Folate deficiency links impaired appetite routing to ovarian dysfunction

Folate deficiency links impaired appetite routing to ovarian dysfunction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Folate deficiency links impaired appetite routing to ovarian dysfunction Afridi Shaikh, Bharti Choudhary, Mukund Chhatpar, Dhaval Fefar, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3860953/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The long-established link between nutrition and reproduction is known to have critical consequences for reproductive function. However, the availability of experimental data on effect of folate deficiency on ovarian health is scarce and uncertain. Our objective was to establish a proof for association between folate deficiency, hormone dynamics, and health of the ovary through in vivo model organism. Folate-deprived female zebrafishes were developed using intraperitoneal administration of methotrexate (MTX) and they were used to study the possible implications of folate deprivation on ovarian health. Changes in the expression of transcripts regulating appetite and ovarian function was assessed by qRT-PCR. ELISA based methods were utilised to quantify and evaluate changes in hormone levels regulating reproductive function. Histology of ovarian tissue was performed to support the study. Folate deprivation resulted in impaired appetite behaviour and alters its regulatory gene expression. Due to folate deficiency, the neuroendocrine function of the brain was affected that resulted in altered reproductive hormone levels. Histological parameter of ovary was performed wherein the follicles are arrested in primary oocyte stage and abundance of scarring of tissue is seen. Furthermore, elevated lipid peroxidation and catalase enzyme activity indicates folate deficiency induced oxidative stress in ovary as one of the responsible mechanisms to aide ovarian dysfunction. Our study provides experiment proof with in vivo folate deficient model of fish that suggests B9 non-availability resulted into loss of appetite and, female gonadal dysfunction, which developed as cumulative effect of deficiency and altered appetite. The intricate interplay between folate deficiency and appetite along with the consequential implications for the synthesis and release of female reproductive hormones, warrant a thorough and in-depth exploration through further research. Folate deficiency Appetite Leptin Hormones Ovarian dysfunction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Vitamins and minerals play vital roles in metabolic pathways that support fundamental cellular functions. In particular, essential vitamin such as folate due to its involvement in energy-yielding metabolism, ovarian stimulation and neuronal function makes its availability critical to maintain body physiology [ 1 ], [ 2 ]. Folate is important in female reproduction for oocyte quality and maturation, implantation, placentation, fetal growth and organ development [ 3 ]. Beyond the familiar functional role of the brain, it plays critical roles in regulating female reproduction during puberty, gametogenesis and childbirth [ 4 ]–[ 6 ]. Evidence suggest that brain impairments are associated with problems in appetite control, which are likely to contribute to weight changes, but the extent to which they associated with wider functional impacts is unclear [ 7 ]. In females, reproductive function and body weight control is largely mediated by the steroid sex hormones, estradiol and progesterone. Estradiol, in particular, by acting at the cortex, hypothalamus and brainstem level helps to regulate the body's appetite and energy use by reducing food intake and increasing energy expenditure [ 8 ], [ 9 ]. Moreover, the estrogen receptors (ERs) and leptin receptors (LPs) are co-expressed in neurons within the areas known to coordinate metabolism and gonadal function [ 10 ]. Studies indicate direct interaction of estrogen and leptin signals at the level of STAT3 pathway [ 11 ]. Our previous study and literature suggest that folate deficiency is associated with mood changes and depression symptoms, which may affect appetite regulation through complex neurotransmitter interactions [ 12 ], [ 13 ]. Depression can in turn impact basic functions like appetite, sleep, and mood due to its diverse effects [ 14 ]. This interplay of folate deficiency, depression, dysregulated leptin signaling and altered appetite presents a captivating pathway for elucidating their collective impact on steroidogenesis and the pathogenesis of ovarian dysfunction. Regarding reproduction, literature majorly reflects upon the birth and developmental defects due to folate deficiency during pregnancy. It is very well known that folate supplementation has proved to be fruitful in preventing neonatal abnormalities [ 15 ], [ 16 ]. But the mechanism responsible for long-term changes in appetite under conditions of folate deficiency remains a topic of ongoing research. Furthermore, the nature of the involvement of folate deficiency in molecular and cellular reactions that translate into ovarian dysfunction is poorly understood. The ramifications of folate deficiency extend beyond gestational considerations, influencing not only brain neurotransmitter levels but also, inciting oxidative stress [ 17 ]. This can potentially disturb the intricate process of follicular development and ovarian function. With the exception of correlational studies, the lack of experimental data demonstrating the impact of folate deficiency on appetite and ovarian function is evident. In this context, our objective was to evaluate the association between folate deficiency, appetite regulation, hormone dynamics, and ovarian function. Close degree of similarity in reproductive regulation systems between human and zebrafish permits us to carry this research in zebrafish. Identification of important neurons involved in regulation of reproductive system, similar regulative hormones and responses in zebrafish as compared to humans adds to the robustness of this animal model to assess reproductive complications [ 18 ]–[ 20 ]. Material and methodologies Animal maintenance Sexually matured healthy female adult Zebrafishes ( Danio rerio ) (weighing 700–800 mg) were utilised. Fishes were housed in 10 L aquarium tanks and water temperature was maintained at 27 ± 2 ᵒC. Fishes were kept under a 14-hour light/10-hour dark cycle and fed with commercial fish food flakes 2 times a day. Treatment We have previously developed a method to induce folate deprivation in zebrafish using methotrexate (MTX), a known DHFR antagonist, which alters the folate mediated one-carbon metabolism [ 13 ]. Fishes were administered methotrexate (MTX) dose (37.5 µg/gm bodyweight) intraperitonially for a time period of 15 days. Fishes were divided in control and folate deprived (FD) groups consisting of 5 individuals each. Tissue collection After subjecting the fishes to a 15-day treatment with MTX, blood samples were collected from the tail vein using heparin-coated vials to obtain plasma. Subsequently, the fishes were euthanized by inducing hypothermia, and their brain, ovary, and liver were dissected for further analysis. qRT-PCR Total RNA from the tissues were extracted with TRIzol reagent (Invitrogen) and cDNA was synthesized using the cDNA synthesis kit by Bio-Rad. qRT-PCR was carried out on cDNA using iTaq Universal SYBR Green Supermix by Bio-Rad. Primers for lepa , lepr , gnrh3 , lhb , fshb , esr1 , cyp19a , hsd3b , nrg1 and gapdh were designed using the Primer-BLAST tool NCBI. For internal control gapdh was used, and fold change in mRNA expression was analysed using the 2 −ΔΔCT method. Melatonin estimation Melatonin level in the brain was determined using method described by [ 21 ]. Detailed procedure is provided in supplementary material. Steroid hormone estimation Plasma Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) were measured via commercial Chemiluminescence Immunoassay (CLIA) kit (Elabscience). Plasma Estradiol and testosterone was measured using commercial enzyme linked fluorescence assay (ELFA) kit. Estimation of Catalase activity Catalase activity was measured according to Sinha, 1972. Detailed procedure is provided in supplementary material. Estimation of Lipid Peroxidation (LPO) LPO was measured by the estimation of malonaldehyde (MDA) level; according to [ 23 ]. Detailed procedure is provided in supplementary material. Histopathological studies Dissected ovaries were washed with Phosphate Buffered saline (PBS) followed by fixation in 10% formalin. After fixation, the tissues were dehydrated using increasing concentrations of ethanol. The dehydrated ovary tissues were then embedded in paraffin (Fischer Scientific), and tissue sections of 5 µm thickness were obtained using a microtome. These sections were subsequently stained with Haematoxylin and Eosin stain. The stained tissue sections from five different ovaries were examined under a microscope to observe any histopathological changes. Statistical analysis Data values were expressed as Mean ± SEM. Graphpad Prism software version 8.4.2 (GraphPad Software, Inc., La Jolla, CA, USA) was used to perform statistical data analysis. Student’s t-test was used to determine the statistical significance between experimental groups. Statistical significance was set at p < 0.05.* denotes p ≤ 0.05, ** denotes p ≤ 0.01, *** denotes p ≤ 0.001, **** denotes p ≤ 0.0001. Results Deficiency of folate leads to changes in the expression of genes related to appetite and reproductive function : In our previous study we observed that folate deprivation induces depressive-like behaviours and alters the appetite of the zebrafish [ 13 ]. We sought to evaluate if folate deficiency changes the transcript of appetite controlling genes. Gene expression analysis showed upregulation of lepa gene in the liver by 1.58-fold. The expression of leptin receptor, lepr , was found to be upregulated by over 15-fold in the brain. Whereas, the expression of Neuropeptide Y ( npy ), an orexigenic factor, was found to be downregulated. Conversely, the expression of cocaine- and amphetamine-regulated transcript ( cart ), an anorexigenic factor, was found to be upregulated in brain by over 4-fold (Fig. 1 a). We further evaluated the expression of transcripts crucial for the reproductive functioning. It is evident from the gene expression study (Fig. 1 b) that in the brain, the genes important for the synthesis of reproductive hormones such as gonadotropin releasing hormone (GnRH), luteinizing hormone (LH) and follicle stimulating hormone are downregulated. This suggests that when subjected to a folate-deficient environment, the overall levels of these hormones might be diminished, potentially leading to repercussions on ovarian function and the development of follicles. Subsequently, it was found that the genes such as cyp19a , esr1 , hsd3b and nrg1 , which are required for proper ovarian function and follicular development are downregulated significantly in the ovary of folate deficient fishes (Fig. 1 c). Estimation of hormones involved in reproductive function: Given that ovulation is an outcome arising from intricate hormonal equilibrium and interplay, any alterations in these mechanisms has the potential to affect its physiological processes [ 24 ]. It can be thus speculated that hormonal levels are a reflection of proper brain and ovarian function. We estimated melatonin levels in brain as it is known to play role in the regulation of many reproductive functions [ 25 ]. Upon estimation, the level of melatonin hormone was found to be elevated in the brain of folate deprived fishes (Fig. 2 a). Furthermore, it was found that the plasma levels of LH (Fig. 2 c) and FSH (Fig. 2 c) were low in the folate deprived fishes, whereas the plasma levels of estradiol (Fig. 2 d) and testosterone (Fig. 2 e) was elevated. Folate deficiency induces histopathological changes and oxidative stress in the ovary: Apart from altered gene expression and hormonal changes, ovarian dysfunction can also be reflected in the form of morphological alterations. Zebrafish oocyte can be divided into four stages i.e., primary growth (PO), cortical alveolus (CO), vitellogenic (VO) and mature oocyte (MO) [ 26 ]. Displayed in Figs. 3 and 4 are the histological sections of zebrafish ovary. In the control group, all the stages of ovarian development along with normally surrounded zona radiata can be evidently observed. On the other hand, in folate deprived fishes most of the oocytes can be seen arrested in primary stage (Fig. 4 a and 4 b). Mature oocytes are scarce and the yolk granules are depleted. Transformation of the yolk granules into eosinophilic granular mass can be observed as well in Fig. 4 c. The view also shows disrupted vitelline membrane (DVM). Furthermore, ovarian photomicrograph (Fig. 4 d) of folate deprived fishes shows infiltration of inflammatory cells and proliferation of interstitial connective tissue. The proliferation of interstitial connective tissue and possible implication of tissue scarring could be due to oxidative stress, as it is an important molecular mechanism underlying fibrosis in variety of organs [ 27 ]. Therefore, we estimated the activity of catalase enzyme and lipid peroxidation (LPO) in the ovarian tissue homogenate as elevated levels of these enzymes can represent oxidative stress in tissues [ 28 ]. This was done to evaluate the hypothesis that folate deficiency can induce oxidative stress in the ovaries, which could be one of the probable mechanisms for ovarian dysfunction. The LPO activity was found to be significantly elevated in the ovary of folate deprived fishes (Fig. 5a), whereas, although not statistically significant, the catalase activity was found to be elevated (Fig. 5b). Fig: 5: Folate deficiency induces oxidative stress in ovarian tissue. (a) Estimation of LPO and (b) Catalase in the ovary. All values are expressed as Mean ± SEM; N = 5. *, p ≤ 0.05; FD, folate deprived. Discussion The association between nutrition and reproduction has long been known to have crucial implications for the reproductive functioning. Inadequate nutrition can lead to depletion of body weight and overall physical condition, leading to a postponement in the onset of puberty [ 29 ]. Among various components of diet, folate is one the important vitamin implicated in reproductive health [ 30 ]. Nonetheless, there remains a substantial knowledge gap concerning the potential correlation that exists between folate and ovarian functionality, as well as an incomplete understanding of the potential consequences resulting from folate deprivation on ovarian function. In our previous study, we have documented that zebrafish deficient in folate, manifest symptoms reminiscent of depressive-like behavior. These manifestations are characterized by a discernible decrease in their appetite, alterations in neurotransmitter activity, and a notable decline in their overall body weight [ 13 ]. We sought to evaluate if folate deficiency affected appetite has any possible implications on the ovarian reproductive dynamics. To develop folate insufficient model, adult female zebrafishes were injected with methotrexate (MTX), a folate antagonist, which works by inhibiting DHFR enzyme (EC 1.5.1.3.) [ 13 ]. Transcript expression study revealed a significant upregulation of lepa gene in the liver. lepa gene encodes for leptin hormone, which is crucial for regulation of body weight, appetite and metabolism and is important for reproductive system [ 31 ]. Leptin hormone is known to supress food intake and thereby induces weight loss [ 32 ]. In zebrafish, lepa gene is predominantly expressed in the liver [ 33 ], [ 34 ]. Elevated serum level of leptin hormone is reported in PCOS patients as well [ 35 ]. Furthermore, transcript expression of lepr was found to be elevated in the brain of folate deficient zebrafishes. lepr gene encodes for leptin receptor and in zebrafish, its expression is reported to be strongest in the brain [ 36 ]. Apart from lepr transcript expression, npy and cart transcript expression was found to be altered in brain. Neuropeptide Y (NPY) is a potent orexigenic peptide found in the brain which stimulates food intake [ 37 ]. In contrast, cocaine- and amphetamine-regulated transcript (CART) is an anorexic peptide, which reduces appetite, resulting in weight loss [ 38 ]. Subsequently, we have reported a significant npy downregulation and a significant cart upregulation in the brain of folate deficient zebrafish. These results suggest that folate deficiency alters the expression of these appetite controlling genes through an unknown mechanism. This explains why folate deficient zebrafishes have reduced appetite and body weight. The reproductive axis is closely associated with nutritional status. The limited availability of nutrients, leading to declined energy reserves, can alter many finely tuned crosstalk between energy metabolism and reproduction, thereby affecting female fertility [ 39 ]. Not just malnutrition, but eating disorders can also affect the reproductive function [ 40 ]. It can be thus postulated that altered leptin signalling, reduced appetite and subsequent weight loss affects the neuroendocrine function of the brain. To validate our speculation, we studied the transcript expression of gnrh , lh and fsh . The expression of these transcripts was significantly downregulated in folate deficient zebrafishes. Gonadotropin releasing hormone (GnRH) is crucial for the synthesis and secretion of luteinizing hormone (LH) and follicular stimulation hormone (FSH) in the brain [ 41 ]. Decreased expression of gnrh , lh and fsh is also reflected in the plasma of fishes, where a significant downregulation of plasma LH and FSH hormone was estimated. Additionally, we observed significant elevation of Melatonin hormone in the folate deficient fish brain. Melatonin is known to play role in the regulation of many reproductive functions including oocyte maturation [ 25 ]. Elevated levels of melatonin in people with eating disorder such as anorexia nervosa has been previously reported as well [ 42 ], [ 43 ], suggesting a correlation between melatonin and folate deprivation induced appetite changes. The impact of melatonin on the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) has generated divergent findings across various studies, highlighting the dual role of melatonin which needs to be characterised further [ 44 ], [ 45 ]. Moreover, it has been observed that women grappling with reproductive disorders like Polycystic Ovary Syndrome (PCOS) exhibit notably elevated levels of melatonin within their serum. This intriguing correlation has led to the proposition of elevated melatonin levels potentially serving as a promising biomarker for prediction of PCOS [ 46 ]. Additionally, we report elevation of plasma estradiol and total testosterone in folate deprived fishes. Proper levels of these hormones are instrumental for the normal functioning of female reproductive system [ 47 ], [ 48 ]. Ovarian steroidogenesis is known to be affected by dyslipidaemia in mice [ 49 ]. Moreover, from previous studies we know that condition such as PCOS displays altered lipid profiles, which includes reduced high-density lipoprotein cholesterol (HDL-C), elevated triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C), along with significantly increased lipoprotein levels [ 50 ]–[ 52 ]. Upon evaluating the hepatic cholesterol, triglyceride and HDL-cholesterol we observed that cholesterol and triglyceride levels were elevated, whereas the HDL-cholesterol levels were decreased (data provided in supplementary). Although not conclusive, these results suggest the possibility of dyslipidaemia under folate deprived condition which alters steroidogenesis and could potentially lead to ovarian dysfunction. Even the Rotterdam guidelines suggest measure of HDL-cholesterol and triglyceride for evaluation of metabolic syndromes [ 53 ], [ 54 ]. We further looked into the functioning of the ovary under folate deficient conditions by the gene transcript study. It is evident from the results that the expression of cyp19a , esr1 , hsd3b and nrg1 is significantly downregulated in the ovarian tissue. Women with PCOS are reported to have decreased expression of CYP19A1 (aromatase) mRNA, which contributes to an increase in testosterone level [ 55 ]. 3β-HSDs catalyse the conversion of pregnenolone, 17α-hydroxy pregnenolone, and dehydroepiandrosterone to progesterone [ 56 ] and its downregulation can be explained by diminished FSH levels as previous study has reported decreased expression in 3βHSD mRNA, possibly due to the downregulation of FSH hormone [ 57 ]. Neuregulin-1 (NRG1) is a member of the EGF-like factor family, and is important for oocyte maturation [ 58 ]. NRG1 is known to be induced by LH in mice [ 59 ] and this could be the possible reason of its low expression in zebrafish ovary. The histological assessment of the control ovary shows follicles at various stages of development (Fig. 3 ), illustrating a well-orchestrated progression. In contrast, upon examining ovarian histology under folate deprivation, a distinct pattern emerges as clearly depicted in Fig. 4 . Notably, maturation of oocytes seems to be affected, as a substantial number of follicles are arrested in primary stages. Concomitantly, a discernible infiltration of inflammatory cells, proliferation of interstitial connective tissue and scarring is observed in the ovarian histology. We have previously demonstrated that folate deficiency can in fact, cause systemic inflammation. Scarring of the ovarian tissue under folate deprivation can partly be explained by oxidative stress. Oxidative stress plays a crucial role as a contributing factor in the initiation of follicular atresia and the process of ovarian aging. This oxidative burden ultimately gives rise to a decline in the reproductive potential of females [ 60 ]. To mitigate the elevated levels of oxidants and prevent oxidative stress, tissues produce enzymes such as catalase and lipid peroxidase, thus elevated levels of these enzymes can represent oxidative stress in tissues [ 28 ]. We report elevation of catalase activity and a significant elevation of lipid peroxidation activity in the ovary under folate deprived conditions. Conclusion The incurable nature of ovarian dysfunction reinforces the importance of understanding the molecular mechanisms behind folate deficiency induced ovarian dysfunction. We have observed altered expression of key genes involved in appetite regulation ( lepa , lepr , cart and npy ) and reproductive function ( gnrh , lh , fsh, cyp19a, esr1, hsd3b and nrg1 ). Furthermore, we have documented histological changes in the ovary, including follicular arrest, infiltration of inflammatory cells, and proliferation of interstitial connective tissue. Our study implies the potential role of leptin and melatonin in mediating the effects of folate deficiency on ovarian function. Further research is needed to elucidate the underlying mechanisms and to determine the long-term consequences of folate deficiency on ovarian health and fertility. Declarations Acknowledgment: Authors would like to express their gratitude to DBT-MSUB-ILSPARE Project funded Dr. Vikram Sarabhai Institute for Cell and Molecular Biology, The Maharaja Sayajirao University of Baroda for necessary lab facilities. Funding: This work is supported by the seed grant of Research & Consultancy Cell, The Maharaja Sayajirao University of Baroda and the Department of Biotechnology (DBT), Government of India for the M.Sc. Biotechnology teaching Program. Ethics Statement: Animals were maintained and handled as per the guidelines of “Committee for Control and Supervision of Experiment on Animals (CCSEA)”. Animals were housed in the animal house facility of the Department of Zoology, The Maharaja Sayajirao University of Baroda (Reg. No. 827/GO/Re/S/04/CPCSEA). Experimental protocol was approved by Institutional Animal Ethics Committee of the Department of Zoology, Faculty of Science, The Maharaja Sayajirao University of Baroda (MSU-Z/IAEC07/08-2023). Data availability: All data generated or analysed during this study are included in this article. Further enquiries can be directed to the corresponding author. Supplementary data is available at the repository which can be accessed by the following DOI: https://doi.org/10.17632/bhmthf6vhd.1. CRediT authorship contribution statement: Afridi Shaikh: Methodology, Formal analysis, Investigation, Writing: original draft. Bharti: Methodology, Formal analysis, Investigation. Mukund Chattpar: Methodology, Formal analysis, Investigation. Dhaval Fefar: Histological analysis. Hetal Roy: Conceptualization, Resources, Supervision, Validation, Writing: review and editing. Declaration of Competing Interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution A.S. - Methodology, Formal analysis, Investigation, Writing: original draft. B.C. - Methodology, Formal analysis, Investigation. M.C. - Methodology, Formal analysis, Investigation. D.F. - Histological analysis. H.R. - Conceptualization, Resources, Supervision, Validation, Writing: review and editing. References Tardy A-L, Pouteau E, Marquez D, Yilmaz C, Scholey A (Jan. 2020) Vitamins and Minerals for Energy, Fatigue and Cognition: A Narrative Review of the Biochemical and Clinical Evidence. 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Hum Reprod Update 12(3):193–207. 10.1093/humupd/dmk003 Tsutsumi R, Webster NJG (2009) GnRH Pulsatility, the Pituitary Response and Reproductive Dysfunction. Endocr J 56(6):729. 10.1507/ENDOCRJ.K09E-185 Jain M, Jain S, Singh T, Haldar C, Jain P (2013) Melatonin and its correlation with testosterone in polycystic ovarian syndrome. J Hum Reprod Sci 6(4):253. 10.4103/0974-1208.126295 Luboshitzky R, Qupti G, Ishay A, Shen-Orr Z, Futerman B, Linn S (2001) Increased 6-sulfatoxymelatonin excretion in women with polycystic ovary syndrome. Fertil Steril 76(3):506–510. 10.1016/S0015-0282(01)01930-6 Díaz López B, Díaz Rodríguez E, Urquijo C, Fernández Álvarez C (2005) Melatonin influences on the neuroendocrine-reproductive axis. Ann N Y Acad Sci 1057:337–364. 10.1196/ANNALS.1356.026 Diaz E, Pazo D, Esquifino AI, Diaz B (2000) Effects of ageing and exogenous melatonin on pituitary responsiveness to GnRH in rats. J Reprod Fertil 119(1):151–156. 10.1530/JRF.0.1190151 Mojaverrostami S, Asghari N, Khamisabadi M, Khoei HH (Dec. 2019) The role of melatonin in polycystic ovary syndrome: A review. Int J Reprod Biomed 17(12):865. 10.18502/IJRM.V17I12.5789 Yu Z, Jiao Y, Zhao Y, Gu W (2022) Level of Estrogen in Females—The Different Impacts at Different Life Stages, J Pers Med , vol. 12, no. 12, Dec. 10.3390/JPM12121995 Nassar GN, Leslie SW (2023) Physiology, Testosterone, StatPearls , Jan. Accessed: Aug. 08, 2023. [Online]. Available: https://www.ncbi.nlm.nih.gov/books/NBK526128/ Abreu JM et al (2021) Dyslipidemia’s influence on the secretion ovarian’s steroids in female mice, Liu Q, jie Xie Y, hua Qu L, Zhang M, Mo Zcheng (2019) Dyslipidemia involvement in the development of polycystic ovary syndrome, Taiwan J Obstet Gynecol , vol. 58, no. 4, pp. 447–453, Jul. 10.1016/J.TJOG.2019.05.003 Ghaffarzad A, Amani R, M.D.3 MMS, Darabi M, Cheraghian B (2016) Correlation of Serum Lipoprotein Ratios with Insulin Resistance in Infertile Women with Polycystic Ovarian Syndrome: A Case Control Study. Int J Fertil Steril 10(1):29. 10.22074/IJFS.2016.4765 I. TSOUMA et al., Lipid Lipoprotein Profile Alterations in Greek Infertile Women with Polycystic Ovaries: Influence of Adipocytokines Levels, In Vivo (Brooklyn) , vol. 28, no. 5, p. 935, Sep. 2014, [Online]. Available: http://iv.iiarjournals.org/content/28/5/935.abstract Wild RA, Rizzo M, Clifton S, Carmina E (Mar. 2011) Lipid levels in polycystic ovary syndrome: systematic review and meta-analysis. Fertil Steril 95(3). 10.1016/J.FERTNSTERT.2010.12.027 Fauser BCJM et al (2004) Jan., Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS), Hum Reprod , vol. 19, no. 1, pp. 41–47, 10.1093/HUMREP/DEH098 Panghiyangani R et al (2020) Apr., CYP19A1 Gene Expression in Patients with Polycystic Ovarian Syndrome, J Hum Reprod Sci , vol. 13, no. 2, p. 100, 10.4103/JHRS.JHRS_142_18 Ye L, Guo J, Ge RS (Jan. 2014) Environmental Pollutants and Hydroxysteroid Dehydrogenases. Vitam Horm 94:349–390. 10.1016/B978-0-12-800095-3.00013-4 Mirϲ F, Smyth CD, Whitelaw PF, Milne M, Hillier SG (1995) Regulation of 3 beta-hydroxysteroid dehydrogenase delta 5/delta 4-isomerase and cholesterol side-chain cleavage cytochrome P450 by activin in rat granulosa cells, Endocrinology , vol. 136, no. 8, pp. 3247–3252, Aug. 10.1210/ENDO.136.8.7628357 Chowdhury I, Branch A, Mehrabi S, Ford BD, Thompson WE (2017) Gonadotropin-Dependent Neuregulin-1 Signaling Regulates Female Rat Ovarian Granulosa Cell Survival, Endocrinology , vol. 158, no. 10, pp. 3647–3660, Oct. 10.1210/EN.2017-00065 Kawashima I et al (2014) Targeted disruption of Nrg1 in granulosa cells alters the temporal progression of oocyte maturation. Mol Endocrinol 28(5):706–721. 10.1210/ME.2013-1316 Bao T et al (Jul. 2022) Naringin prevents follicular atresia by inhibiting oxidative stress in the aging chicken. Poult Sci 101(7):101891. 10.1016/J.PSJ.2022.101891 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies 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-3860953","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":267354589,"identity":"2886822d-b96b-4e9b-a987-4283db62fe4e","order_by":0,"name":"Afridi Shaikh","email":"","orcid":"","institution":"The Maharaja Sayajirao University of Baroda","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Afridi","middleName":"","lastName":"Shaikh","suffix":""},{"id":267354590,"identity":"105fb4bc-72d6-4be8-b7fe-ebae5ca7ecb8","order_by":1,"name":"Bharti Choudhary","email":"","orcid":"","institution":"The Maharaja Sayajirao University of Baroda","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bharti","middleName":"","lastName":"Choudhary","suffix":""},{"id":267354591,"identity":"c1bc2c47-7187-43a0-9b59-9bab3d6e969b","order_by":2,"name":"Mukund Chhatpar","email":"","orcid":"","institution":"The Maharaja Sayajirao University of Baroda","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mukund","middleName":"","lastName":"Chhatpar","suffix":""},{"id":267354592,"identity":"94ad4cdd-00f7-4137-975a-86a40a52134e","order_by":3,"name":"Dhaval Fefar","email":"","orcid":"","institution":"Kamdhenu University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dhaval","middleName":"","lastName":"Fefar","suffix":""},{"id":267354593,"identity":"d983093d-9ea1-435a-9736-d3a1386df78c","order_by":4,"name":"Hetal Roy","email":"data:image/png;base64,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","orcid":"","institution":"The Maharaja Sayajirao University of Baroda","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hetal","middleName":"","lastName":"Roy","suffix":""}],"badges":[],"createdAt":"2024-01-13 18:29:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3860953/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3860953/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49745896,"identity":"6b571f0d-3922-44df-a1a1-9ba0267cd145","added_by":"auto","created_at":"2024-01-17 10:47:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":596248,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFolate deficiency alters the transcript expression of appetite and reproductive function genes.\u003c/strong\u003e (a) Fold change in expression of appetite regulating genes: \u003cem\u003elepa\u003c/em\u003ein liver, \u003cem\u003elepr, npy \u003c/em\u003eand\u003cem\u003e cart\u003c/em\u003e in brain tissue as determined by qRT-PCR. Fold change in expression of reproductive functioning genes: \u003cem\u003egnrh, lh\u003c/em\u003e and \u003cem\u003efsh\u003c/em\u003e in brain (b) and \u003cem\u003ecyp19a1, esr1, hsd3b\u003c/em\u003e and \u003cem\u003enrg1\u003c/em\u003ein ovary (c). All values are expressed as Mean ± SEM; N=5. **, \u003cem\u003ep\u003c/em\u003e £ 0.01; ***, \u003cem\u003ep\u003c/em\u003e £ 0.001; ****, \u003cem\u003ep\u003c/em\u003e £ 0.0001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3860953/v1/128b28208774a503ae43fd7e.png"},{"id":49745899,"identity":"aa820d3f-b31a-4264-9508-d2a3c3198ac6","added_by":"auto","created_at":"2024-01-17 10:47:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":754730,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFolate deficiency impacts reproductive hormone parameters. \u003c/strong\u003e(a) Estimation of Melatonin in the brain. Estimation of plasma LH (b), FSH (c), Estradiol (d) and Testosterone (e) hormone. All values are expressed as Mean ± SEM; N=5. *, \u003cem\u003ep\u003c/em\u003e £ 0.05; FD, folate deprived.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3860953/v1/614836c4d5ffdcf9b0ff8c16.png"},{"id":49745901,"identity":"e7c72dfa-5b80-42ca-81ea-dce8186f7386","added_by":"auto","created_at":"2024-01-17 10:47:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":11636515,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneral view of the normal zebrafish ovary.\u003c/strong\u003e (a) Normal appearance of all stages of ovarian development along with normally surrounded zona radiata. Photomicrographs showing normal appearance of primary oocyte (PO) (stage I) and mature oocyte (MO) (stage IV) (b), cortical alveolus oocyte (stage II) (c) sand vitellogenic oocyte (VO) (stage III) (d). H\u0026amp;E, Haematoxylin and Eosin.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3860953/v1/f561dd54b711d1fa22edee47.png"},{"id":49746243,"identity":"f715f647-cd58-4da0-a198-0d44e51ce66d","added_by":"auto","created_at":"2024-01-17 10:55:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":11928527,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFolate insufficiency induced histopathological alterations in the ovary.\u003c/strong\u003e(a) Most of oocytes arrested in primary oocyte (APO) stage along with absence of mature stage. Detachment of cortical alveolar cell from the vitelline membrane (DCV) is also observed. (b) Oocytes arrested in primary oocyte (APO) stage. (c) Photomicrograph showing transformation of yolk granules into eosinophilic granular mass (EGM) as well as depletion of yolk granules (DYG). The view also shows disrupted vitelline membrane (DVM). (d) Infiltration of inflammatory cells (I) and proliferation of interstitial connective tissue (PICT). H\u0026amp;E, Haematoxylin and Eosin.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3860953/v1/64cb71827dd5a03a4d74fdcc.png"},{"id":49746242,"identity":"45e8b9cf-f427-406a-98e2-96abf9bb08e9","added_by":"auto","created_at":"2024-01-17 10:55:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":319476,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFolate deficiency induces oxidative stress in ovarian tissue. \u003c/strong\u003e(a) Estimation of LPO and (b) Catalase in the ovary. All values are expressed as Mean ± SEM; N=5. *, \u003cem\u003ep\u003c/em\u003e £ 0.05; FD, folate deprived.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3860953/v1/93870e0ae7c0d9f15940e300.png"},{"id":50230309,"identity":"cf8a9c17-2bab-416e-b36c-ab717b54f7d1","added_by":"auto","created_at":"2024-01-26 20:37:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5877025,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3860953/v1/06e7ebe8-6cb6-4974-b9f4-a6358edccd35.pdf"},{"id":49745897,"identity":"f5dc134d-e0f1-4137-bbc8-3afaab88b863","added_by":"auto","created_at":"2024-01-17 10:47:11","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":111752,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalfileforreview.docx","url":"https://assets-eu.researchsquare.com/files/rs-3860953/v1/d58fd54fe3cc3a519e4ae4ef.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Folate deficiency links impaired appetite routing to ovarian dysfunction","fulltext":[{"header":"Introduction","content":"\u003cp\u003eVitamins and minerals play vital roles in metabolic pathways that support fundamental cellular functions. In particular, essential vitamin such as folate due to its involvement in energy-yielding metabolism, ovarian stimulation and neuronal function makes its availability critical to maintain body physiology [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Folate is important in female reproduction for oocyte quality and maturation, implantation, placentation, fetal growth and organ development [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Beyond the familiar functional role of the brain, it plays critical roles in regulating female reproduction during puberty, gametogenesis and childbirth [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u0026ndash;[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEvidence suggest that brain impairments are associated with problems in appetite control, which are likely to contribute to weight changes, but the extent to which they associated with wider functional impacts is unclear [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In females, reproductive function and body weight control is largely mediated by the steroid sex hormones, estradiol and progesterone. Estradiol, in particular, by acting at the cortex, hypothalamus and brainstem level helps to regulate the body's appetite and energy use by reducing food intake and increasing energy expenditure [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Moreover, the estrogen receptors (ERs) and leptin receptors (LPs) are co-expressed in neurons within the areas known to coordinate metabolism and gonadal function [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Studies indicate direct interaction of estrogen and leptin signals at the level of STAT3 pathway [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Our previous study and literature suggest that folate deficiency is associated with mood changes and depression symptoms, which may affect appetite regulation through complex neurotransmitter interactions [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Depression can in turn impact basic functions like appetite, sleep, and mood due to its diverse effects [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This interplay of folate deficiency, depression, dysregulated leptin signaling and altered appetite presents a captivating pathway for elucidating their collective impact on steroidogenesis and the pathogenesis of ovarian dysfunction.\u003c/p\u003e \u003cp\u003eRegarding reproduction, literature majorly reflects upon the birth and developmental defects due to folate deficiency during pregnancy. It is very well known that folate supplementation has proved to be fruitful in preventing neonatal abnormalities [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. But the mechanism responsible for long-term changes in appetite under conditions of folate deficiency remains a topic of ongoing research. Furthermore, the nature of the involvement of folate deficiency in molecular and cellular reactions that translate into ovarian dysfunction is poorly understood. The ramifications of folate deficiency extend beyond gestational considerations, influencing not only brain neurotransmitter levels but also, inciting oxidative stress [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This can potentially disturb the intricate process of follicular development and ovarian function. With the exception of correlational studies, the lack of experimental data demonstrating the impact of folate deficiency on appetite and ovarian function is evident. In this context, our objective was to evaluate the association between folate deficiency, appetite regulation, hormone dynamics, and ovarian function. Close degree of similarity in reproductive regulation systems between human and zebrafish permits us to carry this research in zebrafish. Identification of important neurons involved in regulation of reproductive system, similar regulative hormones and responses in zebrafish as compared to humans adds to the robustness of this animal model to assess reproductive complications [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u0026ndash;[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e"},{"header":"Material and methodologies","content":"\u003cp\u003e \u003cstrong\u003eAnimal maintenance\u003c/strong\u003e \u003cp\u003eSexually matured healthy female adult Zebrafishes (\u003cem\u003eDanio rerio\u003c/em\u003e) (weighing 700\u0026ndash;800 mg) were utilised. Fishes were housed in 10 L aquarium tanks and water temperature was maintained at 27\u0026thinsp;\u0026plusmn;\u0026thinsp;2 ᵒC. Fishes were kept under a 14-hour light/10-hour dark cycle and fed with commercial fish food flakes 2 times a day.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eTreatment\u003c/strong\u003e \u003cp\u003eWe have previously developed a method to induce folate deprivation in zebrafish using methotrexate (MTX), a known DHFR antagonist, which alters the folate mediated one-carbon metabolism [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Fishes were administered methotrexate (MTX) dose (37.5 \u0026micro;g/gm bodyweight) intraperitonially for a time period of 15 days. Fishes were divided in control and folate deprived (FD) groups consisting of 5 individuals each.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eTissue collection\u003c/strong\u003e \u003cp\u003eAfter subjecting the fishes to a 15-day treatment with MTX, blood samples were collected from the tail vein using heparin-coated vials to obtain plasma. Subsequently, the fishes were euthanized by inducing hypothermia, and their brain, ovary, and liver were dissected for further analysis.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eqRT-PCR\u003c/strong\u003e \u003cp\u003eTotal RNA from the tissues were extracted with TRIzol reagent (Invitrogen) and cDNA was synthesized using the cDNA synthesis kit by Bio-Rad. qRT-PCR was carried out on cDNA using iTaq Universal SYBR Green Supermix by Bio-Rad. Primers for \u003cem\u003elepa\u003c/em\u003e, \u003cem\u003elepr\u003c/em\u003e, \u003cem\u003egnrh3\u003c/em\u003e, \u003cem\u003elhb\u003c/em\u003e, \u003cem\u003efshb\u003c/em\u003e, \u003cem\u003eesr1\u003c/em\u003e, \u003cem\u003ecyp19a\u003c/em\u003e, \u003cem\u003ehsd3b\u003c/em\u003e, \u003cem\u003enrg1\u003c/em\u003eand \u003cem\u003egapdh\u003c/em\u003e were designed using the Primer-BLAST tool NCBI. For internal control \u003cem\u003egapdh\u003c/em\u003e was used, and fold change in mRNA expression was analysed using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMelatonin estimation\u003c/strong\u003e \u003cp\u003eMelatonin level in the brain was determined using method described by [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Detailed procedure is provided in supplementary material.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSteroid hormone estimation\u003c/strong\u003e \u003cp\u003ePlasma Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) were measured via commercial Chemiluminescence Immunoassay (CLIA) kit (Elabscience). Plasma Estradiol and testosterone was measured using commercial enzyme linked fluorescence assay (ELFA) kit.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEstimation of Catalase activity\u003c/strong\u003e \u003cp\u003eCatalase activity was measured according to Sinha, 1972. Detailed procedure is provided in supplementary material.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEstimation of Lipid Peroxidation (LPO)\u003c/strong\u003e \u003cp\u003eLPO was measured by the estimation of malonaldehyde (MDA) level; according to [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Detailed procedure is provided in supplementary material.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eHistopathological studies\u003c/strong\u003e \u003cp\u003eDissected ovaries were washed with Phosphate Buffered saline (PBS) followed by fixation in 10% formalin. After fixation, the tissues were dehydrated using increasing concentrations of ethanol. The dehydrated ovary tissues were then embedded in paraffin (Fischer Scientific), and tissue sections of 5 \u0026micro;m thickness were obtained using a microtome. These sections were subsequently stained with Haematoxylin and Eosin stain. The stained tissue sections from five different ovaries were examined under a microscope to observe any histopathological changes.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eStatistical analysis\u003c/strong\u003e \u003cp\u003eData values were expressed as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Graphpad Prism software version 8.4.2 (GraphPad Software, Inc., La Jolla, CA, USA) was used to perform statistical data analysis. Student\u0026rsquo;s t-test was used to determine the statistical significance between experimental groups. Statistical significance was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.* denotes \u003cem\u003ep\u003c/em\u003e \u0026le; 0.05, ** denotes \u003cem\u003ep\u003c/em\u003e \u0026le; 0.01, *** denotes \u003cem\u003ep\u003c/em\u003e \u0026le; 0.001, **** denotes \u003cem\u003ep\u003c/em\u003e \u0026le; 0.0001.\u003c/p\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eDeficiency of folate leads to changes in the expression of genes related to appetite and reproductive function\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eIn our previous study we observed that folate deprivation induces depressive-like behaviours and alters the appetite of the zebrafish [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. We sought to evaluate if folate deficiency changes the transcript of appetite controlling genes. Gene expression analysis showed upregulation of \u003cem\u003elepa\u003c/em\u003e gene in the liver by 1.58-fold. The expression of leptin receptor, \u003cem\u003elepr\u003c/em\u003e, was found to be upregulated by over 15-fold in the brain. Whereas, the expression of Neuropeptide Y (\u003cem\u003enpy\u003c/em\u003e), an orexigenic factor, was found to be downregulated. Conversely, the expression of cocaine- and amphetamine-regulated transcript (\u003cem\u003ecart\u003c/em\u003e), an anorexigenic factor, was found to be upregulated in brain by over 4-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eWe further evaluated the expression of transcripts crucial for the reproductive functioning. It is evident from the gene expression study (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) that in the brain, the genes important for the synthesis of reproductive hormones such as gonadotropin releasing hormone (GnRH), luteinizing hormone (LH) and follicle stimulating hormone are downregulated. This suggests that when subjected to a folate-deficient environment, the overall levels of these hormones might be diminished, potentially leading to repercussions on ovarian function and the development of follicles. Subsequently, it was found that the genes such as \u003cem\u003ecyp19a\u003c/em\u003e, \u003cem\u003eesr1\u003c/em\u003e, \u003cem\u003ehsd3b\u003c/em\u003e and \u003cem\u003enrg1\u003c/em\u003e, which are required for proper ovarian function and follicular development are downregulated significantly in the ovary of folate deficient fishes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of hormones involved in reproductive function:\u003c/h2\u003e \u003cp\u003eGiven that ovulation is an outcome arising from intricate hormonal equilibrium and interplay, any alterations in these mechanisms has the potential to affect its physiological processes [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. It can be thus speculated that hormonal levels are a reflection of proper brain and ovarian function. We estimated melatonin levels in brain as it is known to play role in the regulation of many reproductive functions [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Upon estimation, the level of melatonin hormone was found to be elevated in the brain of folate deprived fishes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Furthermore, it was found that the plasma levels of LH (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) and FSH (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) were low in the folate deprived fishes, whereas the plasma levels of estradiol (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed) and testosterone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee) was elevated.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eFolate deficiency induces histopathological changes and oxidative stress in the ovary:\u003c/h2\u003e \u003cp\u003eApart from altered gene expression and hormonal changes, ovarian dysfunction can also be reflected in the form of morphological alterations. Zebrafish oocyte can be divided into four stages i.e., primary growth (PO), cortical alveolus (CO), vitellogenic (VO) and mature oocyte (MO) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Displayed in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e are the histological sections of zebrafish ovary. In the control group, all the stages of ovarian development along with normally surrounded zona radiata can be evidently observed. On the other hand, in folate deprived fishes most of the oocytes can be seen arrested in primary stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Mature oocytes are scarce and the yolk granules are depleted. Transformation of the yolk granules into eosinophilic granular mass can be observed as well in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec. The view also shows disrupted vitelline membrane (DVM). Furthermore, ovarian photomicrograph (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed) of folate deprived fishes shows infiltration of inflammatory cells and proliferation of interstitial connective tissue.\u003c/p\u003e \u003cp\u003eThe proliferation of interstitial connective tissue and possible implication of tissue scarring could be due to oxidative stress, as it is an important molecular mechanism underlying fibrosis in variety of organs [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Therefore, we estimated the activity of catalase enzyme and lipid peroxidation (LPO) in the ovarian tissue homogenate as elevated levels of these enzymes can represent oxidative stress in tissues [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This was done to evaluate the hypothesis that folate deficiency can induce oxidative stress in the ovaries, which could be one of the probable mechanisms for ovarian dysfunction. The LPO activity was found to be significantly elevated in the ovary of folate deprived fishes (Fig.\u0026nbsp;5a), whereas, although not statistically significant, the catalase activity was found to be elevated (Fig.\u0026nbsp;5b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFig: 5: Folate deficiency induces oxidative stress in ovarian tissue.\u003c/b\u003e (a) Estimation of LPO and (b) Catalase in the ovary. All values are expressed as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM; N\u0026thinsp;=\u0026thinsp;5. *, \u003cem\u003ep\u003c/em\u003e \u0026le; 0.05; FD, folate deprived.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe association between nutrition and reproduction has long been known to have crucial implications for the reproductive functioning. Inadequate nutrition can lead to depletion of body weight and overall physical condition, leading to a postponement in the onset of puberty [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Among various components of diet, folate is one the important vitamin implicated in reproductive health [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Nonetheless, there remains a substantial knowledge gap concerning the potential correlation that exists between folate and ovarian functionality, as well as an incomplete understanding of the potential consequences resulting from folate deprivation on ovarian function. In our previous study, we have documented that zebrafish deficient in folate, manifest symptoms reminiscent of depressive-like behavior. These manifestations are characterized by a discernible decrease in their appetite, alterations in neurotransmitter activity, and a notable decline in their overall body weight [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. We sought to evaluate if folate deficiency affected appetite has any possible implications on the ovarian reproductive dynamics.\u003c/p\u003e \u003cp\u003eTo develop folate insufficient model, adult female zebrafishes were injected with methotrexate (MTX), a folate antagonist, which works by inhibiting DHFR enzyme (EC 1.5.1.3.) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Transcript expression study revealed a significant upregulation of \u003cem\u003elepa\u003c/em\u003e gene in the liver. \u003cem\u003elepa\u003c/em\u003e gene encodes for leptin hormone, which is crucial for regulation of body weight, appetite and metabolism and is important for reproductive system [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Leptin hormone is known to supress food intake and thereby induces weight loss [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In zebrafish, \u003cem\u003elepa\u003c/em\u003e gene is predominantly expressed in the liver [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Elevated serum level of leptin hormone is reported in PCOS patients as well [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Furthermore, transcript expression of \u003cem\u003elepr\u003c/em\u003e was found to be elevated in the brain of folate deficient zebrafishes. \u003cem\u003elepr\u003c/em\u003e gene encodes for leptin receptor and in zebrafish, its expression is reported to be strongest in the brain [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Apart from \u003cem\u003elepr\u003c/em\u003e transcript expression, \u003cem\u003enpy\u003c/em\u003e and \u003cem\u003ecart\u003c/em\u003e transcript expression was found to be altered in brain. Neuropeptide Y (NPY) is a potent orexigenic peptide found in the brain which stimulates food intake [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In contrast, cocaine- and amphetamine-regulated transcript (CART) is an anorexic peptide, which reduces appetite, resulting in weight loss [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Subsequently, we have reported a significant \u003cem\u003enpy\u003c/em\u003e downregulation and a significant \u003cem\u003ecart\u003c/em\u003e upregulation in the brain of folate deficient zebrafish. These results suggest that folate deficiency alters the expression of these appetite controlling genes through an unknown mechanism. This explains why folate deficient zebrafishes have reduced appetite and body weight.\u003c/p\u003e \u003cp\u003eThe reproductive axis is closely associated with nutritional status. The limited availability of nutrients, leading to declined energy reserves, can alter many finely tuned crosstalk between energy metabolism and reproduction, thereby affecting female fertility [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Not just malnutrition, but eating disorders can also affect the reproductive function [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. It can be thus postulated that altered leptin signalling, reduced appetite and subsequent weight loss affects the neuroendocrine function of the brain. To validate our speculation, we studied the transcript expression of \u003cem\u003egnrh\u003c/em\u003e, \u003cem\u003elh\u003c/em\u003e and \u003cem\u003efsh\u003c/em\u003e. The expression of these transcripts was significantly downregulated in folate deficient zebrafishes. Gonadotropin releasing hormone (GnRH) is crucial for the synthesis and secretion of luteinizing hormone (LH) and follicular stimulation hormone (FSH) in the brain [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Decreased expression of \u003cem\u003egnrh\u003c/em\u003e, \u003cem\u003elh\u003c/em\u003e and \u003cem\u003efsh\u003c/em\u003e is also reflected in the plasma of fishes, where a significant downregulation of plasma LH and FSH hormone was estimated. Additionally, we observed significant elevation of Melatonin hormone in the folate deficient fish brain. Melatonin is known to play role in the regulation of many reproductive functions including oocyte maturation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Elevated levels of melatonin in people with eating disorder such as anorexia nervosa has been previously reported as well [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], suggesting a correlation between melatonin and folate deprivation induced appetite changes. The impact of melatonin on the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) has generated divergent findings across various studies, highlighting the dual role of melatonin which needs to be characterised further [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Moreover, it has been observed that women grappling with reproductive disorders like Polycystic Ovary Syndrome (PCOS) exhibit notably elevated levels of melatonin within their serum. This intriguing correlation has led to the proposition of elevated melatonin levels potentially serving as a promising biomarker for prediction of PCOS [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdditionally, we report elevation of plasma estradiol and total testosterone in folate deprived fishes. Proper levels of these hormones are instrumental for the normal functioning of female reproductive system [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Ovarian steroidogenesis is known to be affected by dyslipidaemia in mice [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Moreover, from previous studies we know that condition such as PCOS displays altered lipid profiles, which includes reduced high-density lipoprotein cholesterol (HDL-C), elevated triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C), along with significantly increased lipoprotein levels [\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u0026ndash;[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Upon evaluating the hepatic cholesterol, triglyceride and HDL-cholesterol we observed that cholesterol and triglyceride levels were elevated, whereas the HDL-cholesterol levels were decreased (data provided in supplementary). Although not conclusive, these results suggest the possibility of dyslipidaemia under folate deprived condition which alters steroidogenesis and could potentially lead to ovarian dysfunction. Even the Rotterdam guidelines suggest measure of HDL-cholesterol and triglyceride for evaluation of metabolic syndromes [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe further looked into the functioning of the ovary under folate deficient conditions by the gene transcript study. It is evident from the results that the expression of \u003cem\u003ecyp19a\u003c/em\u003e, \u003cem\u003eesr1\u003c/em\u003e, \u003cem\u003ehsd3b\u003c/em\u003e and \u003cem\u003enrg1\u003c/em\u003e is significantly downregulated in the ovarian tissue. Women with PCOS are reported to have decreased expression of CYP19A1 (aromatase) mRNA, which contributes to an increase in testosterone level [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. 3β-HSDs catalyse the conversion of pregnenolone, 17α-hydroxy pregnenolone, and dehydroepiandrosterone to progesterone [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] and its downregulation can be explained by diminished FSH levels as previous study has reported decreased expression in 3βHSD mRNA, possibly due to the downregulation of FSH hormone [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Neuregulin-1 (NRG1) is a member of the EGF-like factor family, and is important for oocyte maturation [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. NRG1 is known to be induced by LH in mice [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] and this could be the possible reason of its low expression in zebrafish ovary.\u003c/p\u003e \u003cp\u003eThe histological assessment of the control ovary shows follicles at various stages of development (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), illustrating a well-orchestrated progression. In contrast, upon examining ovarian histology under folate deprivation, a distinct pattern emerges as clearly depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Notably, maturation of oocytes seems to be affected, as a substantial number of follicles are arrested in primary stages. Concomitantly, a discernible infiltration of inflammatory cells, proliferation of interstitial connective tissue and scarring is observed in the ovarian histology. We have previously demonstrated that folate deficiency can in fact, cause systemic inflammation. Scarring of the ovarian tissue under folate deprivation can partly be explained by oxidative stress. Oxidative stress plays a crucial role as a contributing factor in the initiation of follicular atresia and the process of ovarian aging. This oxidative burden ultimately gives rise to a decline in the reproductive potential of females [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. To mitigate the elevated levels of oxidants and prevent oxidative stress, tissues produce enzymes such as catalase and lipid peroxidase, thus elevated levels of these enzymes can represent oxidative stress in tissues [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. We report elevation of catalase activity and a significant elevation of lipid peroxidation activity in the ovary under folate deprived conditions.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe incurable nature of ovarian dysfunction reinforces the importance of understanding the molecular mechanisms behind folate deficiency induced ovarian dysfunction.\u0026nbsp;We have observed altered expression of key genes involved in appetite regulation (\u003cem\u003elepa\u003c/em\u003e, \u003cem\u003elepr\u003c/em\u003e, \u003cem\u003ecart\u003c/em\u003e and \u003cem\u003enpy\u003c/em\u003e) and reproductive function (\u003cem\u003egnrh\u003c/em\u003e, \u003cem\u003elh\u003c/em\u003e, \u003cem\u003efsh, cyp19a, esr1, hsd3b and nrg1\u003c/em\u003e). Furthermore, we have documented histological changes in the ovary, including follicular arrest, infiltration of inflammatory cells, and proliferation of interstitial connective tissue. Our study implies the potential role of leptin and melatonin in mediating the effects of folate deficiency on ovarian function. Further research is needed to elucidate the underlying mechanisms and to determine the long-term consequences of folate deficiency on ovarian health and fertility.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors would like to express their gratitude to DBT-MSUB-ILSPARE Project funded Dr. Vikram Sarabhai Institute for Cell and Molecular Biology, The Maharaja Sayajirao University of Baroda for necessary lab facilities.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by the seed grant of Research \u0026amp; Consultancy Cell, The Maharaja Sayajirao University of Baroda and the Department of Biotechnology (DBT), Government of India for the M.Sc. Biotechnology teaching Program.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimals were maintained and handled as per the guidelines of \u0026ldquo;Committee for Control and Supervision of Experiment on Animals (CCSEA)\u0026rdquo;. Animals were housed in the animal house facility of the Department of Zoology, The Maharaja Sayajirao University of Baroda (Reg. No. 827/GO/Re/S/04/CPCSEA). Experimental protocol was approved by Institutional Animal Ethics Committee of the Department of Zoology, Faculty of Science, The Maharaja Sayajirao University of Baroda (MSU-Z/IAEC07/08-2023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this article. Further enquiries can be directed to the corresponding author. Supplementary data is available at the repository which can be accessed by the following DOI: https://doi.org/10.17632/bhmthf6vhd.1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAfridi Shaikh:\u003c/strong\u003e Methodology, Formal analysis, Investigation, Writing: original draft. \u003cstrong\u003eBharti:\u003c/strong\u003e Methodology, Formal analysis, Investigation. \u003cstrong\u003eMukund Chattpar:\u003c/strong\u003e Methodology, Formal analysis, Investigation. \u003cstrong\u003eDhaval Fefar:\u003c/strong\u003e Histological analysis. \u003cstrong\u003eHetal Roy:\u003c/strong\u003e Conceptualization, Resources, Supervision, Validation, Writing: review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.S. - Methodology, Formal analysis, Investigation, Writing: original draft. B.C. - Methodology, Formal analysis, Investigation. M.C. - Methodology, Formal analysis, Investigation. D.F. - Histological analysis. H.R. - Conceptualization, Resources, Supervision, Validation, Writing: review and editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTardy A-L, Pouteau E, Marquez D, Yilmaz C, Scholey A (Jan. 2020) Vitamins and Minerals for Energy, Fatigue and Cognition: A Narrative Review of the Biochemical and Clinical Evidence. 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Poult Sci 101(7):101891. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.PSJ.2022.101891\u003c/span\u003e\u003cspan address=\"10.1016/J.PSJ.2022.101891\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","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":"Folate deficiency, Appetite, Leptin, Hormones, Ovarian dysfunction","lastPublishedDoi":"10.21203/rs.3.rs-3860953/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3860953/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe long-established link between nutrition and reproduction is known to have critical consequences for reproductive function. However, the availability of experimental data on effect of folate deficiency on ovarian health is scarce and uncertain. Our objective was to establish a proof for association between folate deficiency, hormone dynamics, and health of the ovary through in vivo model organism. Folate-deprived female zebrafishes were developed using intraperitoneal administration of methotrexate (MTX) and they were used to study the possible implications of folate deprivation on ovarian health. Changes in the expression of transcripts regulating appetite and ovarian function was assessed by qRT-PCR. ELISA based methods were utilised to quantify and evaluate changes in hormone levels regulating reproductive function. Histology of ovarian tissue was performed to support the study. Folate deprivation resulted in impaired appetite behaviour and alters its regulatory gene expression. Due to folate deficiency, the neuroendocrine function of the brain was affected that resulted in altered reproductive hormone levels. Histological parameter of ovary was performed wherein the follicles are arrested in primary oocyte stage and abundance of scarring of tissue is seen. Furthermore, elevated lipid peroxidation and catalase enzyme activity indicates folate deficiency induced oxidative stress in ovary as one of the responsible mechanisms to aide ovarian dysfunction. Our study provides experiment proof with in vivo folate deficient model of fish that suggests B9 non-availability resulted into loss of appetite and, female gonadal dysfunction, which developed as cumulative effect of deficiency and altered appetite. The intricate interplay between folate deficiency and appetite along with the consequential implications for the synthesis and release of female reproductive hormones, warrant a thorough and in-depth exploration through further research.\u003c/p\u003e","manuscriptTitle":"Folate deficiency links impaired appetite routing to ovarian dysfunction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-17 10:47:06","doi":"10.21203/rs.3.rs-3860953/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":"5be31665-0ea9-4519-ad2d-d51ca7cbed28","owner":[],"postedDate":"January 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-26T20:29:13+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-17 10:47:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3860953","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3860953","identity":"rs-3860953","version":["v1"]},"buildId":"CiT4i_kKBbxQbnFL0ufpk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}