Progress 07/01/23 to 06/30/24
Outputs
Target Audience:We have presented our results at universities, conferences, and Bovine artificial insemination facilities as well as reported our findings in scientific journals. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project trained graduate and undergrad students and provided them with the opportunity to perform basic and translational research. How have the results been disseminated to communities of interest?We have presented the result at several conferences and institutions: 2023 September 14: Select Sires Incorporated "Centriolar Biomarkers of Bull Sperm" 2024 January 16: North Carolina Agricultural & Technical State University - "The unexpected biology of reproductive centrioles - from model animals to human diagnostics". 2024 March 7: Utrecht University, Netherlands - "The unexpected biology of reproductive centrioles - from model animals to human diagnostics" What do you plan to do during the next reporting period to accomplish the goals?We plan to continue studying the fate of the centrioles of subfertile bulls in the early embryo (Activities 3), continue analyzing the sperm samples using superresolution microscopy (Activities 4), and analyze the subfertile bulls we identified using live sperm imaging for third head kinking properties (Activities 5).?
Impacts
What was accomplished under these goals?
New biomarkers We have identified Cep135 and CP110 as sperm neck markers and published this study (Turner, Katerina A., et al. "CP110 and CEP135 localize near the proximal and distal centrioles of cattle and human spermatozoa." Micropublication Biology 2023). Sperm Motility One of our adjectives is assessing spermatozoa centrosome function during sperm transport to fertilize the egg. In the past, we studied that after snap freezing and fixing motile sperm and reconstituting sperm behavior from pictures of multiple sperms with distinct had/neck angles (Khanal et al., 2021). To improve this analysis, we have started developing a more efficient method to assess sperm head/neck angles from live-moving sperm. By analyzing bovine spermatozoa swimming near the glass without rolling in 0.5% methylcellulose, We found that The head-neck angle varies with a consistent frequency with a cycle of 110 ± 25 ms (46 beat cycles total), ranging between 174º ± 3º to 192º ± 2º with an amplitude of 18º ± 4º. Using Kappa as a measure of curvature at each point along the tail, we found that the tail curvature correlated with the head-neck angle. Embryonic development One of our adjectives is to assess the spermatozoa centrosome function posterization in the embryo. We recently found that embryonic cells have atypical structures and that they lack many of the structures and proteins of canonical centrioles (Uzbekov et al., 2023). For that, we have started developing markers for embryonic centrosome function. We have analyzed protein markers for various domains of the centriole throughout early embryogenesis, including CP110, CEP135, POC1B, FAM161A, and SAS-6, using g-tubulin, CEP152 as centrosome markers. We found three patterns of staining: 1. SAS-6 is consistently observed in the centrosome at any embryonic stage we examined. 2. CP110 is consistently observed in the centrosome at any embryonic stage but changes its localization morphology in the centrosome at later stages. 3. CEP135, FAM161A, and POC1B are observed starting at various later stages of embryogenesis. These findings suggest that paternal atypical centriolar transition gradually to canonical centrioles during early embryogenesis.?
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Uzbekov, Rustem, and Tomer Avidor-Reiss. "Comment on:The proximal centriole age in spermatozoa is a potential reason for its different fate in the zygote after fertilizationUzbekov and Avidor-Reiss 2024." Open Biology 14.3 (2024): 230458.
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Avidor-Reiss, Tomer. "Renaissance in sperm cytoplasmic contribution to infertility." Journal of Assisted Reproduction and Genetics 41.2 (2024): 293-296.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Avidor-Reiss, Tomer, and Rustem Uzbekov. "Revisiting the mystery of centrioles at the beginning of mammalian embryogenesis." Journal of Assisted Reproduction and Genetics 40.11 (2023): 2539-2543.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Turner, Katerina A., et al. "Abnormal centriolar biomarker ratios correlate with unexplained bull artificial insemination subfertility: a pilot study." Scientific Reports 13.1 (2023): 18338.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Turner, Katerina A., et al. "CP110 and CEP135 localize near the proximal and distal centrioles of cattle and human spermatozoa." Micropublication Biology 2023 (2023).
- Type:
Books
Status:
Published
Year Published:
2023
Citation:
Royfman, Abigail, Sushil Khanal, and Tomer Avidor-Reiss. "Structural Analysis of Sperm Centrioles Using N-STORM." Cilia: Methods and Protocols. New York, NY: Springer US, 2023. 103-119.
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Progress 07/01/22 to 06/30/23
Outputs
Target Audience:We have presented our results at universities and conferences and reported our findings in scientific journals. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project trained graduate and undergrad students and provide them with the opportunity to perform basic and translational research. How have the results been disseminated to communities of interest?We have presented the result at several conferences and institutions: 2022 July 4-7: European Society of Human Reproduction and Embryology (ESHRE) 2022; Milan, Italy 2022 October 6-9: 13th biennial alpha conference (2022) Sevilla, Spain. 2022 December 8-11: mKARM (Key aspects of reproductive medicine), Russia " Sperm atypical centrioles: new functions and role in infertility" 2022 December 22: Assuta Hospital Ramat Hachayal Israel; "Sperm atypical centrioles' novel functions and fertility roles" 2023 April 7: Online GAF Scientific Writing Webinar on "Common Mistakes During Manuscript Prepetition". 2023 Jun 7: University of Missouri Columbia "Sperm atypical centrioles' novel functions and fertility roles What do you plan to do during the next reporting period to accomplish the goals?We plan to test the cut-off values for fertility and subfertile we found in our first set of bull's semen samples using a new set of 10 fertile bull's semen samples, a sterile bulle, and 11 subfertile bulls. We plan to continue the analysis of the sperm samples using superresolution microscopy (Activities 4) and Image-based flow cytometry. We also plan to study the fate of the centrioles of subfertile bulls in the zygote (Activities 3).
Impacts
What was accomplished under these goals?
We investigated the presence of tubulin post-translational modification in cattle and human sperm by studying their colocalization with known centriolar biomarkers, tubulin, and POC1B. As expected, the anti-tubulin and anti-POC1B antibodies labeled two spots in the sperm neck, the distal centriole and proximal centriole, of cattle and humans. Here, we tested a rabbit monoclonal antibody (Thermo Fisher Scientific, MA533079). Similar to previous studies with antibody 6-11B-1 in sperm, we found that MA533079 labeled the neck and throughout the tail of cattle and human sperm. Also, like in previous studies with antibody 6-11B-1, we found that in the neck, MA533079 labeled two spots, the sperm centrioles, colocalizing with POC1B in cattle (98% of cells; N=272; 4 independent stainings) and human sperm (100% of cells, N= 96; 3 independent stainings). Tubulin acetylation staining is weaker in the midpiece axoneme compared to the centrioles in cattle (the ratio of proximal centriole:(proximal centriole + axoneme), 0.58±0.18, N=119 sperm) and human sperm (the ratio of proximal centriole:(proximal centriole + axoneme), 0.54±0.19, N=96 sperm; 3 independent stainings). We found that anti-glutamylated tubulin antibody GT335 labeled the sperm neck consistently and sometimes the tail midpiece or endpiece in humans and consistently in the endpiece in cattle. In the neck, anti-glutamylated Tubulin antibody GT335 labeled two spots, colocalizing with POC1B in cattle (81.5% of 146 cells, 4 independent stainings) and human sperm (73% of N=90 sperm total from 3 men). This staining is enriched in the proximal centriole compared to the midpiece axoneme in cattle (the ratio of proximal centriole:(proximal centriole + axoneme), 0.85 ± 0.21, N=136 sperm) and in humans (the ratio of proximal centriole:(proximal centriole + axoneme), 0.56 ± 0.18, N=90 sperm). We tested two distinct antibodies that label glycylation and found inconsistent labeling. We found that mouse anti-glycylated Tubulin antibody (EMD Millipore, MABS277) diffusely labeled the neck and the tail at the mid-piece and end-piece of cattle sperm (100%, N=19, 3 independent with increasing antibody concentrations) (Fig. 1G). In human sperm, it did not label the sperm at all (0%, N=10, 3 independent stainings). In contrast, the rabbit anti-glycylated tubulin antibody Adipogen, AG-25B-0034-C100 labeled the cattle sperm neck, midpiece, and end piece (94%, N=102, 2 independent experiments). In humans, the anti-rabbit glycylated tubulin antibody Adipogen, AG-25B-0034-C100 labeled the midpiece and distal centriole. Midpiece labeling was observed in 100% of cells, and distal centriole labeling was observed in 93.3% of cells (N=15). Overall glycylation in the atypical centriole varies depending on species and antibody and therefore is questionable. Altogether, our data suggest that acetylation and glutamylation are present in the atypical distal centriole and can be used as sperm centriolar biomarkers in a species-specific manner. Because acetylated tubulin immunostaining was found to be a strong biomarker for subfertility, we examined its labeling pattern using stimulated emission depletion (STED) super-resolution microscopy (lateral resolution of ~30 nm) and four times expansion microscopy in combination with structured illumination microscopy (SIM) (lateral resolution of ~40 nm) or STED (achievable lateral resolution of ~10 nm). All three approaches yielded similar results; acetylated tubulin signals were present in barrel-shaped PC, a fan-like DC, and the Ax. We presume that acetylated tubulin signals correspond to the microtubules. Cryo-electron microscopy of the DC found that all the bovine sperm axonemes microtubules are continues with the DC microtubules [18]. Consistent with that, the acetylated tubulin immunostaining central lines in the DC appeared continuous with Ax staining in 96% of the analyzed sperm. These lines are probably the microtubule central pair of the Ax that extend into the DC. Unexpectedly, however, in most cases, at the junction of the DC and Ax, the acetylated tubulin signal appeared discontinuous in both the left and right sides of the DC (73% of the analyzed sperm). This staining gap suggests that there is a region of reduced acetylation of the outer microtubules at the DC-Ax junction.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Turner, K.A., Kluczynski, D.F., Hefner, R.J., Moussa, R.B., Slogar, J.N., Thekkethottiyil, J.B., Prine, H.D., Crossley, E.R., Flanagan, L.J., LaBoy, M.M., Moran, M.B., Boyd, T.G., Kujawski, B.A., Ruble, K., Pap, J.M., Jaiswal, A., Shah, T.A., Sindhwani, P., Avidor-Reiss, T., 2022b. Tubulin posttranslational modifications modify the atypical spermatozoon centriole. MicroPubl Biol 2022.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Uzbekov, R., Singina, G.N., Shedova, E.N., Banliat, C., Avidor-Reiss, T., Uzbekova, S., 2023. Centrosome Formation in the Bovine Early Embryo. Cells 12, 1335.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Jaiswal, A; Avidor-Reiss, T (2023). IgG antibodies label the spermatozoan centriole of Drosophila melanogaster. microPublication Biology. 10.17912/micropub.biology.000838.
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Progress 07/01/21 to 06/30/22
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project trained graduate and undergrad students and provide them with the opportunity to perform basic and translational research. How have the results been disseminated to communities of interest?We have presented the result in local conferences and in 2021 SSR. What do you plan to do during the next reporting period to accomplish the goals?We plan to test the cut-of values for fertility and subfertile we found in our first set of bull's semen samples using a new set of 11 bull's semen samples. We plan to start the analysis of the sperm samples using superresolution microscopy (Activities 4).
Impacts
What was accomplished under these goals?
By now, we have analyzed the first set of bullies' sperm and performed Activities 1-3. We have analyzed semen samples from 31 bulls that passed rigorous semen analyses and had a sire conception rate (SCR) ranging between -18 and +3. Of these, 25 were fertile bulls (SCR ≥ -3), and six were subfertile (SCR < -3). We separated each semen sample into high-quality and low-quality sperm fractions using differential gradient centrifugation. We used the high-quality sperm from fertile bulls as our reference population and used Fluorescence-based Ratiometric Analysis of sperm Centrioles (FRAC) with four centriole biomarkers (Tubulin, Acetylated Tubulin, POC1B, and FAM161A) to determine centriole quality. FRAC found statistically significant lower centriole quality in fertile bulls' low-quality sperm (P=0.004) as well as subfertile bulls' high-quality sperm (P=0.0001) and low-quality sperm (P=0.0001). While only a few fertile bulls with high-quality sperm had at least one mean FRAC ratio outside of the high-quality sperm distribution range (4/25, 16%), all the high-quality sperm populations of the subfertile sires (6/6, 100%) had at least one mean ratio outside of the high-quality sperm; a highly significantly different (P=0.000004). This diagnostic criterion has a sensitivity of 1 and specificity of 0.88.Using linear regressions of the mean FRAC ratio of PC Acetylated Tubulin (R=-0.566; P=0.00078), we obtained a cut-off FRAC ratio (0.301) that predicted subfertility within our sample group with a sensitivity of 0.83 and a specificity of 1.00. These data suggest that analysis of centriolar biomarker distribution can identify bull subfertility that avoids detection with current methods.?
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Avidor-Reiss, T., L. Achinger, and R. Uzbekov. 2022. The Centriole's Role in Miscarriages. Front Cell Dev Biol. 10:864692
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Turner, K., N. Solanki, H.O. Salouha, and T. Avidor-Reiss. 2022. Atypical Centriolar Composition Correlates with Internal Fertilization in Fish. Cells. 11:758
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