Katarzyna Chojnowska

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.

The effect of the estrous cycle on the expression of prepro-orexin gene and protein and the levels of orexin A and B in the porcine pituitary

Hypothalamic peptides orexin A (OXA) and orexin B (OXB) are derived from the proteolytic cleavage of a common precursor molecule, prepro-orexin (PPO). They act via two orexin receptors (OX1R and OX2R), which belong to the G-protein coupled receptor superfamily. Orexins are implicated in the regulation of arousal states, energy homeostasis and reproductive neuroendocrine function. The objective of this study was to investigate the presence and changes in orexin expression in the porcine pituitary during the estrous cycle. Adenohypophysis (AP) and neurohypophysis (NP) tissue samples were harvested on days 2 to 3, 10 to 12, 14 to 16, and 17 to 19 of the estrous cycle. The expression of the PPO gene increased in AP and NP during the estrous cycle. The highest PPO protein concentrations in AP were reported on days 2 to 3 (P<0.05), and in NP - on days 10 to 12 and 17 to 19 (P<0.05). The expression of PPO mRNA was lower in AP than in NP, but PPO protein levels were higher in AP. In AP, OXA immunoreactivity was higher (P<0.05) on days 10 to 12 and 14 to 16. In NP, the highest (P<0.05) content of the analyzed protein was observed on days 10 to 12 and the lowest (P<0.05) - on days 14 to 16 and 17 to 19. OXB immunoreactivity in AP reached the highest level (P<0.05) on days 2 to 3, and the lowest level (P<0.05) was determined on days 10 to 12 and 17 to 19. OXB protein concentrations in NP peaked (P<0.05) on days 10 to 12 of the cycle. Our study was the first experiment to demonstrate the expression of the orexin gene and orexin proteins in the porcine pituitary and the correlations between expression levels and the phase of the estrous cycle.

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.

The involvement of peroxisome proliferator activated receptors (PPARs) in prostaglandin F2α production by porcine endometrium.

In the present study, we investigated the in vitro effects of peroxisome proliferator activated receptor (PPAR) ligands on PGF2α secretion and mRNA expression of prostaglandin F synthase (PGFS) in porcine endometrial explants collected on days 10-12 and 14-16 of the estrous cycle or pregnancy. The explants were incubated for 6h with: PPARα ligands - WY-14643 (agonist) and MK 886 (antagonist); PPARβ ligands - l-165,041 (agonist) and GW 9662 (antagonist); PPARγ ligands - 15d-prostaglandin J2 (PGJ2, agonist), rosiglitazone (agonist) and T0070907 (antagonist). The expression of PGFS mRNA in the endometrium and the concentration of PGF2α in culture media were determined by real time RT-PCR and radioimmunoassay, respectively. During the estrous cycle (days 10-12 and 14-16), the agonists - WY-14643 (PPARα), l-165,041 (PPARβ), PGJ2 and rosiglitazone (PPARγ) - increased PGF2α secretion but did not affect PGFS mRNA abundance. During pregnancy (days 10-12 and 14-16), PPARα and PPARγ ligands did not change PGF2α release, whereas PPARβ agonist augmented PGF2α release on days 14-16 of pregnancy. In addition, WY-14643 and l-165,041 increased PGFS mRNA level in both examined periods of pregnancy. PPARγ agonist (PGJ2) and antagonist (T0070907) enhanced PGFS mRNA abundance in the endometrium on days 10-12 and 14-16 of pregnancy, respectively. The results indicate that PPARs are involved in the production of PGF2α by porcine endometrium, and that the sensitivity of the endometrium to PPAR ligands depends on reproductive status of animals.

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.

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.

PPAR ligand association with prostaglandin F2α and E2 synthesis in the pig corpus luteum—An in vitro study

The present study evaluated the involvement of PPARs in prostaglandin (PG) E2 and F2α production in the corpus luteum (CL) of pigs on days 10-12 and 14-16 of the estrous cycle or pregnancy. The tissue explants were incubated for 6h in the presence of PPARα, PPARβ, PPARγ ligands. The concentration of PGs in the incubation media was determined by radioimmunoassay, while mRNA abundance of PG synthetases (PGES and PGFS) was analyzed by quantitative real-time PCR. It was found that L-165,045 and rosiglitazone stimulated PGES synthesis on days 10-12 of the estrous cycle, whereas all factors that were assessed did not affect PGE2 release. The PGFS mRNA abundance in the CL did not change in the presence of PPAR ligands during the assessment periods. However, PPARβ agonist inhibited PGF2α secretion on days 10-12 of the estrous cycle and on days 14-16 of pregnancy. Interestingly, PPAR antagonists, MK 886, GW 9662 or T0070907 decreased PGF2α release by the slices on days 10-12 of the estrous cycle. It is concluded that the CL has a different susceptibility (greatest during mid-luteal phase of the estrous cycle) to the PPAR ligands, which is related to the physiological status of animal. The inhibition of PGF2α release and augmentation of PGES mRNA concentration during mid-luteal phase of the estrous cycle might suggest luteotropic properties of PPAR ligands.

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

Abstract 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.

Prepro-orexin and orexin expression in the hypothalamic–pituitary–adrenal and hypothalamic–pituitary–gonadal axes of free-living Eurasian beavers (Castor fiber L.) depends on season

The Eurasian beaver (Castor fiber L.) is the largest free-living rodent in Eurasia. Beavers show seasonal patterns of reproduction (long-day breeders), stress reactions, and feeding behavior. These phenomena are associated with the function of hormonal regulatory axes: the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG). In mammals, the above processes are also controlled by orexins (OXA and OXB), neuropeptides derived from the same precursor—prepro-orexin (PPO). We hypothesized that in beavers PPO gene expression in tissues creating the HPA-HPG axes and plasma orexin concentrations differ between seasons and sexes. PPO transcript and PPO and orexin proteins were noted in the mediobasal hypothalamus (MBH), pituitary, adrenals, and gonads in April (“breeding, pregnancy”), July (“post-breeding”), and November (“pre-breeding”). In the MBH, seasonal, but not sex-dependent variations were observed in PPO gene expression; however, the transcript content increased in females in July. An interaction between season and sex was noted in the pituitary: PPO gene expression decreased in males in April. The PPO mRNA concentration in adrenals varied between seasons and sexes. Additionally, an interaction between these factors occurred and the highest PPO gene expression was noted in females in April. In the gonads, PPO mRNA levels changed dependent on season, and an interaction between season and sex was found. The PPO transcript increased in November in the testes. Seasonal variations and an interaction between season and sex were observed in plasma OXA. The highest concentration of OXA was noted in July (males) and in November (females). In beavers, PPO gene expression and plasma OXA concentration varied depending on season and these changes were modified by sex. Our results imply that orexin peptides are involved in the regulation of circannual changes of reproductive activity in beavers and seem to be associated with seasonal fluctuations in glucocorticoid secretion and stress reactions.