RESUMO
Incorporation of bovine serum-derived albumin formulation (AlbuMAX) into a basic culture medium, MEMα, enables the completion of in vitro spermatogenesis through testicular tissue culture in mice. However, this medium was not effective in other animals. Therefore, we sought an alternative approach for in vitro spermatogenesis using a synthetic medium without AlbuMAX and aimed to identify its essential components. In addition to factors known to be important for spermatogenesis, such as retinoic acid and reproductive hormones, we found that antioxidants (vitamin E, vitamin C, and glutathione) and lysophospholipids are vital for in vitro spermatogenesis. Moreover, based on our experience with microfluidic devices (MFD), we developed an alternative approach, the PDMS-ceiling method (PC method), which involves simply covering the tissue with a flat chip made of PDMS, a silicone resin material used in MFD. The PC method, while straightforward, integrates the advantages of MFD, enabling improved and uniform oxygen and nutrient supply via tissue flattening. Furthermore, our studies underscored the significance of lowering the oxygen concentration to 10-15%. Using an integrated cultivation method based on these findings, we successfully achieved in vitro spermatogenesis in rats, which has been a long-standing challenge. Further improvements in culture conditions would pave the way for spermatogenesis completion in diverse animal species.
Assuntos
Antioxidantes , Espermatogênese , Masculino , Camundongos , Animais , Ratos , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Testículo/metabolismo , Glutationa/metabolismo , Oxigênio/metabolismoRESUMO
PTBP1, a well-conserved RNA-binding protein, regulates cellular development by tuning posttranscriptional mRNA modification such as alternative splicing (AS) or mRNA stabilization. We previously revealed that the loss of Ptbp1 in spermatogonia causes the dysregulation of spermatogenesis, but the molecular mechanisms by which PTBP1 regulates spermatogonium homeostasis are unclear. In this study, changes of AS or transcriptome in Ptbp1-knockout (KO) germline stem cells (GSC), an in vitro model of proliferating spermatogonia, was determined by next generation sequencing. We identified more than 200 differentially expressed genes, as well as 85 genes with altered AS due to the loss of PTBP1. Surprisingly, no differentially expressed genes overlapped with different AS genes in Ptbp1-KO GSC. In addition, we observed that the mRNA expression of Nanos3, an essential gene for normal spermatogenesis, was significantly decreased in Ptbp1-KO spermatogonia. We also revealed that PTBP1 protein binds to Nanos3 mRNA in spermatogonia. Furthermore, Nanos3+/-;Ptbp1+/- mice exhibited abnormal spermatogenesis, which resembled the effects of germ cell-specific Ptbp1 KO, whereas no significant abnormality was observed in mice heterozygous for either gene alone. These data implied that PTBP1 regulates alternative splicing and transcriptome in spermatogonia under different molecular pathways, and contributes spermatogenesis, at least in part, in concert with NANOS3.
Assuntos
Processamento Alternativo , Regulação da Expressão Gênica , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteínas de Ligação a RNA/metabolismo , Espermatogênese/fisiologia , Espermatogônias/metabolismo , Animais , Deleção de Genes , Genes Reguladores , Células Germinativas/citologia , Heterozigoto , Infertilidade Masculina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a RNA/genética , RNA-Seq , Testículo/metabolismo , TranscriptomaRESUMO
Infertility is considered a global health issue as it currently affects one in every six couples, with female factors reckoned to contribute to partly or solely 50% of all infertility cases. Over a thousand genes are predicted to be highly expressed in the female reproductive system and around 150 genes in the ovary. However, some of their functions in fertility remain to be elucidated. In this study, 13 ovary and/or oocyte-enriched genes (Ccdc58, D930020B18Rik, Elobl, Fbxw15, Oas1h, Nlrp2, Pramel34, Pramel47, Pkd1l2, Sting1, Tspan4, Tubal3, Zar1l) were individually knocked out by the CRISPR/Cas9 system. Mating tests showed that these 13 mutant mouse lines were capable of producing offspring. In addition, we observed the histology section of ovaries and performed in vitro fertilization in five mutant mouse lines. We found no significant anomalies in terms of ovarian development and fertilization ability. In this study, 13 different mutant mouse lines generated by CRISPR/Cas9 genome editing technology revealed that these 13 genes are individually not essential for female fertility in mice.
Assuntos
Sistemas CRISPR-Cas , Fertilidade , Ovário , Animais , Feminino , Ovário/metabolismo , Fertilidade/genética , Camundongos , Sistemas CRISPR-Cas/genética , Oócitos/metabolismo , Masculino , Edição de Genes , Camundongos Knockout , Camundongos Endogâmicos C57BLRESUMO
In mammals, females undergo reproductive cessation with age, whereas male fertility gradually declines but persists almost throughout life. However, the detailed effects of ageing on germ cells during and after spermatogenesis, in the testis and epididymis, respectively, remain unclear. Here we comprehensively examined the in vivo male fertility and the overall organization of the testis and epididymis with age, focusing on spermatogenesis, and sperm function and fertility, in mice. We first found that in vivo male fertility decreased with age, which is independent of mating behaviors and testosterone levels. Second, overall sperm production in aged testes was decreased; about 20% of seminiferous tubules showed abnormalities such as germ cell depletion, sperm release failure, and perturbed germ cell associations, and the remaining 80% of tubules contained lower number of germ cells because of decreased proliferation of spermatogonia. Further, the spermatozoa in aged epididymides exhibited decreased total cell numbers, abnormal morphology/structure, decreased motility, and DNA damage, resulting in low fertilizing and developmental rates. We conclude that these multiple ageing effects on germ cells lead to decreased in vivo male fertility. Our present findings are useful to better understand the basic mechanism behind the ageing effect on male fertility in mammals including humans.
Assuntos
Epididimo , Testículo , Animais , Masculino , Camundongos , Envelhecimento , Fertilidade , Mamíferos , Sêmen , EspermatogôniasRESUMO
Male fertility decreases with aging, with spermatogenic decline being one of its causes. Altered testis environment is suggested as a cause of the phenotype; however, the associated mechanisms remain unclear. Herein, we investigated the age-related changes in testicular somatic cells on spermatogenic activity. The number and proliferation of spermatogonia significantly reduced with aging in mice. Interestingly, senescence-associated ß-galactosidase-positive cells appeared in testicular endothelial cell (EC) populations, but not in germ cell populations, with aging. Transcriptome analysis of ECs indicated that senescence occurred in the ECs of aged mice. Furthermore, the support capacity of ECs for spermatogonial proliferation significantly decreased with aging; however, the senolytic-induced removal of senescent cells from aged ECs restored their supporting capacity to a comparable level as that of young ECs. Our results suggest that the accumulation of senescent ECs in the testis is a potential factor contributing to the age-related decline in spermatogenic activity.
RESUMO
The classical organ culture method, in which tissue is placed at the gasâliquid interphase, is effective at inducing mouse spermatogenesis. However, due to reginal variations in the supply of oxygen and nutrients within a tissue, the progress of spermatogenesis was observed only in limited areas of a tissue. In addition, haploid cell formation and its differentiation to spermatozoon, i.e. spermiogenesis, were infrequent and inefficient. Here, we show that the polydimethylsiloxane (PDMS)-chip ceiling (PC) method, which ensures a uniform supply of nutrients and oxygen throughout the tissue by pressing it into a thin, flat shape, can provide control over the culture space. We used this method to culture testis tissue from neonatal mice, aged 1 to 4 days, and found that modulating the culture space during the experiment by replacing one chip with another that had a higher ceiling effectively increased tissue growth. This adjustment also induced more efficient spermatogenesis, with the process of spermiogenesis being particularly promoted. Meiotic cells were observed from culture day 14 onward, and haploid cells were confirmed at the end of each experiment. This technique was also shown to be a sensitive assay for testicular toxicity. Culture-space control will be a critical regulation parameter for sophisticated tissue culture experiments.
Assuntos
Espermatogênese , Testículo , Masculino , Camundongos , Animais , Animais Recém-Nascidos , Haploidia , Espermatogênese/fisiologia , EspermatozoidesRESUMO
An in vitro spermatogenesis method using mouse testicular tissue to produce fertile sperm was established more than a decade ago. Although this culture method has generally not been effective in other animal species, we recently succeeded in improving the culture condition to induce spermatogenesis of rats up to the round spermatid stage. In the present study, we introduced acrosin-EGFP transgenic rats in order to clearly monitor the production of haploid cells during spermatogenesis in vitro. In addition, a metabolomic analysis of the culture media during cultivation revealed the metabolic dynamics of the testis tissue. By modifying the culture media based on these results, we were able to induce rat spermatogenesis repeatedly up to haploid cell production, including the formation of elongating spermatids, which was confirmed histologically and immunohistochemically. Finally, we performed a microinsemination experiment with in vitro produced spermatids, which resulted in the production of healthy and fertile offspring. This is the first demonstration of the in vitro production of functional haploid cells that yielded offspring in animals other than mice. These results are expected to provide a basis for the development of an in vitro spermatogenesis system applicable to many other mammals.
Assuntos
Espermátides , Testículo , Masculino , Ratos , Camundongos , Animais , Espermátides/metabolismo , Testículo/metabolismo , Sêmen , Espermatogênese/fisiologia , Ratos Transgênicos , Meios de Cultura/farmacologia , MamíferosRESUMO
Gene targeting of embryonic stem (ES) cells followed by chimera production has been conventionally used for developing gene-manipulated mice. Although direct knock-in (KI) using murine zygote via CRISPR/Cas9-mediated genome editing has been reported, ES cell targeting still has merits, e.g., high throughput work can be performed in vitro. In this study, we first compared the KI efficiency of mouse ES cells with CRISPR/Cas9 expression vector and ribonucleoprotein (RNP), and confirmed that KI efficiency was significantly increased by using RNP. Using CRISPR/Cas9 RNP and circular plasmid with homologous arms as a targeting vector, knock-in within ES cell clones could be obtained efficiently without drug selection, thus potentially shortening the vector construction or cell culture period. Moreover, by incorporating a drug-resistant cassette into the targeting vectors, double DNA KI can be simultaneously achieved at high efficiency by a single electroporation. This technique will help to facilitate the production of genetically modified mouse models that are fundamental for exploring topics related to human and mammalian biology.
Assuntos
Sistemas CRISPR-Cas , Ribonucleoproteínas , Animais , Camundongos , Sistemas CRISPR-Cas/genética , DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Edição de Genes/métodos , Técnicas de Introdução de Genes , Plasmídeos/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoRESUMO
Calcium-binding proteins regulate ion metabolism and the necessary signaling pathways for the maturational events of sperm. Our aim is to identify the novel calcium-binding proteins in testis. The gene EFCAB2 (GenBank NM_026626.3, NP_080902.1) was not previously examined, and its properties and exact mechanisms of action are unknown. In this study, we performed phylogenetic and structure prediction analyses of EFCAB2, which displays definitive structural features. Additionally, the distribution, localization, and calcium binding ability of mouse EFCAB2 were investigated. Results revealed extensive conservation of EFCAB2 among different eukaryotic orthologs. The constructed 3D model predicted that mouse EFCAB2 contains seven α-helices and two EF-hand motifs. The first EF-hand motif is located in N-terminal, while the second is located in C-terminal. By aligning the 3D structure of Ca2+-binding loops from EFCAB2 with calmodulin, we predicted six residues that might be involved in Ca2+ binding. The distribution of the Efcab2 mRNA, as determined by northern blotting, was detected only in the testis among mouse tissues. Native and recombinant EFCAB2 protein were detected by western blotting as one band at 20 kDa. In situ hybridization and immunohistochemical analyses showed its localization specifically in spermatogenic cells from primary spermatocytes to elongate spermatids within the seminiferous epithelium, but neither spermatogonia nor somatic cells were expressed. Moreover, EFCAB2 was specifically localized to the principal piece of cauda epididymal sperm flagellum. Furthermore, the analyses of purified recombinant EFCAB2 by Stains-all, ruthenium red staining, and by applying in vitro autoradiography assay showed that the physiological function of this protein is Ca2+ binding. These results suggested that EFCAB2 might be involved in the control of sperm flagellar movement. Altogether, here we describe about EFCAB2 as a novel calcium-binding protein in mouse testis and sperm.