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Updated: May 27, 2025
Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles
Published on: March 17, 2015
Effects of Gonadotropin-Releasing Hormone Analogues on Ovarian Function and Embryogenesis: A Cyclophosphamide-Induced
Qiwang Lin1,2, Mingzhu Cao2, Zijin Xu2
1Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Guangdong Provincial Key Laboratory of Major Obstetric Diseases, Guangdong Provincial Clinical Research Center for Obstetrics and Gynecology, Guangdong Hong Kong Macao Greater Bay Area Higher Education Joint Laboratory of Maternal-Fetal Medicine, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.
Objective:
To clarify the protective effects of gonadotropin-releasing hormone analogues (GnRHas) on cyclophosphamide (CTX)-induced oocyte number loss and development of potential damage.
Design:
Mice model study.
Setting:
Laboratory-based animal study conducted in controlled research facilities.
Population:
Female C57/BL6 mice subjected to CTX-induced ovarian damage.
Methods:
The effects of GnRHa on CTX mice were evaluated in terms of hormones, oocyte count on slices, oocyte count in established three-dimensional-constructed ovaries, in vitro fertilisation, RNA sequencing and microinjection.
Main Outcome Measures:
The main outcome measures were the number of oocytes in intact mouse ovaries and oocyte quality, evaluated using three-dimensional (3D) tissue-clearing methods, oxidative stress markers (reactive oxygen species [ROS] and malondialdehyde [MDT]), mitochondrial function (ATP levels), and embryogenesis rates at the two-cell, four-cell and blastocyst stages.
Results:
In CTX mice, GnRHa pretreatment did not protect endocrine hormone changes, but protected loss of oocyte number on slice counting. A tissue-clearing technique, CUBIC (Clear, Unobstructed Body Imaging Cocktails), was a suitable method for ovaries clearing, and a 3D method for oocyte counting was validated with accuracy of 105.22% ± 3.48%. By this method, GnRHa was also found to protect the loss of oocyte number (597 ± 28 vs. 222 ± 15, p < 0.0001), which may be mediated by upregulated anti-Müllerian hormone (AMH) levels inhibiting primordial follicle development approved by in vitro culture of ovaries. GnRHa also increased the number of retrieved oocytes in CTX mice (19.4 ± 2.1 vs. 15.0 ± 1.6, p < 0.0001) and developmental ability of oocytes (65.0 ± 4.6 vs. 48.1 ± 4.2 for blastocyst, p < 0.0001). RNA sequencing revealed GnRHa pretreatment downregulated pathways of exogenous drug metabolism, oxidative stress and cytochrome P450, validated by detection of adenosine triphosphate (ATP), MDA and ROS levels. The up-expression of Cox17 (cytochrome c oxidase copper chaperone 17) after GnRHa pretreatment was confirmed by PCR and microinjection of siCox17 increased the embryogenesis from CTX mice.
Conclusions:
GnRHa was associated with reduced oocyte loss and improved embryogenesis, likely mediated by AMH and Cox17 upregulation.
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