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We investigated the diagnostic reliability of pregnancy detection using changes in interferon stimulated gene (ISG) messenger RNA (mRNA) levels in circulating immune cells in ewes. Two different groups of ewes (an experimental group, experiment 1 and a farm group, experiment 2) were oestrus-synchronized and blood sampled on day 14 (D0 = day of insemination in control animals, experiment 1) and day 15 (experiment 2). Real-time PCR were performed to evaluate the abundance of different ISG mRNAs. In the experimental group, peripheral blood mononuclear cells of 29 ewes born and bred in experimental facilities were isolated using a Percoll gradient method. Gene expression for Chemokine (C-X-C motif) ligand 10 ( CXCL10), Myxovirus (influenza virus) resistance 1 ( MX1) and Signal transducer and activator of transcription 1 (STAT1) mRNA were, respectively, 8.3-fold, 6.1-fold and 2.7-fold higher ( P < 0.001) in pregnant compared with non-pregnant ewes. The receiver operating characteristic (ROC) curves generated from the real-time PCR data demonstrated that a reliable cut-off could be established for CXCL10, MX1 and STAT1. In the farm group of animals, peripheral blood leucocytes of 37 cross-bred multiparous ewes bought from several herds were isolated using the PAXgene ® procedure. This blood sampling procedure is achievable in farms, whereas the Percoll method is not. No significant differences ( P > 0.10) in CXCL10, STAT1, MX1, Myxovirus (influenza virus) resistance 2 ( MX2) and ISG15 ubiquitin-like modifier ( ISG15) mRNA expression were found between pregnant and non-pregnant ewes. The ROC curves and the hierarchical classification generated from the real-time PCR data failed to discriminate between pregnant and non-pregnant animals. In this group of animals, our results show a strong variability in ISG expression patterns: 17% of animals identified as non-pregnant by the five tests were in fact pregnant, only 52% of pregnant animals had at least two positive results (two genes above threshold), whereas up to five positive results (five genes above threshold) were needed to avoid misclassification. In conclusion, this study illustrates the high variability in ISG expression levels in immune circulating cells during early pregnancy and, therefore, highlights the limits of using ISG expression levels in blood samples, collected on PAXgene ® tubes on farms, for early pregnancy detection in sheep.Keywords: interferon stimulated genes, pregnancy diagnosis, early pregnancy, implantation, sheep ImplicationsThis study aimed to investigate the accuracy of gene transcript quantification in blood (interferon stimulated genes (ISG)) as a method of early pregnancy diagnosis in sheep. This method of pregnancy detection is based on a single blood sample, whereas routine pregnancy diagnosis, namely ultrasonography, requires specific equipment and a skilled operator. Our results show a higher variability in ISG expression patterns in animals from commercial herds than from a controlled research station. Consequently, this prevents th...

Forkhead Box L2 (FOXL2) is a member of the FOXL class of transcription factors, which are essential for ovarian differentiation and function. In the endometrium, FOXL2 is also thought to be important in cattle; however, it is not clear how its expression is regulated. The maternal recognition of pregnancy signal in cattle, interferon-Tau, does not regulate FOXL2 expression. Therefore, in the present study, we examined whether the ovarian steroid hormones that orchestrate implantation regulate FOXL2 gene expression in ruminants. In sheep, we confirmed that FOXL2 mRNA and protein was expressed in the endometrium across the oestrous cycle (day 4 to day 15 post-oestrus). Similar to the bovine endometrium, ovine FOXL2 endometrial expression was low during the luteal phase of the oestrous cycle (4 to 12 days post-oestrus) and at implantation (15 days post-oestrus) while mRNA and protein expression significantly increased during the luteolytic phase (day 15 post-oestrus in cycle). In pregnant ewes, inhibition of progesterone production by trilostane during the day 5 to 16 period prevented the rise in progesterone concentrations and led to a significant increase of FOXL2 expression in caruncles compared with the control group (1.4-fold, p < 0.05). Ovariectomized ewes or cows that were supplemented with exogenous progesterone for 12 days or 6 days, respectively, had lower endometrial FOXL2 expression compared with control ovariectomized females (sheep, mRNA, 1.8-fold; protein, 2.4-fold; cattle; mRNA, 2.2-fold; p < 0.05). Exogenous oestradiol treatments for 12 days in sheep or 2 days in cattle did not affect FOXL2 endometrial expression compared with control ovariectomized females, except at the protein level in both endometrial areas in the sheep. Moreover, treating bovine endometrial explants with exogenous progesterone for 48h reduced FOXL2 expression. Using in vitro assays with COS7 cells we also demonstrated that progesterone regulates the FOXL2 promoter activity through the progesterone receptor. Collectively, our findings imply that endometrial FOXL2 is, as a direct target of progesterone, involved in early pregnancy and implantation.

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In mammals, implantation is associated with major changes in gene profiles in the female reproductive tract. Molecular signatures of the endometrium have also been shown to vary according to the ability of the embryo to develop to term. Nevertheless, analysing endometrial gene patterns during implantation is incompatible with the maintenance of pregnancy. Therefore early determination of pregnancy issue requires a noninvasive method. Peripheral blood mononuclear cells (PBMC) could represent such an alternative but their reaction to the presence of an implanting embryo has to be investigated. The aim of this study was to investigate gene expression profiles of endometrial caruncular tissue (CAR) and PBMC collected from pregnant ewes (n = 4) and nonpregnant ewes inseminated with inactivated sperm (n = 4) at Day 15 after oestrus. Differentially expressed genes (DEG) were identified using an ovine custom-designed array derived from the ovine 15K Agilent array (Ruscanu et al. 2013 J. Virol. 87, 9333–9343). Data were normalized by Loess and analysed by a linear model in the Limma R package. P-values were corrected using the Benjamini and Hochberg procedure. Comparing pregnancy versus nonpregnancy led to the identification of 2826 DEG in CAR (P < 0.05) and 396 DEG in PBMC (P < 0.10; 265 DEG common with CAR). Ingenuity Pathway Analysis (IPA; Ingenuity Inc., Mountain View, CA, USA) analysis of the 396 PBMC-related DEG revealed 72 overrepresented biological functions (P < 0.001). Among the 15 most overrepresented functions, 13 were common between CAR and PBMC and were mostly related to the immune system, as “infectious disease”, “cell-to-cell signalling and interaction”, “immunological disease”, “immune cell trafficking and inflammatory response”. Using the downstream effect analysis (DEA) of IPA, we identified 163 functions predicted to be increased and 8 functions predicted to be decreased for the CAR DEG dataset, whereas 12 functions were predicted to be increased and 40 functions predicted to be decreased for the PBMC DEG dataset. Interferon (IFN) signalling was strongly present in both datasets, with 44% of PBMC DEG and 29% of CAR DEG found to be related to IFN type I response according to the Interferome database (www.interferome.org). A selection of 12 DEG was validated by qRT-PCR in CAR, intercaruncular areas, and PBMC using 8 pregnant and various groups of nonpregnant ewes (n = 7–9/group). Our data show that PBMC transcriptome is influenced by early pregnancy in sheep, including a major impact of IFN type I such as IFN tau, the signal of maternal recognition of pregnancy. Identifying relevant circulating biomarkers reflecting the quality of the embryo will require further investigation. The authors thank UCEA team (INRA), B. Jost (IGBMC) and F. Moreews (Sigenae).

Fertility in cattle includes the ability of the uterus to provide the appropriate environment for pregnancy success, including the transport of spermatozoa before fertilization. Confocal laser endomicroscopy technology (Cellvizio) has been previously used in small ruminants to visualise labelled spermatozoa invivo in the uterine horns of ewes (Druart et al. 2009 Reprod. 138, 15-53). Nevertheless, to the best of our knowledge, no invivo study has reported a live visual analysis of the behaviour of spermatozoa in the bovine uterus upon AI. The aim of this study was to develop an experimental procedure to label bull spermatozoa and to visualise their progression in the uterine horns of dairy cows, using Cellvizio and uterine cervix catheterization. Fresh ejaculated bull spermatozoa were double-labelled with octadecyl rhodamine B chloride and MitoTracker Green FM dyes before dilution and freezing processing. At each step of the process, semen quality was evaluated with a semen quality analyzer (SQA-V b, Medical Electronic System) and compared to nonlabelled bull reference semen. The specificity of the labelling was validated invitro. We also developed a specific device to insert the Cellvizio fibre probe into the genital tract, in order to image vagina, cervix, and uterine body, as well as the proximal, middle, and distal parts of the uterine horn, including the utero-tubal junction (UTJ), before and after AI with fresh and frozen labelled spermatozoa. Then, 10 heifers were oestrus synchronized. Video sequences were recorded at oestrus (n=5), just before AI, at the time of AI (7-12h after the onset of oestrus), 30 and 120min after AI, and 14 days after oestrus (n=5; luteal phase), to investigate the effect of the steroid-primed uterine environment on the distribution and progression of spermatozoa in the genital tract. Using our approach, labelled spermatozoa were detected (1) at the day of oestrus and 14 days after oestrus, (2) in the uterine body at the time of AI, (3) in various parts of the genital tract 30min after AI, and (4) in the UTJ, the cervix, and the vagina 120min after AI. In addition, labelling the spermatozoa did not alter the fertilizing capacity: 4 of the 5 oestrus females were pregnant at Day 35. Additionally, an IVF test showed a blastocyst rate of 45.8% for labelled semen vs. 39.4% for the control group (Carvalho et al. 2017 Reprod. 154, 695-710). In conclusion, our study provides a method to phenotype in situ the transport of spermatozoa in the genital tract of heifers. Further investigations are in progress to optimize video quality, to develop an algorithm for an automatic analysis (spermatozoa count, speed, and path) of the recorded sequences.

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ObjectiveTo investigate the effects of an adjuvant allogenic umbilical cord mesenchymal stromal cell (UC‐MSC) patch applied during fetal surgery on motor and sphincter function in the ovine MMC model.DesignMMC defects were surgically created at 75 days of gestation and repaired 14 days later.PopulationOvine MMC model: fetal lambs.MethodsWe compared lambs that received a UC‐MSC patch with a control group of lambs that received an acellular patch.Main Outcome MeasuresClinical neurological assessment was performed at 2 and 24 hours of life and included determination of the Sheep Locomotor Rating scale (SLR), which has been validated in the ovine MMC model. Electrophysical examinations, spine scans and histological analyses were also performed.ResultsOf the 13 operated lambs, nine were born alive: five had of these had received a UC‐MSC patch and four an acellular patch. At 24 hours of life, lambs in the UC‐MSC group had a significantly higher score (14 versus 5, P = 0.04). Amyotrophy was significantly more common in the control group (75% versus 0%, P = 0.02). All the lambs in the control group and none of those in the UC‐MSC group were incontinent. No significant differences were observed between the UC‐MSC and control groups in terms of the presence of spontaneous EMG activity, nerve conduction or spinal evoked potentials. In the microscopic examination, lambs in the UC‐MSC group had less fibrosis between the spinal cord and the dermis (mean thickness, 453 versus 3921 μm, P = 0.03) and around the spinal cord (mean thickness, 47 versus 158 μm, P < 0.001). Examination of the spinal cord in the area of the MMC defect showed a higher large neuron density in the UC‐MSC group (14.5 versus 5.6 neurons/mm2, P < 0.001). No tumours were observed.ConclusionsFetal repair of MMC using UC‐MSC patches improves motor and sphincter function as well as spinal preservation and reduction of fibrosis.