Progress 07/01/18 to 06/30/23
Outputs
Target Audience:Reproductive Biologists Changes/Problems:Problems with the in vitro testis tissue culture system have persisted in 2022-2023, but success with the ANDY mouse model has proportionally increased during that time frame. What opportunities for training and professional development has the project provided?2018-2023 1. The project allowed for the development and publications of three book chapters by the PI. 2. Altogether, 6 graduate students, 5 undergraduate students, and 2 veterinary professional students were trained using resources afforded by this project. One-on-one training of these students by the PI included research methodology, such as specific wet lab methods, animal handling and rodent husbandry, literature research and management, statistics, science communication and presentation techniques. Personal instruction in the laboratory was supplemented through weekly group meetings and journal clubs. How have the results been disseminated to communities of interest?2018-2023 Book chapters (*corresponding author): 1. Meyer-Ficca ML*, Meyer RG*, Kirkland JB (2022). Niacin. In: Modern Nutrition in Health and Disease (13th ed.), publisher: J.B. Lippincott; *Co-first authors. Invited contribution; in press. 2. Meyer-Ficca, ML*, Meyer RG (2019). Epigenetic Changes in the Paternal Germline; Section II. in Transgenerational Epigenetics, 2nd edition, edited by Trygve Tollefsbol, ISBN-13: 978-0128163634. Invited contribution. 3. Meyer-Ficca ML,Meyer RG * (2018). Circadian Clock, Epigenetic Regulators (Sirtuins) and Metabolism; (2018) in Encyclopedia of Endocrine Diseases, 2nd edition, edited by Ilpo Huhtaniemi, ISBN: 9780128121993. Invited contribution. Research papers (students underlined): 1. Feuz MB, Meyer-Ficca ML, Meyer RG(2023). Beyond Pellagra-Research Models and Strategies Addressing the Enduring Clinical Relevance of NAD Deficiency in Aging and Disease. Cells 12(3):500. PMID: 3676684, PMCID: PMC9913999; DOI: 10.3390/cells 12030500. 2. Zeidler JD, Chini CCS, Kanamori KS, Kashyap S, Espindola-Netto JM, Thompson K, Warner G, Cabral FS, Peclat TR, Gomez LS, Lopez SA, Wandersee MK, Schoon RA, Beckedorff F, Reid JM, Brachs S, Meyer RG, Meyer-Ficca ML, Chini EN (2022). Endogenous metabolism in endothelial and immune cells generates most of the tissue vitamin B3 (nicotinamide). iScience25(11):105431. PMID: 36388973, PMCID: PMC9646960; doi: 10.1016/j.isci.2022.105431. 3. Meyer-Ficca ML, Zwerdling AE, Swanson CA, Tucker AG, Lopez SA, Wandersee MK, Warner GM, Thompson KL, Chini CCS, Chen H, Chini EN, Meyer RG* (2022). Low Testicular NAD+ Levels are Associated With a Decline of Spermatogenesis in Transgenic ANDY and Aging Mice. Frontiers Endocrinol (Lausanne) 13: 896365. PMID: 35600581, PMCID: PMC9120959. DOI: 10.3389/fendo.2022.896356. 4. Meyer RG, Meyer-Ficca ML (2021). NAD Metabolism in Male Reproductive Aging. Adv Geriatr Med Res 3(1):e210005. doi: 10.20900/agmr20210005. Epub 2021 Jan 12. PubMed PMID: 33554222. Presentations at national and international meetings. The following symbols in the author list indicate: * corresponding author, underlined font: undergraduate student author, underlined italic font: veterinary or graduate students. 1. Eggleston M, Zwerdling AE, Lopez SA, Wandersee MK, Meyer-Ficca ML, Meyer RG(2022). Low NAD+ Levels and Associated Metabolic Changes as Potential Factors of Age-Dependent Male Fertility Decline. 47th Annual Meeting of the American Society of Andrology (ASA) and North American Testis Workshop, San Diego, CA. 2. Zwerdling AE*, Meyer-Ficca ML, Park EY, Lopez SA, Tucker AG, Wandersee MK, Warner GM, Thompson KL, Chini CC, Chen H, Chini EN, Meyer RG (2022). Altered testicular glutathione metabolism in mice with low NAD+.47th Annual Meeting of the American Society of Andrology (ASA) and North American Testis Workshop, San Diego, CA. 3. Meyer-Ficca ML, Park EY, Lopez SA, Wandersee MK, Meyer RG(2022). The ANDY mouse - a novel murine model to mimic age-dependent NAD decline and metabolic changes. 2022 Society of Toxicology Annual Meeting, San Diego, CA. 4. Zwerdling AE,Wandersee M, Lopez S, Meyer-Ficca ML, Meyer RG (2021). Quantifying the Effect of NAD Deficiency on DNA Damage in Spermatozoa. ADVS Student Research Symposium. Awarded 3rd prize (Undergraduate Posters). 5. Meyer-Ficca ML,Hoskova R, Forbush M, Lopez S, Wandersee M, Zwerdling A, Meyer RG* (2021). Physiological NAD Deficiency as a Potential Cause of Poor Sperm Quality in the Aging Male. 46th Annual Conference of the American Society of Andrology (ASA) (virtual conference). 6. Lopez SA*, Wandersee MK, Meyer RG, Meyer-Ficca ML (2020). Possible mechanisms behind impaired glucose metabolism in niacin- deficient mice.Mountain West Chapter of the Society of Toxicology (MWSOT) 38th Annual Meeting (online due to COVID-19 travel restrictions) 7. Meyer RG, Bochinclonny A, Swanson CA, Lopez SA, Wandersee MK, Meyer-Ficca ML* (2020). Reproductive Impact of Low NAD+ Levels Typical of the Aging Male. 53rd Society for the Study of Reproduction (SSR) Annual Meeting, Ottawa, ON, Canada (online due to COVID-19 travel restrictions). 8. Meyer-Ficca ML, Bochinclonny A, Mannie C, Wandersee MK, Meyer RG*(2020). Vitamin B3 Requirements of the Testis. 45th Annual Conference of the American Society of Andrology (ASA), Philadelphia, PA (online due to COVID-19 travel restrictions). 9. Meyer RG*, Swanson CA, Wandersee MK,AngelF,Lopez SA, Meyer-Ficca ML (2020). Vitamin B3 as a Molecular Sensor for Metabolic Programming of the Sperm Epigenome and Offspring Physiology. 45th Annual Conference of the American Society of Andrology (ASA), Philadelphia, PA (online due to COVID-19 travel restrictions). 10. Meyer-Ficca ML*, Swanson CA, Mannie C, Wandersee MK, Angel F, Meyer RG(2019).Low NAD levels impair male fertility in mice.Mountain West Chapter of the Society of Toxicology (MWSOT) 37th Annual meeting, Fort Collins, CO. 11. Meyer-Ficca ML*, Swanson CA, Mannie C, Wandersee MK, Angel F, Meyer RG (2019). Low NAD levels impair male fertility in mice. FASEB Science Research Conference (SRC), "The NAD+ Metabolism and Signaling Conference", Dublin, Ireland; (recognized with award for an Exceptional Poster Presentation). 12. Alexie Zwerdling, Mirella L. Meyer-Ficca, Aspen Curtis, Abby Tucker, Sierra Lopez, Miles Wandersee, Ralph G. Meyer (2023). Roles of CD38 and Low NAD in the Decline of Sperm DNA integrity in Aging Males. Poster presentation, American Society of Andrology, ASA 48th Annual Conference, Boston, USA. 13. Morgan Feuz, Alexie E. Zwerdling, Laura Welch, Miles Wandersee, Mirella L. Meyer-Ficca, and Ralph G. Meyer (2023). Single-cell RNA Sequencing of Testicular Cells from NAD+-Deficient Mice Reveals Potential Molecular Changes Associated with Impaired Spermatogenesis. Poster presentation, American Society of Andrology, ASA 48th Annual Conference, Boston, USA. Students involved in the project have also presented their work at the annual local ADVS and USU Student Research Symposia in numerous poster and oral presentations. What do you plan to do during the next reporting period to accomplish the goals?This is the final report.
Impacts
What was accomplished under these goals?
The overarching objective of this project has been to identify processes that are involved in the formation of healthy sperm chromatin as a major factor of male fertility in agricultural animals. Independent from the specific hypotheses formulated as steps toward that larger goal, this project has been quite successful in making major progress towards that goal. Initially, it was proposed to use in vitro tissue culture methods as tools to determine how the exchange of histones for protamines, which is essential for high sperm chromatin quality, is regulated and to identify steps in the process that reduce male fertility if they are impaired. However, recurring technical problems with the proposed tissue culture protocols, where data will still have to be reanalyzed for reproducibility, led to the increased use of the ANDY mouse model of low NAD metabolism over the course of the project. That model allowed for the identification of functions of NAD in spermatogenesis, which has been one of the goals of this project. The work funded by this project has also sparked additional student projects centered on elucidating the impact of chromatin remodeling in spermiogenesis on male fertility, that are still ongoing. The funding provided by this project supported training of 13 graduate, undergraduate and veterinary professional students in the laboratory, and publication of 3 book chapters and 4 research papers with student involvement and authorships. In addition, the project provided opportunities for these students to present their data at local and international scientific meetings.
Publications
- Type:
Journal Articles
Status:
Other
Year Published:
2023
Citation:
Refereed Journal Articles
Feuz, M. B., Meyer-Ficca, M. L., & Meyer, R. G. Beyond Pellagra Ongoing Issues and Models of Niacin and NAD Deficiency. Cells.
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Progress 10/01/21 to 09/30/22
Outputs
Target Audience:Reproductive Biologists Changes/Problems:As described in the Accomplishments section, a change in methodology was implemented to further test the hypotheses. What opportunities for training and professional development has the project provided?This project has allowed for individual training of an undergraduate student and two graduate students (MS) in my laboratory. How have the results been disseminated to communities of interest?In 2022, research associated with this project has been disseminated in two peer-reviewed publications that are associated with, and that acknowledge support by, grant UTA01403 (the following symbols in the author list indicate: underlined font: undergraduate student author, underlined italic font: veterinary or graduate students,bold italic font: PI): 1. Zeidler JD, Chini CCS, Kanamori KS, Kashyap S, Espindola-Netto JM, Thompson K, Warner G, Cabral FS, Peclat TR, Gomez LS, Lopez SA, Wandersee MK, Schoon RA, Beckedorff F, Reid JM, Brachs S, Meyer RG, Meyer-Ficca ML, Chini EN (2022). Endogenous metabolism in endothelial and immune cells is the main source of tissue levels of the vitamin B3 nicotinamide. iScience, accepted for publication. 2. Meyer-Ficca ML, Zwerdling AE, Swanson CA, Tucker AG, Lopez SA, Wandersee MK, Warner GM, Thompson KL, Chini CCS, Chen H, Chini EN, Meyer RG* (2022). Low Testicular NAD+ Levels are Associated With a Decline of Spermatogenesis in Transgenic ANDY and Aging Mice. Frontiers Endocrinol (Lausanne) 13: 896365. PMID: 35600581, PMCID: PMC9120959. DOI: 10.3389/fendo.2022.896356. Students also presented posters acknowledging this grant and that contained data generated with support of UTA01403 at international meetings: 1. Eggleston M, Zwerdling AE, Lopez SA, Wandersee MK, Meyer-Ficca ML, Meyer RG(2022). Low NAD+ Levels and Associated Metabolic Changes as Potential Factors of Age-Dependent Male Fertility Decline. 47th Annual Meeting of the American Society of Andrology (ASA) and North American Testis Workshop, San Diego, CA. 2. Zwerdling AE*, Meyer-Ficca ML, Park EY, Lopez SA, Tucker AG, Wandersee MK, Warner GM, Thompson KL, Chini CC, Chen H, Chini EN,Meyer RG (2022). Altered testicular glutathione metabolism in mice with low NAD+.47th Annual Meeting of the American Society of Andrology (ASA) and North American Testis Workshop, San Diego, CA. 3. Meyer-Ficca ML, Park EY, Lopez SA, Wandersee MK, Meyer RG(2022). The ANDY mouse - a novel murine model to mimic age-dependent NAD decline and metabolic changes. 2022 Society of Toxicology Annual Meeting, San Diego, CA. In addition, students also presented three posters acknowledging UTA01403 support at the local 2022 Utah State University Student Research Symposium. What do you plan to do during the next reporting period to accomplish the goals?The next steps will be to reproduce certain of data sets and finish preparation of an additional manuscript for submission in 2023.
Impacts
What was accomplished under these goals?
This project set out to elucidate developmental processes during spermatid nuclear elongation in postmeiotic germ cell development. Specific Aim 1 has been to test the hypothesis that endogenous DNA strand breaks initiate the histone-to-protamine exchange in spermiogenesis. Specific Aim 2 has been to test the hypothesis that acetylation of H4 histones in position K16 (H4K16ac) is essential for replacement of histones by protamines in developing sperm. If histones are not sufficiently replaced by protamines in sperm, these sperm have normal motility and fertilization capability, but subsequent embryonic development is compromised. We hypothesize that histone H4 hyperacetylation, including in the lysine residue in position 16 of the protein (H4K16ac), is essential for the histone-to-protamine exchange, and that hyperacetylation occurs in response to natural DNA strand breaks created by the cell to relax the DNA, while it binds to protamines. Last year, we cultured mouse testis tissue in vitro, and used topoisomerase II (TOP2) inhibitors to interfere with he known generation of a portion of the natural DNA breaks by this enzyme, which increased H4K16 acetylation, as expected. However, results suggest that testis tissue culture only allowed for investigation of a limited 24 hour time window, so that it remained unclear to what extent elevated H4K16ac increased histone removal as predicted. In addition, using inhibitors of enzymes further downstream of the DNA damage repair (DDR) signaling pathway this year, to further dissect the mechanisms of histone removal, did not yield additional insights, due to culture condition limitations, which presumably increased oxidative stress and DNA damage over time. We therefore used intact mice to test the hypothesis that poly(ADP-ribose) (PAR) metabolism mediated by the NAD-dependent enzymes PARP1 and PARP2 is a crucial step in the initiation of DDR signaling by inducing NAD deficiency in male mice. Lack of NAD in these mice resulted in low testicular NAD levels, lowered PAR synthesis and in lower germ cell H4 hyperacetylation. Sperm from these mice showed elevated frequencies of DNA strand breaks in sperm as determined by a flow cytometry-based sperm TUNEL assay we developed this year. A manuscript reporting these data is in preparation. Our investigations also revealed that long-term NAD deficiency results in complete cessation of spermatogenesis in mice due to inhibition of NAD-dependent testicular aldehyde dehydrogenases (ALDH). These enzymes are essential for the synthesis of retinoic acid, which is an essential regulator of spermatogonial proliferation and differentiation, from retinol (vitamin A).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Meyer-Ficca, M. L., Zwerdling, A. E., Swanson, C. A., Tucker, A. G., Lopez, S. A., Wandersee, M. K., Warner, G. M., Thompson, K. L., Chini, C. C., Haolin, C., Chini, E. N., & Meyer, R. (2022, May 06). Low Testicular NAD+ Levels Cause a Decline of Spermatogenesis in Transgenic ANDY and Aging Mice. Frontiers in Endocrinology, 6;13:896356(6;13:896356).
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Zeidler, J. D., Chini CCS, Kanamori, K. S., Kashyap, S., Espindola-Netto, J. M., Thompson, K., Warner, G., Cabral, F. S., Peclat, T. R., Gomez, L. S., Lopez, S. A., Wandersee, M. K., Schoon, R. A., Reid, K., Menzies, K., Beckedorff, F., Reid, J. M., Brachs, S., Meyer, R., Meyer-Ficca, M., & Chini, E. N. (2022, October 23). Endogenous metabolism in endothelial and immune cells generates most of the tissue vitamin B3 (nicotinamide). iScience, 25(11), 105431.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Zhang, H., Yu, P., Tomar, V. S., Chen, X., Atherton, M. J., Lu, Z., Zhang, H.-G., Li, S., Ortiz, A., Gui, J., Leu, N. A., Yan, F., Blanco, A., Meyer-Ficca, M., Meyer, R., Beiting, D. P., Li, J., Nunez-Cruz, S., O'Connor, R. S., Johnson, L. R., Minn, A. J., George, S. S., Koumenis, C., Diehl, J. A., Milone, M. C., Zheng, H., & Fuchs, S. Y. (2022, July 01). Targeting PARP11 to avert immunosuppression and improve CAR T therapy in solid tumors. Nature cancer, 3(7), 808-820.
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Progress 10/01/20 to 09/30/21
Outputs
Target Audience:Reproductive Biologists Changes/Problems:Covid-19 has caused problems with hiring and retaining student researchers, as well as logistical problems with procuring even basic reagents. We will continue pursuing the specific aims as stated in the grant, using mouse tissues. If fresh and live equine testis tissues should become available, we will perform assays proven in mice using horse tissue. What opportunities for training and professional development has the project provided?Renata Hoskova, the graduate student who was supported by this grant, was trained and mentored until the end of August in the lab in all aspects of the project. She has switched from her PhD track in our group to another research program at USU to pursue a future career in IVF and Assisted Reproductive Techniques in September 2021, and will no longer be available to work on this project in the future. Undergraduate students have been trained to take over experimentation in the project. One-on-one training of these students by the PI included research methodology, such as specific wet lab methods, animal handling and rodent husbandry, literature research and management, statistics, science communication and presentation techniques. Personal instruction in the laboratory was supplemented through weekly group meetings and journal clubs. One of the most outstanding students has been Alexie Zwerdling, who has become an expert in sperm chromatin assessment techniques, graduated with the B.S. in summer 2021. She has been accepted into the USU Master of Public Health program in summer 0f 2021, where she is currently pursuing her MPH degree. She has been continuing her research in our group. Her exposure to laboratory research associated with the current project has confirmed her interest in biomedical research and has sparked her desired to work towards a health-related professional MPH degree. How have the results been disseminated to communities of interest?Results of the research activities supported by this UAES project were disseminated through oral or poster presentations at the following venues, where all presentations credit support by UAES project UTA01403: Sierra Lopez (undergraduate student, now accepted into USU Veterinary Medical Program (WIMU)) Sierra Lopez, Miles Wandersee, Alexie Zwerdling, Mirella L. Meyer-Ficca, Ralph G. Meyer (2021). "Possible Mechanisms of Impaired glucose metabolism in niacin deficient transgenic mice". Oral presentation at the Utah State University Student Research Symposium, April 2021. Sierra Lopez, Miles Wandersee,Courtney Isom,Audrey Lidgard, Renata Hoskova, Ralph G. Meyer, Mirella Meyer-Ficca (2021). "Possible Mechanisms behind Impaired Glucose Metabolism in Niacin-deficient Mice". Poster and Flash Talk at the ADVS Student Research Symposium, Utah State University, August 2021.Sierra won the 2nd place in the Veterinary Student Division for her presentation. Lexie Zwerdling (undergraduate student, now accepted into USU Master in Public Health (MPH) program) Alexie Zwerdling, Miles Wandersee, Sierra Lopez, Mirella L. Meyer-Ficca, Ralph G. Meyer (2021). "Detecting DNA Oxidation in Sperm". Poster and Flash Talk at the ADVS Student Research Symposium, Utah State University, August 2021.Alexie won the 3rd place in the Undergraduate Division for her presentation. Audrey Lidgard (undergraduate student) Audrey Lidgard, Micah Forbush, Sierra Lopez, Renata Hoskova, Ralph G. Meyer, Mirella Meyer-Ficca (2021). Optimal Concentration of Doxycycline to induce ACMSD Expression in ANDY Mice. Poster presentation at the ADVS Student Research Symposium, Utah State University, August 2021. Audrey won the 1st place in the Undergraduate Poster Division for her presentation. Renata Hoskova (graduate student (Ph.D.)) Renata Hoskova, Ralph G. Meyer (2021). "Roles of NAD+ in Testicular Aging and Epigenetic Sperm Modifications" Oral presentation, Utah State University Student Research Symposium, Graduate Student Division, Utah State University, August 2021 Micah Forbush (graduate student (Ph.D.) Micah Forbush, Miles Wandersee, Renata Hoskova, Alexie Zwerdling, Audrey Lidgard, Ralph G. Meyer, and Mirella Meyer-Ficca (2021). "NAD Requirements for DNA repair during spermatogenesis in mice" Oral presentation, Utah State University Student Research Symposium, Graduate Student Division, Utah State University, August 2021. Micah won the 1st place in the Graduate Student Division for her presentation. Ralph G. Meyer (PI) Mirella Meyer-Ficca, Renata Hoskova, Micah Forbush, Sierra Lopez, Alexie Zwerdling, Miles Wandersee, and Ralph G. Meyer (2021). "Physiological NAD Deficiency as a Potential Cause of Poor Sperm Quality in the Aging Male". Poster presentation, American Society of Andrology, ASA 46th Annual Conference. What do you plan to do during the next reporting period to accomplish the goals?A new graduate student currently being hired will perform tissue culture in vitro assays, picking up where Renata has left off, further analyzing spermatid chromatin remodeling, as outlined in the grant proposal.
Impacts
What was accomplished under these goals?
The main focus of this project is the elucidation of developmental processes during the brief phase of spermatid nuclear elongation in postmeiotic germ cell development in males. Specific Aim 1 has been to test the hyspothesis that endogenous DNA strand breaks initiate the histone-to-protamine exchange in spermiogenesis. Specific Aim 2 has been to test the hypothesis that acetylation of H4 histones in position K16 (H4K16ac) is essential for replacement of histones by protamines in developing sperm. Interference with appropriate sperm chromatin development leads to the formation of sperm capable of normal motility and fertilization capacity, but subsequent embryonic development is compromised by DNA strand breaks and genetic, as well as genomic mutations resulting from faulty DNA repair in the zygote. During this developmental step, histones are removed from the developing sperm nucleus and replaced by protamines. This requires hyperacetylation of histone H4 as a key step to remove 97% of all histones from the nucleus. The last acetylation step, and probably the crucial one, is in position K16 of H4 (H4K16ac). We hypothesize that this lysine (K) 16 is acetylated in response to natural DNA strand breaks created by the cell to relax the DNA, while it binds to protamines. We cultured mouse testis freshly isolated from mice, and used topoisomerase II (TOP2) inhibitors to interfere with he known generation of a portion of the natural DNA breaks by this enzyme. two types of TOP2 inhibitors were used: merbarone and etoposide. Both will interfere with TOP2 activity, but at different steps of enzyme activity. Preliminary results of immunoblot analyses showed that both treatments elevated H4K16 acetylation. This result represents an important step towards the accomplishment of both Specific Aims 1 and 2. We are currently using other inhibitors of enzymes involved in the DNA damage repair pathway to further dissect the observed effects. Using whole mice, we also tested the hypothesis that a lack of NAD available for DNA repair in the testis, and therefore the relevant cell type (elongating spermatids) will increase unrepaired DNA strand breaks due to the inhibition of the DNA damage response enzymes PARP1 and PARP2. Our preliminary data indicate that transgenic ANDY mice on a niacin-free diet develop very low testicular NAD values of less than 50% of controls. NAD is the substrate of PARP enzymes, and low levels of NAD could explain a lack of PAR formed by these enzymes, which we documented using immunoblotting, and hence the lack of DNA repair functionality in the spermatids. We are currently following up on these results, which would represent a further step towards accomplishment of the two. specific aims.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Meyer, R. G., & Meyer-Ficca, M. L. (2021, January 12). Metabolism in Male Reproductive Aging. Advances in Geriatric Medicine and Research, 3(1), 2021;3(1):e210005
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Palzer, L., Bader, J. J., Angel, F., Witzel, M., Blaser, S., McNeil, A., Wandersee, M. K., Leu, N. A., Lengner, C. J., Cho, C. E., Welch, K. D., Kirkland, J. B., Meyer, R., & Meyer-Ficca, M. (2018, October 30). Alpha-Amino-Beta-Carboxy-Muconate-Semialdehyde Decarboxylase Controls Dietary Niacin Requirements for NAD+ Synthesis. Cell Reports, 25(5), 1359-1370. 01166,
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2021
Citation:
Presentations
Meyer, R., Hoskova, R. (Author Only), Lopez, S. (Author Only), Wandersee, M. K., Meyer-Ficca, M. (Author Only), Annual Meeting ASA, "PHYSIOLOGICAL NAD DEFICIENCY AS A POTENTIAL CAUSE OF POOR SPERM QUALITY IN THE AGING MALE," American Society of Andrology, Online. (April 2021)
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Progress 10/01/19 to 09/30/20
Outputs
Target Audience:Reproductive Biologists Changes/Problems:There were no significant deviations form the experimental approach, but problems with the availability of equine tissues, and delays caused by an unavailability of students for the envisaged laboratory work. The usage of mouse tissues has been offsetting some of the problems. We will intensify student recruiting efforts throughout the year, and if successful, we do not expect a significant delay in achieving our experimental goals. What opportunities for training and professional development has the project provided?Undergraduate and graduate student training has been an integral component of the proposed project. However, student availability was a major issue during the year of 2020. Two students who had committed to the project in spring 2020 were soon forced to leave campus due to the COVID-19 pandemic. How have the results been disseminated to communities of interest?No manuscripts have been generated from this project, yet. Next year, we will focus on those and on student presentations at local (USU), regional (state of Utah and Intermountain West, as well as national/international events (American Society of Andrology and Society for the Study of Reproduction annual meetings). What do you plan to do during the next reporting period to accomplish the goals?The tissues that were generated in the course of this year will be analyzed in 2021 by students supervised by the PI. The experimental plan initially proposed for this project remains viable, despite the setbacks caused by the COVID-19 pandemic and the lack of suitable horse tissue, which may also be related. We remain confident that we will be able to achieve our set goals in the coming year with the planned analyses.
Impacts
What was accomplished under these goals?
The main focus of this project is the elucidation of developmental processes during the brief phase of spermatid elongation in the testis. This phase of postmeiotic germ cell formation is critical to male fertility, because of the dramatic chromatin remodeling that happens at that time, and which ensures that the male chromosome complement is packaged into a suitable transport form. Interference with appropriate sperm chromatin development leads to the formation of sperm capable of normal motility and fertilization capacity, but subsequent embryonic development is compromised by DNA strand breaks and genetic, as well as genomic mutations resulting from faulty DNA repair in the zygote. One of the key events of sperm chromatin remodeling is the hyperacetylation of histone H4 in different lysine residues (K5, K8, and K12). Adding acetyl groups to H4 reduces the positive charges of the small protein, to weaken its interactions with the negatively charged DNA. The last acetyl group, added to lysine K16 by DNA strand break signaling pathways, is believed to be the most critical one for the removal of nucleosomes from spermatid chromatin, which is a pivotal step to allow the insertion of protamines into the chromatin. While it is known that DNA strand breaks occur naturally during spermatid nuclear elongation, the origin of these DNA strand breaks, that trigger the damage- signaling pathways, is not. Protamine-based chromatin is much more condensed than nucleosomal chromatin. It protects sperm DNA from destructive forces in the environments of the male and female genital tracts. Despite its central importance, H4 K16 acetylation has remained enigmatic, where its control and extent have not been understood. The overarching goal of this project is therefore to determine the steps involved in H4K16 acetylation, and which enzymes are involved in it, using the stallion as a model. Equine spermatogenesis was found to be very similar to human spermatogenesis. However, in the past year, it was not possible to obtain any normal stallion testis tissue for the project, which made it necessary to continue working with mouse tissue. This is a minor issue, because histone acetylation during spermatid elongation is in essence conserved in equine and murine spermatogenesis, which we showed in the course of a previous UAES project. Therefore, a considerable amount of mouse tissue was generated this year, but new experimental results will only be expected after analyses of tissues by students in 2021.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2020
Citation:
Presentations
Lopez, S. (Presenter & Author), Meyer, R. G. (Author Only), Meyer-Ficca, M. L. (Author Only), 38th Annual MWSOT Meeting, "Possible Mechanisms behind Impaired Glucose Metabolism in Niacin-deficient Mice," Mountain West Chapter of the Society of Toxicology (MWSOT), Logan/virtual. (September 18, 2020)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2020
Citation:
Presentations
Lopez, S., Bochinclonny, A. (Author Only), Swanson, C. A. (Author Only), Wandersee, M. (Author Only), Meyer-Ficca, M. L. (Author Only), Meyer, R. G. (Presenter & Author), SSR 52nd Annual Meeting, "Reproductive Impact of Low NAD+ Levels Typical of the Aging Male," Society for the Study of Reproduction, virtual. (July 9, 2020 - July 12, 2020)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2020
Citation:
Presentations
Lopez, S. (Presenter & Author), Meyer-Ficca, M. (Author Only), Meyer, R. (Author Only), 14th annual Utah Conference on Undergraduate Research, "Impaired glucose metabolism in niacin deficient transgenic mice," USU, Logan/virtual. (February 7, 2020)
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:Reproductive biologists and veterinarians Changes/Problems:None anticipated at this point. What opportunities for training and professional development has the project provided?An undergraduate student, Sierra Lopez, is currently being trained in tissue culture techniques in the lab. How have the results been disseminated to communities of interest?Manuscripts are only in preparation at this point. What do you plan to do during the next reporting period to accomplish the goals?Mouse testis tissue will be used to continue dissecting steps of chromatin remodeling in spermiogenesis until stallion tissue becomes available in spring.
Impacts
What was accomplished under these goals?
The overarching objective of this project is to identify processes that are involved in the formation of healthy sperm chromatin as a major factor of male fertility in agricultural animals. The processes under investigation in this project are specifically chromatin remodeling events that are (a) transient, as they happen within about 1-2 days out of the 8.6 days of the murine spermatogenic cycle, and (b) located in postmeiotic cells that are protected by the blood-testis barrier (BTB). Selective enzyme inhibitors that would be useful for functional dissection of the relevant biochemical pathways typically do not cross the BTB, which is one of the main reasons why chromatin remodeling during spermatogenesis has mainly remained enigmatic. In this UAES project, this problem is addressed by the establishment of a protocol for the short-term culture of isolated testis tubules. The establishment and refinement of the culture protocol has therefore been a central focus of the investigations so far. Using murine testes as a model for culturing equine testis material, I spent most of my efforts and funds on mouse per diems, as well as inhibitors and cell culture reagents this year. As outlined in Aim1, experiments on the involvement of topoisomerase 2 beta (TOP2B) in spermatid chromatin condensation have been investigated using the new tissue culture protocol. Merbarone and etoposide, two inhibitors that block different steps of TOP2B enzyme activity, were used to generate frozen and formalin-fixed samples that are currently being evaluated.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2019
Citation:
Meyer-Ficca, M., & Meyer, R. (2019). Epigenetic Changes in the Paternal Germline. Transgenerational Epigenetics, 2nd Edition.
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Progress 07/01/18 to 09/30/18
Outputs
Target Audience:Reproductive biologists and veterinarians Changes/Problems:No problems are anticipated at this point. What opportunities for training and professional development has the project provided?My literature study for this project was basis for two book chapters that were accepted for publication in 2018. How have the results been disseminated to communities of interest?1. Mirella L. Meyer-Ficca, Ralph G. Meyer. Circadian Clock, Epigenetic Regulators and Metabolism. In: Ilpo T. Huhtaniemi (ed.): Encyclopedia of Endocrine Diseases, 2nd. ed. Elsevier. Accepted for publication. 2. Meyer RG, Meyer-Ficca ML: Epigenetic Changes in the Male Germ L; in: T. Tollefsbol (ed.): Transgenerational Epigenetics, 2nd edition, Elsevier, in press, 2018 What do you plan to do during the next reporting period to accomplish the goals?Once a robust tissue culture system has been established using mice (PI), the inhibition of TOP2B using merbarone will be the next milestone. The plan is to engage students in the summer of 2019 to work with the PI on data collection. Leftover tissue from stallion castration surgeries will be used to adapt the tissue culture system for equine tissues as the material becomes available.
Impacts
What was accomplished under these goals?
The overarching objective of this project is to identify processes that are involved in the formation of healthy sperm chromatin as a major factor of male fertility in agricultural animals. Current research in the field of sperm development makes extensive use of rodents as animal models, but our previous work (see section 2) revealed the horse as an untapped, superior model for the investigation of the late steps of spermatogenesis. The late steps in spermiogenesis are arguably the most crucial ones in the development of sperm chromatin but they are still not well understood, in part due to lack of appropriate animal models besides rodents. This project proposes to determine how the exchange of histones for protamines, which is essential for high sperm chromatin quality, is regulated and to identify steps in the process that reduce male fertility if they are impaired. Two hypotheses will be tested in this project: 1. Endogenous DNA strand breaks initiate the histone-to-protamine exchange in spermiogenesis. To test this hypothesis, topoisomerase 2 beta (TOP2B), which is the presumed enzyme that mediates these breaks, will be inhibited using cultured tubules explanted from the animals (stallions). To establish tissue culture conditions, we are currently experimenting with mouse tissue to determine how long we can keep these tubules alive and differentiating. Microdissection of testicular tubule sections are done using a trans-illumination setup, but we have also been breeding a transgenic mouse that expresses an H2B-GFP fusion protein in nuclei of all tissues to help with tubule microdissection. Because histones are eliminated during nuclear elongation, we are currently evaluating whether the disappearance of GFP fluorescence can be followed along the length of the tubule. 2: Acetylation of H4 histones in position K16 (H4K16ac) is essential for replacement of histones by protamines in developing sperm. This hypothesis will be tested later in the project.
Publications
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