Presence of anti-Müllerian hormone (AMH) during follicular development in the porcine ovary - Fig 1

<p>Representative AMH staining (brown) in the porcine ovary: (A) 1. Small preantral follicle, 2. Primary follicle, 3. Recruited primordial follicle, 4. Quiescent primordial follicle in which AMH staining is absent; (B) Large preantral follicle; (C) Small antral follicle. Oocytes are indicated by arrows, AMH positive granulosa cells by asterisks and theca cells by arrowheads. Scale bars represent 15 μm (A), 30 μm (B), 60 μm (C).</p

Comparative analysis of the presence of AMH in the female ovary.

<p>Comparative analysis of the presence of AMH in the female ovary.</p

Presence of anti-Müllerian hormone (AMH) during follicular development in the porcine ovary

<div><p>Background</p><p>Anti-Müllerian hormone (AMH) is expressed by granulosa cells of developing follicles and plays an inhibiting role in the cyclic process of follicular recruitment by determining follicle-stimulating hormone threshold levels. Knowledge of AMH expression in the porcine ovary is important to understand the reproductive efficiency in female pigs.</p><p>Research aim</p><p>In the present study we investigated the expression of AMH during follicular development in prepubertal and adult female pigs by immunohistochemistry, laser capture micro-dissection and RT-qPCR.</p><p>Results and conclusion</p><p>Although in many aspects the immunohistochemical localization of AMH in the porcine ovary does not differ from other species, there are also some striking differences. As in most species, AMH appears for the first time during porcine follicular development in the fusiform granulosa cells of recruited primordial follicles and continues to be present in granulosa cells up to the antral stage. By the time follicles reach the pre-ovulatory stage, AMH staining intensity increases significantly, and both protein and gene expression is not restricted to granulosa cells; theca cells now also express AMH. AMH continues to be expressed after ovulation in the luteal cells of the corpus luteum, a phenomenon unique to the porcine ovary. The physiological function of AMH in the corpus luteum is at present not clear. One can speculate that it may contribute to the regulation of the cyclic recruitment of small antral follicles. By avoiding premature exhaustion of the ovarian follicular reserve, AMH may contribute to optimization of reproductive performance in female pigs.</p></div

Representative AMH staining (brown) in corpora lutea.

<p>(A) Sow ovary, clear staining in the luteal cells–insert higher magnification of AMH positive luteal cells; (B) AMH positive luteal cells is a corpus luteum of a 30-day pregnant sow; (C) Rat ovary, note the absence of AMH staining in the luteal cells; (D) Sow ovary, control incubation in which the primary antibody was replaced by normal goat serum. No staining can be detected in the luteal cells under these conditions. Scale bars represent 80 μm (A), 30 μm (B,C), 20 μm (D).</p

Semi-quantitative analysis of AMH staining during follicular development in the porcine ovary.

<p>AMH staining was quantified in recruited primordial, primary, small preantral, large preantral, small antral, large antral and preovulatory follicles using KS300 software coupled to a Zeiss image analyzer. Low OD values correspond to strong AMH immunostaining. OD values are expressed as grey units (for more details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197894#sec005" target="_blank">Materials and Methods</a>). Values are expressed as mean ± SEM. AMH negative stroma tissue served as a control. Different characters represent significant differences in AMH staining intensity between follicle types (b–P < 0.05; c–P < 0.01).</p

Representative pictures used for LCM capturing of theca cells in preovulatory follicles.

<p>(A) Preovualtory follicle prior to capturing of cells; (B) Same follicle after laser capturing of cells, areas where cells have been captured are indicated by numbered yellow squares; (C) Same follicle stained with the AMH antibody, positive granulosa and theca cells are indicated (brown staining); (D) Agarose gel electrophoresis of AMH cDNA products after RT-PCR (product size—145 bp), lane 1 marker, lane 2—RT (control), lane 3 water (control), lane 4 granulosa cells, lane 5 theca cells, lane 6 luteal cells, lane 7 liver (negative control), lane 8 marker. GC—granulosa layer; black bar—theca layer; ST—stroma. Scale bar represents 30μm (A-C).</p

Representative pictures of hepatic lipid staining with Oil red O.

<p>There were no significant differences in lipid accumulation between the control (A) and the quercetin (B) group. The lipid levels were comparable to mice fed a normal-fat diet (C) and much lower than the positive control of hepatic lipid accumulation from mice fed a high-fat diet (D).</p

Percentages of lipids present in serum per mouse plotted for quercetin mice to control mice.

<p>Lipids were measured with ¹H NMR. Data is presented as the mean ratio of percentages of lipids present in serum per mouse plotted for quercetin-fed (Q) mice over control (C) mice. Total FFA were not changed, while other PUFA than 18∶2 FA, 22∶6 FA, and, w-3 FA were significantly increased. TG were significantly decreased by the quercetin diet. Data is presented as mean ± SEM. Asterisks indicates a significant difference between the control and the quercetin group; * p<0.05, **p<0.01, *** p<0.001. PUFA, poly unsaturated fatty acids; MUFA, mono unsaturated fatty acids; FA, fatty acids; TG, triglycerides; PGLY, phosphoglycerides; PC phosphatidylcholine; EC, esterified cholesterol; TC total cholesterol.</p

Fatty acid composition of the control and quercetin diet in percentages.

<p>Fatty acid composition of the control and quercetin diet in percentages.</p

Regulated hepatic genes with an absolute fold change >1.75 and FDR-adjusted p-value <0.01.

<p>Regulated hepatic genes with an absolute fold change >1.75 and FDR-adjusted p-value <0.01.</p