Anti-Mullerian Hormone (AMH) concentration in follicular fluid and mRNA expression of AMH receptor type II and LH receptor in granulosa cells as predictive markers of good buffalo (Bubalus Bubalis) donors

High individual variability in follicular recruitment and hence in the number of embryos produced is a major factor limiting the application of reproductive technologies in buffalo. Therefore, the identification of reliable markers to select embryo donors is critical to enroll buffaloes in embryo production programs. Better understanding of factors involved in follicular growth is also necessary to improve the response to superovulation in this species. The aim of this work was thus to determine the anti-Mullerian hormone (AMH) concentration in follicular fluid (FF) recovered from different size follicles and evaluate the mRNA expression profiles of development-related (AMHR2, CYP19A1, FSHR, and LHR) and apoptosis-related genes (TP53INP1 and CASP3) in the corresponding granulosa cells (GCs) in buffalo. Another objective was to evaluate whether the AMH concentration in FF and gene expression of GCs is associated with the antral follicular count. Ovaries were collected at the slaughterhouse, and all follicles were counted and classified as small (3–5 mm), medium (5–8 mm), and large (>8 mm). Follicular fluid was recovered for AMH determination, and the mRNA expression of AMHR2, FSHR, LHR, CYP19A1, TP53INP1, and CASP3 was analyzed in GCs. The AMH concentration in FF decreased (P < 0.01) at increasing follicular diameter. The mRNA expression of AMHR2 and FSHR was higher (P<0.05) in small follicles, whereas that of LHR and CYP19A1 was higher (P < 0.05) in large follicles. The intrafollicular AMH concentration was positively correlated with the antral follicular count (r =0.31; P < 0.05). Interestingly, good donors (≥_12 follicles) had a higher (P < 0.05) concentration of AMH and AMHR2 levels in small follicles and higher (P < 0.05) LHR levels in large follicles than bad donors (<12 follicles). These results suggest a potential use of AMH to select buffalo donors to enroll in embryo production programs, laying the basis for further investigations

Mapping of a subset of differentially expressed proteins in pFF+ versus pFF- samples on the complement system canonical pathway.

<p>According to IPA categorization, up-regulated proteins are coloured in red. The assessment of significantly enriched processes networks for differentially expressed proteins was performed by evaluating the probability of a random intersection between the differentially expressed proteins with functional processes by applying the hypergeometric test. The pathway components identified by the algorithm or with no significant differences in their expression levels are reported in white and blue, respectively. Molecules are named according to IPA software.</p

Bar chart of the enriched biological functions of differentially expressed proteins in pFF+ versus pFF- samples.

<p>The assessment of significantly enriched biological functions for differentially expressed proteins was performed by IPA software. p-values and the number (#) of molecules mapped on the enriched categories are reported. </p

Mapping of a subset of differentially expressed proteins in pFF+ versus pFF- samples on the acute phase response signaling canonical pathway.

<p>According to IPA categorization, up-regulated proteins are coloured in red. The assessment of significantly enriched processes networks for differentially expressed proteins was performed by evaluating the probability of a random intersection between the differentially expressed proteins with functional processes by applying the hypergeometric test. The pathway components identified by the algorithm or with no significant differences in their expression levels are reported in white and blue, respectively. Molecules are named according to IPA software.</p

Adenoviral Gene Transfer of PLD1-D4 Enhances Insulin Sensitivity in Mice by Disrupting Phospholipase D1 Interaction with PED/PEA-15

<div><p>Over-expression of phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) causes insulin resistance by interacting with the D4 domain of phospholipase D1 (PLD1). Indeed, the disruption of this association restores insulin sensitivity in cultured cells over-expressing PED/PEA-15. Whether the displacement of PLD1 from PED/PEA-15 improves insulin sensitivity <i>in vivo</i> has not been explored yet. In this work we show that treatment with a recombinant adenoviral vector containing the human D4 cDNA (Ad-D4) restores normal glucose homeostasis in transgenic mice overexpressing PED/PEA-15 (Tg _ped/pea-15) by improving both insulin sensitivity and secretion. In skeletal muscle of these mice, D4 over-expression inhibited PED/PEA-15-PLD1 interaction, decreased Protein Kinase C alpha activation and restored insulin induced Protein Kinase C zeta activation, leading to amelioration of insulin-dependent glucose uptake. Interestingly, Ad-D4 administration improved insulin sensitivity also in high-fat diet treated obese C57Bl/6 mice. We conclude that PED/PEA-15-PLD1 interaction may represent a novel target for interventions aiming at improving glucose tolerance.</p> </div

Expression levels of chemokines in hFF samples.

<p>hFF samples were simultaneously screened for the concentration of the chemokines Eotaxin (A), IP-10 (B), MIP-1α (C), MIP-1β (D), MCP-1 (E) and RANTES (F) by using the Bio-Plex 27-plex human cytokine kit from BioRad according to the manufacturer’s protocol. Measurements were performed on individual hFF samples diluted (1:2) using the standard curves optimization and the calculation of analyte concentrations of the Bio-Plex Manager software. Data are reported as scatter plots and average concentrations. ** p-value≤ 0.05 FF+ <i>versus</i> FF- dataset.</p

Mapping of a subset of differentially expressed proteins in pFF+ versus pFF- samples on the coagulation system pathway.

<p>According to IPA categorization, up-regulated proteins are coloured in red. The assessment of significantly enriched processes networks for differentially expressed proteins was performed by evaluating the probability of a random intersection between the differentially expressed proteins with functional processes by applying the hypergeometric test. The pathway components identified by the algorithm or with no significant differences in their expression levels are reported in white and blue, respectively. Molecules are named according to IPA software.</p

Metabolic characteristics of TgPed/pea-15 mice.

<p>Data are means ± SEM of determinations in at least 10 mice per group. Vehicle TgPed/pea-15 mice vs. Vehicle Wild Type mice;</p>a<p><i>P</i><0.001. Ad-GFP TgPed/pea-15 mice vs. Vehicle Wild Type mice;</p>b<p><i>P</i><0.001. Ad-GFP TgPed/pea-15 mice vs. Vehicle TgPed/pea-15 mice;</p>c<p><i>P</i><0.001. Ad-D4 TgPed/pea-15 mice vs. Vehicle Wild Type mice;</p>d<p><i>P</i><0.001. Ad-D4 TgPed/pea-15 mice vs. Vehicle TgPed/pea-15 mice;</p>e<p><i>P</i><0.01 and</p>f<p><i>P</i><0.001. Ad-D4 TgPed/pea-15 mice vs. Ad-GFP TgPed/pea-15 mice;</p>g<p><i>P</i><0.01 and</p>h<p><i>P</i><0.001.</p

Effect of D4 on PKCalpha and zeta activation in Tg_Ped/pea-15mice.

<p>PKCalpha (<b>A</b>) and zeta (<b>B</b>) activations were determined in the skeletal muscle tissues from Tg_Ped/pea-15mice at one week post Ad-D4 or Ad-GFP infection. Tg_Ped/pea-15 or wild type (Wt) mice injected with PBS (vehicle) were used as control. For the experiment, mice were fasted over night and then i.p. injected or not with insulin (10 U/kg body weight) 10 min before determination. <b>A)</b> The bar graph represents the densitometric quantization of phospho-PKCalpha in three independent immunoblots. ***<i>p</i><0.001 vs. Wt. <b>B)</b> The corresponding blots show the levels of PKCzeta (total and phosphorilated forms) and tubulin in mice as indicated. Blots are representative of three independent experiments.</p