miR-26a-5p and miR-125b-5p affect trophoblast genes and cell functions important during early pregnancy†.

The most critical stage of pregnancy is embryo implantation, which relies on the synchronized developmental capacity of the embryo and uterine receptivity to implantation. In early pregnancy, conceptus and uterus release several factors enabling successful implantation and placentation. Molecules involved in embryo-maternal crosstalk include, but are not limited to, hormones, growth factors, and cytokines. The discovery of microRNAs (small non-coding RNAs regulating gene expression) has revolutionized our understanding of many biological processes, including pregnancy. To date, numerous miRNAs have been detected in different species during pregnancy, both at the endometrial and embryonic sites. Thus, microRNAs are considered important regulators of early pregnancy events. Here, we report miR-26a-5p and miR-125b-5p effects on human and pig trophoblast cell function. Both microRNAs change the level of several genes and proteins important for proper embryo development. Moreover, miR-26a-5p stimulates porcine trophoblast proliferation and has a negative impact on its affinity to laminin. However, miR-125b-5p decreases porcine trophoblast cell migration. Our studies suggest that miR-26a-5p and miR-125b-5p can affect early pregnancy functions by regulating genes and processes important for proper conceptuses' development and progression through the implantation process.

How Cold Shock Affects Ploidy Level and Early Ontogenetic Development of the Sterlet, A. ruthenus L.

The objective of the present research was to study the effect of cold shock (3 °C and 6 °C) on fertilized eggs of the sterlet, Acipenser ruthenus L. Cold shock was applied for various durations (30, 60 and 90 min) and the ploidy levels, survival, and genotypes of the treated embryos/larvae were recorded. Analysis of ploidy levels confirmed the presence of diploid, triploid, and mosaic (1n/2n, 2n/3n, and 1n/2n/3n) genotypes in experimental groups, while it was strictly diploid in control groups. Microsatellite genotyping confirmed both the incidence of polyspermy and retention of the 2nd polar body in experimental groups. However, patterns of inheritance in all diploid offspring in experimental and control groups revealed classical Mendelian disomic inheritance. Interestingly, the observed mosaic sterlets had normal morphology and were alive. However, some larvae had abnormal morphology which may be due to haploid syndrome. In all treatment groups (treatments: 3 °C-30 min; 3 °C-60 min; 3 °C-90 min; 6 °C-60 min), where the percentage of polyploid/mosaic larvae were high, the mortality was also high. Whereas, in the control groups (where there were only diploid (2n) larvae), the mortality was relatively low.

Expression of orexins and their precursor in the porcine ovary and the influence of orexins on ovarian steroidogenesis in pigs.

Orexins A and B are hypothalamic neuropeptides associated with homeostasis and the reproductive system. The aim of the study was to compare the expression of the prepro-orexin gene and the intensity of orexins immunoreactivity in the porcine ovary (corpora lutea, granulosa and theca interna cells) during four different stages of the oestrous cycle (days: 2-3, 10-12, 14-16 and 17-19) and to examine the in vitro effect of orexins on the secretion of steroid hormones by porcine luteal, granulosa and theca interna cells. The highest expression of prepro-orexin mRNA was observed in theca interna cells on days 17-19 of the oestrous cycle. The highest content of immunoreactive orexin A was noted in corpora lutea on days 10-12 and the highest level of immunoreactive orexin B on days 14-16 of the cycle. Immunoreactive orexin A concentrations were higher in theca interna cells than in granulosa cells, whereas similar levels of immunoreactive orexin B were observed in both cell types. Under in vitro conditions, at the concentration of 10 nM, orexins A and B inhibited FSH-induced oestradiol secretion by granulosa cells. The obtained results suggest that the pattern of orexin peptide expression in the porcine ovary is related to the animals' hormonal status. Our findings imply that orexins can affect porcine reproductive functions through modulation of ovarian steroidogenesis.

Adiponectin Expression in the Porcine Ovary during the Oestrous Cycle and Its Effect on Ovarian Steroidogenesis.

Adiponectin is an adipose-secreted hormone that regulates energy homeostasis and is also involved in the control of the reproductive system. The goal of the present study was to investigate changes in adiponectin gene and protein expression in porcine ovarian structures during the oestrous cycle and to examine the effects of in vitro administration of adiponectin on basal and gonadotrophin- and/or insulin-induced secretion of ovarian steroid hormones. Both gene and protein expression of adiponectin were enhanced during the luteal phase of the cycle. Adiponectin affected basal secretion of progesterone by luteal cells, oestradiol by granulosa cells, and testosterone by theca interna cells. The gonadotrophin/insulin-induced release of progesterone from granulosa and theca interna cells and the release of oestradiol and androstenedione from theca cells was also modified by adiponectin. In conclusion, the presence of adiponectin mRNA and protein in the porcine ovary coupled with our previous results indicating adiponectin receptors expression suggest that adiponectin may locally affect ovarian functions. The changes in adiponectin expression throughout the oestrous cycle seem to be dependent on the hormonal status of pigs related to the stage of the oestrous cycle. The effect of adiponectin on ovarian steroidogenesis suggests that this adipokine influences reproductive functions in pigs.

Expression of adiponectin receptors 1 and 2 in the ovary and concentration of plasma adiponectin during the oestrous cycle of the pig.

The aim of this study was to compare the expression levels of adiponectin receptor 1 and adiponectin receptor 2 mRNAs and proteins in porcine ovaries during four stages (days 2 to 3, 10 to 12, 14 to 16, 17 to 19) of the oestrous cycle and to measure adiponectin plasma concentrations during the same phases of the cycle. Higher mRNA expression of adiponectin receptor 1 was detected in porcine granulosa cells than in corpora lutea and theca cells (P < 0.01). In contrast, higher gene expression of adiponectin receptor 2 occurred in newly developed and mature corpora lutea (P < 0.01). The adiponectin receptor 1 protein content was the highest in corpora lutea isolated on days 2 to 3 of the cycle and was the lowest in theca interna cells (P < 0.01). The profile of adiponectin receptor 2 protein was similar to that of adiponectin receptor 1. Adiponectin plasma concentrations were significantly higher throughout the luteal phase than in the follicular phase (P < 0.01). In conclusion, the presence of adiponectin receptor 1 and adiponectin receptor 2 mRNAs and proteins in the porcine ovary suggests that adiponectin may directly affect ovarian functions through its own specific receptors. The expression of both receptors and adiponectin plasma concentration were dependent on hormonal status related to the stage of the cycle.

Expression of adiponectin receptors 1 (AdipoR1) and 2 (AdipoR2) in the porcine pituitary during the oestrous cycle.

BACKGROUND: Adiponectin, protein secreted mainly by white adipose tissue, is an important factor linking the regulation of metabolic homeostasis and reproductive processes. The biological activity of the hormone is mediated via two distinct receptors, termed adiponectin receptor 1(AdipoR1) and adiponectin receptor 2 (AdipoR2). The present study analyzed mRNA and protein expression of AdipoR1 and AdipoR2 in the anterior (AP) and posterior (NP) pituitary of cyclic pigs. METHODS: The total of 20 animals was assigned to one of four experimental groups (n=5 per group) as follows: days 2-3 (early-luteal phase), 10-12 (mid-luteal phase), 14-16 (late-luteal phase), 17-19 (follicular phase) of the oestrous cycle. mRNA and protein expression were analyzed using real-time PCR and Western Blot methods, respectively. RESULTS: The lowest AdipoR1 gene expression was detected in AP on days 10-12 relative to days 2-3 and 14-16 (p<0.05). In NP, AdipoR1 mRNA levels were elevated on days 10-12 and 14-16 (p<0.05). AdipoR2 gene expression in AP was the lowest on days 10-12, and an expression peak occurred on days 2-3 (p<0.05). In NP, the lowest (p<0.05) expression of AdipoR2 mRNA was noted on days 17-19. The AdipoR1 protein content in AP was the lowest on days 17-19 (p<0.05), while in NP the variations in protein expression levels during the oestrous cycle were negligible. AdipoR2 protein in AP was most abundant on days 10-12, and it reached the lowest level on days 2-3 and 17-19 of the cycle (p<0.05). The presence of AdipoR2 protein in NP was more pronounced on days 10-12 (p<0.05). CONCLUSIONS: Our study was the first experiment to demonstrate that AdipoR1 and AdipoR2 mRNAs and proteins are present in the porcine pituitary and that adiponectin receptors expression is dependent on endocrine status of the animals.

Changes in plasma orexin A and orexin B concentrations during the estrous cycle of the pig.

Orexin A (OXA) and orexin B (OXB) are neuropeptides synthesized mainly in the lateral hypothalamus, which are involved in the control of various physiological functions such as energy homeostasis, sleep, wakefulness and feeding behavior. The present study analyzes orexins A and B levels in the porcine plasma during the estrous cycle. The highest plasma concentrations of orexin A were observed on days 2-3 of the estrous cycle (p<0.05 relative to days 10-12 and 14-16) and the lowest (p<0.05) on days 14-16. The highest orexin B levels in the blood plasma were noted on days 17-19 (p<0.05 vs. days 14-16). We demonstrated the presence of OXA and OXB in porcine blood plasma and the impact of the phase of the estrous cycle on the observed changes in plasma orexin levels.

Localization of orexin A and orexin B in the porcine uterus.

The presence of orexins and their receptors in gonads indicate that these hormones participate in the control of reproductive functions. The aim of the study was to compare the expression of the prepro-orexin (PPO) gene in porcine endometrium and myometrium and the intensity of OXA- and OXB-immunoreactivity in the following uterine structures: endometrial glandular and luminal epithelium and stroma as well as the myometrial longitudinal and circular muscles during the four stages (days 2-3, 10-12, 14-16, 17-19) of the estrous cycle. The highest expression of PPO mRNA was observed in the endometrium and the myometrium on days 14-16 of the cycle. The expression of the PPO gene on days 2-3 was more pronounced in the myometrium than in the endometrium, whereas on days 17-19 the gene expression was markedly higher in the endometrium. The OXA signal intensity was highest on days 2-3 in the luminal epithelium and on days 2-3 and 10-12 in the stroma. In circular muscles of the myometrium, the highest immunoreactivity was found on days 2-3 and 10-12, while in longitudinal muscles on days 2-3. OXB-immunoreactivity was highest on days 10-12 in longitudinal muscles, on days 17-19 in glandular epithelium and stroma, and on days 10-12 and 14-16 in luminal epithelium. Our results suggest that orexin A and B are produced in the porcine uterus and that their release is dependent on the hormonal status of animals.

Expression of orexin receptors 1 (OX1R) and 2 (OX2R) in the porcine ovary during the oestrous cycle.

Orexins A and B are neuropeptides which are synthesized mainly in the lateral hypothalamus and are associated with a variety of physiological functions such as energy homeostasis, sleep and wakefulness, feeding behavior, as well as the reproductive system. The orexins activate two G-protein-coupled receptors termed orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). The present study analyses OX1R and OX2R mRNAs and proteins' expression in the porcine ovary on days 2-3, 10-12, 14-16 and 17-19 of the oestrous cycle. Using real-time PCR, higher OX1R mRNA expression was detected in porcine CLs (p<0.01) than in granulosa and theca cells. The expression peak of the OX2R gene occurred in granulosa cells (p<0.05 in comparison with CLs on days 2-3 and 10-12 of the oestrous cycle). The OX1R protein content was higher (p<0.01) in CLs isolated during the luteal phase in comparison with follicular cells. The OX2R protein level was more pronounced (p<0.05) in CLs on days 10-12 and 14-16 than in the remaining periods of the cycle. In conclusion, we demonstrated the presence of OX1R and OX2R genes and proteins in the ovary of the pig and the impact of the hormonal milieu on the expression of both receptors. Our results suggest that orexins may affect reproductive functions.

Expression of orexin receptors 1 (OX1R) and 2 (OX2R) in the porcine pituitary during the oestrous cycle.

Orexin A and B, also termed hypocretin 1 and 2, are associated with the stimulation of food intake and arousal. The biological actions of the hormones are mediated via two distinct G protein-coupled receptors, termed orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). OX1R is selective for orexin A and OX2R binds orexin A and orexin B with similar affinity. The present study analyzed mRNA and protein expressions of OX1R and OX2R in adenohypophysis (AP) and neurohypophysis (NP) of cycling pigs. The tissue samples were harvested on days 2-3, 10-12, 14-16, and 17-19 of the oestrous cycle. Using quantitative real-time PCR higher OX1R gene expression was detected in AP on days 2-3 relative to days 10-12, 14-16 and 17-19 (p<0.05). In NP the OX1R mRNA level was elevated on days 10-12 compared to the remaining stages (p<0.05). OX2R gene expression in AP was the lowest on days 10-12 (p<0.05 compared to days 2-3 and 17-19) and the expression peak occurred on days 17-19 (p<0.05 vs. the all studied stages). In NP the highest (p<0.05) expression of OX2R mRNA was noted on days 17-19 in relation to the remaining periods. OX1R protein content in AP was greatest on days 10-12 (p<0.05), whereas in NP it was greatest on days 2-3 and 14-16 (p<0.05 vs. days 10-12 and 17-19). In both cases the lowest OX1R protein expression was observed during follicular phase (p<0.05 in relation to three remaining studied stages). OX2R protein in AP was lower (p<0.05) on days 2-3 and 14-16 compared to days 10-12 and 17-19. In NP the lowest (p<0.05) expression of this protein was on days 17-19 and the highest on days 10-12 (p<0.05 compared to days 2-3 and 17-19). In summary, the present findings provide the first evidence that OX1R and OX2R mRNAs and proteins occur in the pituitary of the pig and indicate the dependence of orexin receptor expression on the endocrine reproductive state.