The melatonin system is expressed in the ovine uterus: effect of the day of the oestrous cycle and undernutrition.

CONTEXT: Melatonin influences female reproduction, but expression of the melatonin system has not been characterised in the ovine uterus. AIMS: We aimed to determine whether synthesising enzymes (arylalkylamine N-acetyltransferase (AANAT) and N-acetylserotonin-O-methyltransferase (ASMT)), melatonin receptors 1 and 2 (MT1 and MT2), and catabolising enzymes (myeloperoxidase (MPO) and indoleamine 2,3-dioxygenase 1 and 2 (IDO1 and 2)), are expressed in the ovine uterus, and if they are influenced by the oestrous cycle (Experiment 1) or by undernutrition (Experiment 2). METHODS: In Experiment 1, gene and protein expression was determined in sheep endometrium samples collected on days 0 (oestrus), 5, 10 and 14 of the oestrous cycle. In Experiment 2, we studied uterine samples from ewes fed either 1.5 or 0.5times their maintenance requirements. KEY RESULTS: We have demonstrated the expression of AANAT and ASMT in the endometrium of sheep. AANAT and ASMT transcripts, and AANAT protein were more elevated at day 10, then decreased to day 14. A similar pattern was observed for MT2 , IDO1 , and MPO mRNA, which suggests that the endometrial melatonin system might be influenced by ovarian steroid hormones. Undernutrition increased AANAT mRNA expression, but seemed to decrease its protein expression, and increased MT2 and IDO2 transcripts, whereas ASMT expression was unaffected. CONCLUSIONS: The melatonin system is expressed in the ovine uterus and is affected by oestrous cycle and undernutrition. IMPLICATIONS: The results help explain the adverse effects of undernutrition on reproduction in sheep, and the success of exogenous melatonin treatments in improving reproductive outcomes.

Estradiol cypionate administered at the end of a progesterone-based protocol for FTAI induces ovulation and improves postovulatory luteal function and uterine environment in anestrous beef cows.

The objective of this study was to evaluate the effect of the administration of estradiol cypionate (ECP) at the end of an estradiol and progesterone-based protocol for fixed time artificial insemination (FTAI) on ovarian response and uterine function in postpartum anestrous beef cows. Multiparous suckled cows were randomly assigned to receive ECP at doses of 0 (control, n = 15), 0.5 (n = 15) or 1.0 mg (n = 15) im at the time of progesterone intravaginal insert removal. Serum 17ß-estradiol concentrations at 24 h after insert removal were greater (P < 0.05) in both ECP treatments than in controls. No differences in estradiol were found between 0.5 mg and control cows (P > 0.1) from 48 h after insert removal until ovulation, although greater (P < 0.05) concentrations were maintained until ovulation in 1.0 mg ECP treated cows. Maximum 17ß-estradiol concentration attained in each female was greater as ECP dose was greater (10.4 ± 0.4, 11.8 ± 0.5 and 13.5 ± 0.7, for control, 0.5 and 1.0 mg ECP treated cows, respectively; P < 005). Proportion of cows that ovulated tended to be greater (P = 0.06) in ECP treated than in control cows. Ovulation occurred earlier and the size of the ovulatory follicle was smaller (P < 0.05) for 1.0 mg but not for 0.5 mg (P > 0.1) when compared with control cows. After ovulation (Day 13 and 14), serum progesterone concentrations were greater (P < 0.05) in 0.5 and 1.0 mg ECP than control cows. Uterine environment on Day 6 after ovulation was affected by treatment; transcript expression of three of nine evaluated genes (i.e., estrogen, IGF-1 and insulin receptors genes) were upregulated (P < 0.05) after ECP treatment. In conclusion, ECP administration at progesterone insert removal in anestrous cows i) induces greater serum estradiol concentrations and tended to induce greater ovulation rate, ii) acts in a dose-dependent manner, as ECP dose increases ovulation occurs earlier and the size of the ovulatory follicle is smaller, iii) improves postovulatory luteal function and affects uterine gene expression. Altogether, this information contributes with the understanding of the effect of preovulatory estradiol exposure on ovulation and postovulatory ovarian and uterine function in anestrous beef cows.

Effect of equine chorionic gonadotropin (eCG) administration and proestrus length on ovarian response, uterine functionality and pregnancy rate in beef heifers inseminated at a fixed-time.

The objective of the present study was to evaluate the effect of equine chorionic gonadotropin (eCG) administration associated to different proestrus lengths for Fixed-time AI (FTAI) in beef heifers. In Experiment 1, pre-pubertal heifers (n = 46) received a 6-day estradiol/progesterone-based treatment (J-Synch protocol), and were then allocated into four experimental groups in a 2 × 2 factorial design, to receive or not receive eCG (300 IU) at the time of intravaginal progesterone device removal, and to receive GnRH at 48 h or 72 h after device removal (to induce shortened and prolonged proestrus length, respectively). Endometrial samples were obtained 6 d after ovulation from the cranial portion of the uterine horn. The eCG administration induced greater serum estradiol-17ß concentrations before ovulation (P < 0.05) and greater proportion of heifers bearing a competent corpus luteum after ovulation (P = 0.054). Delaying GnRH administration from 48 h to 72 h induced a longer interval from device removal to ovulation (i.e., prolonged proestrus; P < 0.05), larger diameter of the ovulatory follicle, and greater progesterone concentrations on Day 10-11 after ovulation. Heifers in eCG + GnRH72h group had more uterine receptors in luminal epithelium than those in eCG + GnRH48h group (PR and ERα), and than those in No eCG + GnRH72h group (PR) (P < 0.05). No effect of eCG or GnRH treatments was found in endometrial gene expression of progesterone and estrogen receptors. In Experiment 2, a total of 2,598 heifers received the J-Synch protocol associated or not with eCG administration at device removal, followed by FTAI/GnRH at 60 or 72 h after device removal (i.e., prolonged proestrus protocol). Heifers that received eCG had greater P/AI than those not receiving eCG (P < 0.05) and there was an interaction between eCG treatment and time of FTAI. The lowest P/AI was found in those heifers that received FTAI/GnRH at 72 h without eCG treatment at device removal (P < 0.05), and no differences were found between the other experimental groups. In conclusion, prolonging the length of proestrus in J-Synch protocol improves ovulatory follicular diameter and luteal function; and the administration of eCG at device removal improves preovulatory estradiol concentrations and luteal function. Finally, P/AI was enhanced by eCG treatment and the improvement was more evident when FTAI/GnRH was performed at 72 h after device removal.

Local influence of the corpus luteum on the ipsilateral oviduct and early embryo development in the ewe.

The objective of this study was to evaluate the local effect of the corpus luteum (CL) on ipsilateral oviduct-uterus functionality and early embryo development in ewes. A total of 499 embryos were transferred on Day 1 after in vitro fertilization into the ipsilateral (n = 250) and contralateral oviducts (n = 249) of 13 ewes on Day 1 after ovulation (18-20 embryos per oviduct). On Day 6, their reproductive tracts were collected and their uterine horns were flushed for embryo recovery. More recovered embryos, a higher proportion of blastocysts, and more viable embryos were collected when the embryos were transferred into the ipsilateral oviducts (P < 0.05). In addition, almost five times higher P4 concentrations and significantly lower E2 concentrations, with higher P4:E2 ratio, were found in the ipsilateral than contralateral oviductal tissue (P < 0.05). Furthermore, a higher concentration of adiponectin was found in the ipsilateral uterine tissue macerates than in the contralateral side to the CL. The ipsilateral oviductal tissue had a lower expression of PGR and IGFBP5, but the transcript expression of ADIPOR1 was higher in the ipsilateral oviductal tissue. In the uterus, the mRNA expression of ESR1, IGFBP3, IGFBP5, and LEPR was higher or tended to be higher in the ipsilateral than contralateral uterine tissue. Uterine flushing fluid collected from the ipsilateral uterine horn had lower insulin concentrations than the contralateral horn, while no differences were found in the P4 and E2 concentrations. In conclusion, on Day 6 post-ovulation, P4 was elevated in the ipsilateral oviductal tissue, embryo development was advanced, and differential gene expression of PGR, ESR1, IGFBP3, IGFBP5, LEPR, and ADIPOR1 in the oviductal or uterine tissue was found between the ipsilateral and contralateral side. This study demonstrates local regulation of the ovary on the ipsilateral oviduct/uterine horn in the ewe.

Effects of extending the length of pro-oestrus in an oestradiol- and progesterone-based oestrus synchronisation program on ovarian function, uterine environment and pregnancy establishment in beef heifers.

The aim of the present study was to investigate the effects of a strategy for extending pro-oestrus (the interval between luteolysis and ovulation) in an oestrus synchronisation protocol (named J-Synch) in beef heifers on follicular growth, sexual steroid concentrations, the oestrogen receptor ERα and progesterone receptors (PR) in the uterus, insulin-like growth factor (IGF) 1 and pregnancy rates. In Experiment 1, heifers treated with the new J-Synch protocol had a longer pro-oestrus period than those treated with the conventional protocol (mean (±s.e.m.) 93.7±12.9 vs 65.0±13.7h respectively; P<0.05). The rate of dominant follicle growth from the time of progesterone device removal to ovulation was greater in heifers in the J-Synch than conventional group (P<0.05). Luteal area and serum progesterone concentrations were greater in the J-Synch Group (P<0.05) for the 12 days after ovulation. Progesterone receptor (PGR) staining on Day 6 after ovulation in the uterine stroma was lower in the J-Synch than conventional group (P<0.05), and the expression of PR gene (PGR) and IGF1 gene tended to be lower in J-Synch-treated heifers (P<0.1). In Experiment 2 (n=2349), the pregnancy rate 30-35 days after fixed-time AI (FTAI) was greater for heifers in the J-Synch than conventional group (56.1% vs 50.7% respectively). In conclusion, our strategy for extending pro-oestrus (i.e. the J-Synch protocol) significantly improves pregnancy establishment in beef heifers. This improvement was related to an increased rate of growth of the dominant ovulatory follicle, greater progesterone concentrations during the ensuing luteal phase and different uterine patterns of PGR and IGF1, which may have favoured embryo development and pregnancy establishment.

Oocyte developmental competence is improved by relatively greater circulating progesterone concentrations during preovulatory follicular growth.

This study evaluated the effect of progesterone priming during follicular growth on oocyte competence to undergo oocyte cleavage and embryo development in sheep. Two experiments were performed on a total of 195 females that either received or did not receive a progesterone treatment (CIDR-type device) during the first follicular wave, beginning soon after ovulation (i.e., Day 0 of the experiment). On Day 3, the follicular population and oocyte quality (Experiment 1 and 2) and the competence of oocytes for cleavage and embryo development (Experiment 2) were evaluated after laparoscopic ovum pickup (LOPU) and in vitro fertilization. In Experiment 1, in a 2 × 2 factorial study the progesterone priming treatment (treated or not) was or was not associated with a single dose of FSH in a slow-release hyaluronic acid preparation given on Day 0. The follicular population on Day 3 and the number and morphology of recovered cumulus oocyte complexes (COCs) were not affected by the progesterone treatment (P = NS) but were improved by the FSH administration (P <  0.05). An interaction between both treatments was observed (P <  0.05), with more desirable outcome with the females that received both the progesterone and the FSH treatments. In Experiment 2, half of the females received the exogenous progesterone priming, and all females received FSH on Day 0. After follicular aspiration on Day 3, the cleavage rate and the embryo development rate following in vitro fertilization and culture were greater in those females that received the progesterone treatment (P <  0.05). In conclusion, these studies provide evidence that progesterone treatment during follicular growth affects oocyte competence, with the greater progesterone concentrations enhancing the oocyte's capacity to undergo cleavage and embryo development.

Gestation-related gene expression and protein localization in endometrial tissue of Suffolk and Cheviot ewes at gestation Day 19, after transfer of Suffolk or Cheviot embryos.

The objective of this study was to investigate the gene expression of progesterone and estrogen receptor α (PR, ERα), insulin-like growth factor (IGF) 1, IGF-2, their receptor (IGFR1), IGF-binding proteins (BP) 1 to 6, insulin receptor, adiponectin receptors (AdipoR1/2), cyclooxygenase 2 (PTGS2), mucin 1 and to localize PR, ERα, IGF-1, IGFR1, PTGS2, and proliferating cellular nuclear antigen (PCNA) in the endometrium of pregnant (Day 19) Suffolk and Cheviot ewes carrying Suffolk and Cheviot embryos transferred within and reciprocally between breeds. Gene expression was determined by real-time quantitative polymerase chain reaction (RT-qPCR), and antigen determination was measured by immunohistochemistry in the luminal epithelium (LE), superficial and deep glands (SG, DG, respectively) and superficial and deep stroma. Gene expression of PR, IGF-1, IGFBP2, and IGFBP5 was higher in Suffolk than that in Cheviot ewes (P < 0.05). Greater abundance of IGF-2 and IGBP3 expression was found in Cheviot ewes carrying Cheviot embryos than Cheviot ewes carrying Suffolk embryos (P < 0.05). No staining for PR and ERα was observed in the LE, very scarce staining in SG and DG, whereas positive staining was observed in both superficial and deep stroma. No differences were found for PR staining, but Cheviot ewes had higher ERα staining intensity than Suffolk ewes (P < 0.05). Positive staining for IGF-1 was observed in all cell types except DG, and staining of IGFR1 was observed in all cell types. No differences among groups in staining were found for IGF-1 or IGFR1 in any cell type. Positive staining of PTGS2 was observed in LE and SG in all groups. An interaction between ewe and embryo breed affected PTGS2 staining (P < 0.05), whereby Cheviot ewes carrying Suffolk embryos had a lower PTGS2 staining than Suffolk ewes carrying Suffolk embryos. Positive staining of PCNA was found in LE and SG. Suffolk ewes carrying Suffolk embryos showed lower PCNA immunostaining than Cheviot ewes carrying Suffolk embryos (P < 0.05), whereas no differences were observed in ewes carrying Cheviot embryos. This study showed that gestation-related protein expression in the endometrium of Suffolk and Cheviot ewes is affected by both ewe and embryo breed at Day 19 of pregnancy.

Periconceptional undernutrition modifies endocrine profiles and hepatic gene expression in sheep.

This study investigated whether a 22-day period of undernutrition (half maintenance) could affect maternal endocrine responses and liver gene expression during early pregnancy (day 7). Thirty-five ewes were fed 1.5 (n = 15) or 0.5 (n = 20) their maintenance requirements and slaughtered on day 7 of the oestrus cycle or pregnancy (oestrus = day 0). Insulin, IGF, leptin and non-esterified fatty acids (NEFA) were determined on days -14, 0 and 7. Transcripts of the IGF family and adipokines receptors were determined in the liver by real-time RT-PCR. Underfed animals presented lower body weight and body condition, greater plasma concentration of NEFA, and lower plasma concentrations of leptin, insulin and IGF1 compared to adequately fed animals. Underfed ewes presented greater hepatic expression of IGFBP2 than well-fed ewes, but tended to have lesser expression of IGFBP5. While no effect of undernutrition on IGFBP4 and ADIPOR2 mRNA expressions was observed, they were increased by pregnancy in underfed animals. This study shows that undernutrition modifies endocrine profiles and hepatic gene expression of IGFBP2 and 5. The pregnancy status increased hepatic gene expression of IGFBP4 and ADIPOR2 mRNA in undernourished ewes.

Pentoxifylline improves learning and memory in glutamate-lesioned rats.

The present work shows the effects of pentoxifylline (ptx), on learning and memory in rats with hippocampal lesions induced by glutamate (glu). Immediately after stereotaxic procedures and in the absence or presence of glu lesions, animals were treated with ptx (50, 100, or 200 mg/kg, IP) for 6 days. Twenty-four hours after the last injection, behavior and memory tests were performed, animals were sacrificed, and hippocampi dissected for cAMP determination or histopathological studies. Results from the T-maze task showed a less learning ability in the glu-lesioned group compared to other ones. Ptx alone or associated with glu significantly improved memory acquisition, but not memory consolidation compared to glu-lesioned rats. Except for the increased locomotor activity observed in the ptx100+glu-treated group compared to saline, no other difference was detected in the open-field test. A significant impairment in avoidance performance was observed in glu-lesioned group as compared to saline or to other groups in the short as well as in the late phase of memory. All groups showed an improved water-maze performance over time with similar performances on the final day of acquisition. The impairment in memory retention observed in glu-lesioned rats was reversed by the pretreatment with ptx200. Glu induced hippocampal lesion and reduced cAMP levels. Both effects were blocked by ptx, suggesting that its action may be the result of increased cAMP levels and/or inhibition of adenosine A1 receptors.