BCAR4 (Breast Cancer anti-estrogen Resistance 4) gene expression and its role in reproduction of the female rabbit

Le gène BCAR4 (Breast Cancer anti-estrogen Resistance 4) a précédemment été découvert chez le bovin comme un gène exprimé préférentiellement dans l'ovocyte et l'embryon précoce et dont l’inhibition altère le développement embryonnaire in vitro. Cependant, son rôle dans l’ovogenèse, la folliculogenèse et globalement la fertilité in vivo restait inconnu. Le gène est conservé chez divers mammifères mais pas chez les rongeurs, le lapin a donc été choisi pour analyser son expression et sa fonction in vivo. Le transcrit BCAR4 est détecté dans l’ovaire dès la formation des follicules primordiaux, et dans les follicules préantraux et antraux, jusque dans l’ovocyte ovulé. Son abondance diminue après la fécondation et tout au long du développement préimplantatoire pour disparaître dans le blastocyste, un profil typique d’un transcrit maternel.Afin d’appréhender le rôle du gène BCAR4 in vivo dans la reproduction chez la femelle, des lapins porteurs d’une altération du gène BCAR4 ont été créés par édition du génome à l’aide de nucléases effectrices de type activateur de transcription (TALEN), et une lignée a été générée. Les animaux porteurs de la modification à l’état homozygote ou hétérozygote sont viables et en bonne santé. L’efficacité de l’altération génétique a été démontrée par transcription inverse couplée à la PCR : trente fois moindre que chez les animaux de génotype sauvage, l’expression de BCAR4 peut être considérée comme abolie dans les follicules ovariens des individus homozygotes. Les femelles ont été phénotypées sur plusieurs paramètres liés à la reproduction. Le génotype n’a pas d’impact significatif sur la folliculogenèse ou l’activité ovarienne, estimés par le dénombrement folliculaire sur coupes histologiques, la concentration plasmatique en hormone anti-mullérienne ou la réponse à la stimulation ovarienne. Pour évaluer la fertilité et la prolificité, les femelles ont été inséminées trois fois à six semaines d’intervalle. Les femelles homozygotes présentaient un taux de mise-bas significativement plus faible que les femelles hétérozygotes, 22±7% vs 71±11% (moyenne±sem), et une prolificité de 1,5±0,7 vs 5,8±1,5 lapereaux par insémination. En conclusion, le gène BCAR4 n’est pas indispensable pour le développement folliculaire, mais il contribue à une fertilité optimale chez la lapine.The BCAR4 gene (Breast Cancer anti-estrogen Resistance 4) has been previously characterized in cattle as a gene expressed preferentially in the oocyte and early embryo, whose inhibition alters embryonic development in vitro. However, its role in oogenesis, folliculogenesis and overall in fertility in vivo remains unknown. Since this gene is conserved in various mammals but not in rodents, the rabbit has been chosen to investigate its expression and function in vivo. By reverse transcription coupled to PCR, BCAR4 transcript is detected in the ovary when primordial follicles are formed, and in ovarian follicles at the preantral and antral stages, as well as in ovulated oocytes. Its abundance decreases after fertilization and throughout preimplantation development to disappear in the blastocyst, a typical profile for a maternal transcript.In order to elucidate the role of BCAR4 in vivo in female reproduction, rabbits carrying an altered BCAR4 gene were created and a line was generated. Both homozygous and heterozygous carriers of the genetic alteration are viable and appear healthy. The genetic alteration abolishes BCAR4 expression in ovarian follicles of homozygous animals, as the transcript abundance is down thirty-fold as compared to the wild-type phenotype. Females were phenotyped on several parameters related to reproduction. The genotype did not have a significant impact on follicular development or ovarian activity, as estimated by follicular count onto ovarian sections, anti-mullerian hormone concentration in plasma, and the response to ovarian stimulation. To evaluate their fertility and prolificacy, females were inseminated three times every six-weeks. Homozygous females had a significantly lower farrowing rate than heterozygous females, 22±7% vs 71±11% (mean±sem), while prolificacy was 1.5±0.7 vs 5.8±1.5 pups per insemination. In conclusion, BCAR4 is not essential for follicular development but the gene contributes to optimal fertility of female rabbits

Mubritinib, an inhibitor of the EGFR family member ErbB2, blocks oocyte maturation in bovine

Mubritinib, an inhibitor of the EGFR family member ErbB2, blocks oocyte maturation in bovine. 2. Journées du GdR 3606 Repr

KO d’un gène OvocytAire chez le Lapin (KOALA)

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Rôle du gène <em>BCAR4</em> dans la fertilité femelle : expression et approche fonctionnelle <em>in vivo</em> chez le lapin

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Functional investigation of the human oocyte-expressed gene bcar4 using domestic animal models and genome editing.

National audienceWe have previously characterized BCAR4 (Breast Cancer Anti-estrogen Resistant 4) as a gene preferentially expressed in human oocytes as compared to ovarian somatic cells or non-pathologic non-reproductive tissues. BCAR4 is conserved in primates and in various domestic species such as cow, pig, dog, horse or rabbit, but it is not found in the genome of rodents. This pattern coincides with a delayed major activation of the embryonic genome, i.e. after several cleavages. BCAR4 is also an oncogene overexpressed in a subset of breast tumors and other cancers. Altogether, the restricted expression, proliferative properties and phylogeny suggested that BCAR4 may be involved in early embryonic divisions.We have analyzed BCAR4 expression in the cow. While RNA is already transcribed in the oocyte of preovulatory follicles, protein synthesis is delayed until late maturation, peaks in early cleaving embryos, persists until the morula stage to become undetectable in blastocysts. Microinjecting BCAR4-targeting small-interfering RNA significantly decreased BCAR4 expression and compromised in vitro blastocyst development, demonstrating that BCAR4 is a maternal-effect gene.To investigate BCAR4 function in vivo, the rabbit model was chosen for genome editing. Rabbits carrying an altered BCAR4 gene were produced using a transcription activator-like effector nuclease (TALEN). Wild-type, heterozygous and homozygous carrier animals were born following the expected mendelian ratio. They were viable and appeared healthy, as expected for animals with an altered maternal effect-gene. Efficiency of the genetic alteration was evaluated by reverse-transcription coupled to PCR. It showed that BCAR4 RNA expression was abolished in follicles from homozygous carriers as compared to their heterozygous and wild-type littermates. These females will be phenotyped onto various reproductive parameters to assess the role of BCAR4 in fertility in vivo

BCAR4 is involved in in vitro preimplantation development: investigating its role in vivo by genome efditing in rabbit

BCAR4 is involved in [i]in vitro[/i] preimplantation development: investigating its role [i]in vivo[/i] by genome efditing in rabbit. 2. Journées du GdR 3606 Repr

Investigating the role of BCAR4 in ovarian physiology and female fertility by genome editing in rabbit

International audienceBreast Cancer Anti-estrogen Resistance 4 (BCAR4) was previously characterised in bovine species as a gene preferentially expressed in oocytes, whose inhibition is detrimental to in vitro embryo development. But its role in oogenesis, folliculogenesis and globally fertility in vivo remains unknown. Because the gene is not conserved in mice, rabbits were chosen for investigation of BCAR4 expression and function in vivo. BCAR4 displayed preferential expression in the ovary compared to somatic organs, and within the ovarian follicle in the oocyte compared to somatic cells. The transcript was detected in follicles as early as the preantral stage. Abundance decreased throughout embryo development until the blastocyst stage. A lineage of genome-edited rabbits was produced; BCAR4 expression was abolished in follicles from homozygous animals. Females of wild-type, heterozygous and homozygous genotypes were examined for ovarian physiology and reproductive parameters. Follicle growth and the number of ovulations in response to hormonal stimulation were not significantly different between genotypes. Following insemination, homozygous females displayed a significantly lower delivery rate than their heterozygous counterparts (22 ± 7% vs 71 ± 11% (mean ± SEM)), while prolificacy was 1.8 ± 0.7 vs 6.0 ± 1.4 kittens per insemination. In conclusion, BCAR4 is not essential for follicular growth and ovulation but it contributes to optimal fertility in rabbits