Impact of vincristine treatment on the mouse ovary and the potential protective effect of dichloroacetate

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This study investigates the effects of vincristine, a chemotherapy alkaloid, on ovarian function and the potential protective role of dichloroacetic acid (DCA). Adult female mice were divided into four groups receiving vincristine, DCA or both and control. Short- and long-term effects were assessed through histological, immunohistochemical, and functional analyses. Vincristine disrupted estrous cycles, reduced the number of growing follicles, and increased follicular atresia, although the primordial follicle pool remained unaffected. An increase in the activation of primordial follicles and a decrease in AMH signaling were also observed. Long-term outcomes included reduced ovulation and alterations in oocyte diameter and meiotic spindle length. Exposure to both drugs did not fully prevent follicular damage, but it reduced ovarian fibrosis and normalized meiotic spindle morphology. These findings suggest that vincristine, though considered to have low gonadotoxicity, may negatively impact oocyte quality. DCA exhibited a dual role: exacerbating some follicular alterations while improving aspects of oocyte maturation and reducing tissue fibrosis. This highlights the importance of evaluating combined therapies to mitigate adverse reproductive effects in female cancer patients. Sexual & Reproductive Medicine Oncology Ovary Chemoteraphy Gonadotoxicity Primordial follicle Apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION An increase in survival rates for girls and women diagnosed with cancer has shifted attention towards long-term quality of life, particularly in maintaining their reproductive capacity. Therefore, minimizing treatment-induced adverse effects and preserving the largest possible follicular reserve has become a goal of considerable interest. Vincristine, an alkaloid used in some cancer treatments due to its anti-mitotic effect, is indicated as a drug with low or no gonadotoxic activity; however, some studies have shown deleterious effects on the growing follicular population and oocyte maturation [ 1 , 2 ]. Other studies have shown that semi-synthetic vinca alkaloids can induce injury to the ovary. For example, vinorelbine administration to female rats induces effects associated with injury and pyknosis in follicular cells [ 3 ]. More recently, alterations in meiotic progress in oocytes and an increase in aneuploidy were described in mice exposed to this drug [ 4 ]. Although the inhibition of mitosis is the most relevant effect of vinca alkaloids, other activities may be affected. Recently, several studies have demonstrated that vincristine promotes mitochondrial fission, inhibits mitochondrial fusion, and impairs the transport of damaged mitochondria into lysosomes [ 5 – 7 ]. Most chemotherapeutic agents can also impact the ovarian microenvironment, disrupting mechanical forces, altering follicle survival, and ultimately leading to ovarian dysfunction. This imbalance could be manifested by stromal fibrosis that could lead to ovarian dysfunction and eventually accelerate ovarian aging [ 8 , 9 ]. Considering these side effects, we sought to analyze their potential attenuation through the simultaneous use of Dichloroacetic Acid (DCA), a compound whose activity could mitigate the harmful effects of vincristine on the ovary. Dichloroacetic acid (DCA) is a haloacetic acid that inhibits pyruvate dehydrogenase kinase (PDK) and increases pyruvate dehydrogenase (PDH) activity, improving oxidative metabolism by fostering the entrance of pyruvate into the Krebs cycle and the formation of acetyl-CoA and citrate. DCA is formed during the disinfection process of water; it is produced as a by-product during the chlorination of water and may occur in drinking water after chlorine-based disinfection and in swimming pools [ 10 – 12 ]. There are some reports of its activity on reproductive functions, whose results are contradictory. DCA improved the fibrosis and degeneration in the ovaries of diabetic rats [ 13 ]. Also, DCA in vitro exposure of bovine oocytes activates metabolic pathways during the in vitro maturation process [ 14 ]. Several studies have also shown the antifibrotic effect of DCA in several models, such as chronic kidney disease [ 15 ], pulmonary arterial hypertension [ 16 ], or endometrial damage induced by hyperglycemia [ 13 ]. On the other hand, DCA is proposed as a reproductive endocrine-disrupting chemical because it induces a significant reduction of estrogen receptor (alpha and beta) expression in the mouse ovary, increased FSH, and decreased estradiol and progesterone serum levels [ 17 ]. Also, Deng et al [ 18 ] have shown that urinary DCA content was negatively associated with antral follicle count in women. The present study was designed to explore the effects of vincristine treatment on both short-term and long-term reproductive outcomes, as well as the potential protective effects of DCA on these female reproductive parameters using a mouse model. MATERIALS AND METHODS Animals Studies were conducted on eight-week-old C57BL/6 female mice from the breeding colony housed at the Facultad de Medicina (URBE, Montevideo, Uruguay). Animal care and protocols followed international guidelines for the use of laboratory animals. They were approved by the local Experimental Animal Committee (CHEA, Comisión Honoraria de Experimentación Animal, Universidad de la República), Montevideo, Uruguay (protocol number 070151-000016-22). Mice were housed under light-controlled conditions (12-hour light/dark cycle) with free access to food and water and divided randomly into the following groups (n = 9 per group): A) SW - Control group, which was injected with vehicle (saline) and received regular drinking water; B) SD - mice injected with vehicle (saline) and received drinking water containing dichloroacetate (DCA; Sigma, St. Louis, MO); C) VW - mice injected with Vincristine (100 ug/kg) [19] diluted in saline and regular drinking water, D) VD mice Intraperitoneally injected (IP) with Vincristine at the same dose and received DCA in drinking water. DCA was added to tap water in a concentration of 500 mg/L and placed into water bottles. A daily dose of 100 mg/kg was administered, based on a daily water intake of 5 mL [20]. The DCA solution was prepared twice a week, with the total consumed volume measured to ensure a consistent dose. The DCA treatment lasted from day 0 to day 21 (3 weeks). Vincristine was IP injected from Monday to Friday during weeks 2 and 3. Five animals from each group were euthanized at the end of week 3 (short-term experiments), while the remaining mice were maintained for 4 additional weeks to induce ovulation and collect oocytes (long-term experiments) (Fig.1). Vaginal cytology Vaginal epithelial cells were collected daily every morning between 9:00 and 10:00 AM by inserting the tip of a pipette filled with 15 μL of normal saline into the mouse vagina. The recovered liquid was spread on a slide, air-dried, stained with H-E, and analyzed under a light microscope to determine the estrous cycle phases. According to the distribution of present cell types, the corresponding stage was determined. Briefly, diestrus was recognized by the presence of numerous leukocytes, proestrus by the presence of large, round, nucleated cells without leukocytes, and estrus by the predominance of large, irregularly shaped, anucleated cells [21]. Sample collection and histological processing At the end of each experimental period, animals were deeply anesthetized with a combination of ketamine-xylazine (100 mg/kg of body weight and 5 mg/kg, respectively) and killed by cervical dislocation. Ovaries were excised and fixed in a 4% paraformaldehyde solution in PBS for 24 h (4 °C). Samples were dehydrated in increasingly graded alcohols and embedded in paraffin using a standard protocol. Sections of 5 μm thickness were mounted on silanized microscope slides and stained with hematoxylin-eosin or used for immunohistochemical procedures. Follicular dynamics Serially sectioned ovaries (at least 3 from each treatment group) were stained with hematoxylin and eosin. Every fifth serial section was examined, and the number of primordial, healthy growing, and atretic follicles was counted using a 40x objective from a Nikon Eclipse 600 microscope. Primordial follicles were characterized by an oocyte surrounded by a flattened pregranulosa cell layer; growing follicles include preantral and antral follicles. Preantral follicles had one or more layers of cuboidal granulosa cells without discernible antrum surrounding a healthy oocyte; antral follicles were recognized by the presence of an antral cavity and counted when the oocyte nucleus was visualized. Preantral and antral atretic follicles were considered so from the moment when the granulosa layer became loose, showing the presence of pyknotic granulosa cells or the oocyte showed signs of degeneration [22]. Picrosirius Red staining Deparaffinized and rehydrated ovarian tissue sections were stained using Picro Sirius Red Stain Kit (Abcam, Cambridge, UK) following the manufacturer's instructions, dehydrated, and mounted. Images were taken by a scientist who was blinded to experimental groups, using a Nikon digital color camera attached to a Nikon Eclipse 600 microscope with a 20X objective. For each sample, images of at least 5 random areas were analyzed to quantify the average percentage of collagen area, using the Fiji software. Immunohistochemistry Immunohistochemistry was performed using previously reported protocols [22], sections were deparaffinized, rehydrated in ethanol, and rinsed in distilled water. Endogenous peroxidase activity was blocked by 15 min incubation with 0.4% H 2 O 2 in PBS. Sections were microwaved for 2:30 min 4 times in 10 mM sodium citrate buffer (pH 6), with a 2:30 min interval, and then cooled for 20 min. Sections were blocked in PBS containing 0.5% Triton X-100 and 2% BSA for 1 h at room temperature and incubated overnight at 4°C with the primary antibody diluted in blocking solution. The primary antibodies used were rabbit monoclonal anti-cleaved caspase 3 antibody (Cell Signaling Technology, #9664, 1:1200), rabbit monoclonal anti-FoxO3a (Cell Signaling Technology, #12829, 1:1000) and a goat polyclonal anti-AMH (Santa Cruz Biotechnology, sc-6886, 1:300). Then, sections were rinsed in PBS, incubated with the corresponding biotinylated secondary antibody (1:500 in blocking solution) for 1 h, washed in PBS, incubated for 1 h with avidin–HRP (Vector Laboratories, Burlingame, USA), and revealed using 0.02% 3,3-diaminobenzidine (Sigma Chemical Co, St. Louis, USA) solution as chromogen. The reaction was stopped in tap water, and sections were counterstained with hematoxylin, dehydrated in ethanol, cleared in xylene, coverslipped, and examined using a Nikon photomicroscope (Nikon Eclipse E600). Selected sections were incubated in the absence of the primary antibody to confirm the specificity of the immunostaining. Four non-overlapping sections of at least three ovaries per group were immunostained for each antibody. Anti-cleaved caspase 3 antibody was used to detect apoptotic cells. All follicles presented in the section were recorded, and primordial and primary follicles showing at least one follicular cell immunomarked for cleaved caspase 3, and preantral or antral follicles showing at least two granulosa cells marked were considered atretic follicles. The number of atretic follicles was expressed as a percentage of the total follicles counted. Foxo3a labeling percentage in oocytes was performed by counting the total number of primordial follicles present in each section and classifying each according to whether the mark was nuclear, cytoplasmic, or both. The number of primordial follicles showing each signal location was expressed as a percentage of the total primordial follicles counted. The density of the immunoreactive area for AMH was assessed by threshold filtering each image to display darker immunoreactive regions in the field using the Image J thresholding feature, which automatically determines a threshold based on contrast. Sections were analyzed by comparing the overall area occupied by immunoreactive cells in a defined area, resulting in a density of immunoreactivity expressed as a percentage. Induced ovulation and meiotic spindle analysis Four weeks after the end of treatment, animals underwent an induced ovulation test. This waiting time was considered to ensure that the oocytes obtained come from the primordial follicle stage during the treatment period [23]. For this, mice from all experimental groups were injected with 5 IU of PMSG (Novormon, Zoetis SRL, Argentina) and 48 h after with 5 IU of hCG (Chorulon, MSD Animal Health, Argentina). Twenty hours later, animals were deeply anesthetized with a solution of ketamine/xylazine at 100 mg/kg of body weight and 5 mg/kg, respectively, and euthanized by cervical dislocation. Ovulated oocytes were recovered from the oviduct, counted, and collected in Ferticult TM Flushing medium (FertiPro, Belgium), maintained at 37ºC, and treated with 1mg/ml hyaluronidase (Sigma-Aldrich USA) to separate cumulus cells from oocytes. Denuded oocytes were immediately fixed in a fresh solution of 4% PFA in PBS for 1h. Photographs at 40X were obtained to measure the diameter of oocytes. Thereafter, fixed oocytes were washed in PBS, permeabilized in a solution containing 0.25% Triton X-100 and 2% BSA in PBS for 2 h and incubated overnight at 4 °C with an anti-α/β tubulin antibody raised in rabbit (1:100; Cell Signaling Technology, USA). After several washes in PBS for 10 min each, the oocytes were incubated with a goat anti-rabbit secondary antibody conjugated to Alexa Fluor 488 (1:500; Thermo Fisher Scientific, USA) for 1h at room temperature. Chromatin was stained with DAPI for 10 min, washed in PBS, and mounted on glass slides in a drop of antifade medium (Vectashield, Burlingame, USA). All oocytes were examined and photographed using an epifluorescent Nikon Eclipse e600 microscope. The length of spindles was obtained using ImageJ software and determined when a clear definition of spindle organization was detected. RESULTS All animals survived the treatment, and although some had lost weight at the end of week 3, they all recovered their weight and showed no differences from the control group (SW) at the end of the experimental period (week 7). The group exposed to vincristine and drinking water (VW) showed cyclicity alterations during the treatment period; this modification was not observed in the other experimental groups. All animals receiving DCA (SD and VD) showed estral cycles like those of the control group (Fig. 2). Histological analysis of HE stained sections showed that the total number of primordial follicles at the end of treatment (week 3) was similar in all groups studied. Vincristine exposure induced a significant reduction in the number of growing follicles, which was accompanied by an increase in the proportion of atretic follicles, which cannot be corrected with concomitant administration of DCA (Fig. 3). Immunohistochemical analysis of the localization of the FOXO3a transcription factor (Fig. 4A) showed that vincristine exposure induces a significant increase in the proportion of oocytes with a cytoplasmic location of this protein, indicating that a higher number of primordial follicles are activated (Fig. 4B). A substantial decrease in the average percent area of AMH immunoreactivity was also detected after semiquantitative analysis in ovaries from animals treated with vincristine and DCA (Fig. 4C-D). Cleaved caspase 3 immunohistochemical detection showed a significant increase in the percentage of labelled antral follicles in mice exposed to vincristine (Fig. 5). Fibrosis evaluated by Picrosirius Red staining showed that vincristine exposure did not modify the percentage of collagen fiber area, at least at the end of the 3-week treatment period. DCA treatment tended to decrease the percentage of collagen fibers in ovaries compared to the control group, this reduction was significant in mice exposed to both vincristine and DCA (Fig. 6). Evaluation of long-term effects induced by vincristine and DCA treatment showed a significant diminution in the number of ovulated oocytes. Analysis of the oocytes showed that animals treated with vincristine had a significantly larger diameter, accompanied by an increase in the meiotic spindle length. The spindle characteristics of animals in the group that received vincristine together with DCA were similar to those of the control group. The arrangement of chromosomes in the equatorial plane was not affected by any of the treatments (Fig. 7). DISCUSSION Several studies have shown that vincristine and other vinca alkaloids have a low risk of gonadotoxicity [ 24 – 26 ]. Nevertheless, we found that vincristine treatment induces alterations in the estrous cycle during and after treatment completion. This was associated with increased atresia of growing follicles, increased activation of primordial follicles, and decreased AMH-mediated signaling. Furthermore, in the long term, vincristine treatment decreases the ovulatory rate and alters the morphology of the meiotic spindle. On the other hand, the simultaneous presence of DCA and vincristine further reduces the number of growing follicles and the number of ovulated oocytes, although aspects such as meiotic spindle length show values ​​similar to those of controls. Growing follicles are the main ones affected by vincristine treatment. Moreover, cleaved caspase 3 immunohistochemical detection shows that antral follicles are the most sensitive follicle class to vincristine exposure. These results agree with those described by Winship et al [ 2 ], showing that the primordial follicle number was not affected after exposure to vincristine. However, our findings also showed a decrease in AMH signaling produced by preantral follicles after vincristine treatment, and an increase in Foxo3a cytoplasmic localization in oocytes from primordial follicles. These differences could be explained by the different vincristine administration protocols used in both studies. Taken together, these results are indicators of primordial follicular activation, a mechanism proposed to explain the exhaustion of follicular reserve seen with high gonadotoxic drugs, such as cyclophosphamide [ 27 ]. The magnitude of this fact must not be sufficiently important as that provoked by cyclophosphamide and other drugs, since neither in this study nor in similar ones did vincristine produce a decrease in the reserve of primordial follicles. However, it should be considered since chemotherapy treatments are generally carried out with a mixture of drugs that could enhance this effect [ 28 , 29 ]. Primordial follicle dormancy and activation are, in some way, associated with the ovarian environment. Collagen content increases with age, and collagen excess is considered a hallmark of reproductive aging [ 9 , 30 ]. Modifications in the extracellular matrix surrounding primordial follicles were proposed as another mechanism implicated in follicular activation [ 31 , 32 ]. Also, an increase in collagen deposition is induced by most chemotherapy treatments [ 8 , 33 , 34 ]. Our results show that vincristine exposure tends to increase collagen deposition, but the presence of DCA significantly induces a decrease in the collagen content. This is in line with other studies that showed the antifibrotic action of DCA in the ovary and other organs [ 13 , 15 , 16 ]. Its role as a possible regulator of collagen synthesis should be considered, but it goes beyond the objective of this work. An unexpected finding of our study was that oocytes exposed to vincristine during the primordial follicle stage were somehow affected, and this impact was maintained in the long term. This was evidenced by the alterations in the oocyte diameter and the meiotic spindle length. Recently, Chang et al [ 35 ] described alterations in the meiotic spindle in mouse oocytes after in vivo and in vitro vincristine exposure experiments, but in both cases, the analysis was performed during or immediately after. Similar results were presented by Cheng et al [ 4 ] during in vitro maturation of oocytes exposed to vinorelbine, another vinca alkaloid, where altered spindle assembly and chromosome alignment induce aneuploidy in oocytes. Other studies have shown that some mycotoxins, such as nivalenol produced by a Fusarium fungus and present in contaminated food, induce aberrant spindle structure by affecting tubulin acetylation levels, and impairing mitochondrial function, using an in vitro maturation model [ 36 ]. Should vincristine exposure induce mitochondrial dysfunction in our in vivo model, DCA could be modulating mitochondrial function and help to explain our results. Taken together, our results add data about the toxic effects of vincristine on the quality of mammalian oocytes that could persist beyond the end of chemotherapy treatment. 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Chemotherapy-induced damage to ovary: mechanisms and clinical impact. Future Oncol. 2016 Oct;12(20):2333–44. Chang H, Cheng S, Xing G, Huang C, Zhang C, Qian W, Li J. Vincristine exposure impairs mouse oocyte quality by inducing spindle defects and early apoptosis. IUBMB Life. 2024 Jun;76(6):345–56. Wang Y, Pan ZN, Xing CH, Zhang HL, Sun SC. Nivalenol affects spindle formation and organelle functions during mouse oocyte maturation. Toxicol Appl Pharmacol. 2022 Feb 1;436:115882. Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7254488","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":493290315,"identity":"a29976b3-576a-440d-ae9a-1925292921a7","order_by":0,"name":"Toledo, Agustina","email":"","orcid":"","institution":"Unidad Académica de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay","correspondingAuthor":false,"prefix":"","firstName":"Agustina","middleName":"","lastName":"Toledo","suffix":""},{"id":493290316,"identity":"af3fdfac-0981-410b-a434-16073b1b6ee0","order_by":1,"name":"Simoes, Paulina","email":"","orcid":"","institution":"Unidad Académica de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay","correspondingAuthor":false,"prefix":"","firstName":"Paulina","middleName":"","lastName":"Simoes","suffix":""},{"id":493290317,"identity":"6360c8b5-97d3-4d2f-985f-cd915c7c9c6b","order_by":2,"name":"Fernández-Tabó, Clara","email":"","orcid":"","institution":"Unidad Académica de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay","correspondingAuthor":false,"prefix":"","firstName":"Clara","middleName":"","lastName":"Fernández-Tabó","suffix":""},{"id":493290318,"identity":"4cab9cfc-6dde-444e-bf6e-1c474ae41364","order_by":3,"name":"Hernández Mir, Karina","email":"","orcid":"","institution":"Unidad Académica de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay","correspondingAuthor":false,"prefix":"","firstName":"Karina","middleName":"Hernández","lastName":"Mir","suffix":""},{"id":493290319,"identity":"f581c4a4-8adc-418b-8a7e-ac888f1c5e03","order_by":4,"name":"Chávez-Genaro, Rebeca","email":"","orcid":"https://orcid.org/0000-0001-9621-4553","institution":"Unidad Académica de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay","correspondingAuthor":false,"prefix":"","firstName":"Rebeca","middleName":"","lastName":"Chávez-Genaro","suffix":""},{"id":493290320,"identity":"7e9fc5a9-0e15-4702-978a-d07598ca0a50","order_by":5,"name":"Anesetti, Gabriel","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYDADfuYDDAw8DAyMDURqMJCQbEsgVYvBMWK1yLufPfjwR8WfOuNj3IkP3jDYyG44wGMmgU+L4Zm8ZGOeMwYSZsd4NxvOYUgzJqylIcdMmrENqOV+7zZpHobDiYS19L8x//nzn4GEcRvv9t88DP8Ja5GXyDFj4G0Aep+NdxszD8MBwloMJN4YS/McM5acAfSL5ByDZOOZh9mKLfDa0p9j+PFHjRw/fxvvxg9vKuxk+443b7yB15YDqFwgZmZgwe+XBiyCzB/waRkFo2AUjIIRBwBRz0bqkTzijwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-7091-0577","institution":"Unidad Académica de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay","correspondingAuthor":true,"prefix":"","firstName":"Gabriel","middleName":"","lastName":"Anesetti","suffix":""}],"badges":[],"createdAt":"2025-07-30 15:26:03","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-7254488/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7254488/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88038187,"identity":"e4621e23-dd06-48b0-935d-3a0f94395366","added_by":"auto","created_at":"2025-07-31 16:38:10","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":240141,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental design used for short- and long-term experiments. Created in \u0026nbsp;https://BioRender.com\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCreated in BioRender. Toledo, A. (2025) https://BioRender.com/aum6p5c\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7254488/v1/a5e6781b3441a6da576762bf.jpeg"},{"id":88039220,"identity":"341163d4-e9a8-4375-9908-ebd73b884c0b","added_by":"auto","created_at":"2025-07-31 16:46:10","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":403813,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative estrous cycles of two female mice of each group. Vincristine exposure caused irregular and prolonged estrous cycles, which were not observed in the other experimental groups. E= estrus, P = proestrus, D= diestrus; SW= Saline-Water; VW= Vincristine-Water; SD= Saline-DCA; VD= Vincristine-DCA\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7254488/v1/a616627877e52d15b50411ab.jpg"},{"id":88037485,"identity":"f5679d33-5a4d-4c6d-bf7a-13ef40a574ce","added_by":"auto","created_at":"2025-07-31 16:30:10","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":121578,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of total primordial (A) and growing (B) follicles in ovaries of each experimental group. The proportion of atretic follicles with respect to total growing follicles is presented in (C). (*) p\u0026lt;0.05; (**) p\u0026lt;0.01; (****) p\u0026lt;0.0001. SW= Saline-Water; VW= Vincristine-Water; SD= Saline-DCA; VD= Vincristine-DCA\u003c/p\u003e","description":"","filename":"fig3poblacionfolicular.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7254488/v1/3c817237ff5fa50766a7af72.jpg"},{"id":88038189,"identity":"b5df4881-6e1c-43d7-bbfb-35e3a35d4981","added_by":"auto","created_at":"2025-07-31 16:38:10","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":360253,"visible":true,"origin":"","legend":"\u003cp\u003eA- Immunohistochemical detection of FOXO3a. Examples of different subcellular distribution (nuclear or cytoplasmic) of FOXO3a in oocytes from primordial follicles. The inset shows a negative control omitting the primary antibody. Scale bars: 25 μm. B- Percentage of nuclear labelling in each experimental group. C- Representative immunohistochemistry detection of AMH in the ovary. The label is restricted mostly to preantral follicles (brown color). Scale bar: 250 μm. D- Quantification of immunoreactive area occupied in ovaries of each experimental group. (*) p\u0026lt;0.05; (**) p\u0026lt;0.01. SW= Saline-Water; VW= Vincristine-Water; SD= Saline-DCA; VD= Vincristine-DCA\u003c/p\u003e","description":"","filename":"fig4foxoMIS.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7254488/v1/78d4145c2d8ffaf6af9ff52f.jpg"},{"id":88037492,"identity":"e2cf06d8-0af5-42b5-ba8c-00c005c549a3","added_by":"auto","created_at":"2025-07-31 16:30:10","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1491363,"visible":true,"origin":"","legend":"\u003cp\u003eA- Representative images of immunodetection of cleaved-Caspase 3 in ovaries of each experimental group. Inset: negative control incubated without primary antibody. Scale bar: 200 μm. B- Quantification of preantral and antral follicles displaying immunoreactivity to cleaved-caspase 3 in ovaries of each experimental group. (*) p\u0026lt;0.05. SW= Saline-Water; VW= Vincristine-Water; SD= Saline-DCA; VD= Vincristine-DCA\u003c/p\u003e","description":"","filename":"Fig5caspasas.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7254488/v1/5a34eb9a1625be237d5953be.jpg"},{"id":88037494,"identity":"cf1bf655-be2c-44e9-b214-728d4dffd549","added_by":"auto","created_at":"2025-07-31 16:30:10","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1763698,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Representative images of collagen fibres (red) distribution in the ovaries of each experimental group. Picro Sirius red stain. Scale bar: 200 μm. (B) The graph shows the quantification of the area occupied by collagen related to the total ovarian area. (**) p\u0026lt;0.01. SW= Saline-Water; VW= Vincristine-Water; SD= Saline-DCA; VD= Vincristine-DCA\u003c/p\u003e","description":"","filename":"fig6fibrosis.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7254488/v1/d15b3ba4d4425f076c5dee00.jpg"},{"id":88038197,"identity":"6ab9196c-bc39-4f76-a789-9109536a419e","added_by":"auto","created_at":"2025-07-31 16:38:10","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":555794,"visible":true,"origin":"","legend":"\u003cp\u003eGraphs showing ovulation rate (A), oocyte diameter (B), and spindle length (C) of each experimental group. Representative images of the meiotic spindle showing tubulin (green) and DNA (blue) in oocytes of each experimental group (D). Scale bar: 50 μm. (*) p\u0026lt;0.05; (**) p\u0026lt;0.01; (***) p\u0026lt;0.005; (***) p\u0026lt;0.0001. SW= Saline-Water; VW= Vincristine-Water; SD= Saline-DCA; VD= Vincristine-DCA\u003c/p\u003e","description":"","filename":"fig7ovocitosv2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7254488/v1/63839b7a035abbe3d4c8073a.jpg"},{"id":88039222,"identity":"2e904795-ae81-40f1-abfd-b024dff766c7","added_by":"auto","created_at":"2025-07-31 16:46:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5392033,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7254488/v1/7a368a03-0e3a-48be-85d8-a57902d6edc6.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eImpact of vincristine treatment on the mouse ovary and the potential protective effect of dichloroacetate\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAn increase in survival rates for girls and women diagnosed with cancer has shifted attention towards long-term quality of life, particularly in maintaining their reproductive capacity. Therefore, minimizing treatment-induced adverse effects and preserving the largest possible follicular reserve has become a goal of considerable interest. Vincristine, an alkaloid used in some cancer treatments due to its anti-mitotic effect, is indicated as a drug with low or no gonadotoxic activity; however, some studies have shown deleterious effects on the growing follicular population and oocyte maturation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Other studies have shown that semi-synthetic vinca alkaloids can induce injury to the ovary. For example, vinorelbine administration to female rats induces effects associated with injury and pyknosis in follicular cells [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. More recently, alterations in meiotic progress in oocytes and an increase in aneuploidy were described in mice exposed to this drug [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Although the inhibition of mitosis is the most relevant effect of vinca alkaloids, other activities may be affected. Recently, several studies have demonstrated that vincristine promotes mitochondrial fission, inhibits mitochondrial fusion, and impairs the transport of damaged mitochondria into lysosomes [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Most chemotherapeutic agents can also impact the ovarian microenvironment, disrupting mechanical forces, altering follicle survival, and ultimately leading to ovarian dysfunction. This imbalance could be manifested by stromal fibrosis that could lead to ovarian dysfunction and eventually accelerate ovarian aging [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Considering these side effects, we sought to analyze their potential attenuation through the simultaneous use of Dichloroacetic Acid (DCA), a compound whose activity could mitigate the harmful effects of vincristine on the ovary.\u003c/p\u003e\u003cp\u003eDichloroacetic acid (DCA) is a haloacetic acid that inhibits pyruvate dehydrogenase kinase (PDK) and increases pyruvate dehydrogenase (PDH) activity, improving oxidative metabolism by fostering the entrance of pyruvate into the Krebs cycle and the formation of acetyl-CoA and citrate. DCA is formed during the disinfection process of water; it is produced as a by-product during the chlorination of water and may occur in drinking water after chlorine-based disinfection and in swimming pools [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. There are some reports of its activity on reproductive functions, whose results are contradictory. DCA improved the fibrosis and degeneration in the ovaries of diabetic rats [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Also, DCA \u003cem\u003ein vitro\u003c/em\u003e exposure of bovine oocytes activates metabolic pathways during the \u003cem\u003ein vitro\u003c/em\u003e maturation process [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Several studies have also shown the antifibrotic effect of DCA in several models, such as chronic kidney disease [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], pulmonary arterial hypertension [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], or endometrial damage induced by hyperglycemia [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. On the other hand, DCA is proposed as a reproductive endocrine-disrupting chemical because it induces a significant reduction of estrogen receptor (alpha and beta) expression in the mouse ovary, increased FSH, and decreased estradiol and progesterone serum levels [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Also, Deng et al [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] have shown that urinary DCA content was negatively associated with antral follicle count in women.\u003c/p\u003e\u003cp\u003eThe present study was designed to explore the effects of vincristine treatment on both short-term and long-term reproductive outcomes, as well as the potential protective effects of DCA on these female reproductive parameters using a mouse model.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudies were conducted on eight-week-old C57BL/6 female mice from the breeding colony housed at the Facultad de Medicina (URBE, Montevideo, Uruguay). Animal care and protocols followed international guidelines for the use of laboratory animals. They were approved by the local Experimental Animal Committee (CHEA, Comisi\u0026oacute;n Honoraria de Experimentaci\u0026oacute;n Animal, Universidad de la Rep\u0026uacute;blica), Montevideo, Uruguay\u0026nbsp;(protocol number 070151-000016-22).\u003c/p\u003e\n\u003cp\u003eMice were housed under light-controlled conditions (12-hour light/dark cycle) with free access to food and water and divided randomly into the following groups (n = 9 per group): A) \u003cstrong\u003eSW -\u0026nbsp;\u003c/strong\u003eControl group,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewhich was injected with vehicle (saline) and received regular drinking water; B) \u003cstrong\u003eSD -\u0026nbsp;\u003c/strong\u003emice injected with vehicle (saline) and received drinking water containing dichloroacetate (DCA; Sigma, St. Louis, MO); C) \u003cstrong\u003eVW -\u0026nbsp;\u003c/strong\u003e mice injected with Vincristine (100 ug/kg) [19] diluted in saline and regular drinking water, D) \u003cstrong\u003eVD\u003c/strong\u003e mice Intraperitoneally injected (IP) with Vincristine at the same dose and received DCA in drinking water.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDCA was added to tap water in a concentration of 500 mg/L and placed into water bottles. A daily dose of 100 mg/kg was administered, based on a daily water intake of 5 mL [20]. The DCA solution was prepared twice a week, with the total consumed volume measured to ensure a consistent dose. The DCA treatment lasted from day 0 to day 21 (3 weeks). Vincristine was IP injected from Monday to Friday during weeks 2 and 3. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFive animals from each group were euthanized at the end of week 3 (short-term experiments), while the remaining mice were maintained for 4 additional weeks to induce ovulation and collect oocytes (long-term experiments) (Fig.1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVaginal cytology\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVaginal epithelial cells were collected daily every morning between 9:00 and 10:00 AM by inserting the tip of a pipette filled with 15 \u0026mu;L of normal saline into the mouse vagina. The recovered liquid was spread on a slide, air-dried, stained with H-E, and analyzed under a light microscope to determine the estrous cycle phases. According to the distribution of present cell types, the corresponding stage was determined. Briefly, diestrus was recognized by the presence of numerous leukocytes, proestrus by the presence of large, round, nucleated cells without leukocytes, and estrus by the predominance of large, irregularly shaped, anucleated cells [21].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample collection and histological processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the end of each experimental period, animals were deeply anesthetized with a combination of ketamine-xylazine (100 mg/kg of body weight and 5 mg/kg, respectively) and killed by cervical dislocation. Ovaries were excised and fixed in a 4% paraformaldehyde solution in PBS for 24 h (4 \u0026deg;C). Samples were dehydrated in increasingly graded alcohols and embedded in paraffin using a standard protocol. Sections of 5 \u0026mu;m thickness were mounted on silanized microscope slides and stained with hematoxylin-eosin or used for immunohistochemical procedures.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFollicular dynamics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerially sectioned ovaries (at least 3 from each treatment group) were stained with hematoxylin and eosin. Every fifth serial section was examined, and the number of primordial, healthy growing, and atretic follicles was counted using a 40x objective from a Nikon Eclipse 600 microscope. Primordial follicles were characterized by an oocyte surrounded by a flattened pregranulosa cell layer; growing follicles include preantral and antral follicles. Preantral follicles had one or more layers of cuboidal granulosa cells without discernible antrum surrounding a healthy oocyte; antral follicles were recognized by the presence of an antral cavity and counted when the oocyte nucleus was visualized. Preantral and antral atretic follicles were considered so from the moment when the granulosa layer became loose, showing the presence of pyknotic granulosa cells or the oocyte showed signs of degeneration [22].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePicrosirius Red staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDeparaffinized and rehydrated ovarian tissue sections were stained using Picro Sirius Red Stain Kit (Abcam, Cambridge, UK) following the manufacturer\u0026apos;s instructions, dehydrated, and mounted. Images were taken by a scientist who was blinded to experimental groups, using a Nikon digital color camera attached to a Nikon Eclipse 600 microscope with a 20X objective. For each sample, images of at least 5 random areas were analyzed to quantify the average percentage of collagen area, using the Fiji software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemistry was performed using previously reported protocols [22], sections were deparaffinized, rehydrated in ethanol, and rinsed in distilled water. Endogenous peroxidase activity was blocked by 15 min incubation with 0.4% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in PBS. Sections were microwaved for 2:30 min 4 times in 10 mM sodium citrate buffer (pH 6), with a 2:30 min interval, and then cooled for 20 min. Sections were blocked in PBS containing 0.5% Triton X-100 and 2% BSA for 1 h at room temperature and incubated overnight at 4\u0026deg;C with the primary antibody diluted in blocking solution. The primary antibodies used were rabbit monoclonal anti-cleaved caspase 3 antibody (Cell Signaling Technology, #9664, 1:1200), rabbit monoclonal anti-FoxO3a (Cell Signaling Technology, #12829, 1:1000) and a goat polyclonal anti-AMH (Santa Cruz Biotechnology, sc-6886, 1:300). Then, sections were rinsed in PBS, incubated with the corresponding biotinylated secondary antibody (1:500 in blocking solution) for 1 h, washed in PBS, incubated for 1 h with avidin\u0026ndash;HRP (Vector Laboratories, Burlingame, USA), and revealed using 0.02% 3,3-diaminobenzidine (Sigma Chemical Co, St. Louis, USA) solution as chromogen. The reaction was stopped in tap water, and sections were counterstained with hematoxylin, dehydrated in ethanol, cleared in xylene, coverslipped, and examined using a Nikon photomicroscope (Nikon Eclipse E600). Selected sections were incubated in the absence of the primary antibody to confirm the specificity of the immunostaining. Four non-overlapping sections of at least three ovaries per group were immunostained for each antibody. Anti-cleaved caspase 3 antibody was used to detect apoptotic cells. All follicles presented in the section were recorded, and primordial and primary follicles showing at least one follicular cell immunomarked for cleaved caspase 3, and preantral or antral follicles showing at least two granulosa cells marked were considered atretic follicles. The number of atretic follicles was expressed as a percentage of the total follicles counted. Foxo3a labeling percentage in oocytes was performed by counting the total number of primordial follicles present in each section and classifying each according to whether the mark was nuclear, cytoplasmic, or both. The number of primordial follicles showing each signal location was expressed as a percentage of the total primordial follicles counted.\u003c/p\u003e\n\u003cp\u003eThe density of the immunoreactive area for AMH was assessed by threshold filtering each image to display darker immunoreactive regions in the field using the Image J thresholding feature, which automatically determines a threshold based on contrast. Sections were analyzed by comparing the overall area occupied by immunoreactive cells in a defined area, resulting in a density of immunoreactivity expressed as a percentage.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInduced ovulation and meiotic spindle analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour weeks after the end of treatment, animals underwent an induced ovulation test. This waiting time was considered to ensure that the oocytes obtained come from the primordial follicle stage during the treatment period [23]. For this, mice from all experimental groups were injected with 5 IU of PMSG (Novormon, Zoetis SRL, Argentina) and 48 h after with 5 IU of hCG (Chorulon, MSD Animal Health, Argentina). Twenty hours later, animals were deeply anesthetized with a solution of ketamine/xylazine at 100 mg/kg of body weight and 5 mg/kg, respectively, and euthanized by cervical dislocation. Ovulated oocytes were recovered from the oviduct, counted, and collected in Ferticult\u003csup\u003eTM\u003c/sup\u003e Flushing medium (FertiPro, Belgium), maintained at 37\u0026ordm;C, and treated with 1mg/ml hyaluronidase (Sigma-Aldrich USA) to separate cumulus cells from oocytes. Denuded oocytes were immediately fixed in a fresh solution of 4% PFA in PBS for 1h. Photographs at 40X were obtained to measure the diameter of oocytes. Thereafter, fixed oocytes were washed in PBS, permeabilized in a solution containing 0.25% Triton X-100 and 2% BSA in PBS for 2 h and incubated overnight at 4 \u0026deg;C with an anti-\u0026alpha;/\u0026beta; tubulin antibody raised in rabbit (1:100; Cell Signaling Technology, USA). After several washes in PBS for 10 min each, the oocytes were incubated with a goat anti-rabbit secondary antibody conjugated to Alexa Fluor 488 (1:500; Thermo Fisher Scientific, USA) for 1h at room temperature. Chromatin was stained with DAPI for 10 min, washed in PBS, and mounted on glass slides in a drop of antifade medium (Vectashield, Burlingame, USA). All oocytes were examined and photographed using an epifluorescent Nikon Eclipse e600 microscope. The length of spindles was obtained using ImageJ software and determined when a clear definition of spindle organization was detected.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eAll animals survived the treatment, and although some had lost weight at the end of week 3, they all recovered their weight and showed no differences from the control group (SW) at the end of the experimental period (week 7). The group exposed to vincristine and drinking water (VW) showed cyclicity alterations during the treatment period; this modification was not observed in the other experimental groups. All animals receiving DCA (SD and VD) showed estral cycles like those of the control group (Fig. 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHistological analysis of HE stained sections showed that the total number of primordial follicles at the end of treatment (week 3) was similar in all groups studied. Vincristine exposure induced a significant reduction in the number of growing follicles, which was accompanied by an increase in the proportion of atretic follicles, which cannot be corrected with concomitant administration of DCA (Fig. 3).\u003c/p\u003e\n\u003cp\u003eImmunohistochemical analysis of the localization of the FOXO3a transcription factor (Fig. 4A) showed that vincristine exposure induces a significant increase in the proportion of oocytes with a cytoplasmic location of this protein, indicating that a higher number of primordial follicles are activated (Fig. 4B). A substantial decrease in the average percent area of AMH immunoreactivity was also detected after semiquantitative analysis in ovaries from animals treated with vincristine and DCA (Fig. 4C-D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCleaved caspase 3 immunohistochemical detection showed a significant increase in the percentage of labelled antral follicles in mice exposed to vincristine (Fig. 5).\u003c/p\u003e\n\u003cp\u003eFibrosis evaluated by Picrosirius Red staining showed that vincristine exposure did not modify the percentage of collagen fiber area, at least at the end of the 3-week treatment period. DCA treatment tended to decrease the percentage of collagen fibers in ovaries compared to the control group, this reduction was significant in mice exposed to both vincristine and DCA (Fig. 6).\u003c/p\u003e\n\u003cp\u003eEvaluation of long-term effects induced by vincristine and DCA treatment showed a significant diminution in the number of ovulated oocytes. Analysis of the oocytes showed that animals treated with vincristine had a significantly larger diameter, accompanied by an increase in the meiotic spindle length. The spindle characteristics of animals in the group that received vincristine together with DCA were similar to those of the control group. The arrangement of chromosomes in the equatorial plane was not affected by any of the treatments (Fig. 7).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eSeveral studies have shown that vincristine and other vinca alkaloids have a low risk of gonadotoxicity [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Nevertheless, we found that vincristine treatment induces alterations in the estrous cycle during and after treatment completion. This was associated with increased atresia of growing follicles, increased activation of primordial follicles, and decreased AMH-mediated signaling. Furthermore, in the long term, vincristine treatment decreases the ovulatory rate and alters the morphology of the meiotic spindle. On the other hand, the simultaneous presence of DCA and vincristine further reduces the number of growing follicles and the number of ovulated oocytes, although aspects such as meiotic spindle length show values ​​similar to those of controls.\u003c/p\u003e\u003cp\u003eGrowing follicles are the main ones affected by vincristine treatment. Moreover, cleaved caspase 3 immunohistochemical detection shows that antral follicles are the most sensitive follicle class to vincristine exposure. These results agree with those described by Winship et al [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], showing that the primordial follicle number was not affected after exposure to vincristine. However, our findings also showed a decrease in AMH signaling produced by preantral follicles after vincristine treatment, and an increase in Foxo3a cytoplasmic localization in oocytes from primordial follicles. These differences could be explained by the different vincristine administration protocols used in both studies. Taken together, these results are indicators of primordial follicular activation, a mechanism proposed to explain the exhaustion of follicular reserve seen with high gonadotoxic drugs, such as cyclophosphamide [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The magnitude of this fact must not be sufficiently important as that provoked by cyclophosphamide and other drugs, since neither in this study nor in similar ones did vincristine produce a decrease in the reserve of primordial follicles. However, it should be considered since chemotherapy treatments are generally carried out with a mixture of drugs that could enhance this effect [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePrimordial follicle dormancy and activation are, in some way, associated with the ovarian environment. Collagen content increases with age, and collagen excess is considered a hallmark of reproductive aging [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Modifications in the extracellular matrix surrounding primordial follicles were proposed as another mechanism implicated in follicular activation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Also, an increase in collagen deposition is induced by most chemotherapy treatments [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Our results show that vincristine exposure tends to increase collagen deposition, but the presence of DCA significantly induces a decrease in the collagen content. This is in line with other studies that showed the antifibrotic action of DCA in the ovary and other organs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Its role as a possible regulator of collagen synthesis should be considered, but it goes beyond the objective of this work.\u003c/p\u003e\u003cp\u003eAn unexpected finding of our study was that oocytes exposed to vincristine during the primordial follicle stage were somehow affected, and this impact was maintained in the long term. This was evidenced by the alterations in the oocyte diameter and the meiotic spindle length. Recently, Chang et al [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] described alterations in the meiotic spindle in mouse oocytes after \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e vincristine exposure experiments, but in both cases, the analysis was performed during or immediately after. Similar results were presented by Cheng et al [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] during \u003cem\u003ein vitro\u003c/em\u003e maturation of oocytes exposed to vinorelbine, another vinca alkaloid, where altered spindle assembly and chromosome alignment induce aneuploidy in oocytes. Other studies have shown that some mycotoxins, such as nivalenol produced by a \u003cem\u003eFusarium\u003c/em\u003e fungus and present in contaminated food, induce aberrant spindle structure by affecting tubulin acetylation levels, and impairing mitochondrial function, using an \u003cem\u003ein vitro\u003c/em\u003e maturation model [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Should vincristine exposure induce mitochondrial dysfunction in our \u003cem\u003ein vivo\u003c/em\u003e model, DCA could be modulating mitochondrial function and help to explain our results.\u003c/p\u003e\u003cp\u003eTaken together, our results add data about the toxic effects of vincristine on the quality of mammalian oocytes that could persist beyond the end of chemotherapy treatment. Interestingly, it could be suggested that DCA has a dual effect on the follicular population, which may be deleterious in some aspects, since when combined with vincristine it shows greater atresia and lower numbers of ovulated oocytes, but also some beneficial effects, such as the alterations found in ovulated oocytes linked to oocyte size, meiotic spindle length and a reduction in tissue fibrosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding statements\u003c/h2\u003e\u003cp\u003eThis work was supported by Comisi\u0026oacute;n Sectorial de Investigaci\u0026oacute;n Cient\u0026iacute;fica (CSIC 22520220100115UD) and PEDECIBA, Universidad de la Rep\u0026uacute;blica, Montevideo, Uruguay.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u0026Ouml;zalp GR, \u0026Uuml;st\u0026uuml;ner B, Avci G, Bari \u0026Ouml;, Yılmaz MM, Denk B, Aktar A. 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J Mol Histol. 2016 Oct;47(5):491\u0026ndash;501.\u003c/li\u003e\n\u003cli\u003eKano M, Hsu JY, Saatcioglu HD, Nagykery N, Zhang L, Morris Sabatini ME, Donahoe PK, P\u0026eacute;pin D. Neoadjuvant Treatment With M\u0026uuml;llerian-Inhibiting Substance Synchronizes Follicles and Enhances Superovulation Yield. J Endocr Soc. 2019 Nov 1;3(11):2123\u0026ndash;34.\u003c/li\u003e\n\u003cli\u003eLambertini M, Peccatori FA, Demeestere I, Amant F, Wyns C, Stukenborg JB, Paluch-Shimon S, Halaska MJ, Uzan C, Meissner J, von Wolff M, Anderson RA, Jordan K; ESMO Guidelines Committee. Fertility preservation and post-treatment pregnancies in post-pubertal cancer patients: ESMO Clinical Practice Guidelines. Ann Oncol. 2020 Dec;31(12):1664\u0026ndash;78.\u003c/li\u003e\n\u003cli\u003eLee SJ, Schover LR, Partridge AH, Patrizio P, Wallace WH, Hagerty K, Beck LN, Brennan LV, Oktay K; American Society of Clinical Oncology. 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Spatio-temporal remodelling of the composition and architecture of the human ovarian cortical extracellular matrix during in vitro culture. Hum Reprod. 2023 Mar 1;38(3):444\u0026ndash;58.\u003c/li\u003e\n\u003cli\u003eWoodruff TK, Shea LD. A new hypothesis regarding ovarian follicle development: ovarian rigidity as a regulator of selection and health. J Assist Reprod Genet. 2011 Jan;28(1):3\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003eOuni E, Nedbal V, Da Pian M, Cao H, Haas KT, Peaucelle A, Van Kerk O, Herinckx G, Marbaix E, Dolmans MM, Tuuri T, Otala M, Amorim CA, Vertommen D. Proteome-wide and matrisome-specific atlas of the human ovary computes fertility biomarker candidates and open the way for precision oncofertility. Matrix Biol. 2022 May;109:91\u0026ndash;120.\u003c/li\u003e\n\u003cli\u003eBedoschi G, Navarro PA, Oktay K. Chemotherapy-induced damage to ovary: mechanisms and clinical impact. Future Oncol. 2016 Oct;12(20):2333\u0026ndash;44.\u003c/li\u003e\n\u003cli\u003eChang H, Cheng S, Xing G, Huang C, Zhang C, Qian W, Li J. Vincristine exposure impairs mouse oocyte quality by inducing spindle defects and early apoptosis. IUBMB Life. 2024 Jun;76(6):345\u0026ndash;56.\u003c/li\u003e\n\u003cli\u003eWang Y, Pan ZN, Xing CH, Zhang HL, Sun SC. Nivalenol affects spindle formation and organelle functions during mouse oocyte maturation. Toxicol Appl Pharmacol. 2022 Feb 1;436:115882.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Universidad de la República, Montevideo, Uruguay","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ovary, Chemoteraphy, Gonadotoxicity, Primordial follicle, Apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-7254488/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7254488/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe increase in survival rates among women with cancer has shifted focus toward fertility preservation. This study investigates the effects of vincristine, a chemotherapy alkaloid, on ovarian function and the potential protective role of dichloroacetic acid (DCA). Adult female mice were divided into four groups receiving vincristine, DCA or both and control. Short- and long-term effects were assessed through histological, immunohistochemical, and functional analyses.\u003c/p\u003e\u003cp\u003eVincristine disrupted estrous cycles, reduced the number of growing follicles, and increased follicular atresia, although the primordial follicle pool remained unaffected. An increase in the activation of primordial follicles and a decrease in AMH signaling were also observed. Long-term outcomes included reduced ovulation and alterations in oocyte diameter and meiotic spindle length. Exposure to both drugs did not fully prevent follicular damage, but it reduced ovarian fibrosis and normalized meiotic spindle morphology.\u003c/p\u003e\u003cp\u003eThese findings suggest that vincristine, though considered to have low gonadotoxicity, may negatively impact oocyte quality. DCA exhibited a dual role: exacerbating some follicular alterations while improving aspects of oocyte maturation and reducing tissue fibrosis. This highlights the importance of evaluating combined therapies to mitigate adverse reproductive effects in female cancer patients.\u003c/p\u003e","manuscriptTitle":"Impact of vincristine treatment on the mouse ovary and the potential protective effect of dichloroacetate","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-31 16:30:05","doi":"10.21203/rs.3.rs-7254488/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e1f5c8db-bdca-4194-926b-d1e169dc732f","owner":[],"postedDate":"July 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":52405457,"name":"Sexual \u0026 Reproductive Medicine"},{"id":52405458,"name":"Oncology"}],"tags":[],"updatedAt":"2025-07-31T16:30:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-31 16:30:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7254488","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7254488","identity":"rs-7254488","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}