{"title":"亚洲鲈鱼(Lates calcarifer)整个睾丸的冷冻保存及其对凋亡、生殖细胞特异性基因表达、生殖细胞可移植性和 DNA 甲基化的影响","authors":"","doi":"10.1016/j.theriogenology.2024.08.025","DOIUrl":null,"url":null,"abstract":"<div><p>Cryopreservation of spermatogonia could be a useful tool to preserve the genetic resources of fish, which could be further restored via germ cell transplantation. In this study, the protocol for the cryopreservation of the spermatogonia of Asian sea bass (<em>Lates calcarifer</em>), an economically important fishery resource in the Indo-West Pacific, was optimised. The impact of the cryopreservation technique on cell viability and apoptosis, expression of several genes related to immature germ cell markers, transplantability in allogeneic recipients, and global DNA methylation was evaluated. The slow-freezing method was performed for the cryopreservation of immature testis tissue, which contains a high proportion of spermatogonia. The optimal condition that yielded the highest recovery rate of post-thawed spermatogonia included a cryomedium containing Leibovitz's (L-15) medium and 10 % dimethyl sulfoxide, ice equilibration for 60 min before freezing, and subsequent thawing at 4 °C for 8 min. Moreover, a higher number of early and late apoptotic cells was detected in the cryopreserved than in the fresh testes, suggesting that apoptosis could result in reduced viability. The expression levels of <em>dazl</em> decreased in the cryopreserved testes; however, there were no significant differences in the expression levels of <em>nanos2</em> or <em>nanos3</em> between the fresh and cryopreserved testes. Although qRT-PCR showed lower <em>vasa</em> expression in cryopreserved testicular cells, <em>in situ</em> hybridisation showed expressed <em>vasa</em> in the cryopreserved testicular cells. Post-thawed spermatogonia could be incorporated into the genital ridge of allogeneic recipients, suggesting that cryopreserved spermatogonia exhibit transplantability characteristics. Compared with fresh testes, significant changes in the proportion of DNA methylation (decreased 5-mC and 5-caC) were observed in cryomedium-free testicular cells, whereas those of the cryopreserved cells were not significantly different. Therefore, the method we developed for the cryopreservation of the spermatogonia of Asian sea bass enabled post-thaw cells to retain several stemness characteristics and maintain their epigenetic stability.</p></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":null,"pages":null},"PeriodicalIF":2.4000,"publicationDate":"2024-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Cryopreservation of the whole testes of Asian sea bass (Lates calcarifer) and its effects on apoptosis, germ cell-specific gene expression, germ cell transplantability, and DNA methylation\",\"authors\":\"\",\"doi\":\"10.1016/j.theriogenology.2024.08.025\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Cryopreservation of spermatogonia could be a useful tool to preserve the genetic resources of fish, which could be further restored via germ cell transplantation. In this study, the protocol for the cryopreservation of the spermatogonia of Asian sea bass (<em>Lates calcarifer</em>), an economically important fishery resource in the Indo-West Pacific, was optimised. The impact of the cryopreservation technique on cell viability and apoptosis, expression of several genes related to immature germ cell markers, transplantability in allogeneic recipients, and global DNA methylation was evaluated. The slow-freezing method was performed for the cryopreservation of immature testis tissue, which contains a high proportion of spermatogonia. The optimal condition that yielded the highest recovery rate of post-thawed spermatogonia included a cryomedium containing Leibovitz's (L-15) medium and 10 % dimethyl sulfoxide, ice equilibration for 60 min before freezing, and subsequent thawing at 4 °C for 8 min. Moreover, a higher number of early and late apoptotic cells was detected in the cryopreserved than in the fresh testes, suggesting that apoptosis could result in reduced viability. The expression levels of <em>dazl</em> decreased in the cryopreserved testes; however, there were no significant differences in the expression levels of <em>nanos2</em> or <em>nanos3</em> between the fresh and cryopreserved testes. Although qRT-PCR showed lower <em>vasa</em> expression in cryopreserved testicular cells, <em>in situ</em> hybridisation showed expressed <em>vasa</em> in the cryopreserved testicular cells. Post-thawed spermatogonia could be incorporated into the genital ridge of allogeneic recipients, suggesting that cryopreserved spermatogonia exhibit transplantability characteristics. Compared with fresh testes, significant changes in the proportion of DNA methylation (decreased 5-mC and 5-caC) were observed in cryomedium-free testicular cells, whereas those of the cryopreserved cells were not significantly different. Therefore, the method we developed for the cryopreservation of the spermatogonia of Asian sea bass enabled post-thaw cells to retain several stemness characteristics and maintain their epigenetic stability.</p></div>\",\"PeriodicalId\":23131,\"journal\":{\"name\":\"Theriogenology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.4000,\"publicationDate\":\"2024-08-22\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Theriogenology\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0093691X24003534\",\"RegionNum\":2,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"REPRODUCTIVE BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Theriogenology","FirstCategoryId":"97","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0093691X24003534","RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"REPRODUCTIVE BIOLOGY","Score":null,"Total":0}
Cryopreservation of the whole testes of Asian sea bass (Lates calcarifer) and its effects on apoptosis, germ cell-specific gene expression, germ cell transplantability, and DNA methylation
Cryopreservation of spermatogonia could be a useful tool to preserve the genetic resources of fish, which could be further restored via germ cell transplantation. In this study, the protocol for the cryopreservation of the spermatogonia of Asian sea bass (Lates calcarifer), an economically important fishery resource in the Indo-West Pacific, was optimised. The impact of the cryopreservation technique on cell viability and apoptosis, expression of several genes related to immature germ cell markers, transplantability in allogeneic recipients, and global DNA methylation was evaluated. The slow-freezing method was performed for the cryopreservation of immature testis tissue, which contains a high proportion of spermatogonia. The optimal condition that yielded the highest recovery rate of post-thawed spermatogonia included a cryomedium containing Leibovitz's (L-15) medium and 10 % dimethyl sulfoxide, ice equilibration for 60 min before freezing, and subsequent thawing at 4 °C for 8 min. Moreover, a higher number of early and late apoptotic cells was detected in the cryopreserved than in the fresh testes, suggesting that apoptosis could result in reduced viability. The expression levels of dazl decreased in the cryopreserved testes; however, there were no significant differences in the expression levels of nanos2 or nanos3 between the fresh and cryopreserved testes. Although qRT-PCR showed lower vasa expression in cryopreserved testicular cells, in situ hybridisation showed expressed vasa in the cryopreserved testicular cells. Post-thawed spermatogonia could be incorporated into the genital ridge of allogeneic recipients, suggesting that cryopreserved spermatogonia exhibit transplantability characteristics. Compared with fresh testes, significant changes in the proportion of DNA methylation (decreased 5-mC and 5-caC) were observed in cryomedium-free testicular cells, whereas those of the cryopreserved cells were not significantly different. Therefore, the method we developed for the cryopreservation of the spermatogonia of Asian sea bass enabled post-thaw cells to retain several stemness characteristics and maintain their epigenetic stability.
期刊介绍:
Theriogenology provides an international forum for researchers, clinicians, and industry professionals in animal reproductive biology. This acclaimed journal publishes articles on a wide range of topics in reproductive and developmental biology, of domestic mammal, avian, and aquatic species as well as wild species which are the object of veterinary care in research or conservation programs.