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 Table of Contents  
Year : 2020  |  Volume : 4  |  Issue : 4  |  Page : 204-211

Benzyl alcohol-benzyl benzoate clearing reveals the dose-dependent effect of cyclophosphamide on follicle damage in mice

1 Department of Gynecology, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, China
2 Department of Hematology, Huashan Hospital, Fudan University, Shanghai 200040, China

Date of Submission22-Sep-2020
Date of Decision16-Nov-2020
Date of Acceptance22-Nov-2020
Date of Web Publication31-Dec-2020

Correspondence Address:
Hua Jiang
Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2096-2924.305934

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Objective: Cyclophosphamide (CTX), which is commonly used in clinical chemotherapy, has a damaging effect on ovarian follicles. This study aimed to establish a new method to count the number of follicles in mouse ovaries using benzyl alcohol–benzyl benzoate (BABB)-based tissue-clearing technology and evaluate the follicle-damaging effects of different doses of CTX.
Methods: C57BL/6 mice were divided into four groups and administered intraperitoneal injections of 0, 40, 80, or 120 mg/kg CTX. The serum levels of estradiol (E2) and follicle-stimulating hormone (FSH) were detected using an ELISA kit. Mouse ovaries were subjected to BABB clearing and labeled with DAPI, β-actin, and DDX4 to observe the ovarian structure and follicles. A three-dimensional software, Imaris, was used to reconstruct the ovarian structure and automatically count the number of follicles. The effects of different CTX doses on the total follicle number and estrous cycle were determined.
Results: As the CTX dose increased, E2 levels in CTX mice declined from 212.3 to 57.7 pg/mL; the FSH levels increased from 3.2 to 29 ng/mL. Mouse ovaries became transparent after BABB treatment. After fixation, microscopy, and Imaris processing, immunofluorescence signals of β-actin and DAPI from all levels in intact ovaries could be obtained and follicle number in half ovaries could be automatically counted using anti-DDX4 antibody labeling. In the NC, CTX40, CTX80, and CTX120 groups, the proportion of mice in the diestrus phase was 26.67%, 51.67%, 73.33%, and 95.00%, respectively, and the total follicle number was 2,603, 1,761, 1,043, and 262, respectively. E2 levels were positively correlated with follicle number and FSH levels were negatively correlated with follicle number, indicating that the damaging effect of CTX on follicle number may be dose dependent.
Conclusion: BABB can be used to clear intact ovaries from adult mice, and follicle number in half ovaries can be automatically counted. The damaging effect of CTX on follicles and the endocrine system is dose dependent.

Keywords: Benzyl Alcohol–Benzyl Benzoate; Cyclophosphamide; Follicle Damage; Tissue-Clearing Technology

How to cite this article:
Lin QW, Fei H, Jin YF, Wu KP, Dai X, Qu Y, Jiang H. Benzyl alcohol-benzyl benzoate clearing reveals the dose-dependent effect of cyclophosphamide on follicle damage in mice. Reprod Dev Med 2020;4:204-11

How to cite this URL:
Lin QW, Fei H, Jin YF, Wu KP, Dai X, Qu Y, Jiang H. Benzyl alcohol-benzyl benzoate clearing reveals the dose-dependent effect of cyclophosphamide on follicle damage in mice. Reprod Dev Med [serial online] 2020 [cited 2021 Jan 25];4:204-11. Available from: https://www.repdevmed.org/text.asp?2020/4/4/204/305934

  Introduction Top

In female patients, chemotherapy drugs cause ovarian damage, reducing the ovarian follicle number and resulting in infertility and other premature ovarian failure symptoms. Recent studies have revealed that different chemotherapy drugs have diverse mechanisms of fertility damage. Cyclophosphamide (CTX), cisplatin, and doxorubicin are commonly used drugs that cause ovarian injury. CTX is widely administered to patients with lymphoma, breast cancer, or lung cancer. Nguyen et al. first found ovarian fibrosis and follicle reduction in female patients who received chemotherapy, but the underlying mechanism and influencing factors have not yet been fully clarified.[1],[2]

Follicles in experimental animals are important objects in the field of reproduction research, as the number of follicles can be used to determine reproductive capacity.[3],[4] Previously, the evaluation of follicle number in experimental animals was mainly conducted by manual counting of hematoxylin and eosin (H and E)-stained sections by researchers.[5],[6] This slice-based method has a marked possibility of causing experimental errors due to the process of sectioning, subjectiveness, and the uneven distribution of follicles in an ovary. Therefore, the results obtained using this method may not accurately reflect the reproductive capacity of mice.

Tissue clearing is a new histological technology that has been developed in recent years.[7],[8],[9] By applying diverse methods to make the whole tissue transparent, the treated tissue presents a “jelly-like” appearance, which promotes the penetration of fluorescence, enabling the use of optical instruments to study the microstructure of the intact tissue.[10] Clearing technology can be generally divided into three categories: the first is based on organic solvents, such as benzyl alcohol–benzyl benzoate (BABB)/iDISCO; the second is based on inorganic solvents, such as CUBIC/Scale; and the third is based on physical methods, such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ-hybridization-compatible Tissue Hydrogen (CLARITY) and electrophoresis. BABB was one of the earliest clearing technologies to be developed. It is an organic solvent that may inactivate antibodies. However, it can achieve a sufficiently clear outcome in a very short period of time, and it is still the simplest and least time-consuming clearing technology, with promising applications. To date, several studies have reported the application of tissue-clearing technology in reproductive science and embryo development. In 2017, Feng et al. used clarity to clear mouse ovaries and determined the number of follicles in mice under different endocrine conditions.[11] In 2018, Tong Ma used CLARITY-treated ovaries from rats to reveal the protective effect of electroacupuncture on a PCOS rat model.[12] These studies first applied tissue-clearing technology to reproduction science and established new research methods. However, BABB-based tissue-clearing method has not been applied to intact mouse ovaries.

To determine the damaging effect of different CTX doses, this study aimed to establish a new reproductive research method to evaluate total follicle number in intact mouse ovaries using BABB and further explore the effects of different CTX doses on the estrous cycle, endocrine system, and follicle number.

  Methods Top

Mouse housing, modeling, and ovary acquisition

Eight-week-old C57BL/6 female mice were obtained from Jiangsu Jicui Yaokang Biological Technology Co. Ltd. (Jiangsu, China) and maintained in the animal facility of Life Science School of Fudan University. A total of five mice were housed per standard cage under a 12-h light/dark cycle at a room temperature of 21°C. All mice had free access to water and food. For establishing CTX models, mice in each group were intraperitoneally injected with CTX (HY-17420, MedChemExpress, USA) at a concentration of 40, 80, or 120 mg/kg. The state of the estrous cycle was determined based on a vaginal cell smear. To obtain the ovaries, the mice were anesthetized and fixed. The chest was opened using ophthalmic scissors, and a needle was inserted along the left ventricle from the apex of the heart. Perfusion was performed using phosphate-buffered saline (PBS) precooled at 4°C, followed by the addition of 4% paraformaldehyde (PFA) precooled at 4°C. After sufficient perfusion, the ovaries were excised and placed in 4% PFA for further analysis. All animal experiments were approved by the Institutional Animal Care and Use Committee of Fudan University.

Immunolabeling of intact ovaries

The collected ovaries were immersed in 30% sucrose overnight at 37°C and then immersed in PBS containing 2% Triton-X100 (X100, Sigma-Aldrich, USA) (PBST) and incubated in a shaker at 37°C for 48 h to allow the penetration of the antibody (the liquid was changed three times). Subsequently, the ovaries were placed in PBST containing anti-β-actin (ab8227, Abcam, USA, 1:500) or anti-DDX4 antibody (Rabbit Anti-Mouse-DDX4, Abcam, USA, ab13840, 1:500) and DAPI (D9542, Sigma, USA, 1:1,000) and shaken at 37°C overnight. Next, the ovaries were immersed in PBST and incubated in a shaker at 37°C for 2 days (the liquid was changed at least three times). This was followed by immersion in PBST containing Alexa Fluor 488 (Goat Anti-Rabbit-Alexa Flour 488, ab150077 or Donkey Anti-Rabbit-Alexa Flour 647, ab150075, Abcam, USA) and overnight shaking at 37°C. Finally, the ovaries were immersed in PBST and shaken at 37°C for 2 days (the liquid was changed at least three times).

Benzyl alcohol–benzyl benzoate clearing

To ensure complete dehydration, the ovaries were successively shaken in 50%, 75%, and 100% (v/v) methanol/PBS (A506806, Sangon Biotech Co., Ltd, China) for a minimum of 4 h (two changes of absolute methanol to ensure complete dehydration). Agarose became opaque after dehydration, and the methanol was removed from the dish. A mixture of benzyl alcohol (ab13160, Abcam, USA, 1:500) and benzyl benzoate (A502095, Sangon Biotech Co., Ltd., China) at ratio of 1:2 was then added. Within 4 h, the ovaries were optically cleared and fixed for photography (three changes to ensure the removal of residual methanol).

Confocal microscopy

Laser confocal microscopy was performed using a ×10 objective lens to ensure a complete field of view. The sample was observed under a bright field microscope to determine the best position. In LAX software, the following scanning parameters were used: gain = 850–900; PMT1 channel, offset = 0.1%–0.2%; laser parameters: 405 nm and 488 nm lasers, 20% power; scanning mode: mosaic mode = 3 × 3, preview mode to determine the boundary; z-step mode (z-step = 1 nm); and scanning parameters: 1,024 × 1,024, line average = 2, frame average = 2; the original data were saved after scanning for the subsequent processes. The distance between half ovaries in the Z-axis was determined based on the signal of DAPI in intact ovaries.


The concentrations of FSH and E2 in the mouse serum were detected using FSH (AE90998Mu-96T, AMEKO, China) and E2 ELISA kits (K3830-100, Biovision, USA). The blood samples obtained from each group were centrifuged at 3,000 rpm. The serum was separated and stored at - 80°C for later detection. The reagents in the ELISA kits were prepared according to the manufacturer's instructions. The concentrations of FSH and E2 were determined based on the absorbance results of the standard samples, and the detection was performed three times for each sample.

Follicle counting on hematoxylin and eosin-stained ovary slices

The ovaries were fixed with 4% PFA for 48 h, dehydrated with alcohol in xylene, and immersed in wax. The whole wax block was placed on a slicer and cut into thin slices, generally 3–5-μm-thick, and stained with H and E. Three researchers counted the number of follicles in each ovary from all slices. An average value was calculated to determine the number of follicles in each ovary.

Three-dimensional reconstruction and automatic follicle counting

The files obtained from the confocal microscope were transferred to Imaris (version 9, Bitplane AG, Switzerland). Follicles were defined as DDX4-positive cells that were identified by three-dimensional (3D) spot modules. The number of follicles in the intact ovary was calculated by multiplying the number of follicles obtained from half ovaries by 2.

Statistical analysis

The number of follicles in the cleared ovary was calculated using Imaris (Version 9.0.1, Bitplane). Data were presented as mean ± standard deviation, and an unpaired t-test was used to compare the differences between the number obtained using the software and that obtained by the researchers.

  Results Top

Effects of different cyclophosphamide doses on the estrous cycle and endocrine system in mice

To study the effects of different doses of CTX on the estrous cycle and endocrine system in mice, vaginal cell smears and ELISA of E2 and FSH levels were performed on day 5 after chemotherapy. In the NC, CTX40, CTX80, and CTX120 groups, the proportion of mice in the diestrus phase was 26.67%, 51.67%, 73.33%, and 95.00%, respectively [Table 1]; E2 levels were 212.3, 167.5, 103.3, and 57.7, respectively; and FSH levels were 3.2, 5.1, 8.1, and 29, respectively [Figure 1]. As the CTX dose increased, the proportion of mice in the estrus phase increased gradually, E2 levels decreased, and FSH levels increased significantly. The administration of 120 mg/kg CTX stopped the estrous cycle of mice.
Figure 1: Endocrine hormone levels in mice from each group. (a) E2 expression level detected using ELISA (n = 20); (b) FSH expression level detected using ELISA (n = 20). Data are presented as the mean ± standard deviation; *P < 0.001;P < 0.0001. FSH: Follicle-stimulating hormone.

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Table 1: Number of mice from each group in the different estrous cycle phases (n = 40)

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Clearing effect of benzyl alcohol–benzyl benzoate on mouse ovaries

Immunolabeling and BABB clearing processes are depicted in [Figure 2]a. The transparent ovaries were placed at the bottom of the confocal dish and photographed under a stereomicroscope. The captured images showed that the ovary was almost completely transparent and that the boundary of the ovary was invisible [Figure 2]b and [Figure 2]c. The line drawn on the underside of the dish was clearly visible, indicating that BABB was able to clear intact mouse ovaries.
Figure 2: Intact ovaries cleared by BABB. (a) Flowchart of the BABB clearing process in intact mouse ovaries. (b) The cleared ovary was transparent in BABB solvent, and the boundary was almost invisible. (c) The ovary is marked by dashed lines. (d) A chamber prepared using slide, glass cover, and neoprene glue. The ovary was immobilized in the chamber, enabling scanning to continue. (e) Schematic diagram of the scanning process. Scale bar = 0.68 mm. BABB: Benzyl alcohol–benzyl benzoate.

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Ovary immobilization and images from all levels

The mosaic and Z-axis scanning modes on LAX software are essential for scanning intact ovaries, which may lead to the mobilization of ovaries, interrupting the scanning process. To immobilize the ovaries, a small chamber between the slide and slide cover was created during the overall scanning process. Notably, a gap was created in the chamber to discharge air bubbles [Figure 2]d].

All levels of the ovaries labeled with β-actin and DAPI were scanned using a Leica SP8 laser confocal microscope [Figure 2]e. Images from all levels of the ovaries were obtained [Supplemental Videos 1-3]. Images acquired from different distances (100, 225, and 300 μm away from the top surface of the ovary) show the details of the upper, middle, and lower parts of the ovaries [Figure 3]. In the images of all layers, large follicles, blood vessels, small follicles, and fibers can be easily identified, enabling the observation of the ovarian structure.
Figure 3: Images of BABB-treated intact ovaries. The images of an intact ovary acquired at distances of 100 (a), 225 (b), and 350 μm (c) from the top surface are presented. The ovary was labeled with DAPI (left), β-actin (middle), and merge (right). Vessels and follicles are marked. Scale bar = 500 μm. BABB: Benzyl alcohol–benzyl benzoate.

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Automatic follicle counting of oocytes in half ovary

To establish a method to calculate the total follicle number in an intact ovary, an antibody against DDX4, an oocyte specific marker, was added to the cleared ovaries. Images obtained using laser confocal microscopy showed that the follicles in half ovaries could be labeled [Figure 4]a. The Z-axis distance of the half ovary was determined based on the DAPI signal emitted from all ovary levels. The 3D image was reconstructed using the 3D processing software, Imaris, in which the spot function could be used to automatically identify and count the follicles in the half ovaries [Figure 4]b. To determine the accuracy of automatic counting, the follicles were manually counted in the H and E-stained slices [Figure 4]c. The total follicle number obtained using automatic counting was similar to that obtained using the conditional method [Figure 4]d.
Figure 4: Comparison between automatic follicle counting from three-dimensional reconstruction images and manual follicle counting from H and E-stained slices. (a) Images acquired at distances of 52 (left), 107 (middle), and 162 μm (right) from the top surface are presented. The distance of half ovary was determined based on the DAPI signal. Scale bar = 300 μm. (b) Three-dimensional images reconstructed using Imaris (left). Automatically identified DDX4-positive cells (middle) are presented as red or blue spots (right). Scale bar = 300 μm. (c) Standard H and E-stained images of ovarian slices under × 4, ×10, ×20, and × 40 microscope magnifications. Primordial, primary, secondary, or atretic follicles are marked. Scale bar in C = 100 μm. (d) Comparison between automatic and manual counting results revealed no differences. H and E: Hematoxylin and eosin.

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Effect of different cyclophosphamide doses on the total follicle number in intact ovaries

To study the effect of different CTX doses on the total follicle number in mice, C57BL/6 mice were intraperitoneally injected with different doses of CTX (40, 80, and 120 mg/kg). The ovaries were cleared, following which the follicles were counted. The total follicle number in the NC, CTX40, CTX80, and CTX120 groups was 2,603 ± 59, 1,761 ± 50, 1,043 ± 64, and 262 ± 41, respectively, indicating that the total follicle number gradually decreased with an increase in chemotherapy dose [Figure 5] and [Figure 6]a. The total follicle number was inversely proportional to the CTX dose, showing that the damaging effect of CTX on follicle number was dose dependent [Figure 6]b. Correlation analysis of E2 levels and follicle number showed that the Pearson's correlation coefficient was 0.981, indicating that E2 levels were positively correlated with the total follicle number [Figure 6]c. The Pearson's correlation coefficient of FSH levels and follicle number was - 0.860, implying that FSH levels were negatively correlated with the total follicle number [Figure 6]d].
Figure 6: Effects of different CTX doses on the total follicle number, and the correlation of the total follicle number with endocrine hormone levels. (a and b) Correlation analysis of the CTX doses and total follicle number showed that the damaging effect of CTX on follicles was dose dependent. (c) Correlation analysis of E2 levels and the total follicle number. (d) Correlation analysis of FSH levels and the total follicle number. Data are presented as the mean ± standard deviation; n = 32; *P < 0.0001. FSH: Follicle-stimulating hormone; CTX: Cyclophosphamide.

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  Discussion Top

Currently, approximately 5% of cancers affect women younger than 50 years.[13] As cancer patients become long-term survivors, many experience the debilitating complications of chemotherapy. In young female patients receiving CTX treatment, damage to their reproductive ability is one of the most common complications.[11],[14],[15],[16]

CTX is one of the most widely used chemotherapy drugs. It can cleave phosphorus-nitrogen (P-N) bonds, releasing nitrogen mustard, which undergoes intramolecular cyclizations through the elimination of chloride to form a cyclic aziridinium (ethyleneiminium) cation. These cations tend to be attacked by DNA guanine residues, releasing the nitrogen of the alkylating agent and reacting with the second 2chloroethyl side chain, forming a second covalent linkage with another nucleophile and thus interfering with DNA replication. However, this cell death-causing effect also occurs in oocytes, leading to follicle depletion. Plowchalk and Mattison, Meirow et al., and Petrillo et al. reported that the damaging effect of CTX on the number of follicles was dose dependent.[17],[18],[19] However, in contrast to most studies, Kalich-Philosoph et al. reported that mice administered with 150 mg/kg of CTX had more number of oocytes than those administered with 75 mg/kg of CTX, which was found to be a “Burnout” effect, leading to the loss of follicles.[20]

The mechanism of follicle depletion by CTX is not fully understood. In 2017, Jeelani et al. reported that the spindle morphology and chromosome arrangement of oocytes obtained from CTX mice were worse than those of the NC group, and the oocytes showed a shorter survival time than normal.[21] By further adding CTX directly into the culture medium of normal oocytes, they observed a rise in the level of reactive oxygen species, indicating that CTX can directly damage oocytes and affect meiosis. In addition, CTX can damage the blood vessels, cause ovarian interstitial fibrosis, and affect fertility recovery. The findings reported by Jeelani et al. are consistent with routine clinical observations; the quality and quantity of oocytes are abnormal in patients who receive chemotherapy. The age of the patient at treatment, cumulative dose, and administration schedule are major determinants of this adverse effect.[22],[23] Nguyen et al. found that the injection of 300 mg/kg CTX reduced the total follicle number from 6,852 to 720, which is caused by the direct damaging effect of CTX on oocyte DNA. A protein called DNA damage-induced pro-apoptotic protein (PUMA) and its transcriptional activator, TAp63, may play important roles in this process, as the loss of PUMA protects the ovarian reserve from CTX.[1],[24] The mechanism revealed by Nguyen et al. indicated that CTX reduces the number of follicles by directly attacking oocyte DNA. Similarly, Petrillo et al. also observed a dose-dependent increase in histone H2AX phosphorylation (?H2AX), a marker of DNA double-strand breaks, after exposure to the active form of CTX, supporting the theory that CTX causes follicle depletion by damaging the DNA.[19]

In histology, slicing, followed by chemical staining or immunofluorescence, provides access to observe the microstructure of diverse tissues. However, the process of slicing destroys the integrity of tissues, limiting research to the two-dimensional level, which cannot completely reflect what exactly occurs in intact tissues.[25] In addition, the resolution of computed tomography or magnetic resonance images is limited, and the imaging depth of the laser confocal microscope is confined to hundreds of microns.[26] However, tissue-clearing technology, which has emerged in recent years, allows photons to penetrate the intact tissue at a scale of several centimeters and reveal the fine structure inside.[27],[28] BABB is based on an organic solvent, which includes two steps: (1) dehydration with a lipid solvent, usually methanol, hexane, or tetrahydrofuran; and (2) adjustment of the overall refraction, usually with methyl hydrochloride, BABB, dichloromethane, or diphenyl ether.[25] BABB was not applied to the intact ovary in the aforementioned previous studies. In the current study, we applied BABB to the ovaries of mice and found it to be a suitable method to clear intact ovaries.

To determine the effect of different CTX doses on follicle damage, we evaluated the estrous cycle, endocrine hormone levels, and follicle number in mice from four groups. As the CTX dose increased, the proportion of mice in the diestrus phase increased, and 120 mg/kg CTX could significantly disturb the estrous cycle. To accurately count the number of follicles in each ovary, we applied BABB clearing to mouse ovaries, and the intact structure of mouse ovaries was scanned using β-actin, DAPI, and DDX4 labeling by using a confocal microscope. Images of oocytes from half ovaries were obtained and then reconstructed using 3D software. Follicles in the ovaries were automatically identified and counted. In the NC, CTX40, CTX80, and CTX120 groups, the number of follicles was 2,603, 1,761, 1,043, and 262, respectively, revealing the dose-dependent effect of CTX on follicle damage. We observed reduced E2 levels and increased FSH levels in mice due to CTX-induced oocyte depletion. Through clearing technology-based counting, we revealed that the changes in E2 and FSH levels were closely related to the total number of oocytes. E2 levels were positively correlated with the total follicle number, whereas FSH levels were negatively correlated with the total follicle number. In addition, a greater proportion of mice presented with estrous cycle disorder as the CTX dose increased, indicating that the effect of CTX on fertility was dose dependent.

In conclusion, BABB could clear intact mouse ovaries, and the follicles could be automatically counted using 3D software. We found that the damaging effect of CTX on follicles was dose dependent. The higher the dose of CTX, the fewer follicles remained, and the probability of the endocrine hormones and estrous cycle to be affected increased. These findings suggest that tissue-clearing technology may provide a useful method in reproductive science and that the mechanism underlying follicle depletion by CTX should be further investigated.

Supplementary information is linked to the online version of the paper on the Reproductive and Developmental Medicine website.

Financial support and sponsorship

The funding for this research is from Shanghai Wumengchao Medical Science Foundation (No. LJHXM-2019016), Shanghai Wumengchao Medical Science Foundation (No. LJHXM-2019017).

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1]


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