- MeSH
- Radiotherapy Dosage MeSH
- Radiation Injuries, Experimental MeSH
- Mice MeSH
- Spermatozoa radiation effects MeSH
- Testis radiation effects MeSH
- Dose-Response Relationship, Radiation MeSH
- Check Tag
- Mice MeSH
- Publication type
- Comparative Study MeSH
Common carp (Cyprinus carpio) is one of the most cultured fish species over the world with many different breeds and plenty of published protocols for sperm cryopreservation. However, data regarding preservation of gonadal tissue and surrogate production is still missing. A protocol for freezing common carp spermatogonia was developed through varying different factors along a set of serial subsequent experiments. Among the six cryoprotectants tested, the best survival was achieved with dimethyl sulfoxide (Me2SO). In the next experiment, a wide range of cooling rates (0.5-10°C/min) and different concentrations of Me2SO were tested resulting in the highest survival achieved using 2 M Me2SO and cooling rate of -1°C/min. When testing different tissue sizes and incubation times in the cryomedia, the highest viability was observed when incubating 100 mg tissue fragments for 30 min. Finally, sugar supplementation did not yield significant differences. When testing different equilibration (ES) and vitrification solutions (VS) used for needle-immersed vitrification, no significant differences were observed between the tested groups. Additionally, varied exposure time to VS did not improve the vitrification outcome where the viability was 4-fold lower than that of freezing. The functionality of cryopreserved cells was tested by interspecific transplantation into sterilized goldfish recipients. The exogenous origin of the germ cells in gonads of goldfish recipient was confirmed by molecular markers and incorporation rate was over 40% at 3 months post-transplantation. Results of this study can serve for long-term preservation of germplasm in carp which can be recovered in a surrogate recipient.
- MeSH
- Time Factors MeSH
- Dimethyl Sulfoxide pharmacology MeSH
- Carps * MeSH
- Cryopreservation * MeSH
- Spermatogonia * cytology transplantation MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
PIWI-interacting RNAs (piRNAs) support the germline by suppressing retrotransposons. Studies of the pathway in mice have strongly shaped the view that mammalian piRNAs are essential for male but not for female fertility. Here, we report that the role of the piRNA pathway substantially differs in golden hamsters (Mesocricetus auratus), the piRNA pathway setup of which more closely resembles that of other mammals, including humans. The loss of the Mov10l1 RNA helicase-an essential piRNA biogenesis factor-leads to striking phenotypes in both sexes. In contrast to mice, female Mov10l1-/- hamsters are sterile because their oocytes do not sustain zygotic development. Furthermore, Mov10l1-/- male hamsters have impaired establishment of spermatogonia accompanied by transcriptome dysregulation and an expression surge of a young retrotransposon subfamily. Our results show that the mammalian piRNA pathway has essential roles in both sexes and its adaptive nature allows it to manage emerging genomic threats and acquire new critical roles in the germline.
- MeSH
- Cricetinae MeSH
- Mesocricetus metabolism MeSH
- RNA, Small Interfering genetics MeSH
- Oocytes metabolism pathology MeSH
- Retroelements physiology MeSH
- RNA Helicases genetics MeSH
- Spermatogenesis genetics physiology MeSH
- Spermatogonia metabolism pathology MeSH
- Testis metabolism MeSH
- Gene Silencing physiology MeSH
- Animals MeSH
- Check Tag
- Cricetinae MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
This study aimed to provide a molecular signature for enriched adult human stem/progenitor spermatogonia during short-term (<2 weeks) and long-term culture (up to more than 14 months) in comparison to human testicular fibroblasts and human embryonic stem cells. Human spermatogonia were isolated by CD49f magnetic activated cell sorting and collagen(-)/laminin(+) matrix binding from primary testis cultures obtained from ten adult men. For transcriptomic analysis, single spermatogonia-like cells were collected based on their morphology and dimensions using a micromanipulation system from the enriched germ cell cultures. Immunocytochemical, RT-PCR and microarray analyses revealed that the analyzed populations of cells were distinct at the molecular level. The germ- and pluripotency-associated genes and genes of differentiation/spermatogenesis pathway were highly expressed in enriched short-term cultured spermatogonia. After long-term culture, a proportion of cells retained and aggravated the "spermatogonial" gene expression profile with the expression of germ and pluripotency-associated genes, while in the majority of long-term cultured cells this molecular profile, typical for the differentiation pathway, was reduced and more genes related to the extracellular matrix production and attachment were expressed. The approach we provide here to study the molecular status of in vitro cultured spermatogonia may be important to optimize the culture conditions and to evaluate the germ cell plasticity in the future.
- MeSH
- Cell Differentiation genetics MeSH
- Cell Culture Techniques methods MeSH
- Adult MeSH
- Embryonic Stem Cells physiology MeSH
- Fibroblasts physiology MeSH
- Cells, Cultured MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Cell Separation methods MeSH
- Spermatogenesis genetics MeSH
- Spermatogonia physiology MeSH
- Gene Expression Profiling methods MeSH
- Testis physiology MeSH
- Transcriptome genetics MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Male MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
To explore the mechanism whereby cGAS-STING pathway regulates the pyroptosis of cryptorchidism cells, with a view to finding a new strategy for clinically treating cryptorchidism-induced infertility. Spermatogonial GC-1 cells were heat stimulated to simulate the heat hurt microenvironment of cryptorchidism. The cell viability was assayed by CCK-8, and cellular DNA damage was detected by gamma-H2AX immunofluo-rescence assay. Flow cytometry was employed to assess pyroptosis index, while western blot, ELISA and PCR were used to examine the expressions of pyroptosis-related proteins (Caspase-1, IL-1beta, NLRP3) and cGAS-STING pathway proteins (cGAS, STING). After STING silencing by siRNA, the expressions of pyroptosis-related proteins were determined. Pyroptosis occurred after heat stimulation of cells. Morphological detection found cell swelling and karyopyknosis. According to the gamma-H2AX immunofluorescence (IFA) assay, the endonuclear green fluorescence was significantly enhanced, the gamma-H2AX content markedly increased, and the endonuclear DNA was damaged. Flow cytometry revealed a significant increase in pyroptosis index. Western blot and PCR assays showed that the expressions of intracellular pyrogenic proteins like Caspase-1, NLRP3 and GSDMD were elevated. The increased STING protein and gene expressions in cGAS-STING pathway suggested that the pathway was intracellularly activated. Silencing STING protein in cGAS-STING pathway led to significantly inhibited pyroptosis. These results indicate that cGAS-STING pathway plays an important role in heat stress-induced pyroptosis of spermatogonial cells. After heat stimulation of spermatogonial GC-1 cells, pyroptosis was induced and cGAS-STING pathway was activated. This study can further enrich and improve the molecular mechanism of cryptorchidism.
- MeSH
- Acetates * MeSH
- Chromogranin A MeSH
- Phenols * MeSH
- Caspase 1 MeSH
- Cryptorchidism * MeSH
- Humans MeSH
- Nucleotidyltransferases MeSH
- NLR Family, Pyrin Domain-Containing 3 Protein MeSH
- Pyroptosis MeSH
- Signal Transduction MeSH
- Spermatogonia MeSH
- Heat Stroke * MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Publication type
- Journal Article MeSH
Sturgeons are among the most ancient linages of actinopterygians. At present, many sturgeon species are critically endangered. Surrogate production could be used as an affordable and a time-efficient method for endangered sturgeons. Our study established a method for identifying and isolating type A spermatogonia from different developmental stages of testes using flow cytometric cell sorting (FCM). Flow cytometric analysis of a whole testicular cell suspension showed several well-distinguished cell populations formed according to different values of light scatter parameters. FCM of these different cell populations was performed directly on glass slides for further immunocytochemistry to identify germ cells. Results showed that the cell population in gate P1 on a flow cytometry plot (with high forward scatter and high side scatter parameter values) contains the highest amount of type A spermatogonia. The sorted cell populations were characterized by expression profiles of 10 germ cell specific genes. The result confirmed that setting up for the P1 gate could precisely sort type A spermatogonia in all tested testicular developmental stages. The P2 gate, which was with lower forward scatter and side scatter values mostly, contained type B spermatogonia at a later maturing stage. Moreover, expressions of plzf, dnd, boule, and kitr were significantly higher in type A spermatogonia than in later developed germ cells. In addition, plzf was firstly found as a reliable marker to identify type A spermatogonia, which filled the gap of identification of spermatogonial stem cells in sterlet. It is expected to increase the efficiency of germ stem cell culture and transplantation with plzf identification. Our study thus first addressed a phenotypic characterization of a pure type A spermatogonia population in sterlet. FCM strategy can improve the production of sturgeons with surrogate broodstock and further the analysis of the cellular and molecular mechanisms of sturgeon germ cell development.
- Publication type
- Journal Article MeSH
Testis development and ultrastructure of spermatogenic cells and spermatozoa of burbot Lota lota, a commercially important cold freshwater fish, were studied by light and transmission electron microscopy. Spermatogonia, spermatocytes, spermatids, and spermatozoa are distributed along the seminiferous tubules. Electron-dense bodies appear in germ cells from primary spermatogonia to secondary spermatocytes. We identified three distinct stages of spermatid cell differentiation based on chromatin condensation, development of the flagellum, formation of a nuclear fossa, and elimination of excess cytoplasm. Spermatozoa were anacrosomal and characterized by location of the centrioles outside the nuclear fossa and incomplete perpendicular arrangement of the centrioles. The sperm flagellum displayed an axoneme with nine doublets of peripheral microtubules and two central microtubules. These results provide valuable information for burbot taxonomy and may clarify the process of spermatogenesis for this species.
- MeSH
- Cells, Cultured MeSH
- Fishes metabolism MeSH
- Sertoli Cells ultrastructure MeSH
- Spermatids ultrastructure MeSH
- Spermatogenesis * MeSH
- Spermatogonia ultrastructure MeSH
- Spermatozoa ultrastructure MeSH
- Testis cytology ultrastructure MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
