Experimental animal protocols were approved by the Institutional Animal Care and Use Committee at Korea Institute of Oriental Medicine (19-019, Daejeon, Korea). Eight-week-old female C57BL/6 mice (18 – 20 g) were obtained from Narabiotech (Pyeongtaek, Korea) and housed under specific pathogen-free conditions. Animals were randomly divided into two groups and administered intraperitoneal injections of saline without (n = 12) or with 100 mg/kg Cy (Sigma-Aldrich, St. Louis, USA) six times over 2 weeks (n = 12). The mice were sacrificed 4 weeks after the final Cy injection. This timeframe was selected because it provides sufficient time for newly recruited primordial follicles to complete the preantral period . Blood was collected from the inferior vena cava of mice anesthetized with 1.2% avertin (0.6 mL/mouse, Sigma-Aldrich). Each mouse was euthanized by cervical dislocation to collect ovaries and oocytes after blood collection. The ovaries were removed, weighed, and immediately fixed in 4% paraformaldehyde (Biosesang, Seongnam, Korea).
Hormonal assessment by enzyme-linked immunosorbent assay (ELISA)
Sera separated from the blood samples were frozen at -70°C until analysis. The concentration of anti-Müllerian hormone (AMH) was measured by ELISA (MyBiosource, San Diego, CA, USA) in triplicate according to a standard protocol and the manufacturers’ instructions. The inter-assay coefficient of variation was <10% and sensitivity was 0.19 ng/mL.
Histological assessment of ovarian follicles
The whole ovaries were serially sectioned to 5-μm thickness and stained with hematoxylin and eosin. Primordial, primary, secondary, and preovulatory follicles with visible oocytes were counted in every fifth stained section to avoid counting the same follicle twice. The follicle stage was classified as previously described [14, 15]: primordial follicles had a single flat layer of granulosa cells surrounding the oocyte, primary follicles had a single cuboidal granulosa cell layer, secondary follicles had at least two granulosa cell layers and a theca cell layer, and preovulatory follicles had a complete antrum and theca cell layer.
Assessment of oocyte quality
Cyclophosphamide- or saline-injected mice were superovulated via intraperitoneal injection of 5 IU pregnant mare’s serum gonadotropin (Prospec, Rehovot, Israel), followed by 5 IU human chorionic gonadotropin (hCG, Prospec) at 48 h later. Oocytes were collected 18 h post-hCG injection in preincubated Human Tubal Fluid medium (Irvine scientific, CA, USA). Oocytes were fixed in 4% paraformaldehyde and permeabilized in 0.5% Triton X-100 (Sigma-Aldrich) for 10 min. Oocytes were blocked in phosphate-buffered saline containing 3% bovine serum albumin (Genedepot, Katy, TX, USA), and then incubated with rabbit anti-α-tubulin antibody (1:200, Cell Signaling Technologies, Danvers, MA, USA). Oocytes were mounted with VECTASHIELD Antifade Mounting Medium with 4’6’-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA) to visualize the chromosomes, and observed by fluorescence microscopy (Olympus BX51, Tokyo, Japan). Oocytes with well-organized, bipolar spindles and chromosomes that were tightly aligned at the metaphase plate were scored as normal. Oocyte quality was also evaluated by measuring morphometrical parameters, including the complete oocyte, ooplasm, and perivitelline space (PVS) using NIS-elements BR 4.60.00 software (Nikon, Tokyo, Japan) .
Ovaries from Cy- or saline-injected mice were collected, and total RNA was extracted using the RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The purity and integrity of the extracted RNA were evaluated using a NanoDrop ND-1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). All samples were of high purity (optical density (OD)260/OD280 > 2.00) and integrity (RNA integrity number >7.0). Hybridization on GeneChip Mouse Gene 2.0 ST arrays (Affymetrix) was controlled using GeneChip Command Console Software (AGCC, Affymetrix, Santa Clara, CA, USA). We used Affymetrix Expression Console 1.4 Software for basic data extraction (CEL files) and quality control metrics. A fold change value >1.5 and a p-value <0.05 were used as thresholds to identify differentially expressed genes (DEGs). Functional annotation of the DEGs was performed using the Database for Annotation, Visualization and Integrated Discovery version 6.8 (https://david.ncifcrf.gov/hom.jsp). Gene ontology (GO) analysis was performed to identify potential functions of DEGs in the biological process, molecular function, and cellular component categories .
Data are presented as means ± standard deviation (SD). The statistical significance of differences between the two groups was determined by Student’s t-test using GraphPad Prism, version 8.4.0 (GraphPad, Inc., La Jolla, CA, USA). P < 0.05 was considered as statistically significant.