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. 2024 Feb 1;17(1):28.
doi: 10.1186/s13048-024-01345-z.

Sestrin1, 2, and 3 are dispensable for female fertility in mice

Mengchen Wang #  1   2   3   4 , Wenhui Chen #  1   2   3   4 , Xinxin Zeng  1   2   3   4 , Taojun Wang  1   2   3   4 , Yingpu Sun  5   6   7   8 , Qingling Yang #  9   10   11   12
Affiliations

Sestrin1, 2, and 3 are dispensable for female fertility in mice

Mengchen Wang et al. J Ovarian Res. .

Abstract

Background: Sestrins have been implicated in regulating aging in various organs through multiple pathways. However, their roles in ovarian aging remain unrevealed.

Methods: Female Sestrin1-/-, Sestrin2-/-, and Sestrin3-/- mice were generated using the CRISPR-Cas9 system. Body weights, little sizes, ovarian weights, estrous cyclicity, and follicle number in female mice were observed. ELISA was utilized to measure serum anti-Müllerian hormone (AMH) levels. Real time PCR, western blot, immunofluorescence, and Masson trichrome staining were employed for assessment of aging-related change.

Results: The deletion of Sestrin 1, 2, or 3 had no discernible impact on body weights,or serum AMH levels in female mice at the age of 12 months. And there were no discernible differences in litter sizes or estrous cyclicity which were assessed at the age of 8 months. At the age of 12 months, no significant differences were observed in ovarian weights or follicle numbers among the knockout mice. Consistently, the extent of fibrosis within the ovaries remained comparable across all experimental groups at this age. Additionally, autophagy, apoptosis, DNA damage, and inflammation within the ovaries were also found to be comparable to those in wild-type mice of the same age.

Conclusions: The loss of Sestrin 1, 2, or 3 does not exert a noticeable influence on ovarian function during the aging process. Sestrin1, 2, and 3 are not essential for female fertility in mice.

Keywords: Female fertility; Ovarian aging; Sestrin1; Sestrin2; Sestrin3.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Construction of Sesn1−/−, Sesn2−/−, and Sesn3−/− mice. (A) Limiting the expression of gene fragments for Sestrin1, Sestrin2, and Sestrin3 using the CRISPR/Cas9 system. (B) Genotyping of wild-type (WT) and knockout mice. (C) Detection of Sesn1, Sesn2 and Sesn3 mRNA in Sesn1−/−, Sesn2−/−, and Sesn3−/− and WT mice ovaries by Agarose gel electrophoresis of real time PCR. (D) Expression of SESN1, 2, and 3 in WT and knockout mice ovaries detected by IHC. Scale bar = 50 μm
Fig. 2
Fig. 2
Assessment of fertility. (A) Body weights of 3-, 8-, and 12-month-old female mice (n = 6 mice for each group). (B) Mean litter sizes in 6- and 8-month WT, Sesn1−/−, Sesn2−/−, and Sesn3−/− mice (n = 6 mice for each group). (C) Serum AMH levels of 8-month (8 M) and 12-month (12 M) WT, Sesn1−/−, Sesn2−/−, and Sesn3−/− mice (n = 6 mice for each group). (D) Representative estrous cycles from 8 M WT, Sesn1−/−, Sesn2−/−, and Sesn3−/− mice (n = 6 mice for each group). M, metestrus; D, diestrus; P, proestrus; E, estrus. (E) Length of estrous cycles (n = 6 mice for each group). Results are expressed as the mean ± SEM. Statistical differences between WT and knockout mice were measured by an unpaired Student’s t test
Fig. 3
Fig. 3
Comparison of ovarian reserve. (A) Representative micrographs of 12-month-old WT, Sesn1−/−, Sesn2−/−, and Sesn3−/− mice ovarian sections. Scale bar = 500 μm. (B) Ovarian follicle count at developmental stages (primordial follicle (Pri), primary follicle (Prim), secondary follicle (Sec), and antral follicle (Ant) (n = 6 mice for each group). (C) Weights of ovaries in 3 M, 8 M, and 12 M WT, Sesn1−/−, Sesn2−/−, and Sesn3−/− mice (n = 6 mice for each group). (D) Representative image of 12 months ovarian tissues with Masson’s trichrome staining. Scale bar = 500 μm. (E) The fibrosis area per ovarian section measured by ImageJ software. (F) The expression of p16, p21, and p53 in 12 M ovaries (n = 6 mice for each group). Gapdh was used as a loading control. Results are expressed as the mean ± SEM. Statistical differences between WT and knockout mice were measured by an unpaired Student’s t test
Fig. 4
Fig. 4
Analysis of aging processes in ovaries. (A) Relative mRNA levels of Nlrp3, IL-1α, and TNF-α (n = 6 mice for each group). (B) Western blot for p62 protein in ovarian. GAPDH was used as a loading control (n = 6 mice for each group). (C) Representative images of TUNEL and γH2AX staining in 12 M WT, Sesn1−/−, Sesn2−/− and Sesn3−/− mice. Scale bar = 100 μm. (D) Quantitative analysis of TUNEL and γH2AX positive cells (green) per 100 granulosa cells per antral follicle in WT, Sesn1−/−, Sesn2−/− and Sesn3−/− mice in 12 months of age (n = 6 mice for each group). Results are expressed as the mean ± SEM. Statistical differences between WT and knockout mice were measured by an unpaired Student’s t test
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