In vivo imaging of ram spermatozoa in the ewe genital tract using fibered confocal microscopy

National audienceSperm transit in the female tract is one of the key factors in the success of fertilization after artificial insemination in sheep species. However, its study is limited by the absence of in vivo imaging methods. The imaging of ram sperm in the female genital tract was made possible using the confocal fibered microscopy and fluorescent stains adapted to spermatozoa. Our results show the active role of the uterotubal junction in the selection of sperm during their transit

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

Towards the Establishment of a Porcine Model to Study Human Amebiasis

BACKGROUND: Entamoeba histolytica is an important parasite of the human intestine. Its life cycle is monoxenous with two stages: (i) the trophozoite, growing in the intestine and (ii) the cyst corresponding to the dissemination stage. The trophozoite in the intestine can live as a commensal leading to asymptomatic infection or as a tissue invasive form producing mucosal ulcers and liver abscesses. There is no animal model mimicking the whole disease cycle. Most of the biological information on E. histolytica has been obtained from trophozoite adapted to axenic culture. The reproduction of intestinal amebiasis in an animal model is difficult while for liver amebiasis there are well-described rodent models. During this study, we worked on the assessment of pigs as a new potential model to study amebiasis. METHODOLOGY/PRINCIPAL FINDINGS: We first co-cultured trophozoites of E. histolytica with porcine colonic fragments and observed a disruption of the mucosal architecture. Then, we showed that outbred pigs can be used to reproduce some lesions associated with human amebiasis. A detailed analysis was performed using a washed closed-jejunal loops model. In loops inoculated with virulent amebas a severe acute ulcerative jejunitis was observed with large hemorrhagic lesions 14 days post-inoculation associated with the presence of the trophozoites in the depth of the mucosa in two out four animals. Furthermore, typical large sized hepatic abscesses were observed in the liver of one animal 7 days post-injection in the portal vein and the liver parenchyma. CONCLUSIONS: The pig model could help with simultaneously studying intestinal and extraintestinal lesion development

Surgical pinealectomy in ewes

Chapitre 7The pineal gland once regarded as the “seat of the soul” by René Descartes is the structure attached to the caudal part of the roof of the third ventricle. In mammals, the pineal gland ensures the transduction of the photoperiodic information captured by the retina and transmitted to the pineal gland through a transsynaptic network and translated into a hormonal signal through the nocturnal secretion of melatonin. The circadian rhythm of melatonin synthesis (high levels at night, low levels during the day) is triggered by the circadian ‘clock’ located in the suprachiasmatic nuclei of the hypothalamus that projects to the pineal gland via a multi-synaptic pathway. In sheep, the duration of melatonin secretion is proportional to the length of the night and thus providing cues about the time of the year to the animals. Melatonin is involved in several physiological functions including the seasonal control of reproduction in mammals from temperate latitudes. The key role of the pineal gland in this function has been demonstrated by numerous experiments showing that pinealectomy strongly affected the effect of photoperiod on seasonal reproduction. In sheep, the pineal gland is hidden at the bottom of cerebral transverse fissure, in a medial position, close to the cerebral vein. This deep localization makes the surgical excision of the pineal gland very difficult and requires a specific training. In order to avoid pain and side effects both in pre and post surgical care, we designed a protocol of analgesic treatment made of morphine in combination with antiinflammatory compounds. Future improvements of our approach should arise from the use of scanner and magnetic resonance imaging techniques

Les modèles d'animaux de ferme utilisés en chirurgie expérimentale : intérêt et contraintes, aspects éthiques

National audienceThe French agricultural research institute INRA has developed experimental surgery in farm animals in the 1970s. The research programs are aimed at improving theroretical knowledge on major biological functions as well as practical applications on cattle, goats, sheep, pigs, and horses. In this article the research activity of the Reproductive Physiology and Behaviour centre is taken as an example because of its important experimental surgery unit specialized in abdominal and brain surgery. Surgical procedures are performed in a hospital and specimens are collected from a slaughterhouse. This research activity is very strictly controlled by the French regulation, and must be conducted in authorised premises by adequately trained personnel. Special care is given to the alleviation of post-surgical pain. In addition, scientists may seek the opinion of ethics committee on their experimental procedures

Cinq jours à la ferme

Intervention dans un petit reportage "L'agriculture de demain" diffusé sur France 3 mardi 28 février 2012 à 12h55 dans le magazine spécial Salon international de l'Agriculture 201

Surgical transfer of porcine vitrified blastocysts to different uterine sites

International audienc

Disruption of lipid rafts induces gonadotropin release in ovine pituitary and LbetaT2 gonadotroph cells

International audienceIn order to better understand the cellular mechanisms underlying LH and FSH secretion, we have addressed the contribution of lipid rafts to the secretion of gonadotropins. We used methyl-beta-cyclodextrin (MbetaCD), a cholesterol-sequestering agent, on an LbetaT2 murine gonadotroph cell line and on primary cultures of ovine pituitary cells. We found that in both systems, cholesterol depletion by MbetaCD induced a fast and substantial release of LH in the absence of natural stimulation by GnRH. In ovine pituitary cells, MbetaCD-mediated LH release was shown to be independent of protein synthesis. Twenty-four hours after MbetaCD treatment, there was no loss of cell viability and full recovery of LH secretory capabilities, as determined by GnRH or MbetaCD treatment. In addition, our data suggest the existence of a pool of LH that is not released by GnRH treatment but that is released by MbetaCD treatment. Finally, in ovine pituitary cells, MbetaCD treatment induced FSH secretion. Importantly, these in vitro data are supported by in vivo studies, because MbetaCD injected into the pituitary glands of anaesthetized sheep reproducibly induced a peak of LH release

Reproduction related effects of photoperiod on kisspeptin neuronal populations of the ewe diencephalon

International audienc

Suivi coelioscopique des corps jaunes cycliques chez la brebis

National audienc