Transcriptomic analysis of the porcine anterior pituitary gland during the peri-implantation period.

The peri-implantation period is controlled by signals originating from hypothalamic-pituitary-ovarian axis, uterus and developing embryos. The transcriptomic activity of the anterior pituitary gland may be important for the control of the peri-implantation period. The aim of this study was to determine the alternations in the transcriptomic profile of porcine anterior pituitary gland during the peri-implantation period (days 15-16 of pregnancy) in comparison with established for the respective days of the oestrous cycle. Analysis using a microarray approach indicated that the 651 genes (fold-change ˂1.2; p ≤ .05) were differentially expressed (DEGs) in the anterior pituitary of pigs during the peri-implantation period when compared to cyclic females. Of these DEGs, 404 were upregulated and 247 downregulated. Analysis of occurred relationships among DEGs revealed that some of them are involved in steroid-response and oestrogen synthesis, FSH secretion, immune response, PPAR signalling pathway and the potential for DNA methylation. In conclusion, the altered transcriptomic profile of the porcine pituitary gland in pigs during the peri-implantation period indicates the role of embryos presence in the creation of transcriptomic activity of the pituitary gland in pigs.

The role of neurokinin A and its receptor in the regulation of prolactin secretion by the anterior pituitary of cyclic pigs.

In pigs, plasma prolactin concentration markedly changes during the oestrous cycle and the regulation of its secretion is very complex. The contribution of neurokinins in this process has not been sufficiently delineated. The aim of the study was to examine the effects of neurokinin A (NKA) on prolactin synthesis and secretion in cyclic gilts. The expression of NKA precursor (Ppta) and receptor (Tacr2) genes as well as NKA and TACR proteins content in the porcine pituitaries (days 2-3, 9-10, 12-13, 15-16 and 19-20 of the cycle) was determined. Furthermore, the in vitro influence of NKA on the expression of prolactin (Prl), dopamine receptor (D2r), TRH receptor (Trhr) genes and prolactin secretion by the porcine pituitary cells (days 9-10, 15-16 and 19-20 of the cycle) was assessed. The expression of Ppta and Tacr2 as well as NKA and TACR proteins in the pituitary tissue has been changing throughout the oestrous cycle. NKA affected in vitro the expression of studied genes and prolactin secretion depending on the stage of the cycle, dose of NKA and/or duration of the cell incubation. Altogether, the study indicates that NKA is engaged in the modulation of prolactin secretion in the pig during the oestrous cycle.

Effect of kisspeptin and RFamide-related peptide-3 on the synthesis and secretion of LH by pituitary cells of pigs during the estrous cycle.

Actions of kisspeptins (KISSs) and RFamide-related peptide-3 (RFRP-3) at the hypothalamus modulate female reproduction. The action of KISS and RFRP-3 in the pituitary gland of pigs during the estrous cycle has not yet been delineated. The present study was conducted to assess the effect of KISS and RFRP-3 on relative abundance of αGSU and ßLH mRNA transcript and LH secretion in vitro by pituitary cells of gilts during the estrous cycle. The cells were isolated from gilts on Days 2-3, 10-12, 15-16 and 19-20 of the estrous cycle and cultured in vitro without inclusion of GnRH (control) or with GnRH (100 ng/ml), KISS (10-6 M, 10-7 M) and RFRP-3 (10-6 M, 10-7 M) alone or in combination. The relative abundance of α-GSU and ß-LH mRNAs was examined. Treatment with KISS increased the synthesis and/or secretion of LH by pituitary cells and RFRP-3 inhibited the synthesis and secretion of LH in the presence of GnRH on Days 10-12 and 15-16 of the estrous cycle. The synthesis and secretion of LH was greater when there was treatment with KISS and GnRH during the late follicular phase. Treatments with KISS and RFRP-3 affected the synthesis and/or secretion of LH during the luteal phase and luteolysis. In conclusion, KISS and RFRP-3 apparently affects the synthesis and secretion of LH by pituitary cells of estrous cyclic pigs. There appears to be a greater effect of KISS in modulation of LH secretion than RFRP-3 in pigs.

Progesterone, estradiol, arachidonic acid, oxytocin, forskolin and cAMP influence on aquaporin 1 and 5 expression in porcine uterine explants during the mid-luteal phase of the estrous cycle and luteolysis: an in vitro study.

BACKGROUND: The cell membrane water channel protein, aquaporins (AQPs), regulate cellular water transport and cell volume and play a key role in water homeostasis. Recently, AQPs are considered as important players in the field of reproduction. In previous studies, we have established the presence of AQP1 and 5 in porcine uterus. Their expression at protein level altered in distinct tissues of the female reproductive system depending on the phase of the estrous cycle. However, the regulation of aquaporin genes and proteins expression has not been examined in porcine uterine tissue. Therefore, we have designed an in vitro experiment to explain whether steroid hormones, progesterone (P4) and estradiol (E2), and other factors: oxytocine (OT), arachidonic acid (AA; substrate for prostaglandins synthesis) as well as forskolin (FSK; adenylate cyclase activator) and cAMP (second messenger, cyclic adenosine monophosphate) may impact AQPs expression. METHODS: Uterine tissues were collected on Days 10-12 and 14-16 of the estrous cycle representing the mid-luteal phase and luteolysis. Real-time PCR and Western blot analysis were performed to examine the expression of porcine AQP1 and AQP5. Their expression in the uterine explants was also evaluated by immunohistochemistry. RESULTS: The results indicated that uterine expression of AQP1 and AQP5 potentially remains under control of steroid hormones and AA-derived compounds (e.g. prostaglandins). P4, E2, AA, FSK and cAMP cause translocation of AQP5 from apical to the basolateral plasma membrane of the epithelial cells, which might affect the transcellular water movement (through epithelial cells) between uterine lumen and blood vessels. The AC/cAMP pathway is involved in the intracellular signals transduction connected with the regulation of AQPs expression in the pig uterus. CONCLUSIONS: This study documented specific patterns of AQP1 and AQP5 expression in response to P4, E2, AA, FSK and cAMP, thereby providing new indirect evidence of their role in maintaining the local fluid balance within the uterus during the mid-luteal phase of the estrous cycle and luteolysis in pigs.

Expression of proopiomelanocortin, proenkephalin and prodynorphin genes in porcine theca and granulosa cells.

Previous studies have demonstrated the presence of endogenous opioid peptides (EOP) in the ovary and suggested their implication in local interactions within ovarian structures. Nevertheless, data pertaining to the expression of genes, coding for the opioid precursors, in ovarian cells are still rudimentary and not available for the pig. The study was undertaken to test whether genes of the opioid precursors - proopiomelanocortin (POMC), proenkephalin (PENK) and prodynorphin (PDYN) - are expressed in non-treated and gonadotropin-treated theca and granulosa cells isolated from ovarian follicles of the pig. The cells were isolated from small (days 15-16 of the estrous cycle) and large (days 19-20) porcine follicles. Dispersed cells were cultured in Eagle's medium under the water saturated atmosphere of 95% air and 5% CO(2), in the presence or absence of respective gonadotropin; theca cells with LH (100 ng/ml) and granulosa cells with FSH (100 ng/ml). Following 24h-incubation, the cells were harvested and the total RNA was isolated. The expression of genes coding for opioid precursors was estimated by the semi-quantitative RT-PCR technique involving co-amplification of the target cDNA (POMC, PENK or PDYN) and control cDNA (beta-actin or 18S rRNA). Specificities of PCR products were confirmed by Southern analysis and sequencing. In theca cells the expression of opioid precursors appeared to be gonadotropin-dependent except for PENK in the cells isolated from large follicles. In turn, granulosa cells exhibited the expression of POMC and PENK genes independently on treatment with FSH. This gonadotropin induced the expression of PDYN gene in granulosa cells isolated from small and large follicles and significantly increased POMC mRNA content in the cells from the large ones. The present studies indicate that porcine follicular cells (especially granulosa cells) may produce opioid peptides and that gonadotropins may modulate gene expression of their precursors in these cells. Moreover, our results support a participation of opioid peptides in the local regulations within ovarian follicle.

Effects of oxytocin alone and in combination with selected hypothalamic hormones on ACTH, beta-endorphin, LH and PRL secretion by anterior pituitary cells of cyclic pigs.

Oxytocin (OT) is involved in the stimulation of secretion of anterior pituitary hormones in females during the periovulatory and periparturient periods. In the present study we examined the role of OT in control of ACTH, beta-endorphin, LH and PRL secretion in vitro from dispersed anterior pituitary cells collected from gilts during the luteal (Days 10-12; n=6) and follicular (Days 18-20; n=5) phases of the estrous cycle. Isolated anterior pituitary cells (1 x 10(6)/ml) were transferred into 24-well plates, separately for each animal, and were pre-incubated for three days at 37 degrees C in atmosphere of 5% CO(2) and 95% air. The cells which attached to the dishes were incubated (3.5 h, 37 degrees C) in McCoy's medium in the absence (control) or in the presence of the following factors: CRH alone (10(-10), 10(-9), 10(-8), 10(-7) M), OT alone (10(-8), 10(-7), 10(-6) M), LVP alone (10(-7) M), OT (10(-7) M) plus CRH (10(-9) M) and LVP (10(-7) M) plus CRH (10(-9) M) for studying ACTH and beta-endorphin secretion; OT alone (10(-8), 10(-7), 10(-6) M), GnRH alone (100 ng/ml), CRH alone (10(-9) M), OT (10(-7) M) plus GnRH (100 ng/ml) and OT (10(-7) M) plus CRH (10(-9) M) for studying LH and PRL secretion. Concentrations of the studied hormones in media were analyzed by RIA. Oxytocin alone increased ACTH (at doses 10(-7), 10(-6) M), beta-endorphin (at dose 10(-8) M), LH (at dose 10(-8) M) and PRL (at doses 10(-7), 10(-6) M) secretion by pituitary cells isolated only from luteal-phase gilts. None of the studied hormone concentrations in the medium was increased in response to OT when pituitary cells of follicular-phase gilts were examined. Oxytocin in combination with CRH exerted an additive effect on beta-endorphin secretion during the luteal phase. Summarizing, in the present study the stimulatory effect of oxytocin on ACTH, beta-endorphin, LH and PRL secretion by pituitary cells isolated from gilts during the luteal phase was demonstrated. However, the cells collected from follicular-phase gilts appeared to be unresponsive to OT. Moreover, interaction between OT and CRH in affecting beta-endorphin secretion was shown. These results suggest that OT may be transiently involved in the modulation of anterior pituitary hormone secretion in cyclic pigs.

Leptin gene expression in the hypothalamus and pituitary of pregnant pigs.

BACKGROUND: Leptin, the 16-kDa peptide hormone product of the ob gene, is a regulatory hormone secreted mainly by adipose tissue. Recent studies have shown leptin production by other tissues, including rat hypothalamus, rat and human pituitary, rat skeletal muscle, kidney and stomach, human and porcine placenta, human mammary epithelial cells as well as endometrial tissues. This hormone is a central modulator of food intake, metabolism and neuroendocrine functions. OBJECTIVES: The aim of the study was to detect and locate porcine leptin gene expression in the different areas of the hypothalamus and pituitary on days 14-16 and 30-32 of pregnancy in pigs. METHOD: Leptin gene expression was analysed by RT-PCR method. PCR products were subjected to sequencing analysis. RESULTS: Leptin mRNA was expressed in the medial basal hypothalamus, preoptic area, stalk median eminence, anterior pituitary, posterior pituitary and adipose tissue on days 14-16 and 30-32 of pregnancy. Sequence analysis of the 258 bp product from the hypothalamus and pituitary confirmed 99% homology with the corresponding region of porcine leptin cDNA sequence. CONCLUSION: Leptin mRNA expression in the porcine hypothalamus and pituitary gland implies its paracrine and/or autocrine role in the regulation of hypothalamic-pituitary axis activity.

The effect of oxytocin on cortisol and corticosterone secretion in cyclic gilts--in vivo and in vitro studies.

Oxytocin (OT) may be implicated in the modulation of hypothalamo-pituitary-adrenal axis (HPA) at each level. In mature females the influence of OT on the HPA axis appeared to be dependent on ovarian steroid milieu and stress. In cyclic sows, the role of OT in the regulation of corticoid secretion is unknown. In the present study changes in plasma cortisol and corticosterone concentrations in response to exogenous OT (in vivo experiment) and a direct influence of OT on adrenocortical steroidogenesis (in vitro experiment) were determined in luteal- and follicular-phase gilts. In the luteal-phase gilts (n=5), OT injections increased both cortisol (p<0.01) and corticosterone (p<0.05) plasma concentrations, but in the follicular-phase gilts (n=5) only the concentration of cortisol (p<0.05) was elevated in response to the treatment. Areas under the cortisol and corticosterone curves calculated for 30 min period after the OT injection were statistically higher (p<0.05) during the luteal than the follicular phase. In the in vitro experiment, two doses of OT (10(-7) and 10(-6) M) increased (p<0.05) secretion of cortisol by porcine adrenocortical cells representing the luteal phase, but not the follicular phase. However, OT did not affect the release of corticosterone by the cells. Incubation of the cells with the OT-antagonist (10(-5) M) abolished the effects of OT on cortisol secretion. Thus, in the present study, stimulatory effects of OT on the hypothalamo-pituitary-adrenal axis were demonstrated in cyclic gilts. The changes in plasma corticoid concentrations in response to exogenous OT were more prominent during the luteal than the follicular phase of the estrous cycle. Moreover, the in vitro experiment revealed a possibility of direct action of OT on adrenocortical cells isolated from luteal phase gilts. These results suggest that OT may participate in the modulation of HPA axis activity in pigs.

The effects of estradiol on beta-endorphin, GnRH and galanin content in the oviduct and the uterus of ovariectomized gilts.

Steroid hormones are known to affect synthesis and/or release of some peptides in the central nervous system and peripheral tissues. In the present study we determined changes in beta-endorphin, GnRH and galanin contents in uterine and oviductal tissues of ovariectomized (OVX) gilts following treatment with estradiol benzoate (EB) at a dose inducing a preovulatory-like LH surge. Seven month old gilts (90-100 kg of body weight; BW) were used in the study. Four weeks after ovariectomy, experimental animals were injected intramuscularly with EB (15 microg/kg BW) at 24 h (n=5), 48 h (n=6) or 72 h (n=5) before slaughter. Three control gilts received corn oil vehicle. Tissues were sampled from the ampulla and isthmus of the oviduct and from the perioviductal, middle and paracervical regions of the uterine horn for determination of beta-endorphin, GnRH and galanin content. Significant increases of beta-endorphin content were found in all regions of the uterus either 24 h or 48 h after priming with EB. In oviductal tissue, beta-endorphin concentration only tended to increase in response to EB. GnRH content in tissues originating from gilts receiving EB fluctuated from a stimulation in the ampulla of the oviduct and in the paracervical uterus to an inhibition in the middle part of the uterus. A significantly increased concentration of galanin in response to EB was observed exclusively in the paracervical part of the OVX pig uterus. The results suggest an involvement of beta-endorphin, GnRH and galanin in the regulation of uterine function in pigs during the periovulatory period.

Primary antisera against selected steroids or proteins and secondary antisera against gamma-globulins--an available tool for studies of reproductive processes.

This paper presents characteristics of different polyclonal antisera raised against several steroid and protein antigens: 1/ primary antisera against steroid hormones: estradiol-17beta (anti-E2), estrone (anti-E1), testosterone (anti-T), androstendione (anti-A4), cortisol (anti-F) and corticosterone (anti-B); 2/ primary antisera against porcine luteinizing hormone (anti-pLH) and against different forms of porcine pregnancy associated glycoproteins (anti-pPAG) - proteins produced by chorionic tissue; 3/ secondary monovalent antisera raised against rabbit gamma-globulins (Sm-r); 4/ secondary polyvalent antisera against rabbit, pig and quinea pig gamma-globulins mixed at a ratio 1:1:1 (Sp-rpq). All antisera described in the paper present sufficient quality to be routinely used in various RIA, ELISA or Western determinations in physiological and clinical studies of reproductive processes. The antisera against steroid hormones and pLH are available on request.

Effects of mating stimuli and oxytocin on plasma cortisol concentration in gilts.

UNLABELLED: The involvement of oxytocin (OT) in the regulation of glucocorticoid secretion during stress reaction, parturition, and suckling has been documented in various species. In this study four in vivo experiments were conducted on gilts (1) to demonstrate the influence of mating stimuli on plasma cortisol concentration, (2) to test the effect of OT alone and (3) OT combined with OT-antagonist on cortisol secretion and (4) to clarify the role of progesterone and estradiol in cortisol response to exogenous OT. In experiment 1, plasma cortisol concentration in gilts (n=4) increased (p<0.05) from 16.1 +/- 5.3 ng ml(-)1 (control period: 30 min before mating) to 42.8 +/- 11.6 ng ml(-1) and 46.6 +/- 9.6 ng ml(-1) at the time of leaving the pen and during the first visual and olfactory contact with the boar, respectively. During coitus the elevation was maintained (48.8 +/- 9.8 ng ml(-1); p<0.05 vs. control). The plasma cortisol concentration returned to pre-mating levels within 30 min after mating. In experiment 2, gilts (n=7) were treated, according to Latin square design, with saline (2 ml; i.v.) and OT (10, 20, and 30 IU; i.v.). The magnitude of cortisol response (area under cortisol curve) was higher (p<0.01) only after treatments with 20 and 30 IU OT vs. control period (30 min before OT). Gilts (n=3) of experiment 3 were infused with OT-antagonist (Atosiban; 25 mg per gilt per 2 hours; i.v.) and then were injected with OT (20 IU; i.v.) 60 min after the beginning of Atosiban administration. Blockage of OT receptors by Atosiban reversed the stimulatory effect of OT on cortisol secretion. In experiment 4, ovariectomized gilts (n=25) primed (i.m.) with corn oil (n=7), progesterone (P4; n=7), estradiol benzoate (EB; n=4) or EB+P4 (n=7) were treated with OT (20 IU; i.v.). Plasma cortisol concentrations were increased following OT administration in all gilts of experiment 4. The highest cortisol response to OT was noted in gilts primed with EB+P4 (p<0.01 vs. other groups). IN CONCLUSION: (1) leaving the pens, visual and olfactory contact with the boar as well as coitus, increased plasma cortisol concentrations in gilts to similar levels; (2) exogenous OT (20 and 30 IU per gilt) increased cortisol plasma concentration, (3) this effect was abolished by OT-antagonist and (4) E2+P4 elevated cortisol response to OT. Oxytocin may be included to secretagogues of the hypothalamus-pituitary-adrenocortical axis in pigs.

Concentration of neuropeptide Y, galanin, &bgr;-endorphin, vasoactive intestinal peptide and gonadotropin releasing hormone in the hypothalamus of gilts during the estrous cycle.

Numerous reports indicate that peptides isolated from the brain such as beta-endorphin (beta-END), neuropeptide Y (NPY), galanin (GAL) or vasoactive intestinal peptide (VIP), modulate secretion of gonadotropins and prolactin. The objective of the present experiment was to determine concentrations of NPY, GAL, beta-END, VIP and GnRH in the preoptic area (POA), medial basal hypothalamus (MBH) and pituitary stalk-median eminence (SME) during the estrous cycle in the pig. Gilts were slaughtered on Days 5, 10, 15 and 20 of the estrous cycle. Blood samples for analyses of progesterone were taken before slaughter. Neuropeptide concentrations in brain tissues were determined using RIA. The highest concentrations of all determined peptides occurred in SME. GnRH concentration in MBH was lower (p<0.05) in POA and SME on Day 20 than on Day 5. NPY concentration in POA was 5-6 times greater (p<0.05) on Days 10 and 20 than on Day 5. Similarly, concentrations of VIP in POA were greater (p<0.05) on Day 10, Day 15 and Day 20 than on Day 5. The concentration of GAL in POA was higher on Days 10 and 15 (p<0.05) than on Days 5 and 20. The concentration of GAL in SME was lowest on Day 5 and then significantly increased on Days 10, 15 and 20. In SME, concentration of beta-END increased 10 times on Days 15 and 20 when compared to Day 5 of the cycle. The correlation between concentration of GAL in the POA and MBH and progesterone concentration in the peripheral blood was positive, whereas this correlation associated with the SME was negative. These results indicate that considerable changes in various neuropeptide concentrations in different areas of the porcine hypothalamus are associated with stage of the estrous cycle and that GAL may be involved in control of the preovulatory LH surge in pigs.