RESUMO
A simple method for producing pseudopregnant mice supports pup production. In this study, pregnant ICR were obtained mice without mating with vasectomized mice via administration of mouse Kisspeptin-10 (mKp-10) and transferring blastocysts to the uterus. Blastocyst transfer after mKp-10 administration to mice with gapping and reddish pink vagina resulted in 65.2% (15/23) pregnancies, and 39.1% (34/87) of the transferred blastocysts showed full-term growth. Vaginal smears were observed for accurate estrus cycle determination, and subsequent administration of mKp10 to mice during the estrus stage and blastocyst transfer resulted in 95.2% (20/21) pregnancies and 50.7% (104/205) birth rates. Regarding 2-cell transfer after administration of mKp-10, 100% (8/8) of the mice became pregnant, and 45.0% (36/80) of the embryos were born. Administration of mKp-10 to mice during the estrus stage is a convenient way to generate pseudopregnant mice.
Assuntos
Pseudogravidez , Útero , Gravidez , Feminino , Camundongos , Animais , Camundongos Endogâmicos ICR , EstroRESUMO
Ovarian stimulation protocols are widely used to collect oocytes in assisted reproductive technologies (ARTs). Although the influence of ovarian stimulation on embryo quality has been described, this issue remains controversial. Here, we analyzed the influence of ovarian stimulation on developmental speed and chromosome segregation using live cell imaging. Female mice at the proestrus stage were separated by the appearance of the vagina as the non-stimulation (-) group, and other mice were administered pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) as the stimulation (+) groups. The cumulus-oocyte complexes from both groups were inseminated with sperm suspensions from the same male mice. Fertilization rates and developmental capacities were examined, and the developmental speed and frequency of chromosome segregation errors were measured by live-cell imaging using a Histone H2B-mCherry probe. The number of fertilized oocytes obtained was 1.4-fold more frequent in the stimulation (+) group. The developmental rate and chromosome stability did not differ between the groups. Image analysis showed that the mean speed of development in the stimulation (+) group was slightly higher than that in the non-stimulation (-) group. This increase in speed seemed to arise from the slight shortening of the 2- and 4-cell stages and third division lengths and consequent synchronization of cleavage timing in each embryo, not from the emergence of an extremely rapidly developing subpopulation of embryos. In conclusion, ovarian stimulation does not necessarily affect embryo quality but rather increases the chances of obtaining high-quality oocytes in mice.
Assuntos
Blastocisto , Desenvolvimento Embrionário , Oócitos , Indução da Ovulação , Animais , Feminino , Camundongos , Desenvolvimento Embrionário/fisiologia , Blastocisto/fisiologia , Masculino , Oócitos/fisiologia , Gravidez , Gonadotropinas Equinas/farmacologia , Gonadotropina Coriônica/farmacologia , Segregação de Cromossomos , Fertilização in vitro/métodosRESUMO
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
In preimplantation embryos, an abnormal chromosome number causes developmental failure and a reduction in the pregnancy rate. Conventional chromosome testing methods requiring biopsy reduce the risk of associated genetic diseases; nevertheless, the reduction in cell number also reduces the pregnancy rate. Therefore, we attempted to count the chromosomes in mouse embryos using super-resolution live-cell imaging as a new method of chromosome counting that does not reduce the cell number or viability. We counted the 40 chromosomes at the first mitosis by injecting embryos with histone H2B-mCherry mRNA under conditions by which pups could be obtained; however, the results were often an underestimation of chromosome number and varied by embryo and time point. Therefore, we developed a method to count the chromosomes via CRISPR/dCas-mediated live-cell fluorescence in situ hybridization targeting the sequence of the centromere region, enabling us to count the chromosomes more accurately in mouse embryos. The methodology presented here may provide useful information for assisted reproductive technologies, such as those used in livestock animals/humans, as a technique for assessing the chromosomal integrity of embryos prior to transfer.
Assuntos
Aneuploidia , Zigoto , Animais , Blastocisto/patologia , Centrômero/genética , Feminino , Hibridização in Situ Fluorescente , Camundongos , GravidezRESUMO
In assisted reproductive technology (ART)-derived embryos of non-rodent mammals, including humans and cattle, chromosome segregation errors are highly likely to occur during early cleavage division, resulting in aneuploidy, including mosaicism. However, the relationship between chromosomal segregation errors during early cleavage and subsequent embryonic development has not been detailed in these mammals. In the present study, we developed non-invasive live-cell imaging of chromosome segregation dynamics using a histone H2B-mCherry mRNA probe in bovine preimplantation embryos. Chromosome segregation errors in early cleavage affected blastocyst formation. Especially, embryos that underwent abnormal chromosome segregation (ACS) with multiple or large micronucleus formation rarely developed into blastocysts. Embryos with the severe ACS had prolonged cell cycle duration. After transfer of blastocysts with live-cell imaging of chromosome segregation to ten cows, six became pregnant and four of them gave full-term offspring. Interestingly, two of them were derived from blastocysts with ACS. Hence, chromosomal segregation errors with micronucleus formation during early cleavage can be a fatal hallmark of preimplantation embryogenesis in cattle. This technique has shown potential for understanding the relationship between chromosome segregation error and subsequent embryo development, and for selecting viable ART-derived embryos for medical and livestock production.
Assuntos
Aneuploidia , Blastocisto , Animais , Blastocisto/metabolismo , Bovinos , Segregação de Cromossomos , Desenvolvimento Embrionário/genética , Feminino , Mamíferos , Mosaicismo , GravidezRESUMO
Purpose: Our aim is to make an ideal embryo culture medium close to human oviduct fluid (HOF) components, and to evaluate the quality of this medium with embryo quality and clinical outcomes in assisted reproductive technology (ART) by a prospective randomized controlled trial (RCT). Methods: Study I: HOF was collected laparoscopically from patients (n = 28) with normal pelvic findings. According to HOF analysis results, the new medium "HiGROW OVIT®" (OVIT) was designed. Study II: Embryos (2 pronuclei (2PN) = 9633) were assigned from 1435 patients. The blastulation rate (BR), good BR (gBR), utilized (transferred/cryo-preserved) BR (uBR), pregnancy rate (PR), and miscarriage rate (MR) were compared between the OVIT and control groups by RCT. Results: The novel medium 'OVIT' was produced according to 31 HOF components. The concentrations of essential amino acids (e-AAs) were lower in OVIT than in current media, yet the opposite was true for ne-AA concentrations. gBR and uBR were higher in the OVIT group than in the control group. In the older female group, gBT and uBR were significantly higher in the OVIT group. Conclusions: The novel medium 'OVIT' was produced according to HOF data. The OVIT had significantly better embryo quality and clinical outcomes than the current media.
RESUMO
In vitro mouse spermatogenesis using a classical organ culture method became possible by supplementing basal culture medium with only the product of bovine serum albumin purified by chromatography (AlbuMAX), which indicated that AlbuMAX contained every chemical factor necessary for mouse spermatogenesis. However, since the identity of these factors was unclear, improvements in culture media and our understanding of the nutritional and signal substances required for spermatogenesis were hindered. In the present study, chemically defined media (CDM) without AlbuMAX was used to evaluate each supplementary factor and their combinations for the induction of spermatogenesis. Similar to in vivo conditions, retinoic acid, triiodothyronine (T3 ), and testosterone (T) were needed. Based on differences in spermatogenic competence between AlbuMAX, fetal bovine serum, and adult bovine serum, we identified α-tocopherol, which strongly promoted spermatogenesis when combined with ascorbic acid and glutathione. Differences were also observed in the abilities of lipids extracted from AlbuMAX using two different methods to induce spermatogenesis. This led to the identification of lysophospholipids, particularly lysophosphatidylcholine, lysophosphatidic acid, and lysophosphatidylserine, as important molecules for spermatogenesis. New CDM formulated based on these results induced and promoted spermatogenesis as efficiently as AlbuMAX-containing medium. In vitro spermatogenesis with CDM may provide a unique experimental system for research on spermatogenesis that cannot be performed in in vivo experiments.
Assuntos
Antioxidantes/farmacologia , Lisofosfolipídeos/farmacologia , Técnicas de Cultura de Órgãos/métodos , Espermatogênese , Testículo/citologia , Vitaminas/farmacologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Testículo/efeitos dos fármacos , Testículo/metabolismoRESUMO
Background: Cell culture technology has spread prolifically within a century, a variety of culture media has been designed. This review goes through the history, characteristics and current issues of animal-cell culture media. Methods: A literature search was performed on PubMed and Google Scholar between 1880 and May 2016 using appropriate keywords. Results: At the dawn of cell culture technology, the major components of media were naturally derived products such as serum. The field then gradually shifted to the use of chemical-based synthetic media because naturally derived ingredients have their disadvantages such as large batch-to-batch variation. Today, industrially important cells can be cultured in synthetic media. Nevertheless, the combinations and concentrations of the components in these media remain to be optimized. In addition, serum-containing media are still in general use in the field of basic research. In the fields of assisted reproductive technologies and regenerative medicine, some of the medium components are naturally derived in nearly all instances. Conclusions: Further improvements of culture media are desirable, which will certainly contribute to a reduction in the experimental variation, enhance productivity among biopharmaceuticals, improve treatment outcomes of assisted reproductive technologies, and facilitate implementation and popularization of regenerative medicine.
RESUMO
Direct cleavage, a type of abnormal cleavage in which one zygote divides into three or more blastomeres, has been reported in mammals. The incidence of direct cleavage increases in zygotes with three or more pronuclei (multi-PN) and those showing abnormal pronuclei migration. However, there are few reports on the relationship between pronuclei and direct cleavage, and the effects of these relationships on subsequent embryogenesis have not been clarified. It is difficult to observe pronuclei under visible light, especially in bovine zygotes, because of abundant dark lipid droplets in the cytoplasm. We visualized pronuclei by removing lipid droplets from bovine zygotes and analyzed the relationship between the number of pronuclei and direct cleavage using time-lapse cinematography. The direct cleavage rate of multi-PN zygotes was 78.6%, which was significantly higher than that of zygotes with one pronucleus (1 PN, 0.0%) and two pronuclei (2 PN, 8.2%). Observation of pronuclei migration in 2 PN zygotes showed that 3.1% of 2 PN zygotes had non-apposed pronuclei. The direct cleavage rate of zygotes with non-apposed pronuclei was 66.7%, which was significantly higher than that of zygotes with apposed pronuclei (6.4%). Among multi-PN zygotes, the proportions of zygotes with apposed pronuclei and non-apposed pronuclei were 37.5% and 64.3%, respectively. The direct cleavage rate of multi-PN zygotes with non-apposed pronuclei was 100.0%, which was significantly higher than that of zygotes with apposed pronuclei (40.0%). Three-dimensional live-cell imaging of bovine zygotes injected with the mRNA-encoding histone H2B-mCherry showed that the direct cleavage rates of 2 PN and multi-PN zygotes bypassing syngamy were 63.2% and 75.5%, respectively. These rates were significantly higher than that of 2 PN and multi-PN zygotes that underwent syngamy (5.6% and 20.0%, respectively). Regardless of the number of pronuclei, a high frequency of direct cleavage was observed in zygotes in which the pronuclei did not migrate inward the cytoplasm and bypassed syngamy. These results suggest that abnormal fertilization such as multi-PN and migration error of pronuclei in cattle is the primary reason for direct cleavage during the first mitosis. Assessment of direct cleavage during the first mitosis allows exclusion of embryos with abnormal fertilization and may contribute to in vitro produced embryo transfer success.
Assuntos
Desenvolvimento Embrionário , Fertilização in vitro , Animais , Bovinos , Fertilização in vitro/veterinária , Zigoto , Mitose , Núcleo Celular , Fertilização , MamíferosRESUMO
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
To improve the performance of assisted reproductive technology, it is necessary to find an indicator that can identify and select embryos that will be born or be aborted. We searched for indicators of embryo selection by comparing born/abort mouse embryos. We found that asynchronous embryos during the 4-8-cell stage were predisposed to be aborted. In asynchronous mouse embryos, the nuclear translocation of YAP1 in some blastomeres and compaction were delayed, and the number of ICMs was reduced. Hence, it is possible that asynchronous embryos have abnormal differentiation. When the synchrony of human embryos was observed, it was confirmed that embryos that did not reach clinical pregnancy had asynchrony as in mice. This could make synchrony a universal indicator common to all animal species.
Assuntos
Diagnóstico Pré-Implantação , Animais , Blastocisto , Blastômeros , Embrião de Mamíferos , Feminino , Nascido Vivo , Camundongos , GravidezRESUMO
Zygotes require two accurate sets of parental chromosomes, one each from the mother and the father, to undergo normal embryogenesis. However, upon egg-sperm fusion in vertebrates, the zygote has three sets of chromosomes, one from the sperm and two from the egg. The zygote therefore eliminates one set of maternal chromosomes (but not the paternal chromosomes) into the polar body through meiosis, but how the paternal chromosomes are protected from maternal meiosis has been unclear. Here we report that RanGTP and F-actin dynamics prevent egg-sperm fusion in proximity to maternal chromosomes. RanGTP prevents the localization of Juno and CD9, egg membrane proteins that mediate sperm fusion, at the cell surface in proximity to maternal chromosomes. Following egg-sperm fusion, F-actin keeps paternal chromosomes away from maternal chromosomes. Disruption of these mechanisms causes the elimination of paternal chromosomes during maternal meiosis. This study reveals a novel critical mechanism that prevents aneuploidy in zygotes.
Assuntos
Citoesqueleto de Actina/metabolismo , Cromossomos/metabolismo , Fertilização , Proteína ran de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Camundongos , Camundongos EndogâmicosRESUMO
In the pre-implantation embryo, aneuploidy resulting from chromosome segregation error is considered responsible for pregnancy loss. However, only a few studies have examined the relationship between chromosome segregation errors during early cleavage and development. Here, we evaluated this relationship by live-cell imaging using the histone H2B-mCherry probe and subsequent single blastocyst transfer using mouse embryos obtained by in vitro fertilization. We showed that some embryos exhibiting early chromosomal segregation error and formation of micronuclei retained their developmental potential; however, the error affected the blastocyst/arrest ratio. Further, single-cell sequencing after live-cell imaging revealed that all embryos exhibiting micronuclei formation during 1st mitosis showed aneuploidy at the 2-cell stage. These results suggest that early chromosome segregation error causing micronuclei formation affects ploidy and development to blastocyst but does not necessarily cause developmental failure after the blastocyst stage. Our result suggests the importance of the selection of embryos that have reached blastocysts.
Assuntos
Blastocisto , Segregação de Cromossomos , Implantação do Embrião/genética , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Aneuploidia , Animais , Transferência Embrionária , Feminino , Fertilização in vitro , Camundongos , Camundongos Endogâmicos ICR , GravidezRESUMO
OBJECTIVE: p38 mitogen-activated protein (MAP) kinase (p38α) has drawn attention as a new target molecule for the treatment and diagnosis of cancer, and its overexpression and activation have been reported in various types of cancer. In this study, a single photon emission computed tomography (SPECT) imaging probe of p38α was developed to noninvasively image p38α activity for effective qualitative diagnosis of cancer. METHODS: Pyrrolepyridine derivatives, m-YTM and p-YTM, were designed and synthesized based on the structure of the p38α-selective inhibitor. Radioactive iodine-labeled m-YTM, [125I]m-YTM, was synthesized because m-YTM greatly inhibited the phosphorylation of p38α upon examining the inhibitory effects of the compounds. After investigating the binding affinity of [125I]m-YTM to the recombinant p38α, a saturation binding experiment using activated p38α and inactive p38α was performed to determine the binding site. Uptake of [125I]m-YTM into various cancer cell lines was investigated, and the pharmacokinetics was evaluated using tumor-bearing mice. RESULTS: The inhibitory activity of m-YTM was approximately 13 times higher than that of SB203580, a p38α-selective inhibitor. The binding site of [125I]m-YTM was estimated to be the p38α activating site, similar to that of SB203580, because the [125I]m-YTM bound strongly to both activated p38α and inactive p38α. Various different cancer cells incorporated [125I]m-YTM; however, its accumulation was significantly reduced by treatment with SB203580. Pharmacokinetics study of [125I]m-YTM in B-16 tumor-bearing mice was examined which revealed high accumulation of radioactivity in tumor tissues. The ratios of radioactivity in the B-16 tumor to that in blood were 3.1 and 50 after 1 and 24 h, respectively. The ratio of radioactivity in the tumor to that in blood in the tumor-bearing mice generated using other cancer cell lines was also ≥ 1 at 1 h after the administration of the probe. CONCLUSIONS: This study suggests that [123I]m-YTM has potential as a p38α imaging probe effective for various cancer types.
Assuntos
Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Neoplasias/diagnóstico por imagem , Neoplasias/metabolismo , Pirróis/química , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Animais , Transporte Biológico , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Fosforilação/efeitos dos fármacos , Pirróis/metabolismo , Pirróis/farmacocinética , Pirróis/farmacologia , Distribuição TecidualRESUMO
Nuclear/chromosomal integrity is an important prerequisite for the assessment of embryo quality in artificial reproductive technology. However, lipid-rich dark cytoplasm in bovine embryos prevents its observation by visible light microscopy. We performed live-cell imaging using confocal laser microscopy that allowed long-term imaging of nuclear/chromosomal dynamics in bovine in vitro fertilised (IVF) embryos. We analysed the relationship between nuclear/chromosomal aberrations and in vitro embryonic development and morphological blastocyst quality. Three-dimensional live-cell imaging of 369 embryos injected with mRNA encoding histone H2B-mCherry and enhanced green fluorescent protein (EGFP)-α-tubulin was performed from single-cell to blastocyst stage for eight days; 17.9% reached the blastocyst stage. Abnormalities in the number of pronuclei (PN), chromosomal segregation, cytokinesis, and blastomere number at first cleavage were observed at frequencies of 48.0%, 30.6%, 8.1%, and 22.2%, respectively, and 13.0%, 6.2%, 3.3%, and 13.4%, respectively, for abnormal embryos developed into blastocysts. A multivariate analysis showed that abnormal chromosome segregation (ACS) and multiple PN correlated with delayed timing and abnormal blastomere number at first cleavage, respectively. In morphologically transferrable blastocysts, 30-40% of embryos underwent ACS and had abnormal PN. Live-cell imaging may be useful for analysing the association between nuclear/chromosomal dynamics and embryonic development in bovine embryos.
Assuntos
Bovinos/embriologia , Segregação de Cromossomos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/ultraestrutura , Fertilização in vitro , Animais , Bovinos/genética , Sobrevivência Celular , Aberrações Cromossômicas , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/embriologia , Feminino , Proteínas de Fluorescência Verde/análise , Proteínas Luminescentes/análise , Microscopia Confocal/métodos , Imagem Óptica/métodos , Gravidez , Proteína Vermelha FluorescenteRESUMO
We previously reported the successful induction and completion of mouse spermatogenesis by culturing neonatal testis tissues. The culture medium consisted of α-minimum essential medium (α-MEM), supplemented with Knockout serum replacement (KSR) or AlbuMAX, neither of which were defined chemically. In this study, we formulated a chemically defined medium (CDM) that can induce mouse spermatogenesis under organ culture conditions. It was found that bovine serum albumin (BSA) purified through three different procedures had different effects on spermatogenesis. We also confirmed that retinoic acid (RA) played crucial roles in the onset of spermatogonial differentiation and meiotic initiation. The added lipids exhibited weak promoting effects on spermatogenesis. Lastly, luteinizing hormone (LH), follicle stimulating hormone (FSH), triiodothyronine (T3), and testosterone (T) combined together promoted spermatogenesis until round spermatid production. The CDM, however, was not able to produce elongated spermatids. It was also unable to induce spermatogenesis from the very early neonatal period, before 2 days postpartum, leaving certain factors necessary for spermatogenic induction in mice unidentified. Nonetheless, the present study provided important basic information on testis organ culture and spermatogenesis in vitro.
Assuntos
Técnicas de Cultura de Órgãos/métodos , Espermatogênese , Fatores Etários , Animais , Bovinos , Meios de Cultura/química , Hormônio Foliculoestimulante/farmacologia , Técnicas In Vitro , Lipídeos/farmacologia , Hormônio Luteinizante/farmacologia , Masculino , Meiose/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Soroalbumina Bovina/isolamento & purificação , Soroalbumina Bovina/farmacologia , Transdução de Sinais , Espermatogênese/efeitos dos fármacos , Espermatogênese/fisiologia , Espermatogônias/efeitos dos fármacos , Testículo/citologia , Testículo/efeitos dos fármacos , Testículo/fisiologia , Testosterona/farmacologia , Tretinoína/farmacologia , Tri-Iodotironina/farmacologiaRESUMO
Oocytes without a zona pellucida (ZP) often occur as a result of congenital or operational effects, but they are difficult to handle, and embryonic survival is low. Such zona-free (ZF) oocytes are therefore not used in clinics or laboratories. Furthermore, in the laboratory, removal of the ZP is often necessary for genetic manipulation by viral infection or twin production by blastomere separation, but adverse effects on development have been reported. It would therefore be extremely valuable if the embryo could be covered with a structure similar to that of the ZP. In this study, we sought to determine whether an agarose capsule could serve as a substitute for the ZP. Our results indicate that embryos derived from these agarose capsules were able to develop normally, and could be transplanted to obtain viable offspring, without having to remove the agarose capsule. Furthermore, before compaction, the agarose capsule embryos exhibited good freezing tolerance, and survival rate was extremely high compared to ZF embryos. Thus, agarose capsules represent a valuable tool for utilizing oocytes and embryos that lack a ZP, both in a clinical and livestock setting.
Assuntos
Embrião de Mamíferos , Oócitos , Sefarose , Animais , Cápsulas , Transferência Embrionária , Desenvolvimento Embrionário , Feminino , Congelamento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos ICR , GravidezRESUMO
Cellular differentiation is associated with dynamic chromatin remodeling in establishing a cell-type-specific epigenomic landscape. Here, we find that mouse testis-specific and replication-dependent histone H3 variant H3t is essential for very early stages of spermatogenesis. H3t gene deficiency leads to azoospermia because of the loss of haploid germ cells. When differentiating spermatogonia emerge in normal spermatogenesis, H3t appears and replaces the canonical H3 proteins. Structural and biochemical analyses reveal that H3t-containing nucleosomes are more flexible than the canonical nucleosomes. Thus, by incorporating H3t into the genome during spermatogonial differentiation, male germ cells are able to enter meiosis and beyond.
Assuntos
Histonas/genética , Espermatogônias/metabolismo , Testículo/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Azoospermia/etiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Células Cultivadas , Cromatina/química , Cromatina/metabolismo , Células Germinativas/citologia , Células Germinativas/metabolismo , Haploidia , Histonas/química , Histonas/deficiência , Masculino , Meiose , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nucleossomos/química , Nucleossomos/metabolismo , Espermatogênese , Testículo/patologiaRESUMO
OBJECTIVE: To develop an efficient cryopreservation method using a single sperm. DESIGN: Experimental study. SETTING: Laboratory of a private institute. PATIENT(S): A fertile donor. INTERVENTION(S): We produced hollow-core capsules with agarose walls. A single human sperm was injected into each capsule as per the conventional intracytoplasmic sperm injection (ICSI) method. The capsules that contained the spermatozoa were cryopreserved on polycarbonate or nylon mesh sheets using nitrogen vapor. Before their use, the capsules were thawed and recovered. The motile spermatozoa in the capsules were counted. MAIN OUTCOME MEASURE(S): The recovery rates of the agarose capsules and the spermatozoa in these capsules after thawing and the mortality and survival rates of the spermatozoa. RESULT(S): The recovery rates of the capsules were 91.5% (75/82) using polycarbonate sheets (PS) and 98.3% (59/60) using mesh sheets (MS) after thawing. The recovered capsules were not at all damaged. The recovery rates of the spermatozoa were 91.5% (75/82) using PS and 96.7% (58/60) using MS. Sperm motility rates were 85.3% (64/75) and 82.8% (48/58), whereas the survival rates of the immotile spermatozoa by the hypoosmotic swelling test were 81.8% (9/11) and 50.0% (5/10); furthermore, the total survival rates of the spermatozoa were 97.3% (73/75) and 91.4% (53/58) using PS and MS, respectively. There was no significant difference between the results obtained using PS and MS. CONCLUSION(S): A cryopreservation method for a single sperm using an agarose capsule has been developed. The method is expected to be useful in ICSI treatment in patients with few spermatozoa.
Assuntos
Cápsulas , Criopreservação/métodos , Preservação do Sêmen/métodos , Sefarose , Cápsulas/química , Sobrevivência Celular , Criopreservação/instrumentação , Humanos , Masculino , Microinjeções , Preservação do Sêmen/instrumentação , Sefarose/química , Injeções de Esperma Intracitoplásmicas , Espermatozoides/citologiaRESUMO
In mammals, DNA is methylated at CpG sites, which play pivotal roles in gene silencing and chromatin organization. Furthermore, DNA methylation undergoes dynamic changes during development, differentiation, and in pathological processes. The conventional methods represent snapshots; therefore, the dynamics of this marker within living organisms remains unclear. To track this dynamics, we made a knockin mouse that expresses a red fluorescent protein (RFP)-fused methyl-CpG-binding domain (MBD) protein from the ROSA26 locus ubiquitously; we named it MethylRO (methylation probe in ROSA26 locus). Using this mouse, we performed RFP-mediated methylated DNA immunoprecipitation sequencing (MeDIP-seq), whole-body section analysis, and live-cell imaging. We discovered that mobility and pattern of heterochromatin as well as DNA methylation signal intensity inside the nuclei can be markers for cellular differentiation status. Thus, the MethylRO mouse represents a powerful bioresource and technique for DNA methylation dynamics studies in developmental biology, stem cell biology, as well as in disease states.