Accelerated onset of diabetes in non-obese diabetic mice fed a refined high-fat diet.

AIM: Type 1 diabetes results from autoimmune events influenced by environmental variables, including changes in diet. This study investigated how feeding refined versus unrefined (aka 'chow') diets affects the onset and progression of hyperglycaemia in non-obese diabetic (NOD) mice. METHODS: Female NOD mice were fed either unrefined diets or matched refined low- and high-fat diets. The onset of hyperglycaemia, glucose tolerance, food intake, energy expenditure, circulating insulin, liver gene expression and microbiome changes were measured for each dietary group. RESULTS: NOD mice consuming unrefined (chow) diets developed hyperglycaemia at similar frequencies. By contrast, mice consuming the defined high-fat diet had an accelerated onset of hyperglycaemia compared to the matched low-fat diet. There was no change in food intake, energy expenditure, or physical activity within each respective dietary group. Microbiome changes were driven by diet type, with chow diets clustering similarly, while refined low- and high-fat bacterial diversity also grouped closely. In the defined dietary cohort, liver gene expression changes in high-fat-fed mice were consistent with a greater frequency of hyperglycaemia and impaired glucose tolerance. CONCLUSION: Glucose intolerance is associated with an enhanced frequency of hyperglycaemia in female NOD mice fed a defined high-fat diet. Using an appropriate matched control diet is an essential experimental variable when studying changes in microbiome composition and diet as a modifier of disease risk.

Embryonic cell migratory capacity is impaired upon exposure to glucose in vivo and in vitro.

BACKGROUND: Impairments in cell migration during vertebrate gastrulation lead to structural birth defects, such as heart defects and neural tube defects. These defects are more frequent in progeny from diabetic pregnancies, and we have recently provided evidence that maternal diabetes leads to impaired migration of embryonic mesodermal cells in a mouse model of diabetic pregnancy. METHODS: We here report the isolation of primary cell lines from normal and diabetes-exposed embryos of the nonobese diabetic mouse strain, and characterization of their energy metabolism and expression of nutrient transporter genes by quantitative real-time PCR. RESULTS: Expression levels of several genes in the glucose transporter and fatty acid transporter gene families were altered in diabetes-exposed cells. Notably, primary cells from embryos with prior in vivo exposure to maternal diabetes exhibited reduced capacity for cell migration in vitro. CONCLUSIONS: Primary cells isolated from diabetes-exposed embryos retained a "memory" of their in vivo exposure, manifesting in cell migration impairment. Thus, we have successfully established an in vitro experimental model for the mesoderm migration defects observed in diabetes-exposed mouse embryos.

Prolonged proteasome inhibition cyclically upregulates Oct3/4 and Nanog gene expression, but reduces induced pluripotent stem cell colony formation.

There is ample evidence that the ubiquitin-proteasome system is an important regulator of transcription and its activity is necessary for maintaining pluripotency and promoting cellular reprogramming. Moreover, proteasome activity contributes to maintaining the open chromatin structure found in pluripotent stem cells, acting as a transcriptional inhibitor at specific gene loci generally associated with differentiation. The current study was designed to understand further the role of proteasome inhibition in reprogramming and its ability to modulate endogenous expression of pluripotency-related genes and induced pluripotent stem cells (iPSCs) colony formation. Herein, we demonstrate that acute combinatorial treatment with the proteasome inhibitors MG101 or MG132 and the histone deacetylase (HDAC) inhibitor valproic acid (VPA) increases gene expression of the pluripotency marker Oct3/4, and that MG101 alone is as effective as VPA in the induction of Oct3/4 mRNA expression in fibroblasts. Prolonged proteasome inhibition cyclically upregulates gene expression of Oct3/4 and Nanog, but reduces colony formation in the presence of the iPSC induction cocktail. In conclusion, our results demonstrate that the 26S proteasome is an essential modulator in the reprogramming process. Its inhibition enhances expression of pluripotency-related genes; however, efficient colony formation requires proteasome activity. Therefore, discovery of small molecules that increase proteasome activity might lead to more efficient cell reprogramming and generation of pluripotent cells.

Silencing histone deacetylase-specific isoforms enhances expression of pluripotency genes in bovine fibroblasts.

Histone deacetylases (HDACs) catalyze deacetylation of histones that results in altered transcriptional activity. Inhibitors of HDACs have been shown to induce transcriptional changes that contribute positively to reprogramming somatic cells either by nuclear transfer or inducing a pluripotent state. However, the exact molecular mechanisms whereby HDAC inhibitors function and the specificity of the HDAC isoforms in cell reprogramming are not yet fully understood. Herein, we report the ability of individual isoform-specific HDACs to modulate endogenous expression of pluripotency-associated genes in bovine somatic cells. This in vitro study showed that a transient selective depletion of HDACs resulted in elevated mRNA levels of Oct-4, Sox2, and Nanog. In particular, we found that inhibition of specific HDAC isoforms using small interfering (si) RNA significantly increased expression of Nanog, a key factor required for totipotency induced by somatic cell nuclear transfer and for maintaining pluripotency in embryonic and induced pluripotent stem cells. Our study suggests that this gene might be the most susceptible to HDAC activity inhibition. Moreover, a regulatory role of the class III HDAC, SIRT3, on an Oct4-Sox2-Nanog transcriptional network was revealed. We observed the upregulation of pluripotency-related genes by depletion of SIRT3. SIRT3 is localized to mitochondria and is associated with energy metabolism processes, suggesting metabolic changes may be linked to reprogramming in bovine fibroblasts. In conclusion, we show that targeting selective HDACs can potentially be useful to enhance reprogramming and that sirtuins may play a pivotal role in somatic cell reprogramming by upregulating an Oct4-Sox2-Nanog transcriptional network. Dedifferentiating donor somatic cells by upregulating developmentally important genes through specific knockdown of epigenetic targets, in particular HDACs, may provide a path to improving livestock cloning and the in vitro production of pluripotent cells.

Diet-induced obesity in stem cell antigen-1 KO mice.

Stem Cell Antigen-1 (Sca-1) is a member of the lymphocyte-activated protein 6 family and has served as a marker for the identification of stem cells in various tissues, including fat depots. In vitro and in vivo studies suggest the possible involvement of Sca-1 in adipogenic differentiation and link Sca-1 antigenicity with adipocyte progenitors. Previously, we showed that Sca-1-enriched populations of ear mesenchymal stem cells possess enhanced capacity to differentiate into adipocytes. Additionally, we determined the natural frequency and localization of Sca-1-positive progenitor/stem cells in brown and white fat in situ. The present study addressed the question whether Sca-1 deficiency alters the white adipose tissue response to a high-saturated-fat diet. Our results show that Sca-1 null mice (Sca-1(-/-)) fed high-fat diet developed obesity equally well as wild-type mice, suggesting either an indirect in vivo effect of Sca-1 or a compensatory response to Sca-1 deficiency. However, contrary to wild-type mice, high fat diet-fed Sca-1(-/-) mice showed no alterations in serum adipocytokines. The data lead to the conclusion that Sca-1 is either redundant or a nonessential marker of adipose progenitor/stem cells. Nevertheless, since Sca-1-deficient mice displayed elevated blood glucose at fasting and exhibited glucose intolerance and insulin resistance, Sca-1 has subtle effects on adipose function. Thus, the Sca-1-deficient mice may provide a useful model for metabolic studies.

Screening for epigenetic target genes that enhance reprogramming using lentiviral-delivered shRNA.

Small molecules will need to be identified and/or developed that target protein classes limiting reprogramming efficiency. A specific class of proteins includes epigenetic regulators that silence, or minimize expression, of pluripotency genes in differentiated cells. To better understand the role of specific epigenetic modulators in reprogramming, we have used shRNA delivered by lentivirus to assess the significance of individual epi-proteins in reprogramming pluripotent gene expression.

Uncoupling of inflammation and insulin resistance by NF-kappaB in transgenic mice through elevated energy expenditure.

To study the metabolic activity of NF-kappaB, we investigated phenotypes of two different mouse models with elevated NF-kappaB activities. The transcriptional activity of NF-kappaB is enhanced either by overexpression of NF-kappaB p65 (RelA) in aP2-p65 mice or inactivation of NF-kappaB p50 (NF-kappaB1) through gene knock-out. In these models, energy expenditure was elevated in day and night time without a change in locomotion. The mice were resistant to adulthood obesity and diet-induced obesity without reduction in food intake. The adipose tissue growth and adipogenesis were inhibited by the elevated NF-kappaB activity. Peroxisome proliferator-activator receptor gamma expression was reduced by NF-kappaB at the transcriptional level. The two models exhibited elevated inflammatory cytokines (tumor necrosis factor-alpha and interleukin-6) in adipose tissue and serum. However, insulin sensitivity was not reduced by the inflammation in the mice on a chow diet. On a high fat diet, the mice were protected from insulin resistance. The glucose infusion rate was increased more than 30% in the hyperinsulinemic-euglycemic clamp test. Our data suggest that the transcription factor NF-kappaB promotes energy expenditure and inhibits adipose tissue growth. The two effects lead to prevention of adulthood obesity and dietary obesity. The energy expenditure may lead to disassociation of inflammation with insulin resistance. The study indicates that inflammation may prevent insulin resistance by eliminating lipid accumulation.

Cell growth characteristics, differentiation frequency, and immunophenotype of adult ear mesenchymal stem cells.

Ear mesenchymal stem cells (EMSCs) represent a readily accessible population of stem-like cells that are adherent, clonogenic, and have the ability to self-renew. Previously, we have demonstrated that they can be induced to differentiate into adipocyte, osteocyte, chondrocyte, and myocyte lineages. The purpose of the current study was to characterize the growth kinetics of the cells and to determine their ability to form colonies of fibroblasts, adipocytes, osteocytes, and chondrocytes. In addition, the immunophenotypes of freshly isolated and culture-expanded cells were evaluated. From 1 g of tissue, we were able to isolate an average of 7.8 x 10(6) cells exhibiting a cell cycle length of approximately 2-3 days. Colony-forming unit (CFU) assays indicated high proliferation potential, and confirmed previously observed multipotentiality of the cells. Fluorescence-activated cell sorting (FACS) showed that EMSCs were negative for hematopoietic markers (CD4, CD45), proving that they did not derive from circulating hematopoietic cells. The FACS analyses also showed high expression of stem cell antigen-1 (Sca-1) with only a minor population of cells expressing CD117, thus identifying Sca-1 as the more robust stem cell biomarker. Additionally, flow cytometry data revealed that the expression patterns of hematopoietic, stromal, and stem cell markers were maintained in the passaged EMSCs, consistent with the persistence of an undifferentiated state. This study indicates that EMSCs provide an alternative model for in vitro analyses of adult mesenchymal stem cells (MSCs). Further studies will be necessary to determine their utility for tissue engineering and regenerative medical applications.

Flow cytometric and immunohistochemical detection of in vivo BrdU-labeled cells in mouse fat depots.

This study has determined the natural frequency and localization of progenitor/stem cells within fat depots in situ based on their ability to retain DNA nucleotide label (BrdU). Neonate and mature male C57BL6/J mice were injected intraperitoneally with BrdU- and label-retaining cells (LRC) were quantified in fat depots by immunohistochemical, immunofluorescent, and flow cytometric methods. In neonates, LRC constituted 27% of the cells in inguinal fat (iWAT) and 65% in interscapular brown fat (BAT) after Day 10 and 26% of the cells in epididymal fat (eWAT) after Day 28. After 52 days, the LRC accounted for 0.72% of iWAT, 0.53% of eWAT and 1.05% of BAT, respectively. The BrdU-labeled cells localized to two areas: single cells distributed among adipocytes or those adjacent to the blood vessels wall. In mature C57BL6/J mice, flow cytometric analysis determined that a majority of the LRC were also positive for stem cell antigen-1 (Sca-1).

IFATS collection: Stem cell antigen-1-positive ear mesenchymal stem cells display enhanced adipogenic potential.

Hyperplasia is a major contributor to the increase in adipose tissue mass that is characteristic of obesity. However, the identity and characteristics of cells that can be committed into adipocyte lineage remain unclear. Stem cell antigen 1 (Sca-1) has been used recently as a candidate marker in the search for tissue-resident stem cells. In our quest for biomarkers of cells that can become adipocytes, we analyzed ear mesenchymal stem cells (EMSC), which can differentiate into adipocytes, osteocytes, chondrocytes, and myocytes. Our previous studies have demonstrated that EMSC abundantly expressed Sca-1. In the present study, we have analyzed the expression of adipogenic transcription factors and adipocyte-specific genes in Sca-1-enriched and Sca-1-depleted EMSC fractions. Sca-1-enriched EMSC accumulated more lipid droplets during adipogenic differentiation than Sca-1-depleted. Similarly, EMSC isolated from Sca-1(-/-) mice displayed reduced lipid accumulation relative to EMSC from wild-type controls (p < .01). Comparative analysis of the adipogenic differentiation process between Sca-1-enriched and Sca-1-depleted populations of EMSC revealed substantial differences in the gene expression. Preadipocyte factor 1, CCAAT enhancer-binding protein (C/EBP) beta, C/EBPalpha, peroxisome proliferator-activated receptor gamma2, lipoprotein lipase, and adipocyte fatty acid binding protein were expressed at significantly higher levels in the Sca-1-enriched EMSC fraction. However, the most striking observation was that leptin was detected only in the conditioned medium of Sca-1-enriched EMSC. In addition, we performed loss-of-function (Sca-1 morpholino oligonucleotide) experiments. The data presented here suggest that Sca-1 is a biomarker for EMSC with the potential to become functionally active adipocytes. Disclosure of potential conflicts of interest is found at the end of this article.

Chronic consumption of a low-fat diet leads to increased hypothalamic agouti-related protein and reduced leptin.

OBJECTIVE: This study examined the hypothesis that dietary fat under ad libitum feeding conditions influences expression levels (mRNA) of the mouse agouti-related protein (AgRP), leptin, leptin receptor (OBRb), and neuropeptide Y (NPY) at early stages of development. METHODS: C57Bl/6J male mice were placed on a high-fat diet (HFD) or a low-fat diet (LFD) shortly after weaning. Groups of mice were euthanized at various ages and real-time one-step reverse transcriptase polymerase chain reaction was used to analyze gene expression in the hypothalamus (AgRP, NPY, OBRb), the adrenal gland (AgRP), the testis (AgRP), and epididymal fat (leptin). RESULTS: Leptin expression increased linearly with age but only under the HFD despite body weight gain under both diets. This pattern of expression coincided with reduced expression of hypothalamic AgRP under an HFD, whereas OBRb and NPY did not fluctuate in response to diet. By contrast, consumption of an LFD (i.e., high carbohydrate) increased hypothalamic AgRP and suppressed adipose leptin, which is consistent with the notion that leptin could regulate AgRP centrally. In contrast, AgRP expression in the adrenal gland initially decreased and then increased with age under both diets. CONCLUSIONS: Dietary fat can have a tissue-dependent effect on AgRP that may be unfettered by leptin under an HFD.

Circadian expression of genes regulating food intake.

OBJECTIVE: The Agouti-related protein (AgRP), neuropeptide Y (NPY), proopiomelanocortin (POMC), cocaine and amphetamine-regulated transcript (CART), Orexin, melanin concentrating hormone (MCH), leptin, and its hypothalamic receptor (LR) are key regulators of food intake and energy homeostasis. In the present study, we examined the circadian expression profiles of these genes. RESEARCH METHODS AND PROCEDURES: We used quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to measure mRNA levels, spectral analysis to evaluate periodicity, and correlation analysis to examine for associations with diurnal food intake. RESULTS: No gene in particular stood out as a strong candidate, but the overall circadian expression profiles of leptin and its hypothalamic receptor came close to statistically and graphically resembling the diurnal feeding behavior of mice. In mathematical terms, adrenal AgRP exhibited strong circadian expression and had the highest correlation with food intake, followed by leptin. Yet its highest point of expression occurred 8 hours after nocturnal food intake had peaked, suggesting that adrenal AgRP could not play a direct role in the initiation of nocturnal feeding; neither did hypothalamic AgRP, NPY, POMC, CART, Orexin, or MCH. DISCUSSION: These data show that ad libitum feeding in mice is influenced by complex central and peripheral circuits involving orexigenic and anorectic agents of which leptin and its hypothalamic receptor could play more prevalent roles.

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.

Long form leptin receptor mRNA expression in the hypothalamus and pituitary during early pregnancy in the pig.

OBJECTIVE: The aim of this study was the detection and location of long form leptin receptor (OB-Rb) in different area of hypothalamus and pituitary in the pig during early pregnancy. SETTINGS AND DESIGN: Expression of OB-Rb was examined by RT-PCR in the different area of hypothalamus: medial basal hypothalamus (MBH), preoptic area (POA), stalk median eminence (SME), as well as pituitary: the anterior (AP) and posterior (NP) lobe collected from gilts at days 14-16 (n=4) and 30-32 (n=4) of pregnancy. RESULTS: The results showed that OB-Rb mRNA was expressed in the hypothalamus (MBH, POA and SME), pituitary (AP, NP) and adipose tissue in the pig during early pregnancy (at days 14-16 and 30-32). CONCLUSION: These findings support the idea that leptin might play a role in the regulation of the hypothalamic-pituitary axis activity, and consequently in the control of pregnancy during critical period of embryo implantation in the pig.

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.

The influence of estradiol and progesterone on the concentrations of uterine oxytocin receptors and plasma PGFM in response to oxytocin in ovariectomized gilts.

Peripubertal gilts (n = 25) were treated with corn oil (CO) or ovarian steroids, one month following an ovariectomy. The first day of treatment was assigned as the first day of the experiment. The gilts received: Group (Gr) I (n = 4)--CO (2 mL x day(-1) from 1st to 12th day), Gr II (n = 4) and Gr III (n = 4)--progesterone (P4; 10 to 100 mg x day(-1) from 1st to 12th day), Gr IV (n = 5)--estradiol benzoate (EB; 400 microg x day(-1) from 1st to 3rd day), Gr V (n = 4) and Gr VI (n = 4)--EB + P4 (EB 400 microg x day(-1) from 1st to 3rd day, 20 microg x day(-1) at 6th and 9th day, 50 microg at 12th day plus P4 10 to 100 mg from 4th to 15th day). All gilts were injected with oxytocin (OT; 20 IU; i.v.) on the following days of the experiment: 13th (Gr I and Gr II), 15th (Gr III and Gr IV), 16th (Gr V) and 18th (Gr VI). Concentrations of the PGF2alpha metabolite--PGFM were determined in blood samples, collected from 30 min before to 120 min after OT injection. Baseline PGFM concentrations (30 min before OT) differed among treatment groups and were the highest in Gr V and Gr VI (P < 0.01 vs. other groups). The magnitude of the PGFM response to OT increased only in four of the five gilts of Gr IV and in three of the four gilts of Gr VI, and it was higher (P = 0.009) in Gr VI than in Gr IV. In the remaining groups, PGFM concentrations did not increase above the baseline in response to OT. The day after OT injection, oxytocin receptors (OTR) were found in the uterine tissues of all animals studied. The lowest OTR concentrations were in Gr I--75.5 +/- 11.2 fmol x mg protein(-1) and the highest in Gr IV--712.9 +/- 86.7 fmol x mg protein(-1); (P < 0.05 vs. other groups). The values of K of OTR differed among groups (P < 0.001) and ranged from 1.62 +/- 0.44 nM in Gr I to 12. 08 +/- 1.9 nM in Gr VI. A positive correlation (r = 0.54; P < 0.01) between plasma E2 and uterine OTR concentrations was observed. In conclusion, E2 and P4 are involved in both PGF2 synthesis/secretion and OTR formation, however, full PGF response to OT does not develop before puberty. Estrogens are evident stimulators of uterine OTR synthesis ingilts.