CRS induced activation of primordial follicles and up-regulated the level of AMH.

<p>(A)H&E staining morphological features of ovaries derived from the control and CRS groups (scale bar, 100 μm). The red outline showed the enlargements of primordial follicles (d), primary follicles (f) and secondary follicles(b). (B)Effects of 3 or 8 weeks of CRS on different stages of follicles in ovaries removed from adult female BALB/c mice (<i>n</i> = 6). (C) The ratio of primary follicles to primordial follicles in different groups (<i>n</i> = 6). This ratio was 1 for the control group; the data in the CRS group were standardized by the ratio of the control group, and then the odds ratios were calculated and analyzed statistically. (D) The serum AMH level of mice in both the CRS (<i>n</i> = 10) and control groups (<i>n</i> = 15). (E)The mRNA expression level of AMH in mouse ovaries of both the CRS and control groups (<i>n</i> = 6) (*<i>P</i>< 0.05, **<i>P</i>< 0.01, ***<i>P</i>< 0.001, ****<i>P</i>< 0.0001). CRS, Chronic restraint stress.</p

Monthly mean temperature and total precipitation values at Jingtai (1957–2013) and Jingyuan (1951–2013) meteorological stations.

<p>Monthly mean temperature and total precipitation values at Jingtai (1957–2013) and Jingyuan (1951–2013) meteorological stations.</p

Tree-ring-based reconstruction comparisons among three sites.

<p>(a) The reconstructed precipitation in Liancheng. (b) The SPEI_AJ reconstruction in the Taihe Mountains. (c) The reconstructed precipitation in the Changling-Shoulu region.</p

Chronic restraint stress induces excessive activation of primordial follicles in mice ovaries

<div><p>Chronic stress is an important factor influencing people’s health. It usually causes endocrinal disorders and a decline in reproduction in females. Although studies of both human and animals suggest a detrimental effect of stress on reproduction, the influence of chronic stress on the ovarian reservation and follicular development is still not clear. In this study, a chronic restraint stress (CRS) mouse model was used to investigate the effect of stress on ovarian reservation and follicular development and explore the underlying mechanism. In this study, after 8 weeks of CRS, primordial follicles were excessively activated in the ovaries of the CRS group compared with the control group. Further results showed that the activation of primordial follicles induced by CRS was involved in the increasing expression level of Kit ligand and its receptor Kit and the activation of phosphatidylinositol 3-kinase (PI3K)/phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/protein kinase B (Akt) pathway. The corticotropin-releasing hormone (CRH) is a neuropeptide released due to stress, which plays an important role in regulating follicle development. A high level of serum CRH was detected in the CRS mouse model, and the real-time polymerase chain reaction assay showed that the mRNA level of its main receptor CRHR1increased in the ovaries of the CRS mouse group. Moreover, 100nM CRH significantly improved the activation of primordial follicles in newborn mouse ovaries <i>in vitro</i>. These results demonstrated that CRS could induce immoderate activation of primordial follicles accompanied by the activation of Kit-PI3K signaling, in which CRH might be an important endocrine factor.</p></div

Schematic diagram showing the course of CRS inducing abnormal activation of primordial follicles.

<p>CRS leads to the excessive activation of primordial follicles, accompanied by up-regulated expression of Kitl and its receptor Kit, and the activation of PI3K/PTEN/Akt pathway. What is more, the level of serum AMH was increased significantly after CRS. CRH induced by chronic stress might play an important role in the activation and exhaustion of primordial follicles. CRS, Chronic restraint stress.</p

Correlations between the tree-ring STD chronology and monthly mean temperature, and monthly total precipitation (1957–2012 AD).

<p>Horizontal dashed lines represent the 95% C.L.</p

Correlations between the STD chronology and monthly SPEI data at time scales from 1 to 24 months (AD 1951–2011).

<p>Circles represent significant at the 95% C.L.</p

Patterns of field correlation in our study.

<p>(a) Correlations between the gridded (37.25°N, 104.75°E) SPEI_AJ series with the 0.5×0.5 gridded mean April-June SPEI at the 10-month scale. (b) Correlations between reconstructed SPEI_AJ with the 0.5×0.5 gridded mean April-June SPEI at the 10-month scale (1951–2011 AD).</p

CRS reduced the body weight of mice and caused deregulation of the estrous cycle.

<p>(A) CRS reduced the body weight of mice (<i>n</i> = 10, <i>P</i>< 0.05). (B) The ovarian gross morphology of the control and CRS 8w groups. The size of the control group was almost two times larger than that of the CRS group. (C) The ovarian weight was significantly decreased in the CRS 8w group compared with the control group (<i>n</i> = 6, <i>P</i>< 0.001). (D) The morphological characterization of the smears in every substage of the mice estrum cycle. Briefly, three types of cells were present in the smear, including small leukocytes, larger and round epithelial cells, and large, flat, and nonnuclear cornified cells. The relative numbers of leukocytes (L) and epithelial (E) and cornified (C) cells in the vaginal smear indicated the stage of the estrous cycle. (a) Contained L and was classified as diestrum (DE). (b) Contained E and was classified as proestrum(PE). (c) Contained C and was classified as estrum (E). (d) Contained three types of cells and was classified as metaestrum (ME). (Bar = 100μm) (E) CRS caused deregulation of the estrous cycles of mice, and the CRS 8w group remained in the diestrum stage almost throughout the cycle (<i>n</i> = 6; PE, proestrum; E, estrum; ME, metaestrum; DE, diestrum). (F) Serum corticosterone level of mice in both control and CRS 8w groups (<i>n</i> = 10, <i>P</i>< 0.001). (G) Serum concentration of CRH in mice of both control and CRS 8w groups (<i>n</i> = 10, <i>P</i>< 0.05). CRS, Chronic restraint stress.</p

Map of the sampling site.

<p>Map of the sampling site.</p