylated H3K9 accompanies heterochromatin establishment and DNA stabilization in the zygote, both of which benefit the establishment of the pronuclear histone code. Further studies are under way to elucidatee the mechanism of BML-278 during embryonic development and blastocyst formation.
Publications
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2016
Citation:
Oko R., Aarabi M., Mao J, Balakier H, Sutovsky P (2016) Sperm specific WW-domain binding proteins. In: The Sperm Cell: Production, Maturation, Fertilization, Regeneration. Second Edition, DeJonge C, Barrat C, Eds., Cambridge University Press, Cambridge, UK, In Press.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Sutovsky P (2015) New approaches to boar semen evaluation, processing and improvement. Reprod. Dom. Anim. 50 Suppl. 2:11-19.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Lin Y, Xu C, Sutovsky P, Wu D, Che L-Q, Fang Z-F, Xu S-Y, Ren B, Dong H-J (2015)
Impact of intrauterine growth restriction on long-term fertility in male pig model. Reprod. Fert. Dev., doi: 10.1071/RD15130
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Nevoral J, Sutovsky P, Zamostna K, Petelak A, Hoskova K, Zalmanova T, Prokesova S, Jelinkova P, Marynikova V, Petr J (2015) SIRT1 as a key factor for histone code establishment in early embryo, from a perspective of assisted reproduction. Slovak J. Anim. Sci. 48:145-158.
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