Zygote arrest 1 gene in pig, cattle and human: evidence of different transcript variants in male and female germ cells

BACKGROUND: Zygote arrest 1 (ZAR1) is one of the few known oocyte-specific maternal-effect genes essential for the beginning of embryo development discovered in mice. This gene is evolutionary conserved in vertebrates and ZAR1 protein is characterized by the presence of atypical plant homeobox zing finger domain, suggesting its role in transcription regulation. This work was aimed at the study of this gene, which could be one of the key regulators of successful preimplantation development of domestic animals, in pig and cattle, as compared with human. METHODS: Screenings of somatic cell hybrid panels and in silico research were performed to characterize ZAR1 chromosome localization and sequences. Rapid amplification of cDNA ends was used to obtain full-length cDNAs. Spatio-temporal mRNA expression patterns were studied using Northern blot, reverse transcription coupled to polymerase chain reaction and in situ hybridization. RESULTS: We demonstrated that ZAR1 is a single copy gene, positioned on chromosome 8 in pig and 6 in cattle, and several variants of correspondent cDNA were cloned from oocytes. Sequence analysis of ZAR1 cDNAs evidenced numerous short inverted repeats within the coding sequences and putative Pumilio-binding and embryo-deadenylation elements within the 3'-untranslated regions, indicating the potential regulation ways. We showed that ZAR1 expressed exclusively in oocytes in pig ovary, persisted during first cleavages in embryos developed in vivo and declined sharply in morulae and blastocysts. ZAR1 mRNA was also detected in testis, and, at lower level, in hypothalamus and pituitary in both species. For the first time, ZAR1 was localized in testicular germ cells, notably in round spermatids. In addition, in pig, cattle and human only shorter ZAR1 transcript variants resulting from alternative splicing were found in testis as compared to oocyte. CONCLUSION: Our data suggest that in addition to its role in early embryo development highlighted by expression pattern of full-length transcript in oocytes and early embryos, ZAR1 could also be implicated in the regulation of meiosis and post meiotic differentiation of male and female germ cells through expression of shorter splicing variants. Species conservation of ZAR1 expression and regulation underlines the central role of this gene in early reproductive processes

The GoldenBricks assembly: A standardized one-shot cloning technique for complete cassette assembly

BBF RFC 92 proposes a new standard assembly method for the Parts Registry. The method makes one-shot cloning of a complete eukaryotic or prokaryotic cassette possible in one day while keeping compatibility with the BBF RFC 10 BioBrick assembly standard

Genes preferentially expressed in bovine oocytes : transcripts regulation during oocyte maturation and ambryo development

La reprise de méiose et le début du développement embryonnaire nécessitent un contrôle très précis de la stabilité et de la traduction des ARN accumulés dans le cytoplasme de l’ovocyte, y compris des transcrits ovocyte-spécifiques. Les travaux menés dans le cadre de cette thèse visaient à améliorer la connaissance des aspects moléculaires de la qualité de l’ovocyte chez le bovin. D’une part, nous avons caractérisé de nouveaux gènes exprimés préférentiellement dans l’ovocyte, en particulier le gène Stem loop binding protein 2, mis en évidence pour la première fois chez les mammifères, qui pourrait réguler la synthèse des histones comme chez le xénope. D’autre part, en combinant des approches globale et ciblée, nous avons montré que les transcrits maternels subissent une dégradation modérée au cours de la maturation mais présentent des profils de déadénylation contrastés. La plupart des gènes ovocyte-spécifiques ne sont pas activés dans l’embryon. Ces travaux ouvrent la voie à la caractérisation d’un profil d’expression associé à la compétence de l’ovocyte à soutenir le développement de l’embryon.Meiotic resumption and early embryo development rely on the precise control of the stability and translation of transcripts accumulated in the oocyte cytoplasm, including oocyte-specific transcripts. This work was to improve the understanding of molecular aspects of oocyte quality in bovine. In one hand, we have characterized novel genes expressed preferentially in the oocyte. In particular, we report expression of Stem loop binding protein 2 for the first time in mammals; it could be involved in controlling histone synthesis as in Xenopus. In the other hand, using both global and targeted approaches, maternal transcripts were shown to undergo moderate degradation during maturation, but they displayed contrasted deadenylation profiles. Most oocyte-specific genes were not reactivated in the embryo. Overall, this work paves the way for the characterization of an expression profile associated with the oocyte competence to support the embryo development

Gènes préférentiellement exprimés dans l'ovocyte bovin : régulation des transcrits au cours de la maturation ovocytaire et du développement embryonnaire préimplantatoire

Diplôme : Dr. d'Universit

Comparison of T7E1 and Surveyor Mismatch Cleavage Assays to Detect Mutations Triggered by Engineered Nucleases

International audienc

Phenotyping of reproductive cells in domestic birds through an-omics approach

National audienc

Phénotypage moléculaire par ICM-MS de la semence

déclaration d’invention et de résultats valorisables DI-RV-16-0010Phénotypage moléculaire par ICM-MS de la semenc

Semen biotechnology optimization for successful fertilization in Japanese quail (Coturnix japonica)

International audienceAmong the reproductive biotechnologies needed to improve Japanese quail conservation and valorization, optimized conditions of semen methodologies including sampling, treatment, and artificial insemination are a prerequisite. However, they have been poorly developed due to specific physiological and behavioral features of the species. The aim of the present study was to optimize them, from semen collection/treatment up to artificial insemination procedures. We studied different parameters including semen preparation (individual/pooled, presence of foam, type and pH of extender) and zootechnical parameters (depth of insemination in the female tract, number of sperm inseminated, insemination frequency). We showed that the separation of semen from individual males was required to optimize fertility, as a prerequisite for future semen cryopreservation. The deleterious effect of mixed foam extract addition on the fertility level was demonstrated. These results highlight parameters involved in male copulatory competitions and in sperm post copulation selection. Furthermore, we took into account extender effects and standardized the zootechnical conditions of insemination. The depth of intravaginal insemination (1 cm) was a key factor, but not the number of sperm inseminated (15-60 million sperm/female). Finally, artificial inseminations with optimized conditions led to successful fertility rates (up to 80%) and a duration of the fertile period equivalent to that obtained by natural mating. (C) 2019 Elsevier Inc. All rights reserved

Des avancées sur les techniques de cryopréservation de la semence et sur son utilisation en insémination animale

Lettre de la Cryobanque NationaleDes avancées sur les techniques de cryopréservation de la semence et sur son utilisation en insémination animal

Sucrose increases the quality and fertilizing ability of cryopreserved chicken sperms in contrast to raffinose

International audienceChicken semen conservation is an important tool for programs of genetic diversity management and of endangered breeds' conservation. However, the method still needs to be improved in order to be applied in a wide variety of environments and breeds. Our objective was to compare the effects of 2 external cryoprotectants saccharides (sucrose and raffinose) on the sperm freezability of a Thai local breed, Pradu Hang Dum, in which semen was frozen with a simple freezing method using nitrogen vapors and dimethyl formamide (DMF). Thirty-six males were selected on their motility vigor score for the experiments. In a first experiment, a large range of sucrose and raffinose doses were tested. Semen quality was evaluated after incubation at 5 degrees C or after cryopreservation in straws in the saline Blumberger Hahnen Sperma Verdunner diluent + DMF (6% v/v) with or without sucrose/raffinose. The best targeted doses of sucrose and raffinose were then kept for experiment 2 that was focused on cryopreserved semen. In this experiment, semen quality was measured on frozen-thawed sperm: different objective motility data evaluated by computer-assisted sperm analysis (CASA), membrane integrity, acrosome integrity, mitochondria function evaluated using flow cytometry, lipid peroxide production assessed by the thiobarbituric acid test. Fertility obtained with frozen-thawed semen supplemented or not with sucrose or raffinose was also evaluated after artificial insemination of laying hens. The presence of sucrose at the osmotically inactive dose 1 mmol significantly increased the vigor motility, membrane integrity, acrosome integrity, and mitochondrial functions of frozen-thawed sperm (P < 0.05), and showed the highest levels of fertility after sperm cryopreservation (91% vs. control 86%, P < 0.001). Raffinose showed negative effects on in vitro semen quality from 1 to 100 mmol. Fertility was also negatively (P < 0.001) affected by raffinose (fertility rate 66 to 70%). We thus showed in the present study the high success of a simple chicken sperm cryopreservation method with an external cryoprotectant easily available and cheap, the sucrose, used at an osmotically inactive low concentration