New insights into the potential effects of PET microplastics on organisms via extracellular vesicle-mediated communication.

Plastics have become an integral part of our daily lives. In the environment, plastics break down into small pieces (<5 mm) that are referred to as microplastics. Microplastics are ubiquitous and widespread in the environment, and all living organisms are exposed to their effects. The present study provides new insights into the potential effects of polyethylene terephthalate (PET) microplastics on organisms via extracellular vesicle (EV)-mediated communication. The study demonstrated that serum-derived EVs are able to transport plastic particles. In addition, PET microplastics alter the content of miRNA in EVs. The identified differentially regulated miRNAs may target genes associated with lifestyle diseases, such as cardiovascular or metabolic diseases, and carcinogenesis. This work expands our understanding of PET microplastics' effects on organisms via EV-mediated communication and identifies directions for further research and strategies.

PPARß/δ Ligands Regulate Oxidative Status and Inflammatory Response in Inflamed Corpus Luteum-An In Vitro Study.

Inflammation in the female reproductive system causes serious health problems including infertility. The aim of this study was to determine the in vitro effects of peroxisome proliferator-activated receptor-beta/delta (PPARß/δ) ligands on the transcriptomic profile of the lipopolysaccharide (LPS)-stimulated pig corpus luteum (CL) in the mid-luteal phase of the estrous cycle using RNA-seq technology. The CL slices were incubated in the presence of LPS or in combination with LPS and the PPARß/δ agonist-GW0724 (1 µmol/L or 10 µmol/L) or the antagonist-GSK3787 (25 µmol/L). We identified 117 differentially expressed genes after treatment with LPS; 102 and 97 differentially expressed genes after treatment, respectively, with the PPARß/δ agonist at a concentration of 1 µmol/L or 10 µmol/L, as well as 88 after the treatment with the PPARß/δ antagonist. In addition, biochemical analyses of oxidative status were performed (total antioxidant capacity and activity of peroxidase, catalase, superoxide dismutase, and glutathione S-transferase). This study revealed that PPARß/δ agonists regulate genes involved in the inflammatory response in a dose-dependent manner. The results indicate that the lower dose of GW0724 showed an anti-inflammatory character, while the higher dose seems to be pro-inflammatory. We propose that GW0724 should be considered for further research to alleviate chronic inflammation (at the lower dose) or to support the natural immune response against pathogens (at the higher dose) in the inflamed corpus luteum.

In vitro effects of PPAR gamma ligands on gene expression in corpus luteum explants in non-pregnant pigs - Transcriptome analysis.

The corpus luteum (CL) is a temporary endocrine structure in the female ovaries that develops cyclically in mature females during luteinization. This study aimed to determine the in vitro effects of peroxisome proliferator-activated receptor gamma (PPARγ) ligands on the transcriptomic profile of the porcine CL in the mid- and late-luteal phase of the estrous cycle using RNA-seq technology. The CL slices were incubated in the presence of PPARγ agonist - pioglitazone or antagonist - T0070907. We identified 40 differentially expressed genes after treatment with pioglitazone and 40 after treatment with T0070907 in the mid-luteal phase as well as 26 after pioglitazone and 29 after T0070907 treatment in the late-luteal phase of the estrous cycle. In addition, we detected differences in gene expression between the mid- and late-luteal phase without treatment (409 differentially expressed genes). This study revealed a number of novel candidate genes that may play a role in controlling the function of CL by regulating signaling pathways related to ovarian steroidogenesis, metabolic processes, cell differentiation, apoptosis, and immune responses. These findings become a basis for further studies to explain the mechanism of PPARγ action in the reproductive system.

Peroxisome proliferator-activated receptor gamma ligands regulate the expression of inflammatory mediators in porcine endometrium during LPS-induced inflammation.

Inflammation in the female reproductive system is one of the most common causes of reproductive dysfunction such as infertility, delay of the reproductive cycle and a reduction in reproductive efficiency. In this study, we aimed to investigate the effect of peroxisome proliferator-activated receptor gamma (PPARγ) ligands on the expression of selected inflammatory mediators: nuclear factor kappa (NF-κB), interleukin (IL)-1ß, IL-6, IL-4, IL-10, leukemia inhibitory factor (LIF) and toll-like receptor 4 (TLR4) in the porcine endometrium treated in vitro with lipopolysaccharide (LPS) on days 10-12 and 18-20 of the estrous cycle. In addition, two experimental protocols were applied to evaluate the role of PPARγ agonists in ongoing and developing inflammation. Endometrial slices were incubated in vitro in the presence of LPS (to induce inflammation) and PPARγ agonists, prostaglandin J2 or pioglitazone (natural or synthetic, respectively). The study showed that PPARγ agonists decreased the expression of pro-inflammatory (NF-κB, TLR4, IL-6) and increased the abundance of anti-inflammatory mediators (IL-10) in the inflamed endometrium of pigs. These findings indicate anti-inflammatory properties of the tested ligands.

Analysis of changes in the proteomic profile of porcine corpus luteum during different stages of the oestrous cycle: effects of PPAR gamma ligands.

CONTEXT: The corpus luteum (CL) is an endocrine gland in the ovary of mature females during the oestrous cycle and pregnancy. There is evidence of a relationship between the secretory function of the CL and PPARs. AIMS: In this study, we investigated the changes in the proteome of the CL in relation to the phase of the oestrous cycle and the impact of PPARγ ligands on the proteomic profile of the CL during the mid- and late-luteal phase of the oestrous cycle. METHODS: The porcine CL explants were incubated in vitro for 6h in the presence of PPARγ ligands (agonist pioglitazone, antagonist T0070907) or without ligands. Global proteomic analysis was performed using the TMT-based LC-MS/MS method. KEY RESULTS: The obtained results showed the disparity in proteomic profile of the untreated CL - different abundance of 23 and 28 proteins for the mid- and late-luteal phase, respectively. Moreover, seven proteins were differentially regulated in the CL tissue treated with PPARγ ligands. In the mid-luteal phase, one protein, CAND1, was downregulated after treatment with T0070907. In the late-luteal phase, the proteins SPTAN1, GOLGB1, TP53BP1, MATR3, RRBP1 and SRRT were upregulated by pioglitazone. CONCLUSIONS: Comparative proteomic analysis revealed that certain proteins constitute a specific proteomic signature for each examined phase. Moreover, the study showed that the effect of PPARγ ligands on the CL proteome was rather limited. IMPLICATIONS: The results provide a broader insight into the processes that may be responsible for the structural luteolysis of the porcine CL, in addition to apoptosis and autophagy.

Tandem Mass Tagging (TMT) Reveals Tissue-Specific Proteome of L4 Larvae of Anisakis simplex s. s.: Enzymes of Energy and/or Carbohydrate Metabolism as Potential Drug Targets in Anisakiasis.

Anisakis simplex s. s. is a parasitic nematode of marine mammals and causative agent of anisakiasis in humans. The cuticle and intestine of the larvae are the tissues most responsible for direct and indirect contact, respectively, of the parasite with the host. At the L4 larval stage, tissues, such as the cuticle and intestine, are fully developed and functional, in contrast to the L3 stage. As such, this work provides for the first time the tissue-specific proteome of A. simplex s. s. larvae in the L4 stage. Statistical analysis (FC ≥ 2; p-value ≤ 0.01) showed that 107 proteins were differentially regulated (DRPs) between the cuticle and the rest of the larval body. In the comparison between the intestine and the rest of the larval body at the L4 stage, 123 proteins were identified as DRPs. Comparison of the individual tissues examined revealed a total of 272 DRPs, with 133 proteins more abundant in the cuticle and 139 proteins more abundant in the intestine. Detailed functional analysis of the identified proteins was performed using bioinformatics tools. Glycolysis and the tricarboxylic acid cycle were the most enriched metabolic pathways by cuticular and intestinal proteins, respectively, in the L4 stage of A. simplex s. s. The presence of two proteins, folliculin (FLCN) and oxoglutarate dehydrogenase (OGDH), was confirmed by Western blot, and their tertiary structure was predicted and compared with other species. In addition, host-pathogen interactions were identified, and potential new allergens were predicted. The result of this manuscript shows the largest number of protein identifications to our knowledge using proteomics tools for different tissues of L4 larvae of A. simplex s. s. The identified tissue-specific proteins could serve as targets for new drugs against anisakiasis.

PPARγ regulates the expression of genes involved in the DNA damage response in an inflamed endometrium.

Inflammation is a biological response of the immune system, which can be triggered by many factors, including pathogens. These factors may induce acute or chronic inflammation in various organs, including the reproductive system, leading to tissue damage or disease. In this study, the RNA-Seq technique was used to determine the in vitro effects of peroxisome proliferator-activated receptor gamma (PPARγ) ligands on the expression of genes and long non-coding RNA, and alternative splicing events (ASEs) in LPS-induced inflammation of the porcine endometrium during the follicular phase of the estrous cycle. Endometrial slices were incubated in the presence of LPS and PPARγ agonists (PGJ2 or pioglitazone) and a PPARγ antagonist (T0070907). We identified 169, 200, 599 and 557 differentially expressed genes after LPS, PGJ2, pioglitazone or T0070907 treatment, respectively. Moreover, changes in differentially expressed long non-coding RNA and differential alternative splicing events were described after the treatments. The study revealed that PPARγ ligands influence the LPS-triggered expression of genes controlling the DNA damage response (GADD45ß, CDK1, CCNA1, CCNG1, ATM). Pioglitazone treatment exerted a considerable effect on the expression of genes regulating the DNA damage response.

A Complex Proteomic Response of the Parasitic Nematode Anisakis simplex s.s. to Escherichia coliLipopolysaccharide.

Helminths are masters at manipulating host's immune response. Especially, parasitic nematodes have evolved strategies that allow them to evade, suppress, or modulate host's immune response to persist and spread in the host's organism. While the immunomodulatory effects of nematodes on their hosts are studied with a great commitment, very little is known about nematodes' own immune system, immune response to their pathogens, and interactions between parasites and bacteria in the host's organism. To illustrate the response of the parasitic nematode Anisakis simplex s.s. during simulated interaction with Escherichia coli, different concentrations of lipopolysaccharide (LPS) were used, and the proteomic analysis with isobaric mass tags for relative and absolute quantification (tandem mass tag-based LC-MS/MS) was performed. In addition, gene expression and biochemical analyses of selected markers of oxidative stress were determined. The results revealed 1148 proteins in a group of which 115 were identified as differentially regulated proteins, for example, peroxiredoxin, thioredoxin, and macrophage migration inhibitory factor. Gene Ontology annotation and Reactome pathway analysis indicated that metabolic pathways related to catalytic activity, oxidation-reduction processes, antioxidant activity, response to stress, and innate immune system were the most common, in which differentially regulated proteins were involved. Further biochemical analyses let us confirm that the LPS induced the oxidative stress response, which plays a key role in the innate immunity of parasitic nematodes. Our findings, to our knowledge, indicate for the first time, the complexity of the interaction of parasitic nematode, A. simplex s.s. with bacterial LPS, which mimics the coexistence of helminth and gut bacteria in the host. The simulation of this crosstalk led us to conclude that the obtained results could be hugely valuable in the integrated systems biology approach to describe a relationship between parasite, host, and its commensal bacteria.

PPARγ ligands modulate the immune response mediators in the pig myometrium - An in vitro study.

The current study was conducted with the aim to investigate effects of PPARγ ligands on synthesis of nuclear receptor κB (NF-κB) and selected cytokines (IL-1ß, IFNγ, TNFα, IL-4, IL-10, LIF) in the pig myometrium on days 14-15 of the estrous cycle (late-luteal phase) and days 14-15 of the gestational period (beginning of embryonic implantation). The myometrial slices were incubated in vitro for 6 h in medium containing PPARγ ligands, agonists: 15d-prostaglandin J2 or pioglitazone, and antagonist - T0070907. The mRNA transcript and protein abundances were evaluated in tissues and culture medium. During the estrous cycle, PPARγ ligands did not have an effect on the mRNA transcript abundance of the immune response mediators used for treatments. The IL-10 protein abundance in the tissue was less when there was inclusions of pioglitazone in the medium, while the treatment with T0070907 resulted in a larger abundance of NF-κB, IL-1ß (in the tissue) and IL-4 (in tissue and culture media). During the gestational period, pioglitazone or PGJ2 suppressed mRNA IFNγ and IL-10 transcript and protein abundances (in the tissue and culture media), whereas there was an enhanced NF-κB protein abundance (in the tissue). Treatment with T0070907 had diverse effects (e.g., for NFκB inhibited mRNA transcript abundance or enhanced protein abundance). The observed changes are related mainly in tissues from pregnant animals. Responses to PPARγ antagonist are indicative of the possible involvement of PPARγ-independent factors as well as ligand-independent activation of the receptor, ligand selectivity/functionality or tissue receptivity to the factors evaluated.

Transcriptome analysis of porcine endometrium after LPS-induced inflammation: effects of the PPAR-gamma ligands in vitro†.

Female fertility depends greatly on the capacity of the uterus to recognize and eliminate microbial infections, a major reason of inflammation in the endometrium in many species. This study aimed to determine the in vitro effect of peroxisome proliferator-activated receptor gamma (PPARγ) ligands on the transcriptome genes expression and alternative splicing in the porcine endometrium in the mid-luteal phase of the estrous cycle during LPS-stimulated inflammation using RNA-seq technology. The endometrial slices were incubated in vitro in the presence of LPS and PPARγ agonists-PGJ2 or pioglitazone and antagonist-T0070907. We identified 222, 3, 4, and 62 differentially expressed genes after LPS, PGJ2, pioglitazone, or T0070907 treatment, respectively. In addition, we detected differentially alternative spliced events: after treatment with LPS-78, PGJ2-60, pioglitazone-52, or T0070907-134. These results should become a basis for further studies explaining the mechanism of PPARγ action in the reproductive system in pigs.

Sex- and season-dependent differences in the expression of adiponectin and adiponectin receptors (AdipoR1 and AdipoR2) in the hypothalamic-pituitary-adrenal axis of the Eurasian beaver (Castor fiber L.).

Adiponectin, a product of the Adipoq gene, is an adipocyte-derived protein hormone of the cytokine family and the most abundantly expressed adipokine. Adiponectin and its receptors AdipoR1 and AdipoR2 (collectively referred to as the adiponectin system) are widely expressed in the central nervous system and other tissues, which suggests that this hormone has pleiotropic effects. Adiponectin could also play a role in the modulation of the hypothalamic-pituitaryadrenal (HPA) hormonal regulatory axis. There is a general scarcity of data on the adiponectin system in wild animals where annual changes in reproductive activity are linked with fluctuations in the activity of the HPA axis. The Eurasian beaver (Castor fiber L.) could be an interesting and suitable model for investigating the above processes. We hypothesized that the expression of the adiponectin system in the tissues of the beaver HPA axis is sex- and season-dependent. The study was performed on adult animals harvested during three different stages of reproductive activity: April ('breeding'), July ('post-breeding') and November ('pre-breeding'). The expression of the adiponectin system was confirmed in all branches (mediobasal hypothalamus, pituitary, adrenal cortex) of the HPA axis in both sexes and during all periods of reproductive activity. The expression of Adipoq, AdipoR1 and AdipoR2 was generally dependent on sex and the period of the reproductive season. The expression of adiponectin system genes was particularly pronounced in the adrenal cortex. These findings suggest that the adiponectin system in the Eurasian beaver could link reproductive processes with stress responses and energy metabolism.

Peroxisome proliferator-activated receptor alpha regulates the expression of the immune response mediators in the porcine endometrium during the estrous cycle and early pregnancy.

PROBLEM: Cytokines are immune response mediators that play an important role in the regulation of reproductive functions. An association between cytokines and peroxisome proliferator receptors (PPARs) has been reported in various tissues, including the endometrium. The present study aimed to evaluate the impact of PPARα ligands on the expression of nuclear factor kappa B (NF-κB) and cytokines (interleukin [IL]-1ß, IL-4, IL-6, IL-8, IL-10, and LIF) in the porcine endometrium in different reproductive stages. METHODS OF STUDY: Endometrial slices were collected from gilts on days 10-12 or 14-16 of the estrous cycle and pregnancy. Endometrial tissue explants were incubated in vitro in the presence or absence of PPARα agonist WY-14643 and antagonist MK886. Expression of mRNA and protein for NF-ĸB and selected cytokines was evaluated by real-time PCR and immunoblot. RESULTS: PPARα agonist WY-14643 decreased the mRNA expression of NF-κB in most of the analyzed stages (excluding days 10-12 of the estrous cycle), but increased the expression of NF-κB protein (excluding days 14-16 of pregnancy). The WY-14643 increased expression of IL-1ß and IL-6 proteins, and the mRNA expression of IL-8 and LIF, decreased IL-4 expression, and did not affect the mRNA and protein expression of IL-10. CONCLUSION: The obtained results demonstrate that PPARα is involved in the regulation of NF-κB and cytokine expression in the porcine endometrium. PPARα ligands exert a varied influence on immune system components, which could be attributed to differences in the receptivity of porcine endometrial tissue during the reproductive cycle.

PPARß/δ ligands regulate the expression of immune response mediators in the porcine endometrium - An in vitro study.

The peroxisome proliferator-activated receptor (PPAR) ß/δ belongs to a group of nuclear receptors that act as transcription factors. PPAR ß/δ plays a significant role in the regulation of female reproductive processes. It has been demonstrated that PPARß/δ is expressed in mouse, rat and porcine endometrium during the estrous cycle and pregnancy. The current study aimed to investigate the effect of selected PPARß/δ ligands on the expression of nuclear factor kappa (NF-κB) and selected cytokines - interleukin (IL)-1ß, IL-6, IL-8, IL-4, IL-10, leukemia inhibitory factor (LIF), in the porcine endometrium on days 10-12 and 14-16 of the estrous cycle (mid- and late-luteal phases corresponding to the full activity and luteolysis of the corpus luteum, respectively) and pregnancy (maternal recognition of pregnancy and beginning of implantation, respectively). Endometrial slices were incubated in vitro in the presence of PPARß/δ agonist L-165,041 (1 or 10 µM) or antagonist GW9662 (10 µM). The expression of mRNA and protein of the immune response mediator in the tissues was determined by real-time PCR and Western Blot. In general, the PPARß/δ agonist inhibited endometrial NF-κB mRNA expression during all analyzed reproductive stages, but it did not change protein expression. In turn, the PPARß/δ antagonist increased NF-κB protein levels on days 10-12 of the estrous cycle or pregnancy. The presence of the PPARß/δ agonist stimulated mRNA expression of LIF, IL-1ß and IL-8 and decreased the expression of IL-6. The presence of PPARß/δ ligands had a varied effect on protein expression in different stages on the analyzed period. The obtained results indicate that PPARß/δ regulates the expression of endometrial NF-κB and selected cytokines in pigs. The effects of PPARß/δ ligands on immune response mediators varied subject to the reproductive status of females and could be associated with differences in endometrial receptivity.

Leptin/leptin receptor system in the regulation of reproductive functions and stress response in the European beaver.

The European beaver (Castor fiber L.) is the largest free-living rodent in Eurasia. The present work aimed to determine sex- and season-related changes in leptin receptor (Ob-R) expression in the hypothalamic-pituitary-gonadal/adrenal axes and uterus of beavers during breeding- (April), post-breeding- (July), and pre-breeding- (November) periods. The expression of Ob-R gene and protein was found in all analyzed tissues. The expression of Ob-R mRNA remained constant in the hypothalamus of both sexes during the analyzed stages. Sex- and season-related changes were found in the pituitary gland; the greatest level was observed in July in both sexes. The same expression pattern was noted in the testis, whereas in the ovary a lack of seasonal changes was found. In uterine tissues, the greatest expression occurred in November. The impact of season was also demonstrated in the adrenal cortex. In females, a higher Ob-R transcript level was noted in April, while in males, an increased mRNA abundance was noted in November than July. Our study suggests that in the beaver, leptin acting via the Ob-R can be an important endocrine factor engaged in the regulation of reproductive functions and stress response.

Peroxisome proliferator-activated receptor gamma ligands affect NF-kB and cytokine synthesis in the porcine endometrium-An in vitro study.

PROBLEM: Cytokines, mediators of the immune response, are involved in the regulation of female reproductive processes during the estrous cycle and pregnancy. The present study aimed to investigate the effect of selected peroxisome proliferator-activated receptor gamma (PPARγ) ligands on the expression of nuclear factor kappa B (NF-κB) and selected cytokines, such as interleukin (IL)-1ß, -4, -6, -8, -10, and the leukemia inhibitory factor, in the porcine endometrium on days 10-12 and 14-16 of the estrous cycle (mid- and late luteal phase, respectively) or pregnancy (maternal recognition of pregnancy and beginning of implantation, respectively). METHOD OF STUDY: Endometrial slices were incubated in vitro in the presence of PPARγ agonists, 15-deoxy-Δ12, 14-prostaglandin J2 or rosiglitazone, and PPARγ antagonist T0070907. mRNA and protein levels in tissues were determined by real-time PCR and Western blot. RESULTS: On days 10-12 of the estrous cycle and days 14-16 of pregnancy, PPARγ ligands enhanced the expression of NF-κB, mRNA cytokines, and/or proteins. During the late luteal phase of the estrous cycle (days 14-16) and maternal recognition of pregnancy (days 10-12), PPARγ ligands inhibited the expression of NF-κB, and they differentially affected the expression of mRNA and proteins of cytokines. CONCLUSION: Our results indicate that PPARγ is engaged in the endometrial synthesis of NF-κB and selected cytokines in pigs. The influence of PPARγ ligands on the tested components of the immune system varied subject to the physiological status of females, and it could be associated with differences in endometrial receptivity.

Seasonal differences in the testicular transcriptome profile of free-living European beavers (Castor fiber L.) determined by the RNA-Seq method.

The European beaver (Castor fiber L.) is an important free-living rodent that inhabits Eurasian temperate forests. Beavers are often referred to as ecosystem engineers because they create or change existing habitats, enhance biodiversity and prepare the environment for diverse plant and animal species. Beavers are protected in most European Union countries, but their genomic background remains unknown. In this study, gene expression patterns in beaver testes and the variations in genetic expression in breeding and non-breeding seasons were determined by high-throughput transcriptome sequencing. Paired-end sequencing in the Illumina HiSeq 2000 sequencer produced a total of 373.06 million of high-quality reads. De novo assembly of contigs yielded 130,741 unigenes with an average length of 1,369.3 nt, N50 value of 1,734, and average GC content of 46.51%. A comprehensive analysis of the testicular transcriptome revealed more than 26,000 highly expressed unigenes which exhibited the highest homology with Rattus norvegicus and Ictidomys tridecemlineatus genomes. More than 8,000 highly expressed genes were found to be involved in fundamental biological processes, cellular components or molecular pathways. The study also revealed 42 genes whose regulation differed between breeding and non-breeding seasons. During the non-breeding period, the expression of 37 genes was up-regulated, and the expression of 5 genes was down-regulated relative to the breeding season. The identified genes encode molecules which are involved in signaling transduction, DNA repair, stress responses, inflammatory processes, metabolism and steroidogenesis. Our results pave the way for further research into season-dependent variations in beaver testes.

Leptin plasma concentrations, leptin gene expression, and protein localization in the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-adrenal axes of the European beaver (Castor fiber).

The European beaver (Castor fiber) is the largest seasonal free-living rodent in Eurasia. Since the physiology and endocrine system of this species remains unknown, the present study aimed to determine plasma leptin concentrations and the expression of the leptin gene and protein in the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-adrenal (HPG and HPA) axes of beavers during breeding (April), postbreeding (July), and prebreeding (November) seasons. Leptin plasma concentrations did not change in females, whereas in males, leptin plasma concentrations were higher in July than those in April. The presence of leptin mRNA and protein was found in all examined tissues. In females, leptin mRNA expression in the hypothalamus, pituitary, ovaries, and myometrium was markedly higher in July than that in April. In males, leptin mRNA levels varied across the examined tissues of the HPG and HPA. Leptin synthesis increased in the hypothalamus during breeding and postbreeding seasons, but seasonal changes were not observed in the pituitary. In turn, testicular leptin levels were higher during breeding and prebreeding stages. Seasonal differences in the concentrations of leptin mRNA were also observed in the adrenal cortex. In males, leptin mRNA levels were higher in November than those in April or July. In females, leptin synthesis increased in the adrenal cortex during pregnancy relative to other seasons. This is the first ever study to demonstrate seasonal differences in leptin expression in beaver tissues, and our results could suggest that leptin is involved in the regulation of the HPG and HPA axes during various stages of the reproductive cycle in beavers.

Orexin receptor expression in the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-gonadal axes of free-living European beavers (Castor fiber L.) in different periods of the reproductive cycle.

Orexins are hypothalamic neuropeptides acting via two G protein-coupled receptors in mammals: orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). In European beavers, which are seasonally breeding animals, the presence and functions of orexins and their receptors remain unknown. Our study aimed to determine the expression of OXR mRNAs and the localization of OXR proteins in hypothalamic-pituitary-adrenal/gonadal (HPA/HPG) axes in free-living beavers. The expression of OXR genes (OX1R, OX2R) and proteins was found in all analysed tissues during three periods of beavers' reproductive cycle (April, July, November). The expression of OXR mRNAs in the beaver HPA axis varied seasonally (P<0.05). The levels of OX1R mRNA also differed between the sexes (P<0.05). In the mediobasal hypothalamus, OX1R transcript content increased in pregnant females in April (P<0.05) and OX2R expression increased in males in July (P<0.05). In the pituitary and adrenals, OX1R mRNA levels were relatively constant in females and peaked in July in males (P<0.05), whereas the OX2R was most highly expressed in males in November and in females in April (P<0.05). In gonads, OX1R expression did not fluctuate between seasons or sexes, but transcript levels were elevated in the testes in November and in the ovaries in July (P<0.05). In turn, OX2R mRNA levels varied between the sexes (P<0.05) and were higher in females (July and November) than in males (P<0.05). The circannual variations in OXR mRNA levels in HPA and HPG axes suggest that the expression of these receptors is associated with sex-specific changes in beavers' reproductive activity and their environmental adaptations.

Maternal effect gene expression in porcine metaphase II oocytes and embryos in vitro: effect of epidermal growth factor, interleukin-1ß and leukemia inhibitory factor.

Maternal effect genes (MEG) play a crucial role in early embryogenesis. In vitro culture conditions may affect MEG expression in porcine oocytes and embryos. We investigated whether in vitro culture medium supplementation with epidermal growth factor (EGF), IL-1ß or LIF (leukemia inhibitory factor) affects the mRNA level of ZAR-1 (zygote arrest 1), NPM2 (nucleoplasmin 2) and DPPA3 (developmental associated protein 3) in porcine MII oocytes and embryos. Cumulus-oocyte complexes (COCs) were matured in NCSU-37 medium (control) or in NCSU-37 with EGF 10 ng/ml, IL-1ß 10 ng/ml or LIF 50 ng/ml. After maturation for 44-46 h, MII oocytes were preserved for the analysis of MEG mRNA levels (experiment 1). In experiment 2, COCs were fertilized, and the presumptive zygotes were cultured in the same groups. Then, 2-, 4-, 8-cell embryos, morulae and blastocysts were collected for the analysis of MEG mRNA levels. LIF addition to the maturation medium increased MII oocyte numbers (P < 0.05), while EGF and IL-1ß did not affect oocyte maturation. Medium supplementation with EGF resulted in lower DPPA3 mRNA levels in MII oocytes and in 2- and 4-cell embryos versus control embryos (P < 0.05). LIF treatment increased DPPA3 mRNA levels in morulae and blastocysts (P < 0.05). Culture with EGF and IL-1ß decreased ZAR-1 and NPM2 mRNA levels in 2-cell embryos (P < 0.05). The inclusion of EGF or IL-1ß in the porcine in vitro production system influences ZAR-1, NPM2 and DPPA3 mRNA in MII oocytes and embryos but not beyond the 4-cell stage. LIF stimulates oocyte maturation and affects DPPA3 mRNA in porcine morulae and blastocysts in vitro.

Peroxisome proliferator-activated receptors in the regulation of female reproductive functions.

Peroxisome proliferator-activated receptors (PPARs) belong to a ligand-dependent nuclear receptor family. In the past decade, numerous studies have revealed the presence and significance of PPARs in the reproductive system. PPARs are expressed at different levels of hypothalamic-pituitary-gonadal (HPG) axis. They are also present in the uterus as well as in the placenta and embryonic tissues of different species. PPARs significance has been reported during the estrous/menstrual cycle and pregnancy with the gamma isoform studied most frequently. Several studies indicate that PPARs regulate proliferation of ovarian cells, tissue remodeling and steroidogenesis. In the endometrium, PPARs are engaged in the regulation of prostaglandins, steroids and cytokines synthesis. The role of PPARs in the trophoblast differentiation, maturation and invasion as well as in the embryo development has also been demonstrated. In this review, we summarize current findings concerning the role of PPARs in the regulation of reproductive functions at different levels of the HPG axis during various physiological statuses of females. In addition, the role of PPARs in the modulation of uterine functions as well as the placenta and embryo development has also been discussed.