Progress 03/01/18 to 02/28/23
Outputs
Target Audience:The research was presented to USDA program directors, scientists, aquacultaure professionals, fish biologista and natural resource managers in 13 talks or posters at local, national and international scientifc meetings. The rsearch will also be presented at a Comparative Endocrinology meeting in Taiwan in October, 2023. The research will be published in four manuscripts to be submitted to several scientifc journals targeting different audiences interested in animal reporduction, commercial aquaculture, comparative endocrinology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Three postdoctoral fellows and a research technician have received training in animal reproduction research techniques on this project How have the results been disseminated to communities of interest?By giving the following thirteen presentations at local, national and international scientific meetings: Peter Thomas, Yefei Pang. Enhancement of male southern flounder broodstock fertility by direct activation of sperm 2nd messenger pathways down stream of mPRalpha.2018 Animal Reproduction Project Director Meeting. New Orleans, USA, July 9-10, 2018. Peter Thomas, Yefei Pang, Wenxian Tan. Multiple signaling pathways mediate progestin hormone induction of sperm motility in southern flounder through membrane progesterone receptor alpha. Annual meeting Society for the Study of Reproduction., New Orleans, USA, July 11-13, 2018. Peter Thomas, Yefei Pang, Wenxian Tan. Progestin induction of sperm hypermotility in southern flounder through mPRα is mediated by activation of multiple signaling pathways.11th International Symposium on Reproductive Physiology of Fish. Manaus, Brazil June 3-0, 2018 Peter Thomas, Yefei Pang, Wenxian Tan. Mechanism of progestin hormone stimulation of hypermotility in Southern Flounder sperm. Fish Reproduction Symposium. 13th International Congress on the Biology of Fish. Calgary, Canada, July 15-19, 2018 Peter Thomas, Yefei Pang, Wenxian Tan. Rapid progestin induction of southern flounder hypermotility through a nongenomic mechanism. Symposium on Physiological Insights Toward Improving Fish Culture. Aquaculture. New Orleans, USA, March 7-12, 2019. Peter Thomas, Yefei Pang, Wenxian Tan. Rapid progestin upregulation of sperm motility in marine fish through membrane progesterone receptor alpha (mPRα). Society for Integrative and Comparative Biology. Austin, TX, USA, January 3-7, 2020. Peter Thomas, Yefei Pang, Wenxian Tan. Rapid progestin induction of southern flounder sperm hypermotility through a nongenomic mechanism. Aquaculture. New Orleans, Louisiana, USA, March 7-11 2019. Peter Thomas. Enhancement of male southern flounder brood stock fertility by direct activation of sperm 2nd messenger pathways downstream of mPRα. Project Directors Meeting REE-NIFA, St. Louis, 14-15 December, 2021. Peter Thomas, Aubrey Converse, Jing Dong, Yefei Pang, Teresa Bennett. Rapid progestogen hormone induction of southern flounder sperm hypermotility through multiple signaling pathways. 2021 Annual Meeting of the Society for the Study of Reproduction (SSR), St. Louis, 15-16 December, 2021. Caroline Matkin, Peter Thomas. Role of Calcium Channels in Sperm Motility in Southern Flounder (Paralichthys lethostigma). Texas Bays and Estuaries Meeting, UTMSI, Port Aransas, TX, September 21-22, 2022. Teresa Bennett, Caroline Matkin, Peter Thomas. Role of second messengers involved in progestin-induced hypermotility through mPRα in southern flounder. Texas Chapter American Fisheries Society, Corpus Christi, Texas, February 23- 25, 2023. Caroline Matkin, Teresa Bennett, Peter Thomas. Calcium signaling in progestin induction of sperm hypermotility in southern flounder (Paralichthys lethostigma). Texas Chapter American Fisheries Society, Corpus Christi, Texas, February 23- 25, 2023. Peter Thomas, Teresa Bennett, Yefei Pang, Caroline Matkin, Aubrey Converse, Jing Dong. Stimulation of southern flounder sperm motility and fertility by direct activation of intracellular signaling pathways. Aquaculture America., New Orleans, February 23-23, 2023. What do you plan to do during the next reporting period to accomplish the goals?Nothing to report
Impacts
What was accomplished under these goals?
Objective 1.Role of Pi3k/Akt/Pde signaling in progestin induction of hypermotility (Experiment 1). Immunocytochemistry (ICC) with a human Akt antibody showed the presence of immunoreactive Akt on flounder midpiece and flagella. Western blot analysis of pAkt on sperm treated with the progestin hormone, 20β-S, showed increase staining intensity compared to the vehicle-treated controls (Experiment 4). Induction of hypermotility by 20β-S and the specific mPR agonist, Org OD 02-0, was significantly attenuated by treatments with two Pi3k inhibitors, LY294002 and wortmannin, and sperm velocity was not significantly different from vehicle-treated controls. Similar results were obtained with two Akt inhibitors, ML-9 and Pf-04691502, which also blocked progestin-induction of sperm hypermotility. In addition, the increase in sperm velocity in response to progestin treatments was blocked by pretreatment with two Pde inhibitors, cilostamide (Pde 3 inhibitor) and rolipram (Pde 4 inhibitor). These results demonstrate an involvement of Pi3k/Akt in progestin induction of hypermotility. Roles of Acy/cAMP and Egfr/Erk1/2 pathways in progestin induction of sperm motility in flounder sperm (Experiment 2). ICC analyses showed strong immunoreactive staining of Golf and PKA on the midpiece and flagella of flounder sperm, consistent with their involvement in the flounder sperm hypermotility response. ICC studies identified Egfr, Erk1/2 and pErk1/2 on flounder sperm. Treatment with 20β-S increased the intensity of pErk1/2 staining, indicating that Erk1/2 is activated by the hormone (Experiment 4). Pretreatment with two Egfr inhibitors, AG1478 (ErbB-1 inhibitor) and AG825 (ErbB-2 inhibitor) and inhibition of matrix metalloproteinase (MMP), a downstream mediator if Egfr activation, with Ilomastat, blocked progestin induction of sperm hypermotility. Pretreatment with two Erk1/2 inhibitors, U1026, and PD980, also blocked the motility responses to the progestins. These experiments support the proposed role ofthe adenyl cyclase (Acy) /cAMP/PKA in flounder sperm motility and demonstrate that the Egfr/Erk1/2 pathway is also involved in progestin induction of sperm motility. Crosstalk by other signaling pathways in progestin upregulation (phosphorylation) of Akt (Experiment 3). Immunoprecipitation of pAkt from sperm membranes on glass beads with subsequent elution of the enriched pAkt sample was necessary for pAkt detection in the Western blot studies. An activator of Acy, forskolin, mimicked the effects of progestins to increase levels of phosphorylated Akt, whereas the Acy inhibitors, dd-Ado and SQ22536, blocked the response to the progestin hormones. Treatment with EGF mimicked the effects of the progestins on pAkt expression, whereas pretreatment with the Egfr inhibitor U1036 and the Mek inhibitor PD98059 blocked phosphorylation of Akt by the progestins. The results show that both the Acy/cAMP and Egfr/Erk1/2 pathways are upstream of the Pi3k/Akt/Pde pathway. These studies on Pi3k/Akt/Pde signaling and crosstalk by other signaling pathways are complete and a manuscript (ms 1) is currently being drafted for publication. Objective 2. Involvement of Ca2+ channels in initiation of flounder sperm motility (additional studies). The results with Ca2+ channel inhibitors suggest voltage-gated L- and T-type Ca2+ channels play a role in the increase in intracellular Ca2+ associated with sperm motility initiation upon release into seawater in southern flounder. The novel Ca2+ channel, CatSper, was identified in flounder and croaker sperm. A draft manuscript (ms3) describing these results is currently undergoing final editing for submission to a scientific journal. Role of Ca++ channels in progestin induction of flounder sperm (Experiment 5). An association of a rapid increase in Ca2+ levels accompanying progestin-induced hypermotility was demonstrated in flounder sperm. The role of voltage-gated L- and T-type Ca2+ channels in progestin-induced sperm hypermotility was examined by pretreatment with either verapamil (L-type inhibitor) or mibefradil (T-type inhibitor) prior to progestin addition. Both inhibitors blocked the progestin-induced intracellular Ca2+ concentration increase and sperm motility. These results show that progestin-induced influx of Ca2+in flounder sperm accompanying the hypermotility response is mediated through both voltage-gated L-type and T-type Ca2+ channels. In addition, treatment with an inhibitor of ABDH, which is involved in CatSper signaling, with MAFP, blocked progestin-induced flounder and croaker sperm motility. In addition, treatment with the CatSper inhibitor HC-056456 also blocked sperm motility in response to the progestins, suggesting that Catsper may also be involved in progestin-dependent upregulation of sperm hypermotility. Role of Acy/cAMP, Pi3k/Akt/Pde, and Egfr/Erk1/2 pathways in progestin-induced Ca2+ increase (Experiment 6). Experiments investigating Ca2+ signaling and second messenger pathways downstream of mPRα (Acy/cAMP, Pi3K/Akt/Pde, and Egfr/Erk1/2) were conducted with two second messenger pharmacological agents. Forskolin and EGF, which activate the Acy/cAMP and Egfr signaling pathways, respectively, mimicked the intracellular Ca2+ increase produced by Org OD 02-0. Inhibition of membrane Acy with dd-ADO blocked the progestin-induced Ca2+ increase. Pretreatment with Pi3K inhibitors, Wortmannin and ML-9, also abrogated the progestin-induced Ca2+ increase. Furthermore, the Pde inhibitor, Rolipram, also blocked the progestin-induced Ca2+ increase. The Egfr/Erk1/2 inhibitors, U0126, AG1478, and AG825 also eliminated the progestin-induced Ca2+ increase. These results suggest Acy/cAMP, Pi3K/Akt/Pde, and Egfr/Erk1/2 pathways are all involved in the progestin-induced intracellular Ca2+ increase in southern flounder sperm. A manuscript (ms4) describing these results is being finalized for submission to a scientific journal. Objective 3. Effects of direct activation of adenylyl cyclase-dependent signaling with forskolin, and direct activation of Egfr signaling with recombinant Egf (Experiments 8 and 9). Acute 1-5 minutes treatment of flounder sperm collected during the spawning season with both 100 nM and 0.1 nM recombinant human EGF mimicked the effects of the progestins to increase sperm motility and also significantly increased sperm fertility, indicating that EGF can directly activate southern flounder sperm to increase motility and fertility. Sperm collected from donors towards the end of the reproductive season were unresponsive to 20β-S and Org OD-02-0 in vitro treatments and sperm motility and fertility was not significantly different for vehicle-treated controls. In contrast, the sperm remained responsive to in vitro treatments with 0.1 nM and 100 nM EGF, significantly increasing sperm motility and fertility. Acute 1-5 minutes in vitro treatment of flounder sperm collected during the reproductive season with the Acy activator, forskolin (10µM), also mimicked the stimulatory effects of the progestins on both sperm motility and fertility, increasing fertilization success significantly above that of the vehicle-treated controls. Acute treatment of sperm collected at the end of the reproductive season with 10 µM forskolin significantly increased sperm motility and fertility compared to that of vehicle-treated controls, whereas the progestins (20 nM) were ineffective. Similar positive results with EGF and forskolin on sperm motility and fertility were obtained with croaker sperm that were unresponsive to progestin treatments, which raises the possibility that these treatments have the potential to increase fertility of male broodstock for a broad range of fish aquaculture species. A manuscript (ms2) reporting these findings is currently undergoing final editing for submission to an aquaculture journal.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Tan, W., Pang, Y., Tubbs, C., Thomas, P. 2019. Induction of sperm hypermotility through membrane progestin receptor alpha (mPR?): a teleost model of rapid, multifaceted, nongenomic progestin signaling. General and Comparative Endocrinology. 279:60-66. https://doi.org/10.1016/j.ygcen.2018.12.002
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Progress 03/01/21 to 02/28/22
Outputs
Target Audience:The research was presented at two scientific conferences. One at the USDA Ree-NIFA Project Diectorsmeeting at St.Louis, in Decmber 2021 and the othe at the Annual Meeting of the Society for the Study of Reproduction at St. Louis, December 2021. Changes/Problems:A bacterila infection caused a marked decrease in the number of acclimated flounder that were to be used in the early stages of the flounder reproductive season, resulting in too few fish to complete all the planned expperiments. To address this additional fish were collected but they were not in reproductive condition. Thse fish will be used in the next reproductive season. What opportunities for training and professional development has the project provided?1-year training for Research Scientist Assisten Teresa Bennett and 6- months training for Postdoctoral Fellow Caroline Matkin How have the results been disseminated to communities of interest?Presentations at naational scientific meetings: Thomas, P. Enhancement of male southern flounder broodstock fertility by direct activvation of sperm 2nd messenger pathways downstream of mPRalpha. Project Directors Meeting REE-NIFA, St. Louis, 145-15 December, 2021. Thomas, P., Converse, A., Dong, J., Pang, Y., Bennett, T. Rapid progestogen hormone induction of southern flounder sperm hypermotility through multiple signaling pathways. 2021 Annual Meeting of the Society for the Study of Reproduction (SSR), St. Louis, 15-16 December, 2021. What do you plan to do during the next reporting period to accomplish the goals?Manuscripts describing the results of the studiew completed to date will be prepared for publication. The role of calcium influx in progestin-induced regulation of Acy/cAMP, Pi3k/Akt/pde, and Egfr/Erk1/2 pathways (Objective 2 ,Exps 5,6,7) will be completed at the beginning of the reproductive season. In vitro forskolin and EGF treatments will be futrther explored, focussing on sperm that are reposnsive to progestin with low basal motilites (Objective 3, Exp. *,9)
Impacts
What was accomplished under these goals?
Renovation of fish culture facilities after damage sustained by Hurricane Harvey (August 2017) is still ongoing. Water quality is still poor resulting in a bacterial infection and the loss of many fish that were being cycled on a strict photoperiod/temperature regime for experiments in the fall of 2021. An additional 130 fish were collected in the November 2021 to make up for this loss, but surprisingly none of them were in reproductive condition. Also none of the flounder collected by Texas Parks and Wildlife Department in 2021 had gonadal development. It has been speculated that the increased water temperatures in the Gulf of Mexico is the cause of impaired reproduction in flounder. The outcome of this is that none of these recently collected were available for experimentation during the 2021-2022 flounder spawning season which severely limited those available for the proposed studies. The recently-collected fish are being acclimated to specific photoperiod and temperature cycles for 6 months prior to use, therefore they will be utilized in the upcoming 2022- 2023 flounder season Only weak signals for phosphorylated Erk were obtained using a variety of antibodies to mammalian pErk, thereby complicating the examination of crosstalk between Erk and other signaling pathways (Objective 1,Exp. 3). Nonetheless, the results showed that Erk phosphorylation was upregulated by exposure to 20β-S, confirming its role in the sperm motility response. The membrane adenylyl cyclase (Acy) inhibitors, dd-Ado and SQ-22536, blocked phosphorylation (activation) of Erk, demonstrating Acy is upstream of Erk. Consistent with these findings, forskolin, which activates Acy, stimulated Erk phosphorylation. Erk phosphorylation was also upregulated by treatment with EGF, indicating that Egfr is also upstream of Erk. Experiments on Erk regulation of Akt phosphorylation were completed with a second Erk (Mek1/2) inhibitor, U0126 These experiments complete the studies in Exp.3 which collectively show that adenylyl cyclase is upstream of both Akt and Erk signaling in flounder sperm. These surprising findings suggest the presence of a complex signaling pathway mediated through Acy regulating sperm motility in southern flounder. The roles of Pi3k/Akt/Pde and Egfr/Erk1/2 pathways in progestin induction of flounder sperm hypermotility were further explored in year 4 (Objective 1). Inhibition of Pi3k using Wortmannin blocked progestin-initiated sperm hypermotility, in agreement with preliminary results from years 2 and 3 (Objective 1; Exp. 1). The sperm motility response to progestin was blocked by pretreatment with the Akt inhibitor ML-9 (Objective 1; Exp.1). Furthermore, pretreatment with the Pde inhibitor, Rolipram, also blocked progestin induced sperm hypermotility (Objective 1; Exp. 1). Collectively these results validate the involvement of Pi3K/Akt/Pde pathway in the mPRα signaling. Additionally, our preliminary results suggest the Egfr pathway is involved in the induction of progestin sperm hypermotility. The Egfr inhibitors, AG1478 and AG825, blocked progestin-induced hypermotility (Objective 1; Exp. 2). Experiments are on-going with Erk1/2 inhibitors, U0126 and PD98059, to determine the role of the Egfr/Erk1/2 pathway. Experiments were performed to investigate the potential roles of Ca2+ channels in flounder sperm hypermotility (Objective 2; Exp. 5). The presence and function of L-and T-type Ca2+ channels were examined using L- type Ca2+ inhibitors (Verapamil, Nifedipine, and Diltiazem) and T-type Ca2+ channel inhibitors (Mibefradil and ML218). Preincubation with the inhibitors resulted in a concentration dependent reduction in sperm motility in all cases, thus confirming the role of L- and T- type Ca2+ channels in motility. Preliminary results suggest Mibefradil blocks progestin induced sperm hypermotility (Objective 2, Exp. 5). Additionally, Verapamil blocked progestin stimulation of flounder sperm hypermotility; however additional experiments are required to confirm these results. Surprisingly, difficulties were experienced in detecting intracellular calcium in flounder sperm using a high-resolution fluorescence microplate reader, although an earlier version of this instrument had detected calcium in croaker sperm. Therefore, a previously successful method used to quantify intracellular calcium in smooth muscle cells using a microscope is being adapted for sperm. Although it was previously thought the specific Ca2+ channel in mammalian sperm, CatSper, was absent in fish, recent studies in salmonids, herring, and barfin flounder confirm the presence of CatSper on the sperm of these fish (Lissabet et al., 2020, Comp. Biochem, Physiol. A 241: 110634; Yanagimachi et al. Biol. Reprod. 2017, 96 780-799). Western blots and immunocytochemistry confirmed our preliminary prior results that CatSper signals are present on the midpiece and tail of flounder sperm. CatSper was also detected on the same regions of Atlantic croaker sperm, with strong immunoreactive bands of the correct size on Western blots. These results suggest CatSper is broadly expressed in the sperm of marine fish. The role of CatSper in the motility response was investigated through motility experiments with a specific CatSper inhibitor (HC-056456, Objective 2; Exp. 5). Preincubation with HC-056456 resulted in a significant decrease in sperm motility at 15 µM, further confirming the presence of CatSper in flounder sperm. Experiments are on-going to examine if progestin induced hypermotility can be abolished by HC-056456. In vitro treatments with the Acy agonist, forskolin, and EGF significantly increased flounder sperm motility (Objective 3; Exp. 8). Preliminary results suggest Forskolin is also effective in increasing flounder sperm motility in sperm that is unresponsive to progestin stimulation (Exp. 9). The effects of osmolarity on the induction of flounder sperm motility showed that a minimum concentration of 240 mM NaCl in the activating solution is required to induce motility. These results are consistent with induction of sperm motility in other marine fish such as the Atlantic croaker (Detweiler and Thomas, 1998). Progress Summary: The remaining experiments in Objective 1, on the roles of the Pi3K/Akt/pde and EGFR/Erk1/2 pathways in sperm motility were successfully completed. Experiments on the cross-talk between Acy and Akt and Erk pathways were completed which showed Acy is upstream of the other signaling pathways and Akt is downstream of all the other pathways (Experiment 3). The likely identification of CatSper on flounder sperm was further explored and the specific CatSper inhibitor, HC-056456. Treatment with this specific CatSper inhibitor blocked the motility of flounder sperm. Additional ICC analyses confirmed the presence of immunoreactive proteins on the midpiece and flagella of flounder sperm. Only weak immunoreactive bands were detected on Western blots of flounder sperm but much stronger bands of the correct size were detected on western blots of Atlantic croaker, another marine teleost, suggesting the widespread presence of CatSper on fish sperm. The COVID-19 pandemic throughout the entire 4th year of the project prevented access to other laboratories to conduct comparative sperm motility experiments (Exp. 10).
Publications
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Progress 03/01/20 to 02/28/21
Outputs
Target Audience:All scientific conferences were canceled during this reporting period due to the COVID-19 pandemic so the results were not reported to the target audience Changes/Problems:Problems: A. COVID -19 1. partial shut down of the University due to COVID-19 delayed research. 2. Required social distancing prevented experiments being conducted which required 2 personnel. 3. Contact with other aquaculture labs to conduct croos-laboraory comparisons not possible. B. Severe weather Two major summer storms in 2020 aand the big freeze ealry in 2021 all caused loss of lighting in the fish holding facilities, disrupting the reproductive cycle of flounder some thaat some chorts failed to reach reproductive condition for sperm motility experiments What opportunities for training and professional development has the project provided?8 months training for a post doc.Laura Jenkins, 3 months training for another post doc. Aubrey Converse How have the results been disseminated to communities of interest?None of the results were dissemeinated to communities of interest because all conferences were canceled due to the COVID-19 pandemic What do you plan to do during the next reporting period to accomplish the goals? Cross-talk between the 2nd messenger pathways (Objective 1, Exp.3) will be completed and cross-talk of these pathways and Ca++ (Objective 2, Exps. 5, 6, 7) will be conducted in the first part of the reproductive season. In particular, the role of CatSper in the 20β-s/mPRα/2nd messenger-mediated induction of sperm hypermotility will be explored. During the second half of the reproductive season efforts will focus on developing practical procedures to enhance sperm motility and fertility through pharmacological treatments with forskolin and EGF (Objective 3, Exps. 8, 9) Progress towards these goals is dependent on fully reopening of the University of Texas and the collaborating laboratories and a return to normal operations.
Impacts
What was accomplished under these goals?
Renovation of fish culture facilities after damage sustained by Hurricane Harvey (August 2017) are still ongoing. In particular, the sea water system has still not been repaired which resulted in poor water quality resulting in the loss of all the fish collected at the beginning of year 1 of the project. New fish were collected in year 2 and acclimated to laboratory conditions but were not available for experimentation at the beginning of the flounder spawning season. This prevented completion of all the Objectives for year 2 so that they had to be completed in year 3. In year 3 the remaining experiments on the roles of Pi3k/Akt/Pde pathway in progestin induction of flounder sperm hypermotility (Objective 1; Exp. 1) were completed with a second series on inhibitors. The enhancement of sperm motility with the progestogen hormone 20β-S was blocked by pretreatment with the Pi3k inhibitor Lys294002, similar to the effects of another Pi3k inhibitor Wortmannin observed in year 2. The sperm motility response to 20β-S was also blocked by pretreatment with the Akt inhibitor, PF-04691502, in agreement with previous experiments conducted in year 2 with another Akt inhibitor, ML9. In addition, sperm hypermotility in response to 20β-S was blocked by pretreatment with Rolipram, confirming a role for phosphodiesterase in motility response which had been suggested from the experiments with cilostamide in year 2. Importantly, none of these inhibitors alone affected sperm motility, indicating they do not exert any nonspecific toxic effects on flounder sperm at these concentrations. The finding that two sets of inhibitors of Pi3k, Akt and pde pathways blocked 20β-S-induced flounder sperm hypermotility provides clear evidence that they are key components of mPRα signaling regulating sperm motility and a sound basis for the remaining studies on this project. Although our results clearly indicated a role of Akt and its phosphorylation in the response to 20β-S in flounder sperm, only weak Akt signals were detected on Western blots using a variety of antibodies, complicating the examination of cross-talk between Akt and other signaling pathways (Exp. 3). A procedure to separate Akt by immunoprecipitation on glass beads was developed which enabled the experiments to be conducted, although this lengthy separation procedure significantly decreased assay throughput. Nevertheless, the results showed that Akt phosphorylation was upregulated by exposure to 20β-S, confirming its role in the sperm motility response. The membrane adenylyl cyclase inhibitor (Ac), dd-Ado, was shown to block activation (phosphorylation) of Akt, demonstrating that mAc is upstream of Akt. This was confirmed in a second series experiments with another mAc inhibitor, SQ22536. The Acy agonist, forskolin, mimicked the effects of 20β-S on Akt phosphorylation, further confirming that Acy is upstream of Akt. In addition, Akt phosphorylation was blocked by treatment with an Erk inhibitor, PD98059. Collectively the results obtained to date suggest that Akt is downstream of other second messengers in the mPRα/20β-S signaling pathway. Experiments on interactions between Acy and Akt on Erk phosphorylation are currently being analyzed. Experiments were conducted on colocalization of interacting signaling components on flounder sperm by immunocytochemistry (Exp. 4), including the potential role of Ca2+ channels in progestin induction of flounder sperm hypermotility (Objective 2, Exp.5). As outlined in our proposal, originally it was thought that the specific Ca++ channel in mammalian sperm, CatSper, was absent in fish (Cai & Chapman, 2011). However, recent studies in salmonids, herring and barfin flounder demonstrate that Catpser is present on the sperm of these fish species, but not in several other fishes (Lissabet et al., 2020, Comp. Biochem, Physiol. A 241: 110634; Yanagimachi et al. Biol. Reprod. 2017, 96 780-799). Therefore, we investigated whether immunoreactive CatSper could be detected on southern flounder sperm using the same antibody as that used in these earlier studies. Immunoreactive bands, the expected size of the CatSper dimer were detected on Western blots of flounder sperm and positive immunoreactive CatSper signals were detected on the midpiece and tail by immunocytochemistry. Additional experiments with a specific CatSper inhibitor, HC-056456, are ongoing to determine whether the Ca++ and sperm motility responses to 20β-S is abrogated by treatment with this CatSper inhibitor. Confirmation that the sperm motility response is dependent on CatSper will greatly advance our proposed model of progestin induction of flounder sperm motility. Progress Summary: The remaing experiments in Objective 1, Experiment 1 on the role of the Pi3K/Akt/pde pathway in sperm motility were successfully completed. Technical difficulties in detecting expression of Akt on sperm were overcome and experiments of the cross-talk between Akt and AC and Erk pathways completed (Experiment 3). Progress continued on Experiments 4 and 5 with the likely identification of CatSper on flounder sperm. The COVID-19 pandemic throughout the entire 3rd year of the project severely curtailed research progress. The University response to the pandemic resulted in partial closure of the laboratory, limiting access to conduct experiments with flounder, and social distancing which prevented many experiments requiring two personnel to be carried out. Moreover, access to other laboratories to conduct comparative sperm motility experiments (Exp. 10) was not possible due to the pandemic. In addition, two severe summer storms in 2020, as well as the severe freeze at the beginning of 2021 all caused power outages and loss of lighting in the fish holding facilities over several days which disrupted the reproductive cycles of several cohorts of flounder, preventing some of the proposed studies in year 3 (e.g. Exp. 9) from being conducted.
Publications
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Progress 03/01/19 to 02/29/20
Outputs
Target Audience:The research was presented at two scientific conferences. One at a symposium to aquaculture researchers and industry represesntatives on applications of fish physiology to improving aqauculture at the World Aquacultute meeting in New Orleans entiled Peter Thomas*, Yefei Pang, Wenxian Tan. Rapid progestin induction of southern flounder sperm hypermotility through a nongenomic mechanism. Aquaculture. New Orleans, Louisiana, USA March 7-11 2019. The other to fish biologists at the Society of Integrative Biology Annual meeting- Peter Thomas*, Yefei Pang, Wenxian Tan. Rapid progestin upregulation of sperm motility in marine fish through membrane progesterone receptor alpha (mPRα). Society for Integrative and Comparative Biology. Austin, TX, USA January 3-7 2020 Presentations at all scientific meetingss this summer wee canceled due to CORVID-19 Changes/Problems:Problems: the shut down of the Univeristy of Texas in mid-March due to the CORVID-19 outbreak prevented further progress with completing the objectives of the project. In particular initiating experiments on cros -talk of the signaling pathways, Experiments 3 and 4, because the sperm motility assays did not permit social distancing. Shut down of the univeristy for 2 months has also slowed progress in analysis of mPR protein concnetraations and activation of second messenger pathways in sperm saamples collected earlier What opportunities for training and professional development has the project provided?• 8 months of postdoctoral training for (1) postdoc, Laura Jenkins How have the results been disseminated to communities of interest?As presentations to scientists and the aquaculture industry- Peter Thomas*, Yefei Pang, Wenxian Tan. Rapid progestin upregulation of sperm motility in marine fish through membrane progesterone receptor alpha (mPR?). Society for Integrative and Comparative Biology. Austin, TX, USA January 3-7 2020. Peter Thomas*, Yefei Pang, Wenxian Tan. Rapid progestin induction of southern flounder sperm hypermotility through a nongenomic mechanism. Aquaculture. New Orleans, Louisiana, USA March 7-11 2019. What do you plan to do during the next reporting period to accomplish the goals?Summary: In the first part of year 3, Western blotting and commercial assays will be used to determine the roles of the Pi3K/Akt/Pde and Egfr/Erk1/2 pathways in progestin induction of flounder sperm motility (Objective 1, Experiments 1-3; Objective 3, Experiment 9A). Additionally, flounder will be set up on 3 photoperiod temperature cycles to further extend the season for sperm motility and fertilization experiments. In the second part of year 3, more fish will be collected to characterize seasonal sperm motility responsiveness to 2nd messenger drug stimulation compared to progestin stimulation of sperm hypermotility (Objective 3, Experiment 8). Sperm motility assays to determine the roles of the Pi3K/Akt/Pde and Egfr/Erk1/2 pathways in progestin induction of flounder sperm motility (Objective 1, Experiments 1-2) will be finalized. Cross talk between the 2nd messenger pathways and calcium will be completed (Objective 2, Experiments 4 and 5), as will experiments to determine inter- and intra-laboratory influences on sperm motility and responsiveness of sperm to drug treatments activating 2nd messenger pathways (Objective 3, Experiments 9 and 10). Note: Progress towards these goals is dependent on reopening of the University of Texas and the collaborating laboratories and a return to normal operations.
Impacts
What was accomplished under these goals?
Efforts early in year 2 served mainly to collect and acclimate new fish. Renovation of fish culture facilities after damage sustained by Hurricane Harvey (August 2017) are ongoing. In particular, the sea water system has not been repaired which resulted in poor water quality resulting in the loss of all the fish collected at the beginning of Year 1 of the project. Over 40 males were collected in the spring in year 2 and an additional 60 male and 20 female flounder were collected and acclimated in the fall of 2019. Fish were set up on 2 photoperiod cycles, one matching the ambient light and temperature and one delayed about two months, in order to extend the sampling season and provide more opportunities to conduct sperm motility experiments. Additionally, Atlantic croaker were cycled over the summer, and new postdoc Dr. Laura Jenkins, was able to train on sperm motility assays prior to experimenting on flounder. By the end of year 2, all captive fish were individually tagged (8mm PIT tags, Biomark, Boise, ID) and measured, with digital records of sampling events traceable over time. Year 2 efforts then focused on determining the roles of Acy/cAMP, Pi3k/Akt/Pde, and Egfr pathways in the progestin induction of sperm hypermotility in flounder sperm (Objective 1, Experiments 1 and 2) and on initiating studies on the efficacy of bypassing mPRa to induce hypermotility through activation of the Egfr- and Acy/cAMP-dependent pathways (Objective 3, Experiment 9). Acy agonist forskolin (1 uM, 10 uM) significantly increased sperm motility and fertilization, respectively, in low motility sperm (~100um/sec) when 20B-S (20 nM) also significantly increased sperm motility and fertilization, supporting the role of the Acy/cAMP pathway in sperm motility. Preincubation (30-min) with Pi3k inhibitor Wortmannin (1nM), AKT inhibitor ML-9 (25uM), or PDE inhibitor Cilostamide (100nM), each significantly inhibited progestin stimulation of sperm hypermotility, supporting the role for the Pi3k/Akt/Pde pathway for sperm motility. Trials were added for Wortmannin and initiated for Pi3K inhibitor LY294002 and PDE inhibitor Rolipram (Objective 1, Experiments 1A, 1B, 1D). Human EGF (100 nM) significantly increased sperm motility in low motility sperm when 20B-S (20 nM) also significantly increase sperm motility (Objective 1, Experiment 2A), confirming that the Egfr pathway is involved in regulating flounder sperm motility. EGF (100 uM) and Forskolin (1 uM) also significantly increased sperm motility in low motility sperm when 20B-S was not effective. This indicates effective induction of hypermotility through activation of the Egfr and Acy/cAMP-dependent pathways while bypassing mPR-alpha (Objective 3, Experiment 9B), supporting a role for second messenger drug treatments for hypermotility. Preincubation with EGFr inhibitor AG1478 significantly inhibited progestin stimulation of sperm motility (Objective 1, Experiment 2A), further supporting the role for the Egfr pathway in induction of sperm hypermotility. Trials with a second inhibitor, AG825, as well as with selective Erk1/2 inhibitors with U0126 (500 nM) and PD98059 (10 ?M) were initiated (Objective 1, Experiment 3A). Additionally, Golf, AKT, and Erk1/2 were localized on sperm by immunocytochemistry using antibodies previously characterized for fish. PKA antibodies were also effectively evaluated for use on flounder sperm (Objective 1, Experiment 4ii). Objective 1 Exp 1A. (a) PI3K inhibitor, LY294002, preincubated with sperm prior to the 20?-S treatments, to determine whether the hypermotility response also involves Pi3K - trials initiated (b) Testing of PI3K inhibitor Wortmannin, tested previously, - completed (replicates added) Exp.1B. (a) AKT inhibitor, ML-9, tested as above, - completed Exp.1D. (a) PDE inhibitor, Rolipram, tested as above - trials initiated Exp.2A. (a) The ability of human EGF (100 nM) will be confirmed to determine if the Egfr pathway is involved in regulating flounder sperm motility - completed Exp.2B. (a) A follow up experiment examining the effects of pretreatment with inhibitors of Egfr transactivation, AG1478 on 20?-S induction of sperm hypermotility will be conducted to confirm the role of the Egfr pathway in this response - completed (b) Inhibitor of Egfr transactivation AG825 (5 ?M) tested as above - trials initiated Exp.2C. The effects of preincubation with U0126 (500 nM) and PD98059 (10 ?M), which selectively inhibit activation of Erk1/2, tested as above - trials initiated Exp.4ii. Localization of Golf, AKT, Egfr, Erk1/2 on sperm will be examined by immunocytochemistry using antibodies we have previously characterized for fish. PKA antibodies also evaluated for use on flounder sperm - completed (Yefei - EGFr?) Objective 3 Exp.9B. The efficacy of bypassing mPRa to induce hypermotility through activation of the EGFr- and Acy/cAMP-dependent pathways by treating low motility sperm with EGF (100nM) and forskolin (10um), respectively, will be investigated at monthly intervals - completed Progress Summary: Most of the proposed experiments in Objective 1, Experiments 1 and 2 were successfully completed. It was planned to begin Experiment 3 in year 2 but they were not conducted because sperm motility experiments had to stop in early March due to closure of the Marine Science Institute in response to the CORVID-19 outbreak. Since the shutdown laboratory research on detection of signaling molecules and mPR? in sperm by Western blot analysis has slowed considerably and will continue to proceed at a much slower pace until the University of Texas opens again.
Publications
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Progress 03/01/18 to 02/28/19
Outputs
Target Audience:Scientific presentations were made at three international scientific meetings as well as at the Animal Reproduction Project Director Meeting Scientists: reproductive and sperm physiologists, endocrinologists, academic finfish aquaculturists Industry: finfish aquaculturists, assisted reproduction (in vitro fertilization) Changes/Problems:Problems: Hurricane Harvey damaged the fish holding facilites which have taken an extended period to repair. New IACUC requirements have delayed approval of many of the fish holding rooms. Consequently the first group of flounder collected in November 2018 were housed under suboptimal conditions and succumbed to a parasitic infection. New fish were collected this year and are now housed in the proper fish holding rooms and have acclimated well. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Presentations Peter Thomas,* Yefei Pang. Enhancement of male southern flounder broodstock fertility by direct activation of sperm 2nd messenger pathways down stream of mPRalpha.2018 Animal Reproduction Project Director Meeting. July 9-10. New Orleans Peter Thomas*, Yefei Pang, Wenxian Tan. Multiple signaling pathways mediate progestin hormone induction of sperm motility in southern flounder through membrane progesterone receptor alpha. Annual meeting Society for the Study of Reproduction. New Orleans, USA. Peter Thomas*, Yefei Pang, Wenxian Tan. Progestin induction of sperm hypermotility in southern flounder through mPRα is mediated by activation of multiple signaling pathways.11th International Symposium on Reproductive Physiology of Fish. Manaus, Brazil June 3-0, 2018 Peter Thomas*Yefei Pang, Wenxian Tan. Mechanism of progestin hormone stimulation of hypermotility in Southern Flounder sperm. Fish Reproduction Symposium. 13th International Congress on the Biology of Fish. Calgary, Canada, July 15-19, 2018 . What do you plan to do during the next reporting period to accomplish the goals?Research will begin in Yr. 2 and will focus on determining the roles of the Pi3k/Akt/Pde and Egfr/Erk1/2 pathways in progestin induction of flounder sperm hypermotility (Objective 1; Exps. 1 & 2). Investigations of cross-talk between the signaling pathways in progestin regulation of sperm motility (Objective 1; Exps. 3 & 4) will be conducted during the second half of Yr.2. Studies to characterize sperm motility and its responsiveness to 2nd messenger drug stimulation compared to progestin stimulation of hypermotility in flounder sperm during the reproductive season (Objective 3, Exp. 8) will also be initiated in Yr. 2
Impacts
What was accomplished under these goals?
Efforts in Year 1 as outlined in the project description were solely to collect southern flounder and acclimate them to laboratory conditions. The fish are being exposed to a normal seasonal temperature/photoperiod regime to promote gametogenesis. As explained in the project description this protracted acclimation period is critical for obtaining reproducible data from sperm motility experiment. Research will begin in the fall of year 2. More than 60 flounder were collected in the fall of 2018 with the help of a commercial fisherman. The fish culture facilities had been closed since Hurricane Harvey (August 2017) and extensive renovations and additional new criteria had to be met before the University of Texas IACUC committee would allow most of the facilities to be used to house the flounder. Only one room which had 8 foot diameter tanks was authorized for use when we collected the fish in November. The flounder were kept in these tanks for several months but did not eat regularly because the tanks were too small. In addition the tanks were overcrowded and had to be maintained on running seawater. Inevitably all the fish became infected with a parasite (Amyloodinium sp.) from the incoming seawater in February 2019 and died overnight due to asphyxiation. Southern flounder migrate from offshore back into the estuaries in the late spring and returned earlier this year due to mild conditions. Our guide was able to help us collect approximately 120 southern flounder in February and we were able to restock the fish tanks. At the end of February approval was granted by IACUC to use several more rooms with larger tanks and the fish were moved to 10 foot and 12 foot diameter tanks. The fish have acclimated to these larger tanks well and have begun to eat dead fish. In addition, the tanks are on recirculation so that re-infestation with Amyloodinum is no longer a concern. We now have sufficient fish for our experiments in year 2 and they should be ready for experimentation by October this year. An additional goal for Year 1 was to advertise the research project at scientific meetings in order to recruit a postdoctoral fellow to work on the project. Presentations were made at three international meetings which generated considerable interest from prospective postdoctoral fellows. A recent Ph.D with extensive experience with fish, Dr. Laura Jenkins, will join the research team on August 1, 2019 to conduct the sperm motility experiments this winter. In addition, a paper reviewing the current state of knowledge on progestin induction of sperm motility in marine fish is in press in General and Comparative Endocrinology.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2019
Citation:
Tan, W., Pang, Y., Tubbs, C., Thomas, P. in press. Induction of sperm hypermotility through membrane progestin receptor alpha (mPR?): a teleost model of rapid, multifaceted, nongenomic progestin signaling. General and Comparative Endocrinology. https://doi.org/10.1016/j.ygcen.2018.12.002
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