Adiponectin, the most abundantly synthesized protein in adipose tissue, has plieotropic effects on liver, muscle, endothelium, placenta, and other tissues. We examined direct effects of recombinant porcine adiponectin on porcine ovarian granulosa cells in vitro. We demonstrate that adiponectin, at physiologically relevant levels (10-25 microg/ml), provokes expression of genes associated with periovulatory remodeling of the ovarian follicle over a time frame of 6-24 h. These include cyclooxygenase-2, prostaglandin E synthase, and vascular endothelial growth factor. Adiponectin modulates steroid synthetic protein gene expression, increasing steroidogenic acute regulatory protein transcript abundance and reducing cytochrome P450aromatase. Adiponectin has antidiabetic properties and sensitizes tissues to insulin. We show that it interacts with both LH and insulin in inducing expression of cyclooxygenase-2 transcripts in granulosa cells. We determined that the MAPK pathway, via phosphorylation of ERK1/2, is involved in mediation of the adiponectin signal in ovarian granulosa cells, rather than protein kinase A or the classic adiponectin transducer, AMP-activated protein kinase. Adiponectin synthesis is reduced in obesity, and our findings suggest that this reduction plays a role in obesity-related ovarian dysfunction.

Adiponectin is an adipocyte-derived hormone that plays an important role in lipid metabolism and glucose homeostasis. Objectives of this study were 1) to determine the presence and distribution of adiponectin and its receptors 1 and 2 (adipoR1 and adipoR2) in porcine tissues; 2) to characterize pig adiponectin, adipoR1, and adipoR2 mRNA levels in various fat depots from three different breeds of pigs; and 3) to study, in stromal-vascular cell culture, the effects of leptin and tumor necrosis factor-alpha (TNFalpha) on pig adiponectin, adipoR1, and adipoR2 gene expression. To this end, fat Chinese Upton Meishan (UM, n = 10), lean Ham Line (HL, n = 10), and Large White (LW, n = 10) gilts were used. We report the isolation of partial cDNA sequences of pig adipoR1 and adipoR2. Porcine-deduced AA sequences share 97 to 100% homology with human and murine sequences. Pig adipoR1 mRNA is abundant in skeletal muscle, visceral fat, and s.c. fat tissues, whereas adipoR2 mRNA is predominantly expressed in liver, heart, skeletal muscle, and visceral and s.c. fat tissues. Pig adiponectin mRNA levels in s.c. and visceral fat tissues were not associated with plasma insulin and glucose in fasting animals. Subcutaneous (r = -0.44, P < 0.05), visceral (r = -0.43, P < 0.05), and total body fat (r = -0.42, P < 0.05) weights were negatively correlated with adiponectin mRNA levels measured in visceral, but not s.c., fat. Pig adipoR1 and adipoR2 mRNA levels, in visceral fat, were less expressed in fat UM gilts than in the lean HL gilts (P < 0.05). Inverse associations were found between s.c. (r = -0.57, P < 0.01), visceral (r = -0.46, P < 0.05), and total body fat (r = -0.56, P < 0.01) weights and adipoR2 mRNA levels in visceral fat only. We were unable to find such associations for adipoR1 mRNA levels in the overall gilt population. The current study demonstrated that TNFalpha downregulates adiponectin and adipoR2, but not adi-poR1, mRNA levels in stromal-vascular cell culture. Moreover, leptin significantly decreased adiponectin mRNA levels, whereas there was no effect on adiponectin receptors. We conclude that adiponectin and adi-poR2 mRNA levels, but not adipoR1, are modulated in pig visceral fat tissues. Furthermore, our results indicate that TNFalpha interferes with adiponectin function by downregulation of adipoR2 but not of adipoR1 mRNA levels in pigs.

Cholesterol is imported and processed to provide substrate for ovarian steroidogenesis. The Niemann Pick type C-1 gene codes for a glycoprotein that processes low-density lipoproteinimported cholesterol. Mutation of this gene causes marked impairment of export of low-density lipoprotein-derived cholesterol from endosomes, and consequent lysosomal accumulation of the sterol. The BALB/c npc(nih-/-) mouse line, bearing spontaneous mutation of the NPC-1 gene, provides a model for investigation of aberrant endosomal cholesterol transfer in the ovary. Female homozygote mutant mice are infertile, with underdeveloped ovarian follicles, reduced steroidogenesis, no ovulation, and no corpora lutea. Mutant ovaries transplanted under wild-type kidney capsules display both ovulation and formation of corpora lutea. Gonadotropin treatment induces ovulation and restores expression of steroidogenic proteins. Pituitary glands of mutants are hypoplastic, and prolactin expression is dramatically reduced compared with wild-type mice. Both long and short splice variants of the dopamine-D2 receptors are overexpressed in the pituitary of BALB/c npc(nih-/-) mice. Chronic treatment of mutant mice with 17beta-estradiol restores pituitary volume, prolactin expression, and folliculogenetic capability. We conclude that inactivating mutation of Niemann Pick C-1 perturbs the hypothalamic-pituitary-ovarian feedback loop. Reduced estrogens attenuate prolactin expression and alter gonadotropin secretion patterns and interfere with normal ovarian follicular development and ovulation.

Expression of vascular endothelial growth factor (VEGF) isoforms and its receptors, Flt-1 and KDR, was investigated during the period of peri-implantation in mink, a species that displays obligate embryonic diapause. Uterine samples were collected during diapause, embryo activation, and implantation from pseudopregnant and anestrous animals and analyzed by semiquantitative reverse transcription polymerase chain reaction and immunohistochemistry. The abundance of mRNA of VEGF isoforms 120, 164, and 188 was highest during late embryo activation and at implantation. VEGF protein was localized to the glandular epithelium at all stages of peri-implantation, whereas the luminal epithelium lacked VEGF reactivity during diapause. Endometrial stroma and luminal and glandular epithelia were positive for VEGF in implanted uteri. The invasive trophoblast cells of the implanting embryo were intensively stained. High levels of VEGF mRNA in pseudopregnant uteri indicates that VEGF upregulation leading to implantation is dependent upon maternal rather than embryonic factors. The abundance of the two receptors, KDR and Flt-1, increased in the uterus during implantation. Low levels of the receptors in pseudopregnant uteri compared with those containing activated or implanted embryos indicates that the embryo regulates receptor expression. These results demonstrate VEGF and VEGF receptor expression during early gestation in mink and suggest that maternal and embryonic input regulates different aspects of the angiogenic process.

Vascular endothelial growth factor (VEGF) is an essential angiogenic signaling element that acts through its two tyrosine kinase receptors, inducing both proliferation of endothelial cells and vascular permeability. Given the importance of vasculogenesis and angiogenesis to early pregnancy, it is of interest to understand the mechanisms regulating vascular development at this stage. We previously demonstrated that VEGF and receptors are up-regulated during embryo implantation in an unique animal model, the mink, a species displaying obligate embryonic diapause. Herein we examined the role of prostaglandin E2 (PGE 2 ) as a regulator of VEGF during early pregnancy and established the mechanisms of this regulation. We demonstrate that activated embryos secrete PGE 2 and that expression of PGE synthase protein in the uterus is dependent upon direct contact with invading trophoblast cells during implantation. Using mink uterine stromal cells transfected with mink VEGF promoter driving the luciferase reporter gene, we show that PGE 2 induces promoter transactivation and that this response can be eliminated by blockade of protein kinase A. Treatment with antagonists to PGE 2 receptors EP2 and EP4 eliminated the PGE 2 -induced response in transfected cells. Deletional studies of the promoter revealed that a region of 99 bp upstream of the transcription start site is required for PGE 2 -induced transactivation. Mutation of an AP2/Sp1 cluster, found within the 99 bp, completely eliminated the PGE 2 response. Furthermore, chromatin immunoprecipitation assays confirmed binding of the AP2 and Sp1 transcription factors to the endogenous mink VEGF promoter in uterine cells. PGE 2 stimulated acetylation of histone H3 associated with the promoter region containing the AP2/Sp1 cluster. Taken together, these results demonstrate that PGE 2 plays an important role in regulating uterine and thus placental vascular development, acting through its receptors EP2 and EP4, provoking protein kinase A activation of AP2 and Sp1 as well as acetylation of histone H3 to transactivate the VEGF promoter.

Recent evidence points to a role for peroxisome proliferator-activated receptors (PPARs) d and g in embryo implantation and survival. In this study, we report the porcine PPARd complete coding sequence and mRNA abundance of PPARd, PPARg1 and g2, angiopoietin-like protein 4 (ANGPTL4) and adipocyte determination and differentiation-dependent factor 1 (ADD1) genes in the pregnant sow endometrium. Real-time PCR analysis was used to study the effect of parity (Yorkshire-Landrace multiparous (YL) and nulliparous (YLn)), site of endometrial tissue sampling (between and at embryo attachment sites) in crossbred Duroc!Yorkshire-Landrace (DYL) sows and stages of pregnancy (non-pregnant, day 15 and day 25 after mating) in Meishan-Landrace (ML) on mRNA levels. Parity effects were observed for PPARd, ANGPTL4, and ADD1, with higher mRNA levels in YL than YLn sows. In DYL sows, lower mRNA levels were present at attachment sites compared to between attachment sites for PPARd, PPARg1, and ANGPTL4. Finally, day 15 pregnant ML sows had lower PPARd mRNA levels compared to day 15 cycling ML sows. A significant increase of PPARg1 mRNA levels was found on day 25 pregnant ML and DYL sows relative to day 15 ML or DYL pregnant sows. PPARd and g immunostaining was detected in endometrial tissue of day 15 cycling sows, day 15 and 25 pregnant sows and epithelial cells of day 25 embryos. Collectively, our results suggest a role for PPARd, PPARg1, and ANGPTL4, but not PPARg2, during the peri-implantation period in pregnant sows. Reproduction (2006) 131 929-942