Actions of anti-Mullerian hormone on the ovarian transcriptome to inhibit primordial to primary follicle transition

Abstract

The oocytes found within the primordial follicles of mammalian ovaries remain quiescent for months to years until they receive the appropriate signals to undergo the primordial to primary follicle transition and initiate folliculogenesis. The molecular mechanisms and extracellular signaling factors that regulate this process remain to be fully elucidated. The current study investigates the mechanisms utilized by anti-Müllerian hormone (AMH; i.e. Müllerian inhibitory substance) to inhibit the primordial to primary follicle transition. Ovaries from 4-day-old rats were placed into organ culture and incubated in the absence or presence of AMH, either alone or in combination with known stimulators of follicle transition, including basic fibroblast growth factor (bFGF), kit ligand (KITL), or keratinocyte growth factor (KGF). Following 10 days of culture, the ovaries were sectioned, stained, and morphologically evaluated to determine the percentage of primordial versus developing follicles. As previously demonstrated, AMH treatment decreased primordial to primary follicle transition. Interestingly, AMH inhibited the stimulatory actions of KITL, bFGF, and KGF. Therefore, AMH can inhibit the basal and stimulated development of primordial follicles. To investigate the mechanism of AMH actions, the influence AMH has on the ovarian transcriptome was analyzed. AMH treatment when compared with controls was found to alter the expression of 707 genes. The overall effect of AMH exposure is to decrease the expression of stimulatory factors, increase the expression of inhibitory factors, and regulate cellular pathways (e.g. transforming growth factor beta signaling pathway) that result in the inhibition of primordial follicle development. Analysis of the regulatory factors and cellular pathways altered by AMH provides a better understanding of the molecular control of primordial follicle development.

Figure 1

Effect of AMH treatment on primordial to primary follicle transition in cultured ovaries. Ovaries from 4-day-old rats were placed into culture for 10 days. Cultured ovaries were treated with 50 ng/ml AMH or were left untreated as controls. After culture, all ovaries were fixed, stained, and subjected to morphological analysis. The follicles per ovary cross-section were categorized as being either primordial or developing (which includes all follicles having undergone the primordial to primary transition). Data are presented as the mean± s.e.m. with data pooled from five separate experiments, n=9–15 per group. Different superscript letters indicate a significant (P<0.01) difference between AMH treated and control by Mann–Whitney test.

Figure 2

Effect of AMH treatment in combination with stimulatory growth factors on primordial to primary follicle transition in cultured ovaries. Cultured ovaries were treated for 10 days with AMH alone or in combination with bFGF, KITL, or KGF, or were left untreated as controls (control). After culture, all ovaries were fixed, stained, and subjected to morphological analysis. The follicles per ovary cross-section were categorized as being either primordial or developing. Data are presented as the mean (± s.e.m.) proportion of developing follicles normalized to each experimental control mean. Data were pooled from three or more separate experiments. One-way ANOVA showed a significant (P<0.01) difference in treated ovaries. Different superscript letters indicate a significant (P<0.05) difference by post hoc Tukey’s test. (A) AMH treatment in combination with bFGF, n=5–19 per group.(B) AMH treatment in combination with KITL, n=6–19 per group.(C) AMH treatment in combination with KGF, n=8–19 per group.

Figure 3

Dendrogram of microarray analysis results showing transcripts that change expression level in AMH-treated versus control ovaries. Seven hundred and seven genes show an expression change as per the criteria described in Materials and Methods between AMH-treated and control ovaries. Compared with controls, 164 genes are increased and 543 genes are decreased in AMH-treated ovaries. Red, increase in expression levels; green, decrease in expression levels. Relative expression changes are according to the scale at right.

Figure 4

Gene transcripts that change expression level in AMH-treated versus control ovaries categorized according to the physiological function. Lighter portion of bar represents a number of transcripts upregulated in AMH-treated ovaries, while darker portion of bar represents transcripts downregulated.

Figure 5

Illustration of TGF-β signaling pathways proposed to be involved in regulating follicle transition using microarray data. Genes that are upregulated by AMH treatment (≥1.5-fold change) are shown in orange. Genes that are downregulated (≥1.5-fold change) are shown in blue. Genes that are not changing in expression level are shown in green. Genes that are not expressed at levels above a raw score of 75 are given in yellow. Genes that are not represented on the Affymetrix RAE 230 2.0 chip are shown in white. Pathways are adapted from KEGG as accessed through Genespring GX 7.3 Expression analysis (Agilent Technologies, Palo Alto, CA, USA).

Figure 6

Illustration of MAPK signaling pathways proposed to be involved in regulating follicle transition using microarray data. Genes that are upregulated by AMH treatment (≥1.5-fold change) are shown in orange. Genes that are downregulated (≥1.5-fold change) are shown in blue. Genes that are not changing in expression level are shown in green. Genes that are not expressed at levels above a raw score of 75 are given in yellow. Genes that are not represented on the Affymetrix RAE 230 2.0 chip are shown in white. Pathways are adapted from KEGG as accessed through Genespring GX 7.3 Expression analysis (Agilent Technologies, Palo Alto, CA, USA).