Progress 03/01/18 to 02/28/23
Outputs
Target Audience:Animal reprodictive physiologists, animal breeders, AI industry, cattle (beef and dairy) and other farm animal producers, as well as biologists in the fields of mammalian spermatogenesis, reproduction and male fertility, comparative genomics, and molecular evolution. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided a training opportunity for a Ph.D. student in 2022. How have the results been disseminated to communities of interest?The results have been disseminated to communities through oral and poster presentation in the university, national and international symposiums/conferences and peer-review publications. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Our research was focused on the role of PRAMEY during fertilization and early embryo development in the last year and the major accomplishments are as follows: The rate of progressive motility was altered when spermatozoa were treated with PRAMEY antibody (ab). We reported earlier that inhibiting PRAMEY in spermatozoa during IVF increased sperm-ZP binding ability and polyspermy rate. The objective of this study was to investigate whether treating spermatozoa with PRAMEY antibody would impact their motility. To do so, we incubated cauda epididymal spermatozoa with PRAMEY antibody, rabbit IgG, or IVF-TALP. The percentage of progressively motile (PM) spermatozoa was 67.3%, 37.6% and 38.9% in the PRAMEY antibody, the rabbit IgG and IVF-TALP, respectively. The PM sperm in the PRAMEY ab treatment group was 1.8-fold higher than the rabbit IgG and IVF-TALP controls (P<0.05). There were close to 3-fold less non-motile (NM) spermatozoa in the PRAMEY ab treatment (11.46%) when compared to the rabbit IgG (28.52%) and IVF-TALP (33.82%) controls (P<0.05). These data suggested that the PRAMEY ab treatment has an effect on the sperm motility. PRAMEY's role in the "testis-epididymis-semen" pathway. Our recent publication (Kern et al., 2022) demonstrated that the PRAMEY protein is present in testis and epididymal fluid, as well as in seminal plasma. We found that exosomes that contain the PRAMEY protein could be the connector/factor in this pathway of "testis-epididymis-semen". Over 400 seminal plasma samples collected from the Pennsylvania Livestock Evaluation Center were analyzed with two different technologies called, Nanosight and Zeta View, to measure size and concentration of exosomes (or small vesicles). Our preliminary data indicated that the number of exosome and the concentration of PRAMEY in seminal plasma vary among bulls. Our goal is to determine if the variations are associated with semen quality and bull fertility traits. PRAMEY's role in DNA methylation during early embryonic development. Our recent findings on PRAMEY's effect on early embryo cleavage leads us to believe that PRAMEY may play an important role in the epigenetic alterations that occur in developing embryos through methylation remodeling. Therefore, we decided to study the effect of PRAMEY on DNA methylation during early embryonic development using bovine IVF. IVF was performed in three technical replicates using bovine matured oocytes and cauda epididymal spermatozoa. A total of 705 oocytes were used for IVF, while 385 of those were fertilized (54.6%) and analyzed for global DNA methylation. Prior to IVF, spermatozoa were treated with anti-PRAMEY ab to determine the consequence of PRAMEY protein inhibition during fertilization, while rabbit IgG was used as a control. For global DNA methylation analysis, paternal and maternal DNA methylation was quantified in 10-, 20-, and 25-hours post fertilization (hpf) zygotes of the PRAMEY ab and rabbit IgG groups using an anti-5-methylcytosine (5-mC) antibody. When comparing paternal and maternal DNA methylation, 5-mC intensity was greater in the paternal DNA than the maternal DNA at 10 hpf in both the PRAMEY ab and control groups (P<0.01). Additionally, 5-mC intensity continued to be greater in paternal DNA at 20 hpf in the PRAMEY ab group (P<0.05), however no difference was observed in the control group (P>0.05). When comparing paternal DNA of the PRAMEY ab and control groups, significantly less 5-mC staining was observed in the paternal DNA of the PRAMEY ab 10 hpf group (P<0.01). No other differences were observed between the PRAMEY ab and rabbit IgG groups in paternal DNA at 20 or 25 hpf (P>0.05). When comparing maternal DNA of the PRAMEY ab and control groups, significantly less 5-mC was observed in the maternal DNA of the PRAMEY ab 25 hpf group (P<0.01). No other differences were observed between the PRAMEY ab and rabbit IgG groups in maternal DNA at 10 or 20 hpf (P>0.05). The decreased amount of methylation observed in the PRAMEY ab treatment group compared to the control at 10 hpf leads us to believe that the process of DNA demethylation may be occurring earlier when the function of the PRAMEY protein is blocked (PRAMEY ab treatment). This finding supports our previous results of advanced embryo cleavage in embryos with blocked PRAMEY protein (Kern et al., 2023). Our data suggested that PRAMEY plays a role in early embryonic development likely through an epigenetic mechanism.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Kern, C, Lu, C., Wu, W.W., Zhang, J.B., Zhao, Y.Q., Ocon-Grove, O.M., Sutovsky, P., Diaz, F., Liu, W.-S. (2021) Role of the bovine PRAMEY protein in sperm function during in vitro fertilization (IVF). Cell and Tissue Research Cell and Tissue Research 391, 577594. DOI: https://doi.org/10.1007/s00441-022-03717-7
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Liu, W.-S., Kern, C. and Yang, M. (2022) The Role of Sex Chromosome-Linked PRAME Genes in Spermatogenesis and Sperm Function. PAG XXIX, January 13-18, 2023, San Diego, CA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Kern, C, Lu, C., Wu, W.W., Zhang, J.B., Zhao, Y.Q., Ocon-Grove, O.M., Sutovsky, P., Diaz, F., Liu, W.-S. The PRAMEY protein in sperm function during in vitro fertilization (IVF), 55th Annual SSR Meeting, 2022. Spokane, Washington.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Kern, C, Lu, C., Wu, W.W., Zhang, J.B., Zhao, Y.Q., Ocon-Grove, O.M., Sutovsky, P., Diaz, F., Liu, W.-S. The PRAMEY protein in sperm function during in vitro fertilization (IVF), Penn State Life Sciences Symposium 2022, University Park, PA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Yang, M., Ma, W., Oatley, J., Liu, W.-S. (2022) Pramel1 is involved in germ cell homing and the initiation of the first round of spermatogenesis through the retinoic acid (RA) signaling pathway in mice. Poster presentation in the 37th annual graduate exhibition organized by the Penn State Graduate School, and in the Penn State Life Sciences Symposium. University Park, PA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Yang, M., Ma, W., Oatley, J., Liu, W.-S. (2022) The mouse Pramel1 gene regulates spermatogonial development through the retinoic acid receptor (RAR) signaling pathway. Oral presentation. The 55th SSR annual meeting, July 24-29, Spokane, WA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Kern, C., Liu, W.-S. (2023) PRAMEY- a sperm-carried protein that influences paternal DNA methylation in zygotes. 38th Annual Graduate Exhibition. University Park, PA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Yang, M., Lei, Y., Liu, W.-S. (2023) Complementary functions of Pramex1 and Pramel1 during spermatogenesis as revealed by Pramex1/Pramel1 double knockout mice. 38th Annual Graduate Exhibition. University Park, PA.
|
Progress 03/01/21 to 02/28/22
Outputs
Target Audience:Animal reprodictive physiologists, animal breeders, AI industry, cattle (beef and dairy) and other farm animal producers, as well as biologists in the fields of mammalian spermatogenesis, reproduction and male fertility, comparative genomics, and molecular evolution. Changes/Problems:In addition to the COVID19 pandemic challenge that has slowed down our research work in IVF analysis, we were/are facing another challenge on sequencing the PRAMEY protein by mass spectrometry (MS). In past years we have tried several rounds of MS in different proteomics core facilities, and we failed to identify the PRAMEY protein although other important acrosome associated proteins, such as Acrosin, were indeed detected in our MS analysis.Even though we do not know why the IP-MS method could not identify the bovine PRAMEY protein in our study, we noticed that other proteomic studies, particular the one performed on the isolated mouse chromatoid body, could not identify the PRAME-related proteins either (Meikar et al. 2014. RNA 20, 483-495). Like the bovine PRAMEY protein, the mouse PRAMEX1 (X-linked; previously known as PRAME) and PRAMEL1 (on Chr 4) are highly enriched in the mouse chromatoid body (see our recent publication, Liu et al. 2021. Cell & Bioscience 11, 102). Failure to identify the bovine and mouse proteins in testis/sperm samples by MS may reveal a challenge in sequencing proteins in the PRAME family. What opportunities for training and professional development has the project provided?This project provided a training opportunity for a Ph.D. student in 2021. How have the results been disseminated to communities of interest?The results have been disseminated to communities through oral and poster presentation in the university, national and international symposiums/conferences and peer-review publications. What do you plan to do during the next reporting period to accomplish the goals?Aim 1: Continue to analyze the PRAMEY protein complex in acrosomal granule and matrix. To study the relationship between PRAMEY and PP1γ2, and PRAMEY and SDS22. Aim 2: We have finished the proposed experiments.
Impacts
What was accomplished under these goals?
Aim 1: We published the data on different isoforms of PRAMEY, i.e. 58, 30, 26 and 13 kDa, in tissues (testis and epididymal caput and cauda), spermatozoa (testicular and epididymal caput and cauda sperm), and fluid (testicular and epididymal caput and cauda fluid, and seminal plasm). By an immunoprecipitation (IP) approach, we isolated and purified the PRAMEY protein complex from the testis tissue and/or cauda sperm for mass spectrometry (MS) with a purpose to sequence the PRAMEY protein. However, we did not identify the PRAMEY protein from the testis and sperm IP products. We are optimizing the MS protocol to repeat the experiments. We reported in past years that the PRAMEY protein was detected by Western blotting (WB) in the exosome of seminal plasma from the freshly ejaculated semen. To further characterize the protein in seminal plasma, we have continuously collected semen samples from yearling bulls during their initial semen quality evaluation in collaboration with the Pennsylvania Livestock Evaluation Center. A total of ~ 400 samples are available for further analysis. A pilot study with 30 bull semen samples indicated that the number of exosome and the PRAMEY concentration (WB data) vary among bulls. We will test if these variations are asociated with semen quality and bull fertility traits. Aim 2: We reported earlier that the number of sperm binding to the egg was 2-fold, and the polyspermy rate was also 2-fold higher in sperm treated with the PRAMEY antibody (compared to the rabbit IgG control). To understanding why the PRAMEY antibody affects sperm-egg binding and polyspermy, we performed two sets of experiments. The first experiment analyzed the sperm motility immediately after the PRAMEY antibody treatment and before using in IVF. We found that the percentage of progressive motile sperm in the PRAMEY antibody treated samples (63%) was significantly higher than that (38%) in the IgG control group. The other experiment was to use the MitoTracker™ green to label mitochondria within the spermatozoal tails prior to IVF. By this approach, we confirmed the presence of sperm tail with the mitochondrial sheath within the fertilized egg after 16 h of fertilization and validated the polyspermy result. Furthermore, the presence of sperm tail with the mitochondrial sheath in the fertilized egg provided indirect evidence that the PRAMEY protein is present in the fertilized egg because PRAMEY is enriched in the mitochondrial sheath based upon our previous immunogold electron microscopy (iEM) data. Therefore, we proposed that PRAMEY may play a role in earlier embryo cleavage (see last year report). In addition to examining the number of spermatozoa bound to the zona pellucida (ZP) and male pronuclear development, we wanted to visualize PRAMEY localization on sperm acrosome during the fertilization process. Therefore, fixed zygotes were stained with PRAMEY antibody using immunofluorescent (IF) staining technique. For the 4 h group, we found that more spermatozoa were bound to the ZP when spermatozoa were treated with PRAMEY antibody prior to fertilization. Additionally, of the spermatozoa that were bound to the ZP, more spermatozoa in the PRAMEY antibody treated group had intact PRAMEY staining in the acrosome (AC) when compared to the rabbit IgG group. The number of spermatozoa bound to the ZP was similar between the 8 h and 16 h PRAMEY antibody and rabbit IgG groups. However, we noticed that very few spermatozoa had intact PRAMEY staining in the AC at either of these time points. Male pronuclear development was only detected in the 16 h PRAMEYantibody treated or rabbit IgG groups. We observed that 36% of the zygotes in the rabbit IgG 16 h group had normal appearing male and female pronuclei, compared to only 15% normal appearing male and female pronuclei in the 16 h PRAMEY antibody treated group. Additionally, 7% of the PRAMEY antibody treated group had more than 2 pronuclei, while no observations of abnormal pronuclei number were recorded for the rabbit IgG group.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Kern, C.H., Yang, M.Y., and Liu, W.-S. (2021) The PRAME family of cancer testis antigens is essential for germline development and gametogenesis. Biol. of Reprod. 105, 290-304. DOI: 10.1093/biolre/ioab074
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Liu, W.-S. Lu, C., Mistry, B. V. (2021) Subcellular localization of the mouse PRAMEL1 and PRAMEX1 reveals multifaceted roles in the nucleus and cytoplasm of germ cells during spermatogenesis. Cell & Bioscience 11, 102. DOI: 10.1186/s13578-021-00612-6
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
Kern, C, Lu, C., Wu, W.W., Zhang, J.B., Zhao, Y.Q., Ocon-Grove, O.M., Sutovsky, P., Diaz, F., Liu, W.-S. (2021) Role of the bovine PRAMEY protein in sperm function during in vitro fertilization (IVF). Cell and Tissue Research. Manuscript under review/revision.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Kern, C.H., Feitosa, W.B., and Liu, W.-S. (2022) The dynamic of PRAMEY isoforms in testis and epididymis suggests their involvement in spermatozoa maturation. Frontiers in Genetics 13, 846345. DOI: https://doi.org/10.3389/fgene.2022.846345
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Liu, W.-S., Kern, C., and Yang, M. (2021) The Role of Sex Chromosome-Linked PRAME Genes in Spermatogenesis and Sperm Function. PAG XXIX (January 8-12, 2022), San Diego, CA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Liu, W.S. (2021) Mammalian sex chromosome-linked cancer/testis antigens (CTAs) and male fertility. European Society of Medicine (ESMED) Virtual Congress, Nov 11-13, 2021.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Chandlar Kern, Chen Lu, Weiwei Wu, Jianbin Zhang, Yaqi Zhao, Olga Maria Ocon-Grove, Francisco Diaz, Wan-sheng Liu. Role of the bovine PRAMEY protein in sperm function during in vitro fertilization (IVF), 36th Penn State Graduate Exhibition 2021 and 5th Penn State Life Sciences Symposium 2021, University Park, PA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Mingyao Yang, Wenzhi Ma, Jon Oatley, Wansheng Liu (2021) Knockout of Pramel1 Impairs Germ Cell Development during Spermatogenesis in Mice through the Retinoic Acid (RA) Signaling Pathway . 54th Annual SSR Meeting, St. Louis, MO. Poster # 2947
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Chandlar Kern, Chen Lu, Weiwei Wu, Jianbin Zhang, Yaqi Zhao, Olga Maria Ocon-Grove, Peter Sutovsky, Francisco Diaz, Wan-sheng Liu (2021) Role of the bovine PRAMEY protein in sperm function during in vitro fertilization (IVF), 54th Annual SSR Meeting, St. Louis, MO.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Hamid Beiki, Clare Gill, Honglin Jiang, Zhihua Jiang, Wansheng Liu, Stephanie McKay, Brenda Murdoch, Gonzalo Rincon, Monique Rijnkels, Tim P.L. Smith, Pablo J. Ross, Huaijun Zhou, James M. Reecy (2021) Additional annotation of bovine genome using integration of multi-omics data. 38th International Society for Animal Genetics (ISAG) Virtual Conference, July 26-30, 2021.
|
Progress 03/01/20 to 02/28/21
Outputs
Target Audience:Animal reprodictive physiologists, animal breeders, AI industry, cattle (beef and dairy) and other farm animal producers, as well as biologists in the fields of mammalian spermatogenesis, reproduction and male fertility, comparative genomics, and molecular evolution. Changes/Problems:Our planned work on in vitro fertilization (IVF) was significantly affected by the COVID-19 pandemics in 2020. In our IVF experimental design, we use cauda epididymal sperm collected from the slaughter house (in Loganton, PA). However, we could not get any testis samples from the slaughter house which was closed to visitors/resarchers for sampling in mid-late 2020. We were lucky to resume the IVF study in spring 2021, and hope to make some good progress for the planned work this year. What opportunities for training and professional development has the project provided?This project provided a training opportunity for a graduate student, Chandlar Kern, who completed her MS degree in the summer of 2020 and became a PhD student in fall 2020. How have the results been disseminated to communities of interest?The results have been disseminated to communities through oral and poster presentation in the international conferences and peer-review publications. What do you plan to do during the next reporting period to accomplish the goals?In 2021, our research will focus on: Specific Aim 1: To analyze the PRAMEY protein complex in acrosomal granule and matrix. To study the relationship between PRAMEY and PP1γ2, and PRAMEY and SDS22. Specific Aim 2: To study the effect of PRAMEY blocking antibody on early fertilization event.
Impacts
What was accomplished under these goals?
For Aim 1, we did a RT-PCR to test whether the bovine PRAMEY gene is expressed in epididymal tissue. We reported in previous years that different isoforms of PRAMEY, i.e. 58, 30, 26 and 13 kDa, were detected in testicular and epididymal tissues as well as in the fluid collected from testis and caput, corpus and cauda epididymis, raising a question on whether or not the PRAMEY gene is testis-specific. Total RNA was extracted from testicular, epididymal (caput, corpus, and cauda), and liver tissues from mature bulls (n=5). The liver tissue was used as a negative control. RT-PCR was performed with a pair of PRAMEY-specific primers. The results indicated that the PRAMEY gene is predominately expressed in the testis tissue, while a very weak band was observed in the caput epididymal tissue, and no expression was detected for the corpus and cauda epididymal tissues or liver tissue. These results suggest that the PRAMEY gene is likely not expressed in epididymal cells. We hypothesize that the weak band detected in the caput epididymis could be the leftover PRAMEY mRNA from the cytoplasmic droplets of immature spermatozoa. We speculate that the cytoplasmic droplet contains the late chromatoid body (CB) that carries large number of mRNAs and small RNAs. Since there was no guarantee to wash away all sperm when collecting caput epididymal tissue, it was likely that the weak band amplified by the PRAMEY primers was from the late CB. Therefore, we tend to believe that the PRAMEY gene is testis (or germ cell)-specific. We reported last year that the PRAMEY protein was detected in the exosome of seminal plasma from the freshly ejaculated semen. To further characterize the protein in seminal plasma, we have collected a total 270 semen samples from yearling bulls during their initial semen quality evaluation in collaboration with the Pennsylvania Livestock Evaluation Center. A pilot study has been recently conducted on 30 semen samples for exosome purification from the seminal plasma and West blotting (WB) analysis with anti-PRAMEY antibody. The WB results are being analyzed in the lab. For Aim 2, our research was focused on the role of PRAMEY in sperm function and fertilization. We have established a bovine in vitro fertilization (IVF) protocol in our lab using cauda epididymal sperm from mature bulls, and oocytes from mature cows, both collected from a local slaughterhouse. To address the question of how PRAMEY plays a role in sperm function and fertilization we began by observing the effect of PRAMEY inhibition during capacitation on cleavage and fertilization rates of embryos. Prior to being used for IVF, spermatozoa were incubated with PRAMEY antibody to inhibit its function during the process of spermatozoa capacitation or with rabbit IgG to serve as a control. Antibody treated spermatozoa were used for fertilization with matured oocytes, and cleavage and fertilization rates were recorded at 45 h post fertilization. We observed that oocytes fertilized with spermatozoa treated with PRAMEY antibody during capacitation did not have a significant difference in embryo cleavage rates for 2 or 4-cell embryos when compared to the control group (P>0.05). Additionally, fertilization rate between the PRAMEY treatment and control groups did not differ (P>0.05). Therefore, we conclude that PRAMEY does not play a role during capacitation of spermatozoa. Back to 2015-2016, we found that PRAMEY may have a role in blockage of polyspermy during fertilization. We repeated the IVF experiments in 2020-2021 to further analyze the effect of PRAMEY antibody treatment on early embryo development. As describe above, sperm were incubated with PRAMEY antibody for 30 min before IVF for the treatment group (103 oocytes), while rabbit IgG was used in the control group (89 oocytes). The embryos were harvested at 45 h post fertilization. The results indicated that the fertilization rate was not significantly different between the PRAMEY treatment (63.1%) and the control group (42.7%) (P>0.05). The rate of embryos developed into 1-cell, 2-cell, and 4-cell was 36.9%, 36.9%, and 26.2% for the treatment group, and 57.3%, 34.8%, and 7.9% for the control group, respectively. The frequency of 1-cell and 4-cell embryo was siginicantly different between the treatment and control groups (P<0.01). This preliminary data suggests that the PRAMEY protein may be involved in embryo cleavage during early embryo development. This finding could be significant as the genome of the bovine early embryo (up to 16-cell stage) is transcriptionally inactive, and the early embryo cleavage event is driven by (protein) signaling from either oocyte or sperm. In this case, the PRAMEY is carried by sperm. We are currently exploring the subcellular localization of PRAMEY by fluorescent staining at different time points during fertilization. Cumulus free oocytes are being fertilized with spermatozoa treated with PRAMEY antibody and rabbit IgG prior to fertilization and then collected at specific time points in the fertilization process, such the acrosome reaction (2 h post IVF), spermatozoa-zona pellucida penetration (4 h post IVF), spermatozoa incorporation into oocyte (8 h post IVF), and pronucleus development and DNA replication (16 h post IVF). By studying PRAMEY subcellular localization at these time points we will have a better understanding of PRAMEY's role throughout the fertilization process.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Kern, C.H., Yang, M.Y., and Liu, W.-S. (2021) The PRAME family of cancer testis antigens is essential for germline development and gametogenesis. Biol. of Reprod. In press. DOI: 10.1093/biolre/ioab074
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
Liu,W.-S., Lu, C., Mistry, B.V. (2021) Subcellular localization of the mouse PRAMEL1 and PRAMEX1 reveals multifaceted roles in the nucleus and cytoplasm of germ cells during spermatogenesis. Cell & Biosciences, revised version, under review.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Yao, Y., Zhang, Y., Liu, W.-S., Deng, X. (2020) Highly efficient synchronization of sheep skin fibroblasts at G2/M phase and isolation of sheep Y chromosomes by flow cytometric sorting. Scientific Reports 10:9933. DOI:10.1038/s41598-020-66905-x
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Chen, X., Zheng, Y., Lei, A., Zhang, H., Niu, H., Li, X., Zhang, P., Liao, M., Lv, Y., Zhu, Z., Pan, C., Dong, W., Chen, H., Wu, D., Liu, W.-S., Hamer, G., Zeng, S., Zeng, W. (2020) Early cleavage of preimplantation embryos is regulated by tRNAGln-TTG-derived small RNAs present in mature spermatozoa. J. Biol. Chem. 295 (32), 10885-10900. DOI: 10.1074/jbc.RA120.013003 jbc.RA120.013003.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Dechow, C.D., Liu, W.-S., Specht, L.W., Blackburn, H. (2020) Reconstitution and modernization of lost Holstein male lineages using samples from a gene bank. J. Dairy Sci. 103, 4510-4516. DOI: https://doi.org/10.3168/jds.2019-17753. This article was selected by the Editor-in-Chief as the Editor's Choice".
- Type:
Journal Articles
Status:
Submitted
Year Published:
2021
Citation:
Kern, C, Lu, C., Wu, W.W., Zhang, J.B., Zhao, Y.Q., Ocon-Grove, O.M., Diaz, F., Liu, W.-S. (2021) Role of the bovine PRAMEY protein in sperm function during in vitro fertilization (IVF). Manuscript in preparation.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Pablo J. Ross, Hamid Beiki, Angela Canovas, Sarah Corum, Clare Gill, Rui Hu, Honglin Jiang, Zhihua Jiang, Chandlar Kern, Colin Kern, Wansheng Liu, Pengcheng Lyu, Wenzhi Ma, Stephanie McKay, Juan Medrano, Jennifer J. Michal, Brenda Murdoch, James M. Reecy Gonzalo Rincon, Monique Rijnkels, Tim P.L. Smith, Milton Thomas, Hongyang Wang, Xiaoqin Xu, Xiaohui Zhang, Yunqi Zhang, Huaijun Zhou (2020) Functional Annotation of the Bovine Genome. Plant and Animal Genome Conference (PAG) XXVIII, Jan 11-15, 2020. San Diego.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Kern, C, and Liu, W.-S. (2020) Role of the Bovine PRAMEY Protein in Sperm Function. SSR, July 9-12, 2020. Virtual meeting.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2020
Citation:
Kern, C.H. (2020) Characterization of PRAMEY isoforms and their involvement in bovine sperm capacitation and acrosome reaction. MS Thesis. The Pennsylvania State University.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Yang, Y., Oatley, J., and Liu, W.-S. (2020) Deletion of the Pramel1 gene leads to germ cell reduction and subfertility in male mice. SSR, July 9-12, 2020. Virtual meeting.
|
Progress 03/01/19 to 02/29/20
Outputs
Target Audience:Animal reprodictive physiologists, animal breeders, AI industry, cattle (beef and dairy) and other farm animal producers, as well as biologists in the fields of mammalian spermatogenesis, reproduction and male fertility, comparative genomics, and molecular evolution. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provides training opportunity for a Master student, Ms. Chandlar Kern. How have the results been disseminated to communities of interest?The results have been disseminated to communities through oral and poster presentation in the international conferences and peer-review publications. What do you plan to do during the next reporting period to accomplish the goals?In this year, we plan to focus on: In this year, we PLan to focus on: Specific Aim 1: To analyze the PRAMEY protein complex in acrosomal granule and matrix. To study the relationship between PRAMEY and PP1γ2, and PRAMEY and SDS22. Specific Aim 2: To determine the fate of the PRAMEY after acrosome reaction and fertilization. To study the effect of PRAMEY blocking antibody on early fertilization event.
Impacts
What was accomplished under these goals?
The research work was focused on Aim 1 and Aim 2 during the second year of the project, by evaluating PRAMEY involvement in capacitation and the acrosome reaction (AR) in ejaculated sperm, as well as in exosomes of seminal plasma. Capacitation is an important physiological pre-requisite before sperm cells can acrosome react and fertilize the oocyte. To understand PRAMEY's function during capacitation, freshly ejaculated sperm were collected from normal Holstein AI bulls (n=5) at 3 different time points for biological and technical replicates. PRAMEY localization was observed by western blot (WB) and immunofluorescent (IF) staining on sperm samples with the following treatments: A. 0 hr. control, B. 4 hr. control, C. capacitated, D. non-capacitated, E. capacitated with PRAMEY antibody, F. capacitated with Rabbit IgG, G. non-capacitated with PRAMEY antibody, and H. non-capacitated with Rabbit IgG. Sperm (1x108) were incubated at 37oC with 5% CO2 and adequate humidity to induce capacitation for 4 hrs. Treatment A had no incubation time, treatment B was incubated for 4 hrs. in PBS, and treatments C, E, and F were treated to induce capacitation using an SP-TALP buffer and heparin, while treatments D, G, and H were incubated in SP-TALP only during that time. It is understood that sperm incubated to induce capacitation will display a reproducible pattern of protein tyrosine phosphorylation that are regulated by a cAMP-dependent pathway. Therefore, we used a tyrosine phosphorylation antibody to confirm the occurrence of capacitation, and we observed an obvious increase in tyrosine phosphorylated protein in sperm that were induced to capacitate, providing evidence that capacitation was successful in the selected sperm. We noticed that the 13 kDa isoform of PRAMEY appears after sperm have went through the hyperactivation process of capacitation, but not present in the control and non-capacitated sperm groups, suggesting that the 13 kDa isoform could be the active PRAMEY isoform for sperm motility. The acrosome reaction (AR) is the fusion of the spermatazoal plasma membrane with the outer acrosomal membrane. This fusion initiates the release of acrosomal enzymes from the acrosome that allow sperm to penetrate the zona pellucida. To understand PRAMEY's function during the AR, freshly ejaculated sperm were collected from normal Holstein AI bulls (n=5) at 3 different time points for biological and technical replicates. PRAMEY localization was observed by western blot (WB) and immunofluorescent (IF) staining on sperm samples with the following treatments: A. 0 hr. control, B. 5 hr. control, C. capacitated and AR, D. capacitated and non-AR, E. capacitated and AR with PRAMEY antibody, F. capacitated and AR with Rabbit IgG, G. capacitated and non- AR with PRAMEY antibody, and H. capacitated and non- AR with Rabbit IgG. Sperm (1x108) were incubated at 37oC with 5% CO2 and humidity to induce capacitation and the acrosome reaction for 4 hrs. and 1 hr., respectively. Treatment A had no incubation time, treatment B was incubated for 5 hrs. in PBS, and treatments C-H were treated to induce capacitation using an SP-TALP buffer and heparin. Treatments C, E, and F were then incubated with an SP-TALP buffer and Lysophosphatidylcholine (LPC) to induce the AR, while treatments D, G, and H were incubated in SP-TALP only during that time. Using fluorescence microscopy with Fluorescein isothiocyanate-labelled pisum sativum agglutinin (FITC-PSA), acrosomal integrity (acrosome-intact or acrosome-reacted sperm) was evaluated to ensure that the AR was occurring under the given conditions. We observed that sperm induced to go through the AR had little to no PSA staining in the acrosome, while sperm that were not induced to go through the AR had perfect PSA staining on the acrosome of the sperm head. This type of staining pattern supports that the AR occurred in the AR treatment as expected. WB analysis for the AR experiments indicated that three PRAMEY isoforms (58, 30, and 13 kDa) were detected. As mentioned above, the 13 kDa isoform was detected in the 5 hr. control and non-AR samples, but not in the 0 hr. control, suggesting that the 13 kDa isoform appears after sperm have went through the hyperactivation process of capacitation, and that the 13 kDa isoform could be the active PRAMEY isoform for sperm motility. The 30 kDa isoform was moderately to highly expressed in all treatments except in the AR sperm. Furthermore, the PRAMEY isoforms (58, 30, and 13 kDa) were not detected in the AR sperm (C, E, and F treatments) except for treatment (E) where the 58 kDa isoform is rescued due to the addition of PRAMEY antibody during the AR process. These results prompt us to hypothesize that PRAMEY is released during the acrosome reaction. To test our hypothesis, IF staining with PRAMEY-specific antibody was performed on fixed sperm from all eight treatments. A typical acrosome-enriched PRAMEY staining pattern was observed in sperm from all non-AR treatments (A, B, D, G, H), whereas little to no PRAMEY staining was observed in the acrosome region of the AR sperm (C, E, F treatments), supporting our hypothesis. Exosomes are membrane bound extracellular vesicles (EVs) that are produced in the endosomal compartment of most eukaryotic cells. Exosomes can transfer molecules from one cell to another via membrane vesicle trafficking. In previous year, we reported that the PRAMEY proteins are present in the testicular, epididymal fluid and seminal plasma. We hypothesize that exosomes could carry the PRAMEY protein through the male to the female reproductive tract. To characterize PRAMEY in exosomes, freshly ejaculated semen was collected from yearling bulls and the sperm and seminal plasma were separated. Exosomes were isolated using a commercial isolation kit and PRAMEY localization was evaluated using WB with the anti-PRAMEY antibody. Our preliminary data have been confirmed by WB and another new technology, Nanosight, that the purified exosomes indeed contain the 30 kDa PRAMEY isoform, and the number of exosomes varies among tested yearling bulls In summary, our results demonstrated that the 13 kDa PRAMEY isoform may play a role in sperm motility during capacitation, and the 58 and 30 kDa PRAMEY isoforms are involved in the acrosome reaction. We speculate that the presence of PRAMEY in exosomes of seminal plasma may be related to semen quality and fertilization.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Liu, W.-S., (2019) Mammalian Sex Chromosome Structure, Gene Content and Function in Male Fertility. July 24-28, 2019. SCBA International Symposium, Kunming, China.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Weber Feitosa, Chandlar Kern, Wan-sheng Liu (2019) Patterns of the PRAMEY expression in the bovine testis and epididymis. SSR2019, July 18-21, San Jose, CA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Liu, W.-S. (2019) What have we learned from the bovine Y-chromosome? ISAG 2019, July 7-12. Lleida, Spain.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Chandlar Kern, Weber Feitosa, Wan-sheng Liu (2019) Subcellular localization of PRAMEY during Bovine Sperm Maturation. SSR2019, July 18-21, San Jose, CA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Mingyao Yang, Weber Feitosa, Wan-sheng Liu (2019) Deletion of the mouse Prame gene affects DDX4 expression in seminiferous tubules during the first wave of spermatogenesis. SSR2019, July 18-21, San Jose, CA.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Hughes, C. K., Maalouf, S.W., Liu, W.-S., Pate, J.L. (2019) Molecular profiling demonstrates modulation of immune cell function and matrix remodeling during luteal rescue. Biology of Reproduction 100(6), 15811596.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Lu C, Yang M, Mossi R, Wang A, Feitosa W, Diaz F, Liu W-S: Deletion of the mouse X-linked Prame gene causes germ cell reduction during the first wave of spermatogenesis. Molecular Reproduction and Development, Feb 2, 2020. online early publication.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2020
Citation:
Dechow, Chad; Liu, Wansheng; Specht, Larry; Blackburn, Harvey. Reconstitution and modernization of lost Holstein male lineages using samples from a gene bank. Journal of Dairy Science. In press.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Liushuai Hua, Gonzalo Rincon, Wansheng Liu. Sequence and Assembly of the Holstein Y Chromosome. PAG2020, Jan 11-15, 2020, San Diego,CA.
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Progress 03/01/18 to 02/28/19
Outputs
Target Audience:Animal reprodictive physiologists, animal breeders, AI industry, cattle (beef and dairy) and other farm animal producers, as well as biologists in the fields of human spermatogenesis, reproduction and male fertility, comparative genomics, and molecular evolution. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provides training opportunity for a Master student, Ms. Chandlar Kern, and a post-doc research associate, Dr. Weber Feitosa. How have the results been disseminated to communities of interest?The results have been disseminated to communities through oral and poster presentation in the international conferences, including the Annual Meeting of SSR 2019, and the ISAG (International Society of Animal Genetics) Conference 2019. What do you plan to do during the next reporting period to accomplish the goals?Research work planned for the second year includes: Specific Aim 1: To determine if PRAMEY is a soluble and/or acrosomal matrix-associated protein, To analyze the PRAMEY protein complex in acrosomal granule and matrix, To study the relationship between PRAMEY and PP1γ2, and PRAMEY and SDS22. Specific Aim 2: To determine the fate of the PRAMEY protein during sperm capacitation, acrosome reaction and fertilization, To study the effect of PRAMEY blocking antibody on early fertilization event.
Impacts
What was accomplished under these goals?
The research work was focused on the Specific Aim 1 during the first year of the project, by characterizing the bovine PRAMEY protein expression in testicular and epididymal tissues and sperm, as well as the ejaculated sperm and fluid (seminal plasma). Using testicular, epididymal and ejaculated sperm from adult bulls, four different isoforms of the PRAMEY protein, including the 58, 30, 26, and 13 kDa isoforms were identified by western blot with a PRAMEY-specific antibody. The 58 kDa that is the predicted intact protein, was observed in a similar level in spermatozoa from testis, caput and cauda of the epididymis and ejaculated sperm. In testicular sperm, the expression of the 58 kDa isoform was higher (p ≤ 0.05) than the 30 kDa isoform. However, it was lower (p ≤ 0.05) than the 30 kDa isoform in epididymal and ejaculated sperm. The 30 kDa isoform had significantly higher (p ≤ 0.05) expression in sperm of epididymis than in testicular and ejaculated sperm. As spermatozoa migrate to epididymis, two adtional isoforms of 26 and 13 kDa were detected. Although there was no difference in the 26 and 13 kDa protein level between caput and cauda sperm, they were higher in epididymal sperm than in ejaculated sperm. The identification of the two additional isoforms in epididymal and ejaculated sperm suggests their involvement in sperm maturation and maybe also in the sperm fertilization capability. However, it is unknown if the 26 and 13 kDa isoforms are new PRAMEY protein variants or if they are products from the 58 and 30 kDa cleavage. To understand the origin of the 26 and 13 kDa isoforms in epididymal and ejaculated sperm, we studied the pattern of PRAMEY expression in testicular, epididymal and ejaculated fluid (seminal plasma). We noticed that the fluids have a different pattern from that of sperm. While the 58 kDa expression level was similar among the fluids from testis and epididymis, its expression level was lower in seminal plasma. The 30 kDa isoform was strongly detected compared to the 58 kDa. Its expression level was higher (p ≤ 0.05) in fluid from testis and caput than in seminal plasma. Interestingly, the 26 kDa isoform was detected only in the fluid from cauda of the epididymis, but not in the fluid from caput. The 13 kDa was weakly detected in fluid from caput epididymis, and highly detected in fluids of cauda epididymis and seminal plasma. Taken together, these results shown that the pattern of the 13 kDa isoform in fluid (caput, cauda and seminal plasma) follows their pattern in sperm (caput, cauda and ejaculated sperm), suggesting that the 13 kDa isoform in epididymal and ejaculated sperm probably is originated from the fluid rather than a cleavage process. In contrast, the 26 kDa isoform in fluid does not follow its pattern in sperm, in which it was weakly detected only in cauda fluid, suggesting that these isoform in sperm maybe is not originated in the epididymal fluid. To understand the presence of the PRAMEY in the testicular and epididymal fluid, we further evaluated the PRAMEY expression in testicular ad epididymal tissues. We found that the 58 kDa was weakly expressed in the adult testicular and epididymal tissue. The expression level of the 30 kDa isoform was higher (p ≤ 0.05) than the 58 kDa, however, no difference was observed between testicular and epididymal tissues. Interestingly, the 26 kDa isoform was not observed in epididymal tissue. The 13 kDa protein expression was weakly detected in caput tissue and its expression was higher (p ≤ 0.05) in caput tissue. Moreover, the 13 kDa isoform was not observed in testicular tissue. The results indicate that the PRAMEY protein may not be germ cell-specific, it is also expressed in somatic cells in the epididymis. In addition, the lack of the 26 kDa expression in epididymal tissue together with its expression in epididymal fluid confirms our hypothesis that this isoform in epididymal sperm is not originated from the epididymal fluid/tissue. Two different custom-made antibodies, i.e. anti-PRAMEY C-terminal and anti-PRAMEY-specific, were used in immunofluorescence in caput and cauda segment of the epididymis. Both antibodies showed the presence of PRAMEY in the spermatozoa agglomerated in center of the epididymal lumen as well as localization of the PRAMEY in the epithelial cells of the epididymis. PRAMEY was observed in a cytoplasmic distribution in the epididymal cells with a diffuse apical and a strong basal expression in the caput epididymal epithelium. Similar to caput, PRAMEY expression in the apical epithelium of the cauda has a diffuse localization. However, its expression in the basal compartment is lower than that observed in the cauda segment of the epididymis. Together with the western blot detection of PRAMEY protein in epididymal tissue, our immunofluorescence analysis confirms that PRAMEY is not a germ cell-specific protein, but it is also expressed in epididymal tissue. We evaluate the dynamic of PRAMEY localization between sperm head and tail during maturation in the epididymis. Among the four PRAMEY isoforms, the 58 kDa isoform was very weakly detected in the separated sperm head and tail, thus it was not evaluated in this experiment. When comparing the 30, 26, and 13 kDa isoforms individually, we found a remarkable decrease of 10.9-, 5.4-, and 3.8-fold respectively, in protein expression from the caput to cauda epididymis in sperm heads, but there was a small decrease in the expression for both the 30 and 26 kDa isoforms in sperm tails (1.9- and 1.2-fold respectively). In contrast, the 13 kDa isoform increased 4-fold in sperm tails from caput to cauda, suggesting this isoform may have a significant role in tail function, but is likely not as important in sperm head function of cauda spermatozoa. When expression levels of all isoforms were combined, PRAMEY expression was nearly equivalent for both caput sperm head and tail. Alternatively, cauda sperm tails have an expression 6-fold higher than cauda sperm heads, suggesting the role of PRAMEY in tail function. To better understand the PRAMEY functional dynamic between head and tail during sperm maturation, we further evaluated the PRAMEY expression in spermatozoa cytosol, nucleus, membrane and mitochondria from spermatozoa during maturation in the epididymis. Again, the 58 kDa isoform was very weakly detected, thus it was not evaluated in this experiment. During bovine sperm maturation in the epididymis, the 30 and 26 kDa isoforms significantly decreased (p ≤ 0.05) in sperm cytosol, nucleus, plasma membrane and mitochondria from caput to cauda, whereas the 13 kDa isoform significantly (p ≤ 0.05) increased. The increase of the 13 kDa in sperm membrane maybe reflect the internalization of this isoform from the fluid by spermatozoa during maturation. The overall increase of the 13 kDa isoform from caput to cauda as evidenced by its expression level in the cytosol, is an indicative of the importance of the 13 kDa protein for sperm maturation, a process in which the sperm acquire motility and fertility. In conclusion, our data showed that PRAMEY is not a germ cell-specific protein, in which it is also expressed in epididymal tissue. The presence of the PRAMEY isoforms in the fluid suggests a possible pathway epididymis-exosome (or epididymosome)-sperm, in which PRAMEY isoforms expressed in epididymal tissue are exported to epididymal fluid and incorporated by sperm cells. Testis (sperm, fluid and tissue) express solely the 58 and 30 kDa, suggesting their involvement in spermatogenesis. Epididymal and ejaculated sperm strongly express the 30 and the 13 kDa isoforms, suggesting their involvement in sperm maturation and fertilization capability. Although the 26 kDa is not expressed in epididymis, it is not clear if this isoform was resulted from a cleavage process, or simply was a dimer of the 13 kDa isoform.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Liu, W.-S. (2019) Mammalian sex chromosome structure, gene content and function in male fertility. Annu Rev Anim Biosci.7,103-124.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Zhang, Y.Y., Han D.P., Dong, X.G., Wang, J.K., Chen, J.F., Yao, Y.Z., Darwish, H.Y.A., Liu, W.-S., Deng, X.M. (2019) Genome-wide profiling of RNA editing sites in sheep. Journal of Animal Science and Biotechnology 10, 31
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Dechow, C., Liu, W.-S. (2018) DNA methylation patterns in peripheral blood mononuclear cells from holstein cattle with variable milk yield. BMC Genomics 19, 744.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Dechow, C., Liu, W.-S., Idun, J., Maness, W. (2018) Two dominant paternal lineages for North American Jersey artificial insemination sires. J. Dairy Sci. 101, 228184.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
8. Liu, W.-S., (2018) The enrichment of testis-specific genes on the mammalian X and Y chromosome. Invited presentation in the International Plant and Animal Genome Research (PAG) XXVI, January 11-15, 2019. San Diego, CA.
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