Molecular Human Reproduction - Advance Access

Effects of natural ligands of PPAR{gamma} on lipid metabolism in placental tissues from healthy and diabetic rats

Capobianco, E., White, V., Higa, R., Martinez, N., Jawerbaum, A. — 2008-07-11

The ligand-dependent nuclear receptor peroxisome proliferators-activated receptors (PPAR) plays an important role in placental development and function. The aim of this study was to analyse the influence of natural PPAR ligands on placental lipid metabolism in diabetic and control rats after midpregnancy, as well as the concentrations of the PPAR endogenous agonist 15deoxy^12,14Prostaglandin J₂ (15dPGJ₂). In vitro experiments showed that 15dPGJ₂ did not regulate placental concentrations of triglycerides, cholesteryl esters, phospholipids and free fatty acids, but decreased the de novo synthesis of these lipid species. PPAR agonists were administered in vivo through dietary supplementation with either 6% olive oil or 6% safflower oil. These treatments led to increases in placental lipid mass in control tissues and more markedly in diabetic tissues. In addition, they led to reductions in the de novo lipid synthesis both in control and diabetic placental tissues. 15dPGJ₂ concentrations were greatly reduced in the placenta from diabetic rats fed with the standard diet. Both dietary supplementations increased the concentrations of 15dPGJ₂ in placentas from control and diabetic rats. These data indicate that, in the placenta, PPAR natural ligands regulate the concentration of their own endogenous ligands. In addition, they increase the placental capacity to accumulate maternal-derived lipids, and reduce the de novo lipid synthesis, thus regulating metabolic pathways that are altered in the placenta from diabetic rats and involved in the lipid transfer to the developing fetus.

Characterization of an acrosome protein VAD1.2/AEP2 which is differentially expressed in spermatogenesis

2008-07-10

The release of enzymes from the acrosome of the sperm head (acrosome reaction) starts the fertilization process and enables the spermatozoa to penetrate the zona pellucida of the oocytes. Defective acrosome reaction is one of the important causes of infertility in men. To investigate the molecular regulation of spermatogenesis in vivo, we used differential display RT-PCR to identify testis-specific genes in a retinol-supplemented vitamin A deficiency (VAD) rat model and identified the VAD1.2 (acrosome-expressing protein 2, AEP2) gene, which was expressed strongly in the rat testis from postnatal day 32 to adult stage. The mouse VAD1.2 mRNA shared 85% and 67% sequence homology, and 74% and 38% amino acid homology, respectively with the rat and human counterparts. VAD1.2 transcript was abundantly expressed in the rat seminiferous tubules at stage VIII-XII and the protein was detected in the acrosome region of the round and elongated spermatids of mouse, human, monkey and pig. VAD1.2 co-localized with lectin-PNA to the acrosome region of spermatids. Interestingly, the expression of VAD1.2 protein in human testis diminished in patients with hypospermatogenesis, maturation arrest, undescended testis and Sertoli cell-only syndrome. Co-immunoprecipitation experiments followed by Western blotting and mass spectrometry (MS-MS) identified syntaxin 1, β-actin and myosin heavy chain (MHC) proteins as putative interacting partners. Taken together, the stage-specific expression of VAD1.2 in the acrosome of spermatids and the binding of VAD1.2 protein with vesicle forming (syntaxin) and structural (β-actin and MHC) proteins suggest that VAD1.2 maybe involved in acrosome formation during spermiogenesis.

Human chorionic gonadotrophin up-regulates hypoxia inducible factor-1 alpha in luteinised granulosa cells: implications for the hormonal regulation of vascular endothelial growth factor A in the human corpus luteum

van den Driesche, S., Myers, M., Gay, E., Thong, K. J., Duncan, W. C. — 2008-06-30

VEGF-dependent angiogenesis is essential for normal luteal development. Although it is believed that hypoxia is the primary inducer of VEGF, in the corpus luteum it is up-regulated by hCG. As HIF1A has been shown to regulate VEGFA under ligand-stimulated conditions, we hypothesised that the effect of hCG on luteal VEGFA was mediated through HIF1A. We studied the effect of hCG on VEGFA and HIF1A expression in human luteinised granulosa cells in vitro and in human corpora lutea in vivo. HCG up-regulated VEGFA (P<0.05) and HIF1A (P<0.001) in vitro and VEGFA (P<0.05) and HIF1A (P<0.05) in vivo. There was a correlation between HIF1A and VEGFA in vivo (P<0.005) and in vitro (P<0.05). Nuclear HIF1A in granulosa-lutein cells was highest during luteal formation and absent from the fully functional corpus luteum (P<0.05). Both VEGFA (P<0.001) and HIF1A (P<0.01) were up-regulated by dibutyryl-cAMP, through a PKA pathway. Hypoxia increased VEGFA (P<0.001) and HIF1A (P<0.05) expression and hCG further augmented VEGFA (P<0.001) and HIF1A (P<0.01) under hypoxic conditions. However progesterone increased hCG-stimulated VEGFA but had no effect on HIF1A expression. The expression of HIF1A is therefore hormonally regulated in luteal cells in vitro and in vivo and may regulate VEGFA expression under normoxic and hypoxic conditions. However the differential effects of progesterone suggest that not all regulation of VEGFA is associated with an up-regulation of HIF1A.

Regulation of Mitochondrial Polarity in Mouse and Human Oocytes: The Influence of Cumulus Derived Nitric Oxide

Van Blerkom, J., Davis, P., Thalhammer, V. — 2008-06-30

Whether exogenous factors influenced the level of mitochondrial polarity (m) in the subplasmalemmal cytoplasm of the oocyte was investigated with denuded and cumulus-enclosed human and mouse oocytes between the germinal vesicle and metaphase II stage. Co-culture of denuded oocytes with cumulus masses or primary cumulus cell cultures demonstrated a ‘proximity’ effect with respect to the detectable level of m in the oocyte. The specificity and reversibility of this effect on subplasmalemmal mitochondria were shown by repeated repositioning between cellular and acellular regions, which sequentially down- or upregulated m. Experimental studies with a nitric oxide (NO) donor and inhibitor of NO synthase indicate that NO produced by cumulus cells has a regulatory influence on m in the subplasmalemmal cytoplasm of the corresponding oocyte. Culture of denuded and cumulus-enclosed (intact) oocytes in low and high oxygen atmospheres suggests that competition between oxygen and NO at the mitochondrial level may regulate the level of m and maintain mitochondria homeostasis in the preovulatory oocyte, with a shift to higher polarity occurring after ovulation. The role of exogenous influences on oocyte mitochondrial polarity is discussed with respect to the regulation of developmental processes in the oocyte and early embryo.

Insulin-like Growth Factor Binding Protein-1 (IGFBP-1) Induces Decidualization of Human Endometrial Stromal Cells via {alpha}5{beta}1 Integrin

Matsumoto, H., Sakai, K., Iwashita, M. — 2008-06-26

Progesterone is known to induce decidualization of human endometrial stromal cells in vitro. Decidualized stromal cells produce insulin-like growth factor binding protein-1 (IGFBP-1) as well as prolactin (PRL). In this study, we tested the possibility that IGFBP-1 directly stimulates endometrial stromal cell decidualization. Endometrial stromal cells were obtained from normal menstruating patients with uterine myoma at hysterectomy. Stromal cells were cultured for up to 4 weeks with estradiol (E2) and/or medroxy progesterone acetate (MPA) in the presence or the absence of IGFBP-1 and, LR³-IGF-I (an IGF-I analogue) that binds to the IGF-I receptor but has reduced affinity for IGFBPs. Decidualization of endometrial stromal cells was evaluated by morphological changes and PRL release into culture media. The binding of IGFBP-1 to endometrial cells was analyzed using a biosensor.

MPA and E2 induced decidualization of stromal cells, while LR³-IGF-I inhibited decidualization by MPA and E2 as well as PRL and IGFBP-1 secretion into medium. IGFBP-1 induced decidualization of stromal cells in the absence of MPA and E2 in the medium. IGFBP-1-induced decidualization was not inhibited by the addition of LR³IGF-1 but was inhibited by the addition of an RGD peptide, however, the RGD peptide had no effect on decidualization when added alone. The binding analysis showed that IGFBP-1 bound to the surface of endometrial stromal cells and an anti-5β1 integrin antibody inhibited its binding.

These results suggest that IGFBP-1 produced by endometrium can mediate progesterone-induced decidualization possibly by interacting with 5β1 integrin on the surface of endometrial stromal cells.

Gene expression in cultured endometrium from women with different outcomes following IVF.

Bersinger, N. A., Wunder, D. M., Birkhauser, M. H., Mueller, M. D. — 2008-06-06

Estradiol and progesterone are crucial for the acquisition of receptivity and the change in transcriptional activity of target genes in the implantation window. The aim of this study was to differentiate the regulation of genes in the endometrium of patients with recurrent implantation failure (IF) versus those who became pregnant after IVF treatment. Moreover, the effect of embryo-derived factors on endometrial transcriptional activity was studied. Nine women with known IVF outcome (IF, M=Miscarriage, OP=ongoing pregnancy) and undergoing hysteroscopy with endometrial biopsy were enrolled. Biopsies were taken during the midluteal phase. After culture in presence of embryo-conditioned IVF media, total RNA was extracted and submitted to reverse transcription, target cDNA synthesis, biotin labelling, fragmentation, and hybridisation using the Affymetrix Human Genome U133A 2.0 Chip. Differential expression of selected genes was re-analysed by quantitative PCR, in which the results were calculated as threshold cycle differences between the groups and normalised to GAPDH and β-actin. Differences were seen for several genes from endometrial tissue between the IF and the pregnancy groups, and when comparing OP with M, 1875 up- and 1807 downregulated genes were returned. Real-time PCR analysis confirmed upregulation for somatostatin, PLAP-2, mucin 4, and CD163, and downregulation of glycodelin, IL-24, CD69, leukaemia inhibitory factor, and prolactin receptor between Op and M. When the different embryo conditioned media were compared, no significant differential regulation could be demonstrated. Although microarray profiling may currently not be sensitive enough for studying the effects of embryo-derived factors on the endometrium, the observed differences in gene expression between M and OP, suggest that it will become an interesting tool for the identification of fertility relevant markers produced by the endometrium.

Permanent Embryo Arrest: Molecular and Cellular Concepts

Betts, D.H., Madan, P. — 2008-05-29

Developmental arrest is one of the mechanisms responsible for the elevated levels of embryo demise during the first week of in vitro development. Approximately 10-15% of IVF embryos permanently arrest in mitosis at the 2-4-cell cleavage stage showing no indication of apoptosis. Reactive oxygen species (ROS) are implicated in this process and must be controlled in order to optimize embryo production. A stress sensor that can provide a key understanding of permanent cell cycle arrest and link ROS with cellular signaling pathway(s) is p66Shc, an adaptor protein for apoptotic response to oxidative stress. Deletion of the p66Shc gene in mice results in extended lifespan, which is linked to their enhanced resistance to oxidative stress and reduced levels of apoptosis. P66Shc has been shown to generate mitochondrial H₂O₂ to trigger apoptosis, but may also serve as an integration point for many signaling pathways that affect mitochondrial function. We have detected elevated levels of p66Shc and ROS within arrested embryos and believe that p66Shc plays a central role in regulating permanent embryo arrest. In this paper we review the cellular and molecular aspects of permanent embryo arrest and speculate on the mechanism(s) and etiology of this method of embryo demise.