Joint MiRNA/mRNA expression profiling reveals rhanges consistent with development of dysfunctional corpus luteum after weight gain

<div><p>Obese women exhibit decreased fertility, high miscarriage rates and dysfunctional corpus luteum (CL), but molecular mechanisms are poorly defined. We hypothesized that weight gain induces alterations in CL gene expression. RNA sequencing was used to identify changes in the CL transcriptome in the vervet monkey (<i>Chlorocebus aethiops</i>) during weight gain. 10 months of high-fat, high-fructose diet (HFHF) resulted in a 20% weight gain for HFHF animals vs. 2% for controls (p = 0.03) and a 66% increase in percent fat mass for HFHF group. Ovulation was confirmed at baseline and after intervention in all animals. CL were collected on luteal day 7–9 based on follicular phase estradiol peak. 432 mRNAs and 9 miRNAs were differentially expressed in response to HFHF diet. Specifically, miR-28, miR-26, and let-7b previously shown to inhibit sex steroid production in human granulosa cells, were up-regulated. Using integrated miRNA and gene expression analysis, we demonstrated changes in 52 coordinately regulated mRNA targets corresponding to opposite changes in miRNA. Specifically, 2 targets of miR-28 and 10 targets of miR-26 were down-regulated, including genes linked to follicular development, steroidogenesis, granulosa cell proliferation and survival. To the best of our knowledge, this is the first report of dietary-induced responses of the ovulating ovary to developing adiposity. The observed HFHF diet-induced changes were consistent with development of a dysfunctional CL and provide new mechanistic insights for decreased sex steroid production characteristic of obese women. MiRNAs may represent novel biomarkers of obesity-related subfertility and potential new avenues for therapeutic intervention.</p></div

A Peptide-binding Motif for I-Ag7, the Class II Major Histocompatibility Complex (MHC) Molecule of NOD and Biozzi AB/H Mice

The class II major histocompatibility complex molecule I-Ag7 is strongly linked to the development of spontaneous insulin-dependent diabetes mellitus (IDDM) in non obese diabetic mice and to the induction of experimental allergic encephalomyelitis in Biozzi AB/H mice. Structurally, it resembles the HLA-DQ molecules associated with human IDDM, in having a non-Asp residue at position 57 in its β chain. To identify the requirements for peptide binding to I-Ag7 and thereby potentially pathogenic T cell epitopes, we analyzed a known I-Ag7-restricted T cell epitope, hen egg white lysozyme (HEL) amino acids 9–27. NH2- and COOH-terminal truncations demonstrated that the minimal epitope for activation of the T cell hybridoma 2D12.1 was M12-R21 and the minimum sequence for direct binding to purified I-Ag7 M12-Y20/ K13-R21. Alanine (A) scanning revealed two primary anchors for binding at relative positions (p) 6 (L) and 9 (Y) in the HEL epitope. The critical role of both anchors was demonstrated by incorporating L and Y in poly(A) backbones at the same relative positions as in the HEL epitope. Well-tolerated, weakly tolerated, and nontolerated residues were identified by analyzing the binding of peptides containing multiple substitutions at individual positions. Optimally, p6 was a large, hydrophobic residue (L, I, V, M), whereas p9 was aromatic and hydrophobic (Y or F) or positively charged (K, R). Specific residues were not tolerated at these and some other positions. A motif for binding to I-Ag7 deduced from analysis of the model HEL epitope was present in 27/30 (90%) of peptides reported to be I-Ag7–restricted T cell epitopes or eluted from I-Ag7. Scanning a set of overlapping peptides encompassing human proinsulin revealed the motif in 6/6 good binders (sensitivity = 100%) and 4/13 weak or non-binders (specificity = 70%). This motif should facilitate identification of autoantigenic epitopes relevant to the pathogenesis and immunotherapy of IDDM

Corpus luteum as a novel target of weight changes that contribute to impaired female reproductive physiology and function

<p>Obesity and malnutrition are associated with decreased fecundity in women. Impaired reproductive capacity in obese women is often attributed to anovulation. However, obese women with ovulatory cycles also have reduced fertility, but the etiology of their impaired reproduction is only partially understood. Accumulating evidence suggests that obesity directly impairs oocyte and embryo quality as well as endometrial receptivity. In obese women, urinary progesterone metabolite excretion is decreased, but in excess of what can be explained by suppressed gonadotropin secretion, suggesting that apart from its central effect obesity may directly affect progesterone (P4) production. These observations have led to the novel hypothesis that obesity directly affects corpus luteum (CL) function. Similarly, we hypothesize that weight loss may contribute to luteal dysfunction. Here, we propose a non-human primate model, the vervet monkey, to examine the effect of weight gain and loss on menstrual cycle parameters and CL gene expression. In this model, weight gain and loss did not significantly alter menstrual cyclicity; however, both induced alterations in the CL transcriptome. In the weight gain monkey, we observed that impaired mid-luteal P4 secretion was associated with downregulation of steroidogenic pathways in CL. Collectively, these preliminary findings support our hypothesis that weight gain and loss may contribute to CL dysfunction. The vervet model described and preliminary observations provide a basis for a larger study to address this important question. Understanding the mechanisms by which weight gain and loss contribute to reproductive dysfunction can assist in the development of targeted treatments to enhance women’s reproductive capability when it is desired.</p> <p><b>Abbreviations:</b> CL: corpus luteum; P4: progesterone; E2: estradiol; PDG: pregnanediol 3-glucoronide; LH: luteinizing hormone; FSH: follicle-stimulating hormone; GnRH: gonadotropin releasing hormone; BMI: body mass index; qrtPCR: quantitative real-time PCR; PGR: progesterone receptor; ART: assisted reproductive technology; IVF: <i>in vitro</i> fertilization; HPO: hypothalamic-pituitary-ovarian axis; MMPs: matrix metalloproteinases</p> <p><b>Gene symbols:</b> LH receptor <i>(LHGCR)</i>; cholesterol side-chain cleavage enzyme <i>(CYP11A1)</i>; 3 beta-hydroxysteroid dehydrogenase type II <i>(HSD3B2)</i>; steroidogenic acute regulatory protein <i>(STAR)</i>; LDL receptor <i>(LDLR)</i>; scavenger receptor B1 <i>(SCARB1)</i>; ATP-binding cassette sub-family A member 1 <i>(ABCA1)</i>; ATP-binding cassette sub-family G member 1 <i>(ABCG1)</i>; apolipoprotein A <i>(APOA1)</i>; 24 dehydrocholesterol reductase <i>(DHCR24)</i>; 3-hydroxy-3-methylglytaryl-CoA reductase <i>(HMGCR)</i>; vascular endothelial growth factor A <i>(VEGFA)</i>; vascular endothelial growth factor C <i>(VEGFC)</i>; vascular endothelial growth factor receptor 1 <i>(VEGFR1)</i>; and TIMP metallopeptidase inhibitor 1 <i>(TIMP1)</i>; amphiregulin <i>(AREG)</i>; epiregulin <i>(EREG)</i>; CCAAT/enhancer binding protein alpha <i>(CEBPBA)</i>; cAMP responsive element binding protein 3-like 1 <i>(CREB3L1)</i>; ADAM metallopeptidase with thrombospodin type 1 motif 1 <i>(ADAMTS1)</i>; matrix metallopeptidase 9 <i>(MMP9)</i>; cytochrome b-245 beta polypeptide <i>(CYBB or NOX2)</i>; NADH oxidase <i>(NCF2 or NOXA2)</i>; Fc fragment of IgG receptor IIb (<i>FCGR2B</i>); Fc fragment of IgG receptor IIb (<i>FCGR2C</i>); ectonucleotide pyrophosphatase/phosphodiesterase 1 <i>(ENPP1)</i>; RAB27A member RAS oncofamily <i>(RAB27A)</i>; hydroxyprostaglandin dehydrogenase <i>(HPGD)</i>; prostaglandin-endoperoxidase synthase 1 <i>(PTGS1)</i>; integrin B2 <i>(ITGB2)</i>; leukotriene A4 hydrolase <i>(LTA4H)</i>; radixin <i>(RDX)</i>; ezrin <i>(EZR)</i>; nuclear receptor subfamily 5 group A member 2 (<i>NR5A2</i>)</p

Venn Diagrams for Total Differentially Expressed Genes by Diet, Weight Gain and Fat Mass.

<p>A. all vervet mRNAs. B. all mRNAs that were annotated to human genes. (p,0.05, FDR<0.15).</p

Differentially Expressed Corpus Luteum miRNAs after High Fat High Fructose Diet.

<p>Differentially Expressed Corpus Luteum miRNAs after High Fat High Fructose Diet.</p

Composition of Control and High Fat High Fructose (HFHF) Experimental Diets.

<p>Monkeys were fed 120 Kcal of diet / kg of body weight per day plus 10% to account for waste. Simple sugars were derived from sucrose (3% for both diets) and high fructose corn syrup (HFCS; 2.4% for control and 10% for HFHF). In addition to the diet, monkeys in the HFHF group were given daily access to a Kool-Aid drink containing 15ml of HFCS / 100ml of water, providing 150–250 additional Kcal per day.</p><p>^† Kcal/g of diet</p><p>* Kcal/ml</p><p>Composition of Control and High Fat High Fructose (HFHF) Experimental Diets.</p