scholarly journals 322 DIFFERENTIAL DEVELOPMENT OF RABBIT EMBRYOS FOLLOWING MICROINSEMINATION USING SPERM AND SPERMATIDS

2005 ◽  
Vol 17 (2) ◽  
pp. 312
Author(s):  
N. Ogonuki ◽  
K. Inoue ◽  
H. Miki ◽  
Y. Hirose ◽  
H. Okada ◽  
...  
Keyword(s):  
Embryo Development ◽  
Germ Cells ◽  
Round Spermatid ◽  
Nuclear Staining ◽  
Male Germ Cells ◽  
Average Cell ◽  
Mouse Oocytes ◽  
Round Spermatid Injection ◽  
Rabbit Embryos

Microinsemination is a technique that delivers male germ cells directly into the ooplasm. The efficiency of fertilization and subsequent embryo development after microinsemination varies with species and the male germ cells used. This study examined the developmental ability of rabbit embryos in vitro and in vivo following microinsemination using haploid male germ cells at different stages. First, we injected rabbit spermatozoa, elongated spermatids, and round spermatids into mouse oocytes to assess their oocyte-activating capacity. Mouse oocytes are a good experimental model for assessing the oocyte-activating capacity of male germ cells from different species. The majority of mouse oocytes were activated irrespective of the stage of rabbit male germ cells injected (77, 61, and 73% for spermatozoa, elongated spermatids, and round spermatids, respectively). By contrast, these male germ cells activated homologous rabbit oocytes at rates of 100, 59, and 29%, respectively. After 120 h in culture, 69, 55, and 13% of these activated rabbit oocytes (pronuclear eggs) developed into blastocysts, respectively. The rate of embryo development into blastocysts following round spermatid injection was significantly improved when oocytes were activated by an electric pulse shortly before microinsemination. The total number of cells was counted in embryos that reached the morula/blastocyst stages in culture using nuclear-staining with propidium iodide. The average cell number of embryos derived from elongated (89 ± 41; mean ± SD) or round spermatid (98 ± 34) injection was significantly lower than that of control embryos (in vivo fertilization) (211 ± 44) (P < 0.01). After 24 h in culture, some four- to eight-cell-stage embryos were transferred into the oviducts of pseudopregnant females. Normal pups were born from embryos involving sperm (4 offspring/16 transfers; 25%) and elongated spermatid (3/26; 12%) injection, but none from those involving round spermatid injection (0/68). These findings indicate that rabbit male germ cells acquire the ability to activate oocytes and to support subsequent embryo development as they undergo spermiogenesis. Immaturity of the nuclear genome or difficulty in coordinating the behavior of the male and female chromosomes might compromise embryo development.

scholarly journals The 28 + 28 day design is an effective sampling time for analyzing mutant frequencies in rapidly proliferating tissues of MutaMouse animals

2021 ◽  
Vol 95 (3) ◽  
pp. 1103-1116
Author(s):  
Francesco Marchetti ◽  
Gu Zhou ◽  
Danielle LeBlanc ◽  
Paul A. White ◽  
Andrew Williams ◽  
...  
Keyword(s):  
Germ Cells ◽  
False Negative ◽  
Sampling Time ◽  
Male Germ Cells ◽  
Negative Results ◽  
False Negative Results ◽  
Detection Of Mutations ◽  
The Impact ◽  
Sampling Times

AbstractThe Organisation for Economic Co-Operation and Development Test Guideline 488 (TG 488) uses transgenic rodent models to generate in vivo mutagenesis data for regulatory submission. The recommended design in TG 488, 28 consecutive daily exposures with tissue sampling three days later (28 + 3d), is optimized for rapidly proliferating tissues such as bone marrow (BM). A sampling time of 28 days (28 + 28d) is considered more appropriate for slowly proliferating tissues (e.g., liver) and male germ cells. We evaluated the impact of the sampling time on mutant frequencies (MF) in the BM of MutaMouse males exposed for 28 days to benzo[a]pyrene (BaP), procarbazine (PRC), isopropyl methanesulfonate (iPMS), or triethylenemelamine (TEM) in dose–response studies. BM samples were collected + 3d, + 28d, + 42d or + 70d post exposure and MF quantified using the lacZ assay. All chemicals significantly increased MF with maximum fold increases at 28 + 3d of 162.9, 6.6, 4.7 and 2.8 for BaP, PRC, iPMS and TEM, respectively. MF were relatively stable over the time period investigated, although they were significantly increased only at 28 + 3d and 28 + 28d for TEM. Benchmark dose (BMD) modelling generated overlapping BMD confidence intervals among the four sampling times for each chemical. These results demonstrate that the sampling time does not affect the detection of mutations for strong mutagens. However, for mutagens that produce small increases in MF, sampling times greater than 28 days may produce false-negative results. Thus, the 28 + 28d protocol represents a unifying protocol for simultaneously assessing mutations in rapidly and slowly proliferating somatic tissues and male germ cells.

scholarly journals Fertilization of mouse oocytes using somatic cells as male germ cells

2001 ◽  
Vol 3 (3) ◽  
pp. 205-211 ◽  
Author(s):  
Orly Lacham-Kaplan ◽  
Rob Daniels ◽  
Alan Trounson
Keyword(s):  
Germ Cells ◽  
Somatic Cells ◽  
Male Germ Cells ◽  
Mouse Oocytes

scholarly journals Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study

2012 ◽  
Vol 14 (4) ◽  
pp. 574-579 ◽  
Author(s):  
Yong Zhu ◽  
Hong-Liang Hu ◽  
Peng Li ◽  
Shi Yang ◽  
Wei Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Ips Cells ◽  
In Vivo Study ◽  
Male Germ Cells ◽  
Induced Pluripotent

Round spermatids stained with MitoTracker can be used to produce offspring more simply

Zygote ◽  
2005 ◽  
Vol 13 (1) ◽  
pp. 55-61 ◽  
Author(s):  
Takafusa Hikichi ◽  
Satoshi Kishigami ◽  
Nguyen Van Thuan ◽  
Hiroshi Ohta ◽  
Eiji Mizutani ◽  
...  
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Round Spermatid ◽  
Testicular Cells ◽  
Infertility Treatments ◽  
Developmental Capacity ◽  
20 Nm ◽  
Harmful Effects ◽  
Round Spermatid Injection ◽  
Selection Of

Although both intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI) are used in infertility treatments, the rate of offspring achieved with ROSI is low compared with that achieved with ICSI. The difficulty in correctly selecting round spermatids from testicular cells is one of the causes of this phenomenon. We easily selected live round spermatids from testicular cells stained with 20 nM MitoTracker, which visualizes mitochondria without killing the cell. Using this method, we divided round spermatids into three groups based on the polarization of their mitochondria, and performed ROSI. The rate of successful offspring achieved with MitoTracker-stained ROSI was the same in all groups. This indicates that changes in the polarization of mitochondria in round spermatids are not directly related to the developmental capacity of subsequently fertilized embryos. Because this staining has no harmful effects on embryo development, the selection of spermatids by MitoTracker under a fluorescence microscope should be useful in research into and the treatment of infertility.

scholarly journals miRNA-dependent poly(A) length control in uncoupling transcription and translation of haploid male germ cells

2021 ◽  
Author(s):  
Chong Tang ◽  
Mei Guo ◽  
Zhuoxing Shi ◽  
Zhuqing Wang ◽  
Chunhai Luo ◽  
...  
Keyword(s):  
Germ Cells ◽  
Regulatory Mechanisms ◽  
Translational Repression ◽  
Dynamic Changes ◽  
Male Germ Cells ◽  
Haploid Male ◽  
Transcriptional Regulatory ◽  
Delayed Translation ◽  
Transcriptional Regulatory Mechanisms

AbstractAs one of the post-transcriptional regulatory mechanisms, transcription and translation’s uncoupling plays an essential role in development and adulthood physiology. However, it remains elusive how thousands of mRNAs get translationally silenced while stability is maintained for up to hours or even days before translation. In addition to oocytes and neurons, developing spermatids have significant uncoupling of transcription and translation for delayed translation. Therefore, spermiogenesis represents an excellent in vivo model for investigating the mechanism underlying uncoupled transcription and translation. Through full-length poly(A) deep sequencing, we discovered dynamic changes in poly(A) length through deadenylation and re-polyadenylation. Deadenylation appeared to be mediated by microRNAs (miRNAs), and transcripts with shorter poly(A) tails tend to be sequestered into ribonucleoproteins (RNPs) for translational repression and stabilization. In contrast, re-polyadenylation allows for translocation of the translationally repressed transcripts from RNPs to polysomes for translation. Overall, our data suggest that miRNA-dependent poly(A) length control represents a novel mechanism underlying uncoupled translation and transcription in haploid male germ cells.

scholarly journals Partial Sperm beta1 Integrin Subunit Deletion Proves Its Involvement in Mouse Gamete Adhesion/Fusion

2020 ◽  
Vol 21 (22) ◽  
pp. 8494
Author(s):  
Virginie Barraud-Lange ◽  
Côme Ialy-Radio ◽  
Céline Chalas ◽  
Isabelle Holtzmann ◽  
Jean-Philippe Wolf ◽  
...  
Keyword(s):  
Western Blot ◽  
Germ Cells ◽  
Blot Analysis ◽  
Integrin Subunit ◽  
Perivitelline Space ◽  
Male Germ Cells ◽  
Beta1 Integrin ◽  
First Time

We have previously shown, using antibodies, that the sperm alpha6beta1 integrin is involved in mouse gamete fusion in vitro. Here we report the conditional knockdown of the sperm Itgb1 gene. It induced a drastic failure of sperm fusogenic ability with sperm accumulation in the perivitelline space of in vitro inseminated oocytes deleted or not for the Itgb1 gene. These data demonstrate that sperm, but not oocyte, beta1 integrin subunit is involved in gamete adhesion/fusion. Curiously, knockdown males were fertile in vivo probably because of the incomplete Cre-mediated deletion of the sperm Itgb1 floxed gene. Indeed, this was shown by Western blot analysis and confirmed by both the viability and litter size of pups obtained by mating partially sperm Itgb1 deleted males with females producing completely deleted Itgb1 oocytes. Because of the total peri-implantation lethality of Itgb1 deletion in mice, we assume that sperm that escaped the Itgb1 excision seemed to be preferentially used to fertilize in vivo. Here, we showed for the first time that the deletion, even partial, of the sperm Itgb1 gene makes the sperm unable to normally fertilize oocytes. However, to elucidate the question of the essentiality of its role during fertilization, further investigations using a mouse expressing a recombinase more effective in male germ cells are necessary.

Transient transmission of a transgene in mouse offspring following in vivo transfection of male germ cells

2002 ◽  
Vol 62 (4) ◽  
pp. 477-482 ◽  
Author(s):  
Catherine Celebi ◽  
Pierrick Auvray ◽  
Thierry Benvegnu ◽  
Daniel Plusquellec ◽  
Bernard JÉgou ◽  
...  
Keyword(s):  
Germ Cells ◽  
Male Germ Cells ◽  
In Vivo Transfection

scholarly journals Spermatogonial Stem Cells for In Vitro Spermatogenesis and In Vivo Restoration of Fertility

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 745 ◽  
Author(s):  
Fahar Ibtisham ◽  
Ali Honaramooz
Keyword(s):  
Stem Cells ◽  
Germ Cells ◽  
Recent Progress ◽  
Male Germ Cells ◽  
Culture Systems ◽  
In Vitro Spermatogenesis ◽  
Haploid Male ◽  
Restoration Of Fertility

Spermatogonial stem cells (SSCs) are the only adult stem cells capable of passing genes onto the next generation. SSCs also have the potential to provide important knowledge about stem cells in general and to offer critical in vitro and in vivo applications in assisted reproductive technologies. After century-long research, proof-of-principle culture systems have been introduced to support the in vitro differentiation of SSCs from rodent models into haploid male germ cells. Despite recent progress in organotypic testicular tissue culture and two-dimensional or three-dimensional cell culture systems, to achieve complete in vitro spermatogenesis (IVS) using non-rodent species remains challenging. Successful in vitro production of human haploid male germ cells will foster hopes of preserving the fertility potential of prepubertal cancer patients who frequently face infertility due to the gonadotoxic side-effects of cancer treatment. Moreover, the development of optimal systems for IVS would allow designing experiments that are otherwise difficult or impossible to be performed directly in vivo, such as genetic manipulation of germ cells or correction of genetic disorders. This review outlines the recent progress in the use of SSCs for IVS and potential in vivo applications for the restoration of fertility.

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