Cypermethrin exposure during perinatal period affects fetal development and impairs reproductive functions of F1 female rats

Journal Pre-proof Cypermethrin exposure during perinatal period affects fetal development and impairs reproductive functions of F1 female rats

Dipty Singh, Delna Irani, Sharad Bhagat, Geeta Vanage PII:

S0048-9697(19)35940-6

DOI:

https://doi.org/10.1016/j.scitotenv.2019.135945

Reference:

STOTEN 135945

To appear in:

Science of the Total Environment

Received date:

12 September 2019

Revised date:

15 November 2019

Accepted date:

3 December 2019

Please cite this article as: D. Singh, D. Irani, S. Bhagat, et al., Cypermethrin exposure during perinatal period affects fetal development and impairs reproductive functions of F1 female rats, Science of the Total Environment (2019), https://doi.org/10.1016/ j.scitotenv.2019.135945

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© 2019 Published by Elsevier.

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Cypermethrin exposure during perinatal period affects fetal development and impairs reproductive functions of F1 female rats Dipty Singh1*, Delna Irani1, Sharad Bhagat2, Geeta Vanage3* 1. Department of Neuroendocrinology, ICMR-National Institute for Research in Reproductive Health, Parel, Mumbai, 400012, India

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Reproductive Health, Parel, Mumbai, 400012, India

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2. Department of Biochemistry and Virology, ICMR-National Institute for Research in

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3. Department of Preclinical Reproductive and Genetic Toxicology, ICMR-National Institute for

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Research in Reproductive Health, Parel, Mumbai, 400012, India

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*Corresponding authors Dr. Dipty Singh

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Dr. GeetaVanage

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Email: [email protected]; [email protected]

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Email: [email protected]

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Abstract: Cypermethrin (CYP) is a ubiquitously present synthetic pyrethroid insecticide. It has endocrine disrupting activitieswhich may adversely affect reproductive development and functions of offspring if exposed during critical developmental period. Thepresent study was undertaken to delineate the effects of CYP exposure in pregnant female rats during perinatalperiodon the

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sexual maturation, hormonal regulation, reproductive development and fertility of F1 female

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offspring and its molecular mechanism of action. Pregnant rats (F0) were gavaged daily with 0,

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1, 10, 25 mg/kg bw/day CYP and 10 µg/kg bw/day Diethylestilbestrol (DES; positive control)

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from gestation day 6 to postnatal day 21. The reproductive development and function parameters

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were evaluated at postnatal days 45 and 75. Reduced body weight, delayed vaginal opening, and disrupted estrous cyclicity were observed at 25 mg/kg CYP dose. CYP exposure significantly

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affected the reproductive organ development and their functions at all doses. Significant

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alterations in ovarian and uterine histology such as luteinization, reduction of primordial follicular reserves, presence of multi-oocyte follicles and thin degenerative luminal and glandular

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uterine epithelium were observed at adulthood. Altered circulatory steroid hormone levels and expression of ovarian and uterine steroid hormone receptorswere observed at PND 75 in the F1 female offspring. Expression of HOXA10 and α-SMA which are important for uterine integrity and functions, were found to be altered at PND 75. Increased pre-implantation loss (PIL%), postimplantation loss (POL%), and reduced litter size in F1 females when cohabitated with unexposed fertile male rats were observed. Overall, perinatal exposure of pregnant rats to CYP led to significant long lasting effects on the reproductive functions of F1 female offspring. The adverse effects were passed on to F2 generation via female germ line and posed developmental

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Journal Pre-proof anomalies. The present finding necessitates additional molecular studies to understand its transgenerational mechanism of action via female germline. Keywords: Cypermethrin, Endocrine disruptor, Fertility, Reproductive toxicant, Vaginal

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opening, Estrus cyclicity, Ovarian and uterine dysfunction

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Journal Pre-proof 1. Introduction Synthetic pyrethroids are widely being used as less hazardous alternative to organophosphorus, organochlorines and carbamates. Cypermethrin (CYP) is one such pyrethroid which is commonly being applied in households and in agriculture as an insecticide (Solati et al., 2010). Cypermethrin induces neurotoxicity by prolonged opening of sodium channel, leading to hyperexcitation of the central nervous system. It also modulates chloride, voltage-gated calcium and

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potassium channels which in turn induces DNA damage and oxidative stress (Kumar Singh et al.,

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2012). CYP has been identified as an endocrine disruptor with previous studies documenting it to

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have anti-androgenic as well as estrogenic effects which might elicit unintended effects in non-

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target organisms (Chen et al., 2003; McCarthy et al., 2006; Du et al., 2010).

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In animal models, the reproductive effects of CYP exposure were majorly studied on males such

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as poor sperm quality, sperm DNA damage, reproductive hormone alterations, decreased weight of testosterone sensitive organs etc. (Wang et al., 2009; Solati et al., 2010;Li et al., 2013; Huang

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and Li, 2014; Sharma et al., 2014; Marettova et al., 2017). Our previous report also demonstrated

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for the first time that perinatal CYP exposure affects sexual maturation of F1 males even at low concentration and leads to teratogenic and embryotoxic effects in F2 offspring (Singh et al., 2017).

The effects of CYP on female reproductive functions were less often studied possibly due tocomplexity of female reproductive systemwhich involves a series of interdependent and interrelated functions. However, few previous animal studies have shown the effect of CYP on female reproduction. CYP exposed (50 mg/kg bw) adult albino female rats displayed altered 4

Journal Pre-proof body weight, organ weight and thickened myometrium compared to the controls. Degenerative ovarian changes were also reported such as increased follicular atresia and decreased concentration of proteins, lipids, phospholipids and cholesterol in adult CYP exposure (Sangha et al., 2011; Sangha et al., 2013). Fetal derangements were observed from pregnant albino mice treated with different doses of CYP during gestation periods along with pre, peri and postimplantational losses (Raees et al., 2010). A recent study on adult exposure of -CYP in female

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albino rats demonstrated disrupted estrous cycles and alteredreproductive hormone levels

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(Obinna and Kagbo, 2017). The low-dose β-CYP exposure during adulthood showed an adverse

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effects on the uterine endometrium and the embryo implantation in adult mice whose rate was

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found to be decreased along with reduced serum estradiol levels and HOXA10 expression in the

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uterus (Zhou et al., 2018a; Zhou et al., 2018b).

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So far, adult exposure to CYP has been majorly studied and very few reports are available exploring the effects of its exposure during the critical window of development such as the

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gestational and lactation period. Due to its lipophilicity, CYP may accumulate in body fat and

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various other organs such as skin, liver, kidneys, adrenal glands, ovaries and brain (Crawford et al., 1981; Tian et al., 2008; Soliman et al., 2015). Also, residues of CYP have been reported in human breast milk in a study conducted in the countries of Brazil, Columbia and Spain which measured different types of pyrethroids (Corcellas et al., 2012). This indicates infant exposure to CYP while breast-feeding and in utero.

Currently, information on reproductive development and fertility status of perinatally CYP exposed (encompassing gestation and lactation period) F1 female offspring and whether any

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Journal Pre-proof effects can be possibly transmitted via female germline to F2 generation is scarce. In this view, the present study delineates the effects of CYP exposure to pregnant female rats (F0) during perinatal period on the fetal development (F1 and F2) and reproductive functions of F1 female offspring. The various reproductive parameters have been studied at peri-pubertal and adult stages including fertility status of CYP exposed F1 females.

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2. Materials and methods

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2.1 Chemicals and reagents

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CYP was a kind gift from GHARDA chemicals (95% pure; 50% cis and 50% trans isomers;

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technical grade). Diethylstilbestrol (DES; 99% purity) and corn oil was procured from Sigma Chemical Co., St. Louis, MO. Testosterone, Progesterone and Estradiol ELISA kits were

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obtained from DiagnosticBiochem Canada Inc. TRIzol reagent and SuperScript III First-strand

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cDNA synthesis kit were procured from Invitrogen. Brilliant II SYBR Green qPCR Master Mix

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2.2 Dose preparation

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was procured from Agilent, USA.

Stock solution of CYP (100 mg/ml) was prepared in corn oil and desired concentrations viz. 1 mg/kg bw, 10 mg/kg bw and 25 mg/kg bw were prepared by further dilution with same solvent. DES was dissolved in ethanol (99% pure) and further diluted in corn oil to acquire the desired concentration. The content of alcohol in final solution was kept below 0.15%. Control groups were orally gavaged with only corn oil. These dose solutions were kept at 37°C overnight in brown colored bottles and later stored at room temperature for oral dosing.

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Journal Pre-proof 2.3 Animal care and maintenance Holtzman strain rats were used for this study. Male and female rats (9-10 weeks old and weighing ~250 g) were randomly bred in the animal house of the National Institute for Research in Reproductive Health Mumbai (ICMR-NIRRH), Mumbai, India. The animals were kept in polypropylene cages with sterile paddy husk, temperature (23 ± 1 °C), humidity (55 ± 5%), and dark cycle (14 h light/10 h). The animals were kept on ad libitum diet of soy-free rat pellets

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(containing fiber, crude protein and nitrogen-free extract) and water (purified by UV and reverse

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osmosis) and with their quality being routinely monitored by qualitative and quantitative

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proximal analysis. The study protocol was approved by the Institutional Animal Ethics

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Committee (IAEC approval No. 11/13) ICMR-NIRRH. The studies were carried out in agreement with committee for the purpose of control and supervision of experimental animals

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2.4 Experimental design

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(CPCSEA), India guidelines.

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Proven fertile male rats were cohabitated with proestrus female rats in the ratio 1:2 respectively.

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Females showing sperm positive vaginal smear were considered pregnant with day 0 of gestation. The copulated dams (F0) were randomly dispersed into the five groups (10 rats/group) such as: Group 1- vehicle control (corn oil); Group 2- CYP 1 mg/kg bw/day; Group 3- CYP 10 mg/kg bw/day; Group 4- CYP 25 mg/kg bw/day; Group 5- DES 10 µg/kg bw/day (positive control).

The F0 pregnant rats were treated with their respective dosage by gavaging once daily (as per body weight changes) from gestation day 6 (GD 6) till postnatal day 21 (PND 21). The F1 litters

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Journal Pre-proof were weighed and sexed based on their anogenital distance (AGD) post parturition. The F1 female offspring (adjusted 4-5 females/litter) were kept with the F0 lactating mothers until weaning. Thereafter, all pups were weaned on PND 22 and group-housed unisexually. Body weights of the dams and pups were monitored at fixed time intervals during the experimental period.

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By CO2 asphyxiation, randomly selected F1 female rats were sacrificed at different time points:

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PND 22 (juvenile), PND 45 (pre-pubertal) and PND 75 (adult stage). Prior to sacrificing, blood

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was collected via the retro orbital route from which further serum was separated and stored at -

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20°C for hormone analysis. The reproductive and vital organs were also dissected out, weighed and stored in 10% neutral buffered formalin (NBF). Furthermore, for real time PCR analysis,

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ovaries and uterus were snap frozen in liquid nitrogen.

2.5 Sexual maturation

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Two physiological components of sexual maturation in female rats such as vaginal opening (VO)

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(PND 35-42) and estrus cycle following first estrus were evaluated. The CYP treated F1 female pups were monitored for VO from PND 30 onwards. Onset of estrus cycle was monitored by taking vaginal fluid by instilling 10 µl 0.9% saline in the vagina and subsequent aspiration. The fluid smears were prepared on slides and viewed under Zeiss Axioskop40 microscope (Oberkochen, Germany).

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Journal Pre-proof 2.6 Cyclicity The estrous cyclicity of F1 female rats was assessed according to the type of cells viewed in the vaginal smears prepared on glass slide. The 3 consecutive estrus cycles were monitored after first estrous detection. The estrous cycle phases were detected and noted by cytological characteristics such as: Proestrus marked by predominance of nucleated epithelial cells, estrus marked by predominance of cornified epithelial cells and diestrus marked by predominance of

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leukocytes. Frequency of each phase and estrous cycle length were calculated using the data

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obtained.

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2.7 Hormone analysis

Serum samples collected at PND 45 and 75 were used to estimate the levels of serum

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progesterone, estradiol and testosterone in the F1 female offspring using an enzyme-linked

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immunosorbent assay (ELISA) kit as per the manufacturer’s protocol (Diagnostic Biochem

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Canada Inc) and was assessed using ELISA plate reader (µQuant, Biotek Instruments Inc, USA).

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2.8 RNA extraction, cDNA preparation and real time PCR for quantification gene

Total RNA was extracted from ovary and uterus samples of F1 females (collected at PND 75) using TRIzol reagent and was given Dnase I treatment (Thermo Fisher Scientific, USA). cDNA synthesis was carried out using Superscript III First-strand synthesis system for RT-PCR (Thermo Fisher Scientific, USA). The total RNA concentration obtained was approximately 100 µg/ml after Dnase I treatment and cDNA synthesis was carried out as per the kit’s protocol. RTPCR was performed in the iCycler real-time PCR system (Bio-Rad Laboratories) using Brilliant

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Journal Pre-proof III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies, USA). The 20 µl reaction mixture consisted of SYBR Green, template cDNA, respective primers and DEPC-treated water. The following RT-PCR program was used: initial denaturation for 3 min at 95˚C, followed by 45 cycles of denaturation at 95˚C for 15s, primer annealing (Table 1) for 30s and extension at 72˚C for 40s with final extension step at 72˚C for 5 min. The specificity of primer amplification was confirmed by DNA melting curve analysis and followed by checking amplicon size by running

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the products on 1.5% agarose gel electrophoresis. The iCycler software by Bio-Rad was used to

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obtain the standard curve and generate mean Ct values for each experiment set. The difference of

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the Ct valuesof the target gene and reference gene (18S) was calculated as ΔCt. The difference between the control and experimental group was then obtained as ΔΔCt = [ΔCt (experimental) -

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ΔCt (control)]. The n-fold differential expression was compared with control for all target genes

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and was expressed as 2^ΔΔCt relative expression with respect to 18S. The gene expression level

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of each sample was quantified as the mean of triplicate RT-PCR experiments (2 sets).

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2.9 Histological examination of ovary and uterus

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The 10% neutral buffered formalin fixed ovary and uterus tissues (PND 75) were chopped into 5 mm thick tissue pieces which were then refixed in fresh fixative for another 24 h. Using automatic tissue processor (Leica ASP 200, Germany), the tissues were further processed. The sections (5 µm) of paraffin embedded tissue blocks were placed on glass slide and were subjected to Hematoxylin and Eosin (Sigma, USA) staining. Serial sectioning was done in case of ovaries to carry out follicular counting. The Hematoxylin and Eosin stained sections were visualized on a Zeiss Axioskop40 photomicroscope (Oberkochen, Germany) and images were captured using a Zeiss Axiocam MRC camera (Oberkochen, Germany).

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2.10 Counting of ovarian follicles Hematoxylin and eosin stained ovarian sections of F1 female rats (PND 75; n = 3/group) were used to count different type of ovarian follicles; namely, primordial, primary, secondary, tertiary, graafian and atretic follicles. The corpora lutea were also counted. The serial sections were used

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for the counting as per the previously reported protocol (Picut et al., 2014).

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2.11 Fertility assessment of cypermethrin exposed F1 female rats

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The fertility assessment was performed on randomly selected perinatally CYP exposed F1

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females at PND 75 (n = 6) by cohabitating them with unexposed fertile adult males (1 male: 2 females). The vagina was checked once daily in the morning for copulation plug and copulation

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was confirmed by presence of sperm in the vaginal smears. Time taken for copulation for each

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pair was also noted. At GD 20, copulated females were sacrificed and the ovaries and uterine horns were dissected out. The uterine horns were viewed for live and resorbed (dead) fetuses (F2

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generation), the number of implantation sites (IS). The ovaries were examined for the number of

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corpora lutea (CLs). The copulation index, PIL% and POL % were calculated for the control and different dose groups. The F2 fetuses were examinedfor crown-rump length (CRL), weight and other physical anomalies. Developmental effects were also studied for F1 fetuses which were exposed to CYP during GD 6 to GD 19.

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Journal Pre-proof 2.12 Statistical analysis The data was analyzed using the GraphPad Prism™ software version 5.0 (San Diego, USA) and represented as the mean ± SD. Significance of the findings were analyzed using one way ANOVA with Bonferroni's post-test wherelevels of significance was considered at P ≤ 0.05.

3. Results

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3.1 Body and organ weights of F1 female offspring

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CYP and DES treated dams (F0) displayed no dose related mortality and no significant

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difference in the gestational and lactational body weights as compared to control group. Also, no

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significant change was observed in the relative weights of vital organs of the CYP treated dams compared to the control. However, the steroidogenic organs viz. ovaries and adrenal gland of 25

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mg CYP group dams did show a significant decrease in their relative weights as compared to

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treatment.

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control (data not shown). No mortality was observed in the CYP and DES treated dams during

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The body weight of F1 pups did not show any group-wise variation from PND 0 to PND 4. However, 25 mg CYP and DES group female pups displayed significantly decreased body weights from PND 7 which persisted till PND 75 as compared to control pups. 1 mg and 10 mg CYP groups did not display any effects on body weights as compared to control (Fig 1).

The relative vital organ weights seemed to remain unchanged in all treatment groups as compared to the controls at all studied time points. Nonetheless, the spleen and adrenal gland of the F1 females of all treatments groups exhibited significant decrease in relative weight at PND

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Journal Pre-proof 22 compared to control. Decreased relative weight of the adrenal gland persisted till PND 75 in the 25 mg CYP group whereas the spleen weights of all treatment groups appeared comparable to the control at PND 45 and 75. The relative weights of ovary and uterus organs of F1 females exhibited changes in the CYP and DES groups. A decrease in the relative ovary weights compared to control was observed in the 10 mg, 25 mg CYP and DES groups at PND 22 and this effect was persistently observed till adulthood (PND 75) only in the 25 mg CYP and DES

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groups. Decreased relative uterine weights were noted only in the 25 mg CYP group; with effect

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persisting till adulthood. 1 mg CYP group seemed to exhibit no effects on its ovarian and uterine

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weights (Table 2). No mortality was observed in the CYP and DES treated F1 female rats till

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sacrifice.

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3.2 Sexual maturation and estrous cyclicity of F1 female offspring

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Delayed vaginal opening (VO) was observed in F1 females of all treatment groups as compared to control however, the results were found significant in only 25 mg CYP and DES groups. In

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control group, VO was observed between PND 38 to 40 whereas in 1 mg and 10 mg CYP groups

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VO varied between PND 38 to 47. 25 mg CYP and DES groups exhibited VO between PND 43 to 46 and PND 43 to 48 respectively (Fig. 2A).

A disrupted estrous cycle was noted in all F1 females of the treatment groups as compared to control. However, it was only the 25 mg CYP group F1 females which showed a significant reduction in the estrous cycle length compared to the control group (Fig. 2B). In case of 1 mg and 10 mg CYP groups, there was anon-significant decrease in the length of diestrus and estrus phase and a non-significant increase in case of their proestrus phase. The DES group showed a

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Journal Pre-proof non-significant increase in diestrus and proestrus phase and a decrease in estrus phase. The 25 mg CYP group displayed a non-significantly reduced diestrus, significantly reduced proestrus and significant prolonged estrus days (Fig. 2C).

3.3 Histological changes in ovary and uterus of F1 female offspring On histological examination at PND 75, all the treatment group ovaries presented with larger and

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greater number of corpora lutea as compared to the control group (Fig. 3A a, b, c, d, e). Ovarian

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defects such as multi-oocyte follicles (MOFs) were observed in the 10 mg and 25 mg CYP

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groups as well as in the DES group (Fig. 3A c, d, e, i, j, m, n, o). Control and 1 mg CYP group

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showed no such defects (Fig. 3A a, b). Furthermore, the ovarian follicles at different development stages were counted at PND 75. The CYP and DES treated F1 female rats showed

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altered follicular counts. Decrease in the primordial follicular reserves was observed in all the

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treatment groups in comparison to the control. The number of atretic follicles also seemed to be more in the treatment groups with the 1 mg CYP and DES groups displaying much higher atresia

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(Fig. 3A g, i). Also, fewer mature ovarian graafian follicles were noted in the 10 mg, 25 mg CYP

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and DES groups. Large corpora lutea were mainly observed in 1 mg CYP and DES group ovaries and their numbers were much higher too compared to the control group (Fig. 3A b, e).

The control F1 females showed normal uterine histology with regular lumen with intact luminal epithelium, large number of glands having healthy glandular epithelium (Fig. 3B a, b, c).The 25 mg CYP group uterine histology displayed irregular uterine lumen (Fig. 3B e) with incomplete luminal epithelium. Also, there was a decrease in the number of uterine glands and thinner

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3.4 Ovarian follicle counts The ovarian follicles at different development stages were counted at PND 75. The CYP and DES treated F1 female rats showed altered follicular counts. Decrease in the primordial follicular

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reserves was observed in all the treatment groups in comparison to the control whereas only the 1

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mg CYP group displayed significantly decreased primary follicular counts. Secondary and

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tertiary follicle counts were observed to be lower in all CYP and DES groups. Also, fewer

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mature ovarian graafian follicles were noted in the 10 mg, 25 mg CYP and DES groups. An increase in atretic follicular counts was observed in the 10 mg and 25 mg CYP and DES groups.

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Large and increased corpora lutea were mainly observed in 1 mg CYP and DES group ovaries

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to the control group (Fig. 4).

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whereas the 10 mg and 25 mg CYP group displayed lower number of corpora lutea as compared

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3.5 Serum hormone concentrations of F1 female offspring Perinatal CYP exposure affected the hormone homeostasis of F1 females which was observed at the peri-pubertal and adult stages. At PND 45, the serum estradiol levels seemed to have decreased in case of all CYP groups, except the 10 mg CYP group as compared to the control group (Fig. 5A). The opposite was observed at PND 75, where there was an increase in all CYP groups except the 10 mg CYP group. The DES group also displayed decreased estradiol levels at PND 45 and increased levels at PND 75 as compared to the control group. The 10 mg CYP group showed least affected estradiol levels and was comparable to the control group (Fig. 5A).

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The serum progesterone levels seemed to be altered at both PND 45 and 75 in the CYP treated groups and DES. At PND 45, the 1 mg CYP group displayed comparable levels of serum progesterone to control; whereas, 10 mg CYP group showed a non-significant decrease while 25 mg CYP and DES groups displayed a significant decrease as compared to the control group. At PND 75, the 1 mg CYP group displayed significantly higher levels of the serum progesterone

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levels whereas non-significant increased levels were observed in the 10 mg CYP and DES

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progesterone levels as compared to control (Fig. 5B).

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groups as compared to control. The 25 mg CYP group displayed non-significantly lower

In case of the serum testosterone levels, at PND 45 the 1 mg CYP and DES groups showed

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significantly reduced levels as compared to control and 25 mg CYP group displayed a non-

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significant increase in the serum testosterone levels. At PND 75, the 1mg and 25 mg CYP group exhibited significantly increased serum testosterone levels. DES group displayed non-significant

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increased levels as compared to control. However, 10 mg CYP group’s serum testosterone levels

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remained comparable to control at both PND 45 and PND 75 (Fig. 5C).

3.6 mRNA expression levels of steroid receptors of F1 female offspring The ovarian AR, PR, ERα and ERβ mRNA expression levels were studied at PND 75 in F1 female offspring and were found to be altered in case of all the CYP groups and as well as DES group as compared to control (Fig. 6). In case of the 1 mg CYP group, significant down regulationwas observed for the steroid receptors AR, ERα and ERβ but non-significant change was observed in case of PR. As for the 10 mg CYP group, all the steroid receptors accounted for

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Journal Pre-proof decreased mRNA expression levels. On the contrary, in case of the 25 mg CYP group, all the steroid receptors displayed significantly increased expression levels. Furthermore, DES group displayed significantly decreased expression levels for all the steroid receptors except in case of PR, where a significant over-expression was observed.

The uterine ERβ and PR mRNA expression levels were studied at PND 75 in F1 female

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offspring and were found to be altered in case of all the CYP groups and as well as DES group as

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compared to control (Fig. 6). A significant increase in the PR mRNA expression was observed in

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the 10 mg and 25 mg CYP groups and a non-significant increase was observed in the case of the 1 mg CYP group. A significant decrease in the PR mRNA expression levels was observed in the

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DES group as compared to control (Fig. 6A). Significant increase in the ERβ mRNA expression

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levels was observed in the 1 mg CYP and DES groups with a non–significant up regulation

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observed in the 10 mg CYP group. The ERβ mRNA expression was found to be significantly

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down regulated in the 25 mg CYP group as compared to the control group (Fig. 6B).

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3.7 mRNA expression levels of HOXA10 and α-SMA in uterus of F1 female offspring HOXA10 and α-SMA, being important for uterine functioning, their relative mRNA expressions in F1 female uterus were studied at PND 75. A significantly high expression of HOXA10 was observed in the 10 mg CYP group; whereas, non-significant down regulation was observed in the 25 mg CYP and DES groups and the 1 mg CYP HOXA10 expression levels were similar to the control group (Fig. 7A). α-SMA expression levels seem unchanged in the 1 mg and 10 mg CYP groups as compared to control; whereas, the 25 mg CYP and DES groups show significant down regulation of α-SMA in uterus (Fig. 7B).

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3.8 Effect of perinatal CYP exposure on fertility of F1 females and the developmental effects on their offspring Fertility assessment data of F1 female rats displayed various alterations in the perinataly exposed CYP groups as compared to controls (Table 3). The copulation indices of the 10 mg and 25 mg CYP and DES groups were found to be significantly reduced. Also, the time taken for copulation

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was significantly higher in the CYP and DES groups as compared to controls. The F1 female rats

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of 25 mg CYP and DES groups copulated with the untreated male rats displayed a significant

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increase in the number of resorptions. Also, a phenomena where only one uterine horn had

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fetuses while there were no fetuses in the other uterine horn was observed in the 25 mg CYP group (Fig. 8A). The mean number of corpora lutea per litter observed in the F1 female ovaries

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did not show any such significant changes in the CYP and DES groups and was found

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comparable to control. Mean number of implantation sites per litter was non-significant reduced in the CYP and DES groups. Therefore, a higher PIL%/litter was noted in the CYP and DES

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group females with the 25 mg CYP group females displaying the most significant increase. Also,

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significantly higher POL%/litter was observed in the 25 mg CYP and DES groups with the 10 mg CYP group displaying a non-significant increase as compared to control.

The sired F1 females had reduced litter sizes as compared to control (Fig. 8B). The CRLs of the F2 male and female fetuses of 10 mg CYP and DES groups were observed to be significantly reduced as compared to control; whereas the 25 mg CYP F2 fetuses showed a non-significant reduction of the mean CRLs (Fig. 8C). The mean weights of the F2 male and female fetuses of CYP and DES groups were found to be significantly reduced as compared to the control F2 pups

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Journal Pre-proof (Fig. 8D). The F1 and F2 fetus showed physical malformations such as an under developed body and tail deformities (Fig. 9).

4. Discussion The literature available on effects of CYP exposure during the critical window period of embryonic and fetal development is scarce. This critical window period has been found to be

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very sensitive to any perturbations by environmental toxicants, especially endocrine disruptor

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chemicals (EDCs). CYP being ubiquitously present in our environment; the effects of its

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perinatal exposure on the reproductive functions in female rodents have been undertaken.

This study reports the adverse effects of CYP on the organ weight, histology, serum steroid

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hormone concentrations, sexual maturation, fertility status, in F1 female rats and the

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transgenerational consequences observed in the F2 generation. As per no observed adverse effect level (5 mg/kg bw/day) and low observed adverse effect level (25 mg/kg bw/day) levels reported

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in a 3 generation reproductive study via oral route by EPA, reregistration eligibility decision for

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cypermethrin (Edwards, 2006), CYP doses were selected for this study. Also, human breast milk has been reported to have CYP residues in the range of 945.1e1443.8 ppb in a previous study (Bedi et al., 2013) which further helped formulating the lowest dose range. DES was chosen as a positive control based on available literature where it showed adverse reproductive effects in rodents at 10 µg/kg bw (Sharpe et al., 1998; Goyal et al., 2003).

The higher dose group F1 females displayed reduced body weights which persisted till adulthood indicating detrimental effects of highest dosage on body weight. Organ weight reductions were

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Journal Pre-proof also observed in the same dose group where the steroid hormone producing or sensitive organs viz. ovaries, uterus and adrenal gland showed persistently reduced organ weights at peri-pubertal and adult stages. Even though the lower CYP treated groups displayed reduced relative organ weights at the juvenile stage, the effects were transient; but not the case in the highest dose group which means that at this dosage, there is a significant growth defect in hormone sensitive organs. The effects induced by CYP exposure was quite comparable to the effects induced by DES

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which is a very potent estrogen.

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Our study indicated that maternal CYP exposure during perinatal period may affect the

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reproductive development of F1 female offspring. The high dose of 25 mg of CYP delayed the vaginal opening, decreased proestrus period and increased estrus period with an overall decrease

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in the estrous cycle lengthat adulthood. These perturbations might be due to altered serum

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hormones levels, especially estradiol levels, which govern the estrus cycle. Notable serum progesterone level alterations at adult-hood could be attributed to the increased number of large

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corpora lutea observed during histological examination (Rina Aritonang et al., 2017). Serum

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testosterone levels were also found to be notably high during adulthood. So, overall, the effects of CYP on serum reproductive hormones vary with varying doses.

In rodent models, primordial follicle formation has been shown to be inhibited by action of estrogen and progesterone by inhibition of apoptosis (Kezele and Skinner, 2003). Primordial follicle transition to primary follicle (the initial recruitment) is tightly regulated by interactions between transcription factors, paracrine factors and steroid hormones while being independent of gonadotropins (Zama et al., 2016). As the success of female reproduction is determined by such

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Journal Pre-proof important processes, endocrine disruption by EDCs such as CYP can lead to early depletion of ovarian follicular reserves and therefore, may result in early reproductive senescence (Skinner, 2005). DES has previously been proven to show increased luteinization and formation of multioocyte follicles (Iguchi et al., 1990). This is attributed by the estrogenic effects of DES. So, a similar phenomenon was observed in the CYP treated groups which exhibited increased estradiol levels even though there was no direct exposure at the adult stage to the F1 offspring. The 1 mg

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CYP group ovaries displayed increased luteinization whereas the 25 mg CYP and DES female

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rat ovaries displayed luteinization as well as MOFs. However, the 10 mg CYP rats also displayed

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MOFs but no significant luteinization. Increased atresia of the follicles can be attributed to the

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high androgen levels observed in the CYP and DES treated adult F1 rats (Thomson et al., 1993). The varying follicular counts of different population could be contributing to the altering steroid

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receptor expression in the ovaries. A study has reported that neonatal exposure to DES induced

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MOFs which is a process mediated by ERβ and not ERα (Kirigaya et al., 2009). CYP could also induce this effect via ERβ which is the dominant ER type in ovary (Kyriakidis and

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Papaioannidou, 2016). However, the possibility of its action via ERα cannot be ruled out. The

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mRNA levels of both the ERs showed a similar level of expression in each CYP dosage group. Downregulation was observed in all the treatment groups except 25 mg CYP group which showed a significant upregulation of ERs. PRs are mainly expressed by the granulosa cells of pre-ovulatory follicles (Iwai et al., 1990; Park and Mayo, 1991); and in our study the 1 mg and 10 mg CYP group showed lower mRNA expression levels of PR but the 25 mg CYP and DES group showed an increased expression of PR mRNA even though their pre-ovulatory follicle counts were lower when compared to control. This could be due to effects of perinatal exposure

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Journal Pre-proof of CYP and DES on the gene regulatory mechanisms in the granulosa cells or a defective hormone feedback mechanism or due to the presence of many MOFs in these two groups.

The presence of ERs and PR makes the uterus susceptible to the actions of EDCs (Zama et al., 2016). Also, with the altered endogenous hormone levels observed in the CYP treated groups, detrimental effects on the uterine structure and functions are expected. Increased ERβ expression

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levels were only observed in the 1 mg CYP and DES groups and the PR expression levels were

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notably increased in all the CYP groups. The 25 mg CYP treatment group showed the most

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drastic histological abnormalities in the uterus as well as increased PR and decreased ERβ

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expression levels. The abnormalities observed were increased uterine diameter with an irregular lumen, a degenerative luminal epithelium and thin glandular epithelium. Large and fewer glands

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were observed in the endometrium of the 25 mg CYP group. PR levels in the epithelial cells are

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mainly down-regulated by ERβ signals (Sahlin et al., 2006) and in case of ERβ knock-out studies, estradiol has shown to increase PR expression in uterine stromal cells (Weihua et al.,

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2000). Similar to this, the 25 mg CYP group displayed significantly lower ERβ expression levels

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and higher PR levels along with increased serum estradiol levels.

Embryo implantation is greatly dependent on the uterine receptivity, which in turn is regulated by estradiol levels (Wang and Dey, 2006) and increased estradiol levels have been known to rapidly close the window of implantation (Zhang et al., 2013). Also, a recent study showed increased rate of pre implantation losses after β-CYP treatment (Zhou et al., 2018a). The CYP groups clearly displayed altered steroid hormone levels, which could be the cause of the pre and post implantation losses, increased copulation time; and hence, impaired fertility. In case of ERβ

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Journal Pre-proof knockout studies in mice, when the uteri were examined during pregnancy, implantation was usually observed in only one horn, and there were usually several dead or resorbed fetuses (Weihua et al., 2000). So a similar observation was seen in case of the 25 mg CYP pregnant F1 female rats which could be attributed to the significantly low expression of ERβ in their respective uteri and further lead to reduced litter size.

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DES was broadly prescribed to pregnant women to sustain pregnancy during 1940s to the 1970s.

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The female babies born after this kind of exposure to DES in utero (DES daughters) exhibited

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genital tract abnormalities later in life (Herbst et al., 1971). Perinatal exposure of laboratory

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rodents to DES has generated a spectrum of reproductive tract lesions which were found to be similar to those observed in humans (McLachlan et al., 1980). HOXA10 is an important

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regulator of uterine stromal cell proliferation and local immunosuppression which are

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implantation-associated events. It is highly expressed in the proliferating and differentiated uterine stroma during the peri-implantation period with progesterone as the primary inducer of

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HOXA10 in this tissue (Yao et al., 2003). HOXA10 is the downstream effector of estrogen and

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progesterone receptors and can also regulate the differentiation and proliferation of stromal cells in the mouse endometrium leading to endometrial receptivity modulation (Das, 2010). Also, effect of β-CYP on embryo implantation in adult female mice (F0) had been studied recently which showed that it alters HOXA10 mRNA levels and reduces implantation success (Zhou et al., 2018b) and during the secretory phase of the menstrual cycle, progesterone-mediated upregulation of HOXA10 in the endometrium is required for embryo implantation and therefore, fertility (Daftary and Taylor, 2006). Hence, HOXA10 showcased a great dysregulation further leading to the reduced uterine receptivity and contributing to increase in the PIL and POL%. α-

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Journal Pre-proof SMA is expressed by the smooth muscle cells in the uterine myometrium and periglandular cells and is a part of the tissue remodeling machinery (Czernobilsky et al., 1993; Shynlova et al., 2005). α-SMA levels showed significant decrease in the 25 mg CYP group which could possibly explain the distended uterine morphology and histological abnormalities.

Earlier reports have indicated the teratogenic and embryotoxic effects of CYP in F1

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generationrats (Gupta, 1990; Madu, 2015; Singh et al., 2017), however transmission of these

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effects via female germline have not been reported. Our study not only confirmed previous

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findings of developmental effects with respect to F1 offspring but also demonstrated for the first

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time that these effects can also be transmitted to next generation (F2) via female germline.

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5. Conclusion

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Humans are constantly being exposed to many estrogenic EDCs like CYP. The present findings raise a concern where CYP exposure during critical window of development may lead to

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reproductive and developmental defects across the generations. Also, evident by our

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observations that CYP exposure shows non-monotonic dose response where the lowest dose (1 mg CYP) is able to disrupt the endocrine homeostasis of F1 female offspring and has a profound effect on reproductive functions at adulthood. Hence, it can be concluded that CYP exposure during critical window of development affects not only reproductive functions of F1 generation but these effects are transformed to next generation (F2). Further mechanistic studies are warranted to understand these effects.

Acknowledgement

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Journal Pre-proof The assistance rendered by Dr. Vikas Dighe, Ms. Priyanka Maske, Mr. Mahadeo Pawar, Mr. Jayant Tare, Mr. Pravin Salunkhe, Mr. Suryakant Mandavkar, Mr. Nilkanth Shelar and Mr. Swapnil Lokhande is gratefully acknowledged.

Funding The present work (RA/796/08-2019) was supported by intramural and extramural funds received

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(SERB-DST; Grant No: EMR/2016/000336) respectively.

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from Indian Council of Medical Research (ICMR) and Science and Engineering Research Board

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Table 1: Sequences of primers, annealing temperature and amplified products of PCR

Table 2: Relative organ weights (mg) of F1 female offspring perinatally exposed to CYP

Table 3: Fertility assessment of F1 female offspring perinatally exposed to CYP

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Journal Pre-proof Figure 1: Body weight of F1 female rats (n = 14) exposed perinatally to CYP at PND 1 to 22, PND 45, and PND 75. The values are expressed as Mean ± SD; asterisks (*) indicate statistical significance compared to vehicle control (*P ≤ 0.05)

Figure 2: Sexual maturation and estrus cyclicity in F1 females exposed perinataly to CYP (A) Age at vaginal opening; (B) Length of estrus cycle; (C) Individual estrus cycle stages.

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Mean ± SD; (n = 14).*Control vs CYP exposed (*P ≤ 0.05, **P ≤ 0.01)

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Figure 3: Histopathological changes observed at PND 75 in the ovary and uterus of F1

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female rat upon perinatal exposure to CYP. (A) Microphotograph of control ovary (a, f, k) and CYP (1, 10, 25 mg) exposed ovary (b, g, l, c, h, m, d, I, n) and (e, i, o) represents DES

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exposed ovary; ► indicates CL, * indicates atretic follicles and arrows indicate MOFs. (B)

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Uterine histological sections of control (a, b, c) and 25 mg CYP exposed (d, e, f); ► indicates large endometrial gland, * indicates degenerative luminal epithelium and black

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arrows indicate thin glandular epithelium.

Figure 4: Effect of perinatal exposure of female rats to CYP on the ovarian follicular count at PND 75. The number of follicles are expressed as Mean ± SD (n = 3). *Control vs CYP exposed (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001)

Figure 5: Effect of perinatal exposure of female rats to CYP on reproductive hormone levels in F1 females. Serum (A) Estradiol, (B) Progesterone and (C) Testosterone levels at

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Journal Pre-proof PND 45 and 75. Mean ± SD (n = 6). *Control vs CYP exposed (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001)

Figure 6: Effect of CYP exposure during perinatal period on mRNA expression levels of steroid receptors (AR, PR, ERα and ERβ) in ovary and uterus of F1 female offspring at PND 75; (A) Ovarian androgen and progesterone receptor; (B) Ovarian estrogen receptor

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α and β; (C) Uterine progesterone and estrogen receptor β. The results are normalized to

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the housekeeping 18S rRNA and compared with vehicle control group female rats. Mean ±

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SD (n = 6). *Control vs CYP (*p ≤ 0.05, **p ≤ 0.01, ***P ≤ 0.001)

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Figure 7: Effect of perinatal exposure to CYP in F1 female offspring at PND 75 on mRNA expression levels of (A) Hoxa10 (B) α-SMA in uterus. Relative expressions with respect to

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18S (housekeeping gene). Mean ± SD (n = 6). *Control vs CYP exposed (***P ≤ 0.001)

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Figure 8: Fertility assessment of F1 females perinatally exposed to CYP: (A) 25 mg CYP exposed F1 female showed implantation in one uterine horn; (B) Litter size of F2 fetuses (C) Crown-rump length (CRL) of F2 male and female fetuses; (D) Body weight of F2 male and female fetuses. Mean ± SD (n = 6). *Control vs CYP exposed (*p ≤ 0.05, **p ≤ 0.01, ***P ≤ 0.001)

Figure 9: Photograph showing tail-less feature (arrow) in harvested F1 and F2 fetuses of perinatally exposed 25 mg CYP group in comparison with control F1 fetus respectively.

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Journal Pre-proof Gene name ERα

Primers

Annealing temp (OC) 59

Product size (bp) 234

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F: 5’-CGT GTG CAA TGA CTA TGC CTC-3’ R: 5’-TTT CAT CAT GCC CAC TTC GTA A-3’ ERβ F: 5’-AAC CTC AAA AGA GTC CTT GGT GTG -3’ 62 327 R: 5’-AAC ACT TGC GAA GTC GGC AG-3’ AR F: 5’-CCC ATC GAC TAT TAC TTC CCA CC-3’ 59 247 R: 5’-TTC TCC TTC TTC CTC TTA GTT TGA-3’ PR F: 5’-TGG TTC CGC CAC TCA TCA-3’ 61 102 R: 5’-TGG TCA GCA AAG AGC TGG AAG-3’ HOXA10 F: 5’-CCT AAG GTC TTG CTT GCC TG-3’ 57 167 R: 5’-GGG AGA ATT GTG GTG TGC TT-3’ α-SMA F: 5’- TGT GCT GGA CTC TGG AGA TG-3’ 57 144 R: 5’- GAA GGA ATA GCC ACG CTC AG-3’ 18S F: 5’-CAT TCG AAC GTC TGC CCT AT-3’ 59-62 243 R: 5’-GTT TCT CAG GCT CCC TCT CC-3’ Table 1: Sequences of primers, annealing temperature and amplified products of PCR

Table 2: Relative organ weights (mg) of F1 female offspring perinatally exposed to CYP Groups PND 22

Ovary

0 mg 1 mg 10 mg 25 mg DES

0.56 ± 0.11

0.48 ± 0.03

0.68 ± 0.06

0.54 ± 0.08

0.53 ± 0.09**

0.48 ± 0.06**

0.40 ± 0.07*

0.55 ± 0.10*

0.47 ± 0.05*

0.44 ± 0.06*

1.50 ± 0.39

1.92 ± 0.67

2.29 ± 0.44

1.12 ± 0.47

2.10 ± 0.61

1.88 ± 0.33

1.61 ± 0.81

1.48 ± 0.17

2.48 ± 0.67

0.69 ± 0.11**

1.31 ± 0.15*

1.71 ± 0.13*

1.00 ± 0.41

2.10 ± 0.44

1.98 ± 0.20

0.96 ± 0.25

0.39 ± 0.08

0.32 ± 0.10

0.48 ± 0.14**

0.30 ± 0.02

0.28 ± 0.02

0.66 ± 0.12**

0.32 ± 0.07

0.26 ± 0.07

0.36 ± 0.09***

0.28 ± 0.03*

0.23 ± 0.03**

0.44 ± 0.08***

0.29 ± 0.09*

0.27 ± 0.02

6.49 ± 0.50

3.56 ± 0.29

2.94 ± 0.27

0.66 ± 0.17

na

0.57 ± 0.15*

lP

0.59 ± 0.07

ur

0 mg 1 mg 10 mg 25 mg DES

Adrenal gland

0 mg 1 mg

PND 75

0.67 ± 0.05

Jo

Uterus

0.84 ± 0.19

PND 45

re

Organs

10 mg 25 mg DES

Spleen

0 mg

35

Journal Pre-proof 1 mg 10 mg

4.17 ± 1.10**

3.62 ± 0.36

3.10 ± 0.29

4.26 ± 1.00**

3.45 ± 0.19

3.12 ± 0.23

5.03 ± 0.08*

3.96 ± 0.27

2.99 ± 0.26

3.97 ± 0.05**

3.23 ± 0.36

2.76 ± 0.42

25 mg DES

*Control vs CYP exposed *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; (N = 10)

Table 3: Fertility assessment of F1 female offspring perinatally exposed to CYP

b

100 83 83 66

6±2 12 ± 2** 13 ± 3** 14 ± 1**

Mean no. of implantation sites

PIL%/L c

POL %/L d

14.50 ± 1.5 10.76 ± 1.5 11.90 ± 2.5 11.50 ± 1.5

17.15 ± 1 25.74 ± 3* 32.34 ± 1** 28.61 ± 2*

0 9±1 16 ± 2* 17 ± 2*

of

Mean no. of corpora lutea

ro

Time taken for copulation

-p

Copul ation Index %a

17.50 ± 1 14.49 ± 2 17.59 ± 2 16.11 ± 1

Copulation Index % = (Number of females copulated / Number of females cohabitated × 100).

b

na

a

6 6 6 6

No. of female s cohabi tated/ copula ted 6/6 6/5 6/5 6/4

re

Corn oil 10 mg CYP 25 mg CYP DES

N

lP

Concentration

c

ur

Time taken for Copulation (the interval between the first day of cohabitation and GD 0 i.e the day of sperm positive vaginal smears). PIL %/Litter = (Number of CLs − Number of implantations) / (Number of CLs) × 100.

d

Jo

POL %/Litter = (Number of implants − Number of viable fetuses) / (Number of Implantations) × 100 N represents total number of CYP exposed F1 female cohabitated with fertile control male / group (*P ≤ 0.05, **P ≤ 0.01)

36

Jo

ur

na

lP

re

-p

ro

of

Journal Pre-proof

37

ro

of

Journal Pre-proof

Jo

ur

na

lP

re

-p

Graphical abstract

38

Journal Pre-proof Highlights:

of ro -p re lP na



ur

  

Perinatally CYP-exposedF1 females exhibited delayed vaginal opening and disrupted estrous cycle CYP exposure led to adverse effects on ovarian and uterine functions in F1 females CYP-exposed F1 females were sub-fertile and had reduced litter size CYP exposure altered ovarian and uterine expression of AR, PR, ERα, ERβ, HOXA10 and α-SMA in F1 females Effects induced by CYP were transgenerational leading to embryotoxic effects in F1 and F2 fetuses

Jo



39