Progress 01/01/24 to 12/31/24
Outputs
Target Audience:The target audience were students, scientists and IVF companies. In 2024, our results and findings were presented at the 21st Annual Gilbert S. Greenwald Symposium on Reproduction and the Animal Science Graduate Student Forum, University of Missouri. Moreover, part of the project's findings was published in "Nature Communications" journal (impact factor= 14.7). Also, a book chapter related to DNA damage repair was published in Advances in Anatomy, Embryology and Cell Biology series (Springer Nature). @font-face { panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic- mso-font-pitch:variable; mso-font-signature:-536870145 1107305727 0 0 415 0;}@font-face { panose-1:2 11 0 4 2 2 2 2 2 4; mso-font-charset:0; mso-generic- mso-font-pitch:variable; mso-font-signature:536871559 3 0 0 415 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin-top:0in; margin-right:0in; margin-bottom:8.0pt; margin-left:0in; line-height:115%; mso-pagination:widow-orphan; ; mso-ascii- mso-ascii-theme-font:minor-latin; mso-fareast- mso-fareast-theme-font:minor-latin; mso-hansi- mso-hansi-theme-font:minor-latin; mso-bidi- mso-bidi-theme-font:minor-bidi; mso-font-kerning:1.0pt; mso-ligatures:standardcontextual;}p.p1, li.p1, div.p1 {mso-style-name:p1; mso-style-unhide:no; mso-margin-top-alt:auto; margin-right:0in; mso-margin-bottom-alt:auto; margin-left:0in; mso-pagination:widow-orphan; ; mso-fareast-}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; mso-ascii- mso-ascii-theme-font:minor-latin; mso-fareast- mso-fareast-theme-font:minor-latin; mso-hansi- mso-hansi-theme-font:minor-latin; mso-bidi- mso-bidi-theme-font:minor-bidi;}.MsoPapDefault {mso-style-type:export-only; margin-bottom:8.0pt; line-height:115%;}div.WordSection1 {page:WordSection1;} p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 10.0px Helvetica; color: #000000} Changes/Problems:We lost the access to the nearest slaughterhouse for obtaining bovine ovaries since 2023. However, we were able to made alternative arrangements to consistently purchase bovine ovaries from an IVF company. However, due to the long transportation distance, the quality of oocytes and embryos were relatively low. This challenge led us to reassess our priorities, postponing some experiments that require very high quality embryos. It is expected to get access to a new slaughterhouse in Missouri in 2025. What opportunities for training and professional development has the project provided?A postdoctoral researcher received a high-quality training and now he became an expert in the field of oocyte and early embryo development. Dr. Ezz presented his findings at several conferences either as a talk or a poster at scientific meetings including the annual meeting of the Greenwald symposium. Dr. Ezz submitted his recent data for the presentation at the annual meeting of Society for Study of Reproduction. We also hired a new PhD student who is learning new techniques and acquired the necessary skills to carry out the experiments. How have the results been disseminated to communities of interest?In 2024, our results and findings were presented at the 21st Annual Gilbert S. Greenwald Symposium on Reproduction and the Animal Science Graduate Student Forum, University of Missouri. Moreover, part of the project's findings was published in "Nature Communications" journal (impact factor= 14.7). Also, a book chapter related to DNA damage repair was published in Advances in Anatomy, Embryology and Cell Biology series (Springer Nature). What do you plan to do during the next reporting period to accomplish the goals?1- Keep working on dissecting the molecular mechanism(s) through which CTSB and CTSL influence embryo developmental competence. 2- Generating CTSB and CTSL knockout embryos to confirm the relationship between cathepsins B/L and embryo development.
Impacts
What was accomplished under these goals?
1- Identify cathepsins (CTSs) that compromise bovine preimplantation embryo quality. Mass Spec and proteomic analysis: Based on bovine preimplantation embryo quality, four groups of bovine embryos were sampled: (1) 2-4 cell stage embryos (slow developing), (2) 6-8 cell stage embryos with good morphology, (3) 6-8 cell stage embryos with poor morphology, and (4) 16-cell stage embryos (fast developing). The embryos from each group were pooled together. Proteins were extracted and separated on a C18 column with a 44-minute gradient and analyzed using the Evosep One LC system connected to a timsTOF Pro2 mass spectrometer in DIA-PASEF mode, employing 16 scans per cycle over an m/z range of 400-1200. Data analyses were conducted using Spectronaut (v19.3) with Uniprot databases. Protein quantification was done based on MaxLFQ with cross-run normalization, and differential abundance testing to identify significant candidates based on stringent statistical thresholds. Importantly, we identified 7 cathepsins that are differentially regulated. In general, identified cathepsins were increased in poor-quality and slow-developing embryos when compared to good-quality and fast developing bovine embryos. Ongoing experiments are being conducted to validate the relationship between these newly identified cathepsins and embryo quality. 2- Determine the underlying molecular mechanisms whereby CTSB compromises preimplantation embryo developmental competence and quality. In the previous reporting periods, we identified CTSL and showed that it correlates with embryo developmental competence. However, the underlying mechanism through which CTSB and CTSL influences embryo developmental competence is unknown. We previously found that inducing DNA damage in early-stage bovine embryos using etoposide resulted in lower CTSL and higher CTSB activities than controls. Moreover, we found that inhibition of CTSL increased DNA damage levels in preimplantation bovine embryos, whereas increasing CTSL or inhibiting CTSB reduced DNA damage levels when compared to control embryos. Importantly, CTSL upregulation or CTSB inhibition decreased DNA damage levels and increased the blastocyst rates in etoposide-treated embryos. In this current reporting period, we revealed the underlying mechanism through which cathepsins regulate DNA damage. We found that autophagy activity is directly correlated with DNA damage levels. Autophagy induction decreased DNA damage levels in bovine embryos, whereas autophagy inhibition increased DNA damage levels. Importantly, we found that cathepsin B inhibition or cathepsin L upregulation improved DNA damage repair in bovine preimplantation embryos by activating autophagy pathway.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Accepted
Year Published:
2024
Citation:
F. Sun, N.N. Ali, D. Londo�o-V�squez, C.A. Simintiras, H. Qiao, M.S. Ortega, Y. Agca, M.M. Takahashi, R.M. Rivera, A.M. Kelleher, P. Sutovsky, A.L. Patterson, A.Z. Balboula*. (2024). Increased DNA damage in full-grown oocytes is correlated with diminished autophagy activation. Nature Communications, 15, 9463.
- Type:
Book Chapters
Status:
Accepted
Year Published:
2024
Citation:
F. Sun, P. Sutovsky, A.L. Patterson, A.Z. Balboula AZ*. (2023). Mechanisms of DNA damage response in mammalian oocytes, Advances in Anatomy, Embryology and Cell Biology series. 238:47-68.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
M. Aboul Ezz, A.Z. Balboula, The role of cathepsin in DNA damage repair, 21st Annual Gilbert S. Greenwald Symposium on Reproduction. November 7-8, 2024
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Progress 01/01/23 to 12/31/23
Outputs
Target Audience:The target audience were students, scientists and IVF companies. In 2023, our results and findings were presented at the 20th Annual Gilbert S. Greenwald Symposium on Reproduction and the Animal Science Graduate Student Forum, University of Missouri. Moreover, part of the project's findings was published in "Cell Proliferation" journal (impact factor= 8.5). Changes/Problems:A major challenge we faced during the reporting period is the loss of access to the slaughterhouse for obtaining bovine ovaries since the last year. However, after 6 months, we were able tomade alternative arrangementsto consistently purchase bovine ovaries. This challenge led us to reassess our priorities, resulting in a reduction in the number of experiments conducted during the reporting period. What opportunities for training and professional development has the project provided?We hired a postdoctoral researcher who is still learning new techniques and acquired the necessary skills to carry out all the experiments. Dr. Ezz presented his findings at several conferences either as a talk or a poster including the annual meeting of the Society for Study of Reproduction. Importantly, he received several awards while presenting the findings of this project. We also hired a MS student who is learning new techniques and acquired the necessary skills to carry out the experiments. How have the results been disseminated to communities of interest?In 2023, our results and findings were presented at the 20th Annual Gilbert S. Greenwald Symposium on Reproduction and the Animal Science Graduate Student Forum, University of Missouri. Moreover, part of the project's findings was published in "Cell Proliferation" journal (impact factor= 8.5). What do you plan to do during the next reporting period to accomplish the goals?1- Dissecting the molecular mechanism(s), in detail, through which CTSB compromises embryo developmental competence. 2- Dissecting the molecular mechanism(s), in detail, through which CTSL influences embryo developmental competence in detail.
Impacts
What was accomplished under these goals?
1- Identify cathepsins (CTSs) that affect bovine preimplantation embryo quality. We previously demonstrated that decreasing the activity of lysosomal cathepsin B (CTSB) improves bovine oocyte and preimplantation embryo developmental competence. Whether other cathepsins, in addition to CTSB, regulateembryo developmental competence was unknown. We successfully identified lysosomal cathepsin L (CTSL) as a crucial regulator for oocyte and embryo developmental competence and quality. We employed a specific CTSL detection assay in live cells to show that CTSL activity correlates with early embryo development. Inhibiting CTSL activity during early embryo development significantly impaired embryo developmental competence as evidenced by lower cleavage, blastocyst, and hatched blastocyst rates in cattle. Moreover, enhancing CTSL activity, using recombinant CTSL (rCTSL), during oocyte maturation or early embryo development significantly improved oocyte and embryo developmental competence. Importantly, recombinant CTSL supplementation during oocyte maturation and early embryo development significantly rescued, at least in part, the developmental competence of heat-shocked oocytes/embryos which are notoriously known for reduced quality. Altogether, our findings provide novel evidence that CTSL plays a pivotal role in regulating early embryonic development, offering a promising approach to improve the efficiency of assisted reproductive technologies and to reduce the incidence of early embryonic loss. 2- Determine the underlying molecular mechanisms whereby CTSB compromises preimplantation embryo developmental competence and quality. We previously demonstrated that CTSB is inversely correlated with embryo developmental competence. In major goal 1, we identified CTSL and showed that it correlates with embryo developmental competence. However, the underlying mechanism(s) through which CTSB and CTSL influences embryo developmental competence is unknown. DNA damage is a serious problem affecting all cell types. To restore the cellular homeostasis, cells activate DNA damage repair (DDR) mechanisms. How preimplantation embryos sense and repair DNA damage is largely unknown. We found that inducing DNA damage in early-stage embryos using etoposide resulted in lower CTSL and higher CTSB activities than controls. Moreover, we found that inhibition of CTSL increased DNA damage levels in preimplantation bovine embryos, whereas increasing CTSL or inhibiting CTSB reduced DNA damage levels when compared to control embryos. Importantly, CTSL upregulation or CTSB inhibition decreased DNA damage levels and increased the blastocyst rates in etoposide-treated embryos. In conclusion, our data strongly indicate that lysosomal cathepsins influence embryo developmental competence, at least in part, through regulating the efficiency of DDR in preimplantation embryos.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Ezz MA, Takahashi M, Rivera RM, Balboula AZ. (2023). Cathepsin L regulates oocyte meiosis and early embryonic development, Cell Proliferation, e13526. (selected for cover art issue)
- Type:
Book Chapters
Status:
Accepted
Year Published:
2024
Citation:
Sun F, Sutovsky P, Patterson AL, Balboula AZ. (2023). Mechanisms of DNA damage response in mammalian oocytes, Advances in Anatomy, Embryology and Cell Biology series. (In Press, Book Chapter)
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Progress 01/01/22 to 12/31/22
Outputs
Target Audience: The target audience werestudents,scientists and IVF companies. Our results and findings were shared with students and faculty members through departmental and collegeseminars and conferences. The results were presented at (1) the annual meeting for the Society for the Studyof Reproduction (July 2022), (2) the19th Annual Gilbert S. Greenwald Symposium on Reproduction (October 13-14, 2022),(3)College of Agriculture, Food and Natural ResourcesResearch Symposium (2022) and (4) at the2023 Animal Reproduction ProjectDirector's (PD)meeting. Moreover, our findings were submitted for publication in top-tier journal (currently under review in Cell Proliferation journal). Changes/Problems:A major challenge that faced us during the reporting period is that we no longer have access to the slaghterhouse to get bovine ovaries since January 2023. However, we already made arrangments to purchase bovine ovaries starting this May. What opportunities for training and professional development has the project provided?We hired a postdoctoral researcher (M.A. Ezz) who learned a lot of techniques and acquired the necessary skills to carry out all the experiments. Dr. Ezz presented his findings at several conferences either as a talk or a poster including the annual meeting of the Society for Study of Reproduction. Importantly, he received the Greenwald symposium travel award as well as the first place prize for presenting his results at the first College of Agriculture, Nultrition and Food Resource Research Conference. How have the results been disseminated to communities of interest?Our results and findings werepresented at (1) the annual meeting for the Society for the Studyof Reproduction (July 2022), (2) the19th Annual Gilbert S. Greenwald Symposium on Reproduction (October 13-14, 2022),(3)College of Agriculture, Food and Natural ResourcesResearch Symposium (2022) and (4) at the2023 Animal Reproduction ProjectDirector's (PD)meeting. Moreover, our findings were submitted for publication in top-tier journal (currently under review in Cell Proliferation journal). What do you plan to do during the next reporting period to accomplish the goals?1-Studying the mechanism(s) through which Cathepsin L improves bovineoocyte and embryo development. 2- Studying the mechanism(s) through which Cathepsin B compromises bovine oocyte and embryo development. 3- Identifying other cathepsins that influence bovine oocyte and embryo development.
Impacts
What was accomplished under these goals?
1- Identify cathepsins (CTSs) that affect bovine preimplantation embryo quality. Early embryonic loss, caused by reduced embryo developmental competence, is the major cause of subfertility inanimals. This embryo developmental competence is determined during oocyte maturation and the first embryo divisions.We successfully identified cathepsin L (CTSL) as a crucial regulator for oocyte and embryo developmental competence and quality.Cathepsin L, a lysosomal cysteine protease, is involved in regulating cell cycle progression, proliferation, and invasion of different cell types.However, its role in oocyte and embryo development was unknown. We employed a specific CTSL detection assay in live cells to show that CTSL activity correlates with meiotic progression and early embryo development.InhibitingCTSL activity during oocyte maturation or early embryo development significantly impaired oocyte and embryo developmental competence as evidenced by lower cleavage, blastocyst, and hatched blastocyst rates in cattle. Moreover, enhancing CTSL activity, using recombinant CTSL (rCTSL), during oocyte maturation or early embryo development significantly improved oocyte and embryo developmental competence.Importantly, recombinant CTSL supplementation during oocyte maturation and early embryo development significantly rescued, at least in part, the developmental competence of heat-shocked oocytes/embryos which are notoriously known for reduced quality. Altogether, our findings provide novel evidence that CTSL plays a pivotal role in regulating oocyte meiosis and early embryonic development, offering a promising approach to improve the efficiency of assisted reproductive technologies and to reduce the incidence of early embryonic loss.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
M.A. Ezz, R.M. Rivera, A.Z. Balboula. Cathepsin L is crucial for oocyte maturation, 19th Annual Gilbert S. Greenwald Symposium on Reproduction. October 13-14, 2022.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
M.A. Ezz, R.M. Rivera, A.Z. Balboula. Characterization of Cathepsin L role in bovine oocyte maturation and preimplantation embryo development. 55th annual Meeting of the society for the study of reproduction, Spokane, Washington, USA, 26-29 July 2022.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
M.A. Ezz, R.M. Rivera, A.Z. Balboula. The role of Cathepsin L in bovine preimplantation embryo development. 1st College of Agriculture, Food and Natural Resources Research Conference, Columbia, MO, USA, 12-13 October 2022.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
M.A. Ezz, M. Takahashi, R.M. Rivera, A.Z. Balboula. Cathepsin L regulates oocyte meiosis and early embryonic development (Cell Proliferation).
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