scholarly journals Structurally similar Drosophila alpha-tubulins are functionally distinct in vivo.

1997 ◽  
Vol 8 (3) ◽  
pp. 481-500 ◽  
Author(s):  
J A Hutchens ◽  
H D Hoyle ◽  
F R Turner ◽  
E C Raff
Keyword(s):  
Germ Cells ◽  
Microtubule Assembly ◽  
Meiotic Spindle ◽  
Dominant Male ◽  
Developmentally Regulated ◽  
Male Germ Cells ◽  
Alpha Tubulin ◽  
Tubulin Isoforms ◽  
Tubulin Structure

We used transgenic analysis in Drosophila to compare the ability of two structurally similar alpha-tubulin isoforms to support microtubule assembly in vivo. Our data revealed that even closely related alpha-tubulin isoforms have different functional capacities. Thus, in multicellular organisms, even small changes in tubulin structure may have important consequences for regulation of the microtubule cytoskeleton. In spermatogenesis, all microtubule functions in the postmitotic male germ cells are carried out by a single tubulin heterodimer composed of the major Drosophila alpha-84B tubulin isoform and the testis-specific beta 2-tubulin isoform. We tested the ability of the developmentally regulated alpha 85E-tubulin isoform to replace alpha 84B in spermatogenesis. Even though it is 98% similar in sequence, alpha 85E is not functionally equivalent to alpha 84B. alpha 85E can support some functional microtubules in the male germ cells, but alpha 85E causes dominant male sterility if it makes up more than one-half of the total alpha-tubulin pool in the spermatids. alpha 85E does not disrupt meiotic spindle or cytoplasmic microtubules but causes defects in morphogenesis of the two classes of singlet microtubules in the sperm tail axoneme, the central pair and the accessory microtubules. Axonemal defects caused by alpha 85E are precisely reciprocal to dominant defects in doublet microtubules we observed in a previous study of ectopic germ-line expression of the developmentally regulated beta 3-tubulin isoform. These data demonstrate that the doublet and singlet axoneme microtubules have different requirements for alpha- and beta-tubulin structure. In their normal sites of expression, alpha 85E and beta 3 are coexpressed during differentiation of several somatic cell types, suggesting that alpha 85E and beta 3 might form a specialized heterodimer. Our tests of different alpha-beta pairs in spermatogenesis did not support this model. We conclude that if alpha 85E and beta 3 have specialized properties required for their normal functions, they act independently to modulate the properties of microtubules into which they are incorporated.

scholarly journals Structure and expression of murine mgcRacGAP: its developmental regulation suggests a role for the Rac/MgcRacGAP signalling pathway in neurogenesis

1999 ◽  
Vol 343 (1) ◽  
pp. 225-230 ◽  
Author(s):  
Chantal ARAR ◽  
Marie-Odile OTT ◽  
Aminata TOURÉ ◽  
Gérard GACON
Keyword(s):  
Germ Cells ◽  
Developmental Regulation ◽  
Ventricular Zone ◽  
Developmentally Regulated ◽  
Gene Encoding ◽  
Male Germ Cells ◽  
Preferential Expression ◽  
Wide Range ◽  
Developing Neurons

Rho-family GTPases regulate a wide range of biological functions including cell migration, cell adhesion and cell growth. Recently, results from studies in vivo in Drosophila, mouse and humans have demonstrated the involvement of these GTPases in mechanisms controlling neuronal differentiation and the development of the central nervous system (CNS). However, the signalling pathways underlying these functions and the proteins directly regulating RhoGTPases in developing neurons are poorly defined. Here we report the structure and expression pattern of the murine orthologue of mgcRacGAP, a human gene encoding a RacGTPase partner expressed in male germ cells [Touré, Dorseuil, Morin, Timmons, Jegou, Reibel and Gacon (1998) J. Biol. Chem. 273, 6019-6023]. In contrast with that from humans, murine mgcRacGAP encodes two distinct transcripts. Both are developmentally regulated. A 2.2 kb transcript is strongly expressed in mature testis and is up-regulated with spermatogenesis. A 3 kb RNA is predominant in the embryo and is expressed primarily in the CNS during the neurogenic phase, decreasing after birth. In situ hybridization analysis in embryonic-day 14.5 mouse embryos demonstrates a preferential expression of mgcRacGAP in the proliferative ventricular zone of the cortex. In addition to the expression of mgcRacGAP in male germ cells already reported in humans and suggesting an involvement in spermatogenesis, we characterize an embryonic transcript whose expression is closely correlated with neurogenesis. This result addresses the question of the role of Rac/MgcRacGAP pathway in neuronal proliferation.

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.

Insertions of up to 17 amino acids into a region of alpha-tubulin do not disrupt function in vivo

1987 ◽  
Vol 7 (10) ◽  
pp. 3799-3805
Author(s):  
P J Schatz ◽  
G E Georges ◽  
F Solomon ◽  
D Botstein
Keyword(s):  
Amino Acids ◽  
Variable Region ◽  
Yeast Cells ◽  
Meiotic Spindle ◽  
Nuclear Movement ◽  
Yeast Saccharomyces Cerevisiae ◽  
Alpha Tubulin ◽  
Essential Components ◽  
Altered Proteins

Microtubules in yeasts are essential components of the mitotic and meiotic spindle and are necessary for nuclear movement during cell division and mating. The yeast Saccharomyces cerevisiae has two alpha-tubulin genes, TUB1 and TUB3, either of which alone is sufficient for these processes when present in a high enough copy number. Comparisons of sequences from several species reveals the presence of a variable region near the amino terminus of alpha-tubulin proteins. We perturbed the structure of this region in TUB3 by inserting into it 3, 9, or 17 amino acids and tested the ability of these altered proteins to function as the only alpha-tubulin protein in yeast cells. We found that each of these altered proteins was sufficient on its own for mitotic growth, mating, and methods of yeast. We conclude that this region can tolerate considerable variation without losing any of the highly conserved functions of alpha-tubulin. Our results suggest that variability in this region occurs because it can be tolerated, not because it specifies an important function for the protein.

Immunodetection of acetylated alpha-tubulin in stony corals: Evidence for the existence of flagella in coral male germ cells

2017 ◽  
Vol 84 (12) ◽  
pp. 1285-1295 ◽  
Author(s):  
Shinya Shikina ◽  
Yi-Ling Chiu ◽  
Chieh-Jhen Chen ◽  
Shih-Han Yang ◽  
Jack I-Chen Yao ◽  
...  
Keyword(s):  
Germ Cells ◽  
Male Germ Cells ◽  
Alpha Tubulin ◽  
Stony Corals

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

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.

Sto1p, a fission yeast protein similar to tubulin folding cofactor E, plays an essential role in mitotic microtubule assembly

1999 ◽  
Vol 112 (12) ◽  
pp. 1979-1988 ◽  
Author(s):  
E.L. Grishchuk ◽  
J.R. McIntosh
Keyword(s):  
Fission Yeast ◽  
Microtubule Assembly ◽  
Wild Type ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Cold Sensitive ◽  
Alpha Beta ◽  
Sensitive Allele ◽  
Delta Cells

The proper functioning of microtubules depends crucially on the availability of polymerizable alpha/beta tubulin dimers. Their production occurs concomitant with the folding of the tubulin polypeptides and is accomplished in part by proteins known as Cofactors A through E. In the fission yeast, Schizosaccharomyces pombe, this tubulin folding pathway is essential. We have taken advantage of the excellent cytology available in S. pombe to examine the phenotypic consequences of a deletion of sto1(+), a gene that encodes a protein similar to Cofactor E, which is required for the folding of alpha-tubulin. The interphase microtubule cytoskeleton in sto1-delta cells is severely disrupted, and as cells enter mitosis their spindles fail to form. After a transient arrest with condensed chromosomes, the cells exit mitosis and resume DNA synthesis, whereupon they septate abnormally and die. Overexpression of Spo1p is toxic to cells carrying a cold-sensitive allele of the alpha- but not the beta-tubulin gene, consistent with the suggestion that this protein plays a role like that of Cofactor E. Unlike its presumptive partner Cofactor D (Alp1p), however, Sto1p does not localize to microtubules but is found throughout the cell. Overexpression of Sto1p has no toxic effects in wild-type cells, suggesting that it is unable to disrupt alpha/beta tubulin dimers in vivo.

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
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