Leptin is a polypeptide hormone produced predominantly in adipocytes. It has been found to be implicated in the regulation of satiety and energy homeostasis. A role for leptin in reproduction was later suggested by findings that this hormone may be involved in the regulation of the hypothalamic- pituitary-gonadal axis via endocrine, paracrine and/or autocrine pathways. The objective of the study was to investigate the ontogeny of the long isoform of leptin receptor (OB-Rb) gene in porcine ovarian follicles. The expression of OB-Rb gene was detected in porcine primordial, primary, secondary and antral follicles by in situ hybridization. In summary, our data suggest that leptin might have a direct effect on porcine follicles and plays an important role in the follicular development.
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Leptin is a multifunctional regulator in numerous tissues, including the pituitary. It is not known, whether the porcine pituitary is a source of leptin synthesis and possesses the leptin receptor protein. It is also unknown, if a relationship exists between expression levels of these proteins in the pituitary and physiological status of sows. Therefore, the aim of the study was 1] to examine, by Western-blotting analysis, the expression levels of leptin and the long form of leptin receptor (OB-Rb) in the porcine anterior (AP) and posterior (NP) pituitary gland during mid- and late-luteal phases of the oestrous cycle (days 10 - 12 and 14 - 16) as well as during two stages of early pregnancy (days 14 - 16 and 30 - 32); and 2] to localise, using in situ hybridisation method (ISH), the expression of leptin and OB-Rb genes in the pituitary gland in the above mentioned stages of the cycle and pregnancy. Western-blotting analysis showed that leptin protein expression in AP was higher in the late-luteal phase than in the mid-luteal phase, while OB-Rb protein expression in both lobes was higher in the mid-luteal phase. In turn, during pregnancy leptin protein content in AP and OB-Rb protein content in NP were more pronounced on days 14 - 16 than on days 30 - 32. Comparison of leptin and OB-Rb protein expression levels in AP between the mid-luteal phase and two periods of pregnancy showed, respectively, stimulation of leptin protein and inhibition of OB-Rb protein expressions during both examined stages of pregnancy. Taking AP from late-luteal phase as the point of reference, it was revealed stimulation of leptin expression during earlier period of pregnancy, whereas on days 30 - 32 of pregnancy both the hormone and its receptor expressions were diminished. In turn, comparison of leptin and OB-Rb protein expression levels in NP between the late-luteal phase and days 14 - 16 or 30 - 32 of pregnancy showed inhibition of leptin protein expression and stimulation of OB-Rb protein expression during pregnancy. Moreover, ISH studies localised leptin and OB-Rb mRNAs expression in the cells of AP as well as NP tissue during the two stages of the cycle and pregnancy. In conclusion, our findings suggest that leptin is produced within the pituitary in the pig and may participate in auto/paracrine manner in the regulation of this gland function during the luteal phase of the oestrous cycle and early pregnancy.
Leptin is a 16-kDa protein hormone encoded by the obese (ob) gene and acts on receptors in the hypothalamus to regulate food intake and energy balance. The identification of leptin and its receptor mRNAs and proteins in human and mouse endometrium and placental trophoblast has attracted attention to the potential role of leptin in implantation. Thus, the aim of this study was to compare the expression levels of porcine leptin mRNA and protein in endometrium and myometrium during mid- and late-luteal phases of the oestrous cycle (days 10 - 12 and 14 - 16) as well as during two stages of pregnancy respondent to the beginning (days 14 - 16) and the end (days 30 - 32) of the implantation process, and in trophoblast during both periods of pregnancy. Leptin gene and protein expression in myometrium, and leptin mRNA expression in endometrium was more pronounced in the mid- and late-luteal phases of the cycle in comparison to studied periods of pregnancy, whereas leptin protein concentration in endometrium was either enhanced on days 30 - 32 of pregnancy in relation to days 14 - 16 of the cycle or there were no changes between pregnancy and luteal phase of the cycle. On days 30 - 32 of pregnancy, expression of the leptin gene in the endometrium, and of the leptin gene and protein in the myometrium was more pronounced in comparison to the earlier stage of pregnancy. Moreover, leptin gene expression in porcine trophoblast increased during the beginning of the implantation process compared to days 30 - 32 of pregnancy, while the protein concentration decreased on days 14 - 16 of pregnancy. In conclusion, the finding of leptin gene and protein expression in porcine endometrium, myometrium and trophoblast indicates that locally synthesised leptin can participate in the control of pig reproduction. The fluctuation of the hormone concentration during pregnancy and changes in its level between pregnancy and the oestrous cycle may indicate leptin's involvement in the implantation process.
The aim of the present study was to evaluate the possible direct effects of GnRH, oxytocin (OT) and vasoactive intestinal peptide (VIP) on the release of LH and PRL by dispersed porcine anterior pituitary cells. Pituitary glands were obtained from mature gilts, which were ovariektomized (OVX) one month before slaughter. Gilts randomly assigned to one of the four groups were treated: in Group 1 (n=8) with 1 ml/100 kg b.w. corn oil (placebo); in Group 2 (n=8) and Group 3 (n=8) with estradiol benzoate (EB) at the dose 2.5 mg/100 kg b.w., respectively, 30-36 h and 60-66 h before slaughter; and in Group 4 (n=9) with progesterone (P4) at the dose 120 mg/100 kg b.w. for five consecutive days before slaughter. In gilts of Group 2 and Group 3 treatments with EB have induced the negative and positive feedback in LH secretion, respectively. Isolated anterior pituitary cells (106/well) were cultured in McCoy's 5a medium with horse serum and fetal calf serum for 3 days at 37°C under the atmosphere of 95% air and 5% CO2. Subsequently, the culture plates were rinsed with fresh McCoy's 5A medium and the cells were incubated for 3.5 h at 37°C in the same medium containing one of the following agents: GnRH (100 ng/ml), OT (10-1000 nM) or VIP (1-100 nM). The addition of GnRH to cultured pituitary cells resulted in marked increases in LH release (p<0.001) in all experimental groups. In the presence of OT and VIP we noted significant increases (p<0.001) in LH secretion by pituitary cells derived from gilts representing the positive feedback phase (Group 3). In contrast, OT and VIP were without any effect on LH release in Group 1 (placebo) and Group 2 (the negative feedback). Pituitary cells obtained from OVX gilts primed with P4 produced significantly higher amounts (p<0.001) of LH only after an addition of 100 nM OT. Neuropeptide GnRH did not affect PRL secretion by pituitary cells obtained from gilts of all experimental groups. Oxytocin also failed to alter PRL secretion in Group 1 and Group 2. However, pituitary cells from animals primed with EB 60-66 h before slaughter and P4 produced markedly increased amounts of PRL in the presence of OT. Neuropeptide VIP stimulated PRL release from pituitary cells of OVX gilts primed with EB (Groups 2 and 3) or P4. In contrast, in OVX gilts primed with placebo, VIP was without any effect on PRL secretion. In conclusion, the results of our in vitro studies confirmed the stimulatory effect of GnRH on LH secretion by porcine pituitary cells and also suggest a participation of OT and VIP in modulation of LH and PRL secretion at the pituitary level in a way dependent on hormonal status of animals.
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The study was conducted to determine gene expression of short form of leptin receptor (OB-Rs) using real time RT-PCR in distinct tissues of the central nervous system (medial basal hypothalamus, preoptic area, stalk median eminence), pituitary and reproductive tract (corpus luteum, ovarian stroma, endometrium, myometrium, and trophoblast) in pigs during luteal phase of the cycle and early gestation. The expression of OB-Rs mRNA in SME did not differ between analyzed stages of the cycle and pregnancy. In anterior pituitary, transcript levels were almost identical in mid- and late-luteal periods, but significantly decreased on 30-32 day of gestation when compared with day 14-16. In posterior pituitary, significantly higher expression was observed in two periods of pregnancy when compared with two stages of luteal phase. In corpus luteum the lowest expression was observed during days 10-12 of the cycle, whereas markedly higher levels were detected in late-luteal stage and gestation. In ovarian stroma the expression of Ob-Rs mRNA was markedly diminished during days 14-16 of the cycle when compared with days: 10-12 of the cycle and 30-32 of pregnancy. The expression of Ob-Rs mRNA in endometrium and myometrium reached the lowest levels on 30-32 day of pregnancy in comparison with earlier stage, 14-16 day. Summarizing, the expression of the short form of leptin receptor mRNA was found in majority of tested tissues including hypothalamus, pituitary and reproductive tract and their levels fluctuated depending on the phase (mid- and late-luteal) of the cycle and the day of pregnancy (early and late stage of implantation).
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Leptin is a polypeptide that plays a key role in the regulation of energy homeostasis and is also linked, among others, to mechanisms controlling reproductive processes. Data concerning the involvement of leptin in controlling reproductive functions at the level of hypothalamus and pituitary in the pig are limited. Therefore, in the present study, an expression of genes coding for leptin and long-form leptin receptor (Ob-Rb) was determined by a semiquantitative reverse transcription polymerase chain reaction (RT-PCR) in the discrete areas of porcine hypothalamus (medial basal hypothalamus - MBH, preoptic area - POA, stalk median eminence - SME) and pituitary (anterior - AP and posterior/neural - NP parts) during the luteal phase of the cycle (days 10-12 and 14-16) and two early stages of pregnancy (days 14-16 and 30-32). Leptin gene expression in MBH was found to be higher in the mid- than in the late-luteal phase, whereas in other structures studied it remained unchanged during these periods. More pronounced differences were noted in expression of Ob-Rb gene, which was increased in MBH, AP and NP during the late-luteal phase in comparison to the mid-luteal one, whilst the relationship in the POA was reversed. In turn, during pregnancy, leptin gene expression in all tested areas of hypothalamus as well as Ob-Rb mRNA content in MBH were higher on days 30-32 than on days 14-16. In contrast, in the anterior pituitary, Ob-Rb gene expression was more pronounced on days 14-16 than during later stage of pregnancy. Comparison of leptin and Ob-Rb mRNA content in studied structures between the mid-luteal phase and days 14-16 of pregnancy revealed inhibition of leptin gene expression in almost all examined tissues (MBH, POA, SME, NP) during early pregnancy whereas Ob-Rb gene expression was inhibited in POA but stimulated in both parts of the pituitary during this stage. In summary, obtained results suggest an involvement of leptin in the regulation of hypothalamic-pituitary axis activity during both the luteal phase of the cycle and early pregnancy in pigs.