Biochemical and histopathological investigation to study the impact of pyriproxyfen exposure on ovarian morphology and reproductive function in female rats

Abstract

Background

Pyriproxyfen (PYR) is a pyridine-based broad-spectrum insect growth regulator and pesticide which works as an analogue of juvenile hormone. Its exposure to aquatic animals and crops is linked with various hazardous effects on biological functions. We aimed to find the possible reprotoxic effects of pyriproxyfen in adult female Sprague-Dawley rats through histological and biochemical approaches.

Methods

Adult female rats were assigned to four groups and were administered 0 mg/kg (Control), 62 mg/kg b.w, 124 mg/kg b.w, and 186 mg/kg b.w., of PYR dissolved in distilled water for 28 consecutive days. Body mass index, blood glucose levels, total protein concentration, lipid profile, ovarian histology and reproductive hormonal profiles were determined.

Results

There were no significant changes in body weight due to PYR exposure; however, slight alterations in ovarian and uterine weights were noted in the treatment groups. The 186 mg/kg b.w. treatment significantly affected estrous cyclicity. Furthermore, a non-significant increase in total protein levels and a significant (p < 0.05) rise in triglyceride and total cholesterol levels were recorded. However, a significant decline in high-density lipids was recorded in the high-dose treatment group (186 mg/kg bw) as compared to the control. A notable reduction in plasma concentration of estradiol, progesterone, and cortisol levels was recorded between the control and all the treated groups. Ovarian histomorphological analysis showed distorted basal membranes, increased empty spaces, tissue decompaction, degenerate follicles, and disassembled epithelium in the high-dose treated group (186 mg/kg b.w).

Conclusion

Oral administration of PYR in adult female rats leads to altered organ weights, disturbed normal estrous cycle, increased triglycerides and total cholesterol, reduced high-density lipids concentrations, and damaged ovarian architecture, affecting biochemical and reproductive function in female rats.

Introduction

Approximately 5.6 billion pounds of pesticides are being used annually in the world [1], and this usage is unexpectedly rising, of which 95% possess the ability to be widely dispersed in the environment and to affect non-target organisms [2]. Due to this prevalent dispersal of pesticide usage, the majority of people may be occupationally subjected to pesticides and in jeopardy of their negative effects. Workers from pesticide manufacturing industries and certain handlers in public health (e.g., destroyers of household pests) are occupationally exposed. In the farming sector, farmers and qualified appliers of pesticides get exposed to pesticides as well [3–6]. Every year, over 150,000 people die from pesticide poisoning, highlighting the need for better safety measures and education. Most deaths result from self-poisoning through ingestion, but occupational and accidental exposures can also be fatal due to skin contact or inhalation. Additionally, certain pesticides can disrupt hormonal functions, altering the balance necessary for the proper functioning of the reproductive system.

Pyriproxyfen (PYR; IUPAC name, 4-phenoxyphenyl (RS)-2-(2-pyridyloxy) propyl ether) is a pyridine-based broad-spectrum insect growth regulator (IGR) pesticide which works as an analogue of juvenile hormone [7–9]. It is an aromatic compound [10], first manufactured in 1990 by Sumitomo Chemicals Co., Ltd. and marketed under the trade name of “Lano^® 10EC” [11, 12]. Being a strong estrogen agonist, PYR is categorized as an endocrine disruptor [9]. It is extensively used in households, horticulture, and agriculture to control various insect species [8, 10], because of its ability to inhibit embryogenesis in insects [13]. PYR is used against whiteflies, thrips, aphids, scales, jassids, mealworms, cutworms, and bollworms [14–18]. It has been proven to be effective against house insects such as houseflies, mosquitos, cat fleas, and cockroaches as well [9]. In Pakistan and India, PYR is used for controlling the spread of certain vectors, including Culex quinquefasciatus and Anopheles stephensi [19]. Due to its extensive use and stable nature, it gets accumulated in the environment leading to detrimental effects on a range of living organisms (plants and animals) [20]. Its exposure in plants is linked with growth retardation of root and shoots of legume plants such as peas, green gram, chickpeas and lentils [21]. Similarly, in animals and humans, exposure to PYR follows dermal, inhalation, or oral routes [22] and its continual exposure may induce growth retardation, hormonal imbalances, impaired reproductive functions, and neurodevelopmental disruptions [8, 23]. In the previous report, the neurodevelopmental toxicity at different dosesof pyriproxyfen (0, 100, 300, and 1000 mg/kg/day) in rat pups was evaluated, and the results revealed arhinencephaly and reduction in brain weight in treated pups as compared to control pups (13). The continuous use of insecticides in some countries can lead to interference with endocrine homeostasis, resulting in the impairment of the male reproductive system [24, 25]. In fishes (Labeo rohita), studies, revealed a decrease in body and organ weight, lowered red blood cell number, disturbances in the structural integrity, DNA damage in the visceral organs and alterations in the antioxidant levels [26]. Additionally, it is known to bring reproductive toxicity in some non-target organisms such as mice; studies suggested that repeated exposure to PYR leads to decreased weight gain in pregnant treated groups, reduced litter size, and increased stillbirths [27]. Another study reported that PYR may damage testicular architecture in male mice by interfering with spermatogenesis [18]. It also interferes with normal steroidogenic pathways, lowering testosterone and estrogen levels in male and female zebrafish [28]. Thus, it is conferred that pyriproxyfen exposure in humans, plants and animals exerts detrimental effects on organizational functions.

Although the literature review suggests the involvement of PYR in damaging the reproductive health of non-target animals, the available data is still insufficient to classify PYR as a potent reprotoxic agent. Furthermore, a considerable body of research has been undertaken to establish a correlation between occupational exposure to pesticides and fertility issues in men [29], but studies among women are scarce. Therefore, the present study was designed to find the possible toxic effects of PYR on the female reproductive system by using adult female Sprague-Dawley rats as an animal model. In the era of excessive chemical use, such preliminary studies are a prerequisite to identify risk factors associated with chemicals which might be harmful to human health in the long run so that their use can be regulated accordingly.

Material and methods

Ethics statement

The study protocol (BAS#345A) was approved by the ethics committee of animal studies, at Quaid-i-Azam University, Islamabad, Pakistan. The study is reported per ARRIVE guidelines [30]. All methods were carried out following relevant guidelines and regulations.

Chemicals and animals

Pyriproxyfen (10.8%EC PYR) (CAS#95737-68-1), manufactured by Nantong Chemical Co., Ltd (China) and imported by Suncorp Pesticides Multan, was purchased from Anqa Agro Multan. The pesticide was diluted in distilled water following the protocol of previous studies [25, 31] and the desired doses of PYR for each group were prepared. The prepared doses of PYR and the distilled water for the control group were administered to the rats daily via oral gavage for 28 consecutive days.

Forty healthy adult female Sprague-Dawley rats (Rattus norvegicus) were collected from the primate facility of the Zoology Department, Quaid-i-Azam University, Islamabad. The rats were placed in a separate stainless-steel cage in a well-ventilated room. The temperature of the room was maintained between 20–25˚C and the rats were subjected to 24-hour light and dark cycles (12-hour light and 12-hour dark cycles). All the rats were given food chaw and tap water during the experiment. The departmental ethical committee approved the number of animals used for the experiment, their handling, and scarification (BAS#345A).

Experimental design and sample collection

Adult female Sprague Dawley rats (average weight 160 ± 10 g, n = 40) were assigned to four groups. We kept 10 animals per group, considering the risk of mortality. Animals in Group 1 (control) were administered 0 mg/kg (control), 62 mg/kg (G1), 124 mg/kg (G2), and 186 mg/kg (G3) of pyriproxyfen orally for 28 consecutive days. The pyriproxyfen effects were determined following OECD guidelines for chemical testing (i.e. the effect on reproduction, survival, individual growth, and endocrine disruption). The doses of PYR used in our experiment were according to the previous studies performed by researchers [18, 23, 27]. Pyriproxyfen (technical) mammalian toxicity is as follows: Oral (rat) LD50 >5000 mg/kg; Dermal (rat) LD50 >2000 mg/kg; Inhalation (rat) LD50 >1000 mg/kg [32]. The sub-lethal doses were selected for the current study because a previous study by Shahid and colleagues reported lethal detrimental effects at doses above 300 mg/per kg bw during a 21-day study in mice [25]. The control group rats were treated with distilled water. No mortality was observed during the experimental period; hence on the 29th day of our experiment, the body weights of all animals were recorded, while 6 animals from each group were randomly selected and euthanized by injecting a ketamine/xylazine mixture (75/2.5 mg/kg, respectively, i.p.) [33]. Anaesthetized rats were secured in a supine position and the blood was withdrawn via cardiac puncture and poured into the heparinized syringes to obtain plasma. The blood was centrifuged at 3000 rpm for 15 min, after that the plasma was stored at -20˚C for various biochemical examinations [34]. Ovaries, uterus, kidneys, liver, and heart were collected, washed in normal saline solution (0.9% NaCl), and weighed. Ovaries were immediately fixed in a 10% formalin solution for histological analysis.

Body weight determination

The body weight of rats in different treatment was determined and noted down on 1st, 14th, and 28th day of the experiment. Top-loading Sartorius Entris II Digital Balance (Germany) was used for weighing purposes.

Organ weight determination

After successful dissections, the rat’s organs (ovaries, uterus, kidneys, liver, and heart) were collected and washed in normal saline solution (0.9% NaCl). After washing, their weights were measured using Sartorius Entris II Digital Balance (Germany).

Body mass index (BMI) determination

For calculating rat’s BMI (g/cm²), body weight and body length were measured [35]. The length was measured using measuring tape, and the subsequent formula was used to determine the BMI previously used by Engelbregt et al. (2001) [36].

Blood glucose determination

The rat’s blood glucose levels were measured using an EasyGluco Auto-coding TM (INFOPIA Co., Ltd. Korea) glucometer. Glucose levels were measured on the empty stomach early in the morning on the 1st, 14th, and 28th day by pricking the tail tip with a sterile needle and then placing the blood drop on the edge of the glucometer’s strip.

Determination of estrous cycle

Vaginal smears were collected and cytology was performed on 1st, 7th, 14th, 21st, and 28th day for cytology staging. Estrous stages were assigned by subjectively interpreting vaginal cytology stained with hematoxylin and eosin (H & E), as per previous investigations [37]. Briefly, rats were restrained, and their tails were raised to visualize the vagina. Next, the vaginal smear was collected and cells were rinsed gently by introducing a slight volume (10–20 µl) of normal saline solution (0.9%) through a pipette (repeat 4 to 5 times). The liquid sample was placed on a glass slide, air-dried, and stained with hematoxylin and eosin (H&E). It was then examined under a microscope (AmScope B120, China) to evaluate the characteristics of the cytoplasm and nucleus to determine the estrous phase.

Total protein estimation

To quantitatively determine the total protein in samples, a total protein estimation kit by AMP Diagnostics (AMEDA Labordiagnostik GmbH, Austria, CAS# RT2341) was used. The absorbance of samples and standard was read against reagent blank at a wavelength of 540 nm using Piccos 05 Chemistry Analyzer (AMP Diagnostics, GmbH, Austria).

Lipid profile total cholesterol estimation

For the estimation of total cholesterol (TC), and triglycerides, AMP diagnostic kits were used manufactured by AMEDA Labordiagnostik GmbH (Graz/Austria, CAS# RT2950). The same procedure was performed as provided by the manufacturer.

HDL-cholesterol estimation

To measure the HDL-C levels in our samples we used kits provided by Bio-active Diagnostic Systems (Voehl, Germany 321673-30-7). The readings were taken at 500 nm.

Hormonal analysis

Progesterone and estradiol concentrations in plasma were determined following the instructions provided by the manufacturers on the Pro ELISA kit obtained from (Bio Check Inc, USA, CAS# E-EL-0154). Using enzyme immunoassay (EIA) kits (Bio check Inc, USA, CAS # 3-CMO-BSA 80-IC10) cortisol concentrations were evaluated quantitatively.

Ovarian histology

Once the ovaries were successfully secured after dissection, they were fixed in 10% Phosphate Buffered saline (PBS) formalin for 24 h, it stabilizes and preserves the tissue for further processing. The tissues were dehydrated with different concentrations of alcohol and then cleared with xylene. After the wax (wax 30 min, wax 30 min, wax 45 min) infiltration and embedding of tissues, the ovarian tissue sections (5 μm) were cut using a Rotary microtome S712 (RWD, China) and stained with hematoxylin and eosin until the tissue sections were blue. After the staining process was done the slides were mounted with Canada balsam. After that, xylene-dipped coverslips were used to cover the tissue sections on the slides.

Microscopy

Slides containing different sections of ovaries were examined using an Olympus CX23 microscope (Tokyo, Japan) with an attached Canon digital camera (Tokyo, Japan) for taking microphotographs. The sections were examined at 10X and 40X magnification. Microphotographs were taken at both magnifications and evaluated.

Histomorphometric analysis

Ovarian histomorphometry was performed on H&E-stained sections from 6 animals per group. To eliminate bias, the analysis was conducted by a researcher blinded to the treatment groups. For each ovary, five non-overlapping fields at 10x magnification were captured and analyzed using ImageJ software (NIH, USA). The following structures were quantified: (1) Healthy Follicles: Primordial, primary, secondary, and antral follicles with intact granulosa cell layers and no pyknotic nuclei were counted; (2) Atretic Follicles: Follicles with disorganized granulosa cells, pyknotic nuclei, or detachment of the oocyte were counted separately; (3) Corpora Lutea (CL): The total number of CL per section was recorded; (4) Cysts: Fluid-filled structures with a thin lining lacking granulosa cells were counted; (5) Area of Empty Spaces: The total area of optical empty spaces, indicative of tissue decompaction and edema, was measured and expressed as a percentage of the total tissue area analyzed.

Statistical analysis

The Sample size for the current study was calculated by the resource equation method [38] by using the following formula:

Here, E is the degree of freedom of analysis of variance (ANOVA). The value of E should not be less than 20 to increase the chance of getting a more significant result. As this method is based on ANOVA, it applies to all animal experiments [39]. In the present study, initially, we made four groups with 10 animals in each. And final analysis was done on 6 animals from each treatment group. This sample size is adequate as the chances of death of animals cannot be ignored. Statistical analysis of all the data was performed using IBM^® SPSS^® Statistics version 25 (IBM Corp.). The data was analyzed by applying a one-way Analysis of Variance (ANOVA) followed by Tukey’s test for comparison of separate groups to each other. Finally, all the data was presented as Mean ± SEM by setting the significance level at p < 0.05.

Results

Effect of sub-acute administration of different doses of PYR on body weight, BMI,and organ weights

We examined behavioral changes, clinical signs of systemic toxicity, and mortality during the experimental period. There were no recorded cases of morbidity, mortality, or changes in behavior during this specific time frame. Furthermore, no noteworthy dissimilarities were monitored in water and food consumption between the experimental groups. The average body weight of animals from each group (Control, G1, G2, and G3) on day 1 was 160 ± 10 g. On day 14 and 28 of treatment, a non-significant (p > 0.05) change in the body weights of control and different treatment groups was recorded (Table 1).

Table 1.

Mean ± SEM body weight (g) of adult female Sprague Dawley rats on different days as a result of treatment with various doses of PYR n = 6 per group

Treatments	Body Weight
	Day1	Day14	Day28
Control	155.3 ± 7.6	158.3 ± 6.2	160.9 ± 4.7
G1 (62 mg/kg)	166.7 ± 2.7	173.6 ± 4.6	161.1 ± 6.5
G2 (124 mg/kg)	160.2 ± 7.9	167.5 ± 8.5	157.3 ± 4.3
G3 (186 mg/kg)	161.3 ± 5.8	168.9 ± 4.4	154.8 ± 9.4

All the treated groups (G1, G2, and G3) showed a non-significant (p > 0.05) decrease in BMI values (g/cm²) when compared to that of the control (Table 2). The ovarian weight in the PYX treatment groups showed a significant decrease (p < 0.001). The lowest ovarian weight was observed in the high-dose group. However, when comparing the treatment groups, G1, G2, and G3 exhibited no significant differences in ovarian weight (p > 0.05). Compared to control G3 treatment showed significantly increased (p < 0.05) uterine weight. Compared to control all the experimental groups (G1, G2, and G3) showed significantly increased (p < 0.05) kidney weight. While only G3 showed a significant increase (p < 0.05) in heart weight in comparison to the control group. The difference in heart weight among experimental groups was non-significant (p > 0.05). Liver weight showed an increase in dose dose-dependent manner. When compared to the control, the increase in G1 liver weight was non-significant, while that of the G2 and G3 was highly significant (p < 0.05 and p < 0.001 respectively) (Table 2).

Table 2.

Mean ± SEM BMI (g/cm²) and organ weight (g) of adult female Sprague Dawley rats treated with different doses of PYR

Treatments	Parameters
	BMI (g/cm2)	Ovary Weight	Uterine Weight	Kidney Weight	Liver weight	Heart weight
Control	0.14 ± 0.067	0.12 ± 0.01	0.31 ± 0.07	0.49 ± 0.03	5.79 ± 0.30	0.66 ± 0.04
G1 (62 mg/kg)	0.12 ± 0.018	0.09 ± 0.01a***	0.43 ± 0.09	0.67 ± 0.07a*	6.27 ± 0.26	0.76 ± 0.04
G2 (124 mg/kg)	0.13 ± 0.062	0.08 ± 0.01a***	0.48 ± 0.09	0.68 ± 0.06a*	7.14 ± 0.43a*	0.75 ± 0.04
G3 (186 mg/kg)	0.12 ± 0.053	0.07 ± 0.01a***	0.57 ± 0.08a*	0.70 ± 0.04a*	8.82 ± 0.51a***b***	0.80 ± 0.03a*

a= (value compared to control), b = (value compared to G1), * (p < 0.05), ** (p < 0.01), *** (p < 0.001)

Effect of sub-acute administration of different doses of PYR on blood glucose, total protein, lipid profile and reproductive hormone levels

Table 3 indicates the blood glucose levels in different treatment groups. The blood glucose levels in the control group showed no significant differences on day 1st, 14th, and 28th day of the experiment whereas G1 (62 mg/kg) showed a significant (p < 0.001) decrease in the blood glucose level on day 28th when compared to the glucose levels on day 1st and day 14th. The G2 group showed a slight decrease on day 28 but it was a statistically non-significant change from day 1 to day 28. In G3 treated group the day 28th of the experiment marked a highly significant decline (p < 0.001) in the blood glucose levels as compared to day 1st and day 14th. Finally, the 28th-day blood glucose levels of all treated groups (G1, G2, and G3) were significantly decreased (p < 0.001) as compared corresponding blood glucose levels of control.

Table 3.

Mean ± SEM blood glucose concentration (g) of adult female Sprague Dawley rats on different days as a result of treatment with various doses of PYR

Treatments	Blood Glucose
	Day 1	Day 14	Day 28
Control	107.4 ± 2.1	111.8 ± 1.5	108 ± 0.9
G1 (62 mg/kg)	114.6 ± 4.1	108.2 ± 2.4	93.4 ± 4.1a***b***
G2 (124 mg/kg)	104 ± 4.1	106 ± 3.3	85.4 ± 2.5a***b**
G3 (186 mg/kg)	96 ± 6.5	107.6 ± 7.2	73.6 ± 3.3a***

a= (value compared to control), b= (value compared to G3), * (p < 0.05), ** (p < 0.01), *** (p < 0.001). n = 6 per group

The effects of PYR on protein concentration, total cholesterol, TGL, estradiol, and progesterone concentration in different treatment groups are presented in Table 4. Non-significant (p > 0.05) upturns in protein concentration were observed in different treatment groups compared to control. A noteworthy rise in TGL levels was seen in dose dose-dependent manner compared to the control group. Our study revealed that PYR administration caused a decrease in plasma HDL concentrations in the G2 and G3 treatment groups. The lowest HDL concentration was recorded in G2-treated rats. The dose-dependent decline in plasma estradiol and progesterone concentration was noted as compared to the control group. A remarkable reduction (p < 0.001) in plasma cortisol levels was measured between the control and all the treated groups. A non-significant change was observed in plasma cortisol levels within all the treated groups.

Table 4.

Mean ± SEM of lipid profile, protein concentration and reproductive hormone profile of adult female Sprague Dawley rats were treated with different doses of PYR

Treatments	Biochemical parameters
	Total Protein concentration	Total Cholesterol (mmol/L)	Total glyceride Level (mmol/L)	High-density lipids Cholesterol (mg/dL)	Progesterone (ng/ml)	Estradiol (ng/ml)	Cortisol (ng/ml)
Control	5.63 ± 0.36	53.07 ± 4.25	74.1 ± 4.25	65.53 ± 2.55	77.30 ± 4.87	50.71 ± 2.23	84.8 ± 8.40
G1(62 mg/kg)	5.72 ± 0.38	62.83 ± 4.77	78.91 ± 4.24b**	63.66 ± 1.13b***	74.96 ± 2.21	32.42 ± 3.71 a*	39.8 ± 2.70 a***
G2 (124 mg/kg)	6.29 ± 0.16	59.53 ± 4.62	86.15 ± 4.05b**	55.71 ± 4.02a*	63.14 ± 3.74 a*	29.66 ± 3.04 a**	33.6 ± 2.44 a***
G3 (186 mg/kg)	6.29 ± 0.28	67.09 ± 2.91s*	93.99 ± 3.93a*	43.72 ± 3.9a***	62.98 ± 4.18 a*	27.90 ± 3.39 a**	31.6 ± 3.47 a***

a = (value compared to control), b = (value compared to G3), * (p < 0.05), ** (p < 0.01), *** (p < 0.001)

n = 6 per group

Quantification of estrous cycle disruption

The estrous cycle was quantitatively assessed by determining the predominant stage for each animal on observation days. The results, presented in Table 5, demonstrate that PYR exposure significantly disrupted cyclicity. While the control group displayed a normal, rotating pattern of stages, the treated groups showed a dose-dependent arrest. Notably, the high-dose group (G3, 186 mg/kg) exhibited a significant prolongation of the diestrus phase, with over 80% of animals in diestrus on day 28 compared to 0% in the control group (Fig. 1).

Table 5.

Percentage of rats in each estrous cycle stage following PYR Treatment. Data represents the percentage of animals within a group observed in a given stage on each sampling day

Group / Stage	Day 1	Day 7	Day 14	Day 21	Day 28
Control
Proestrus	33.3%	16.7%	0%	50.0%	16.7%
Estrus	50.0%	66.7%	83.3%	33.3%	66.7%
Metestrus	16.7%	16.7%	16.7%	16.7%	16.7%
Diestrus	0%	0%	0%	0%	0%
G1 (62 mg/kg)
Proestrus	0%	0%	0%	0%	0%
Estrus	33.3%	16.7%	0%	0%	0%
Metestrus	66.7%	83.3%	100%	100%	83.3%
Diestrus	0%	0%	0%	0%	16.7%
G2 (124 mg/kg)
Proestrus	0%	0%	0%	0%	0%
Estrus	0%	16.7%	0%	0%	0%
Metestrus	16.7%	50.0%	33.3%	33.3%	16.7%
Diestrus	83.3%	33.3%	66.7%	66.7%	83.3%
G3 (186 mg/kg)
Proestrus	0%	0%	0%	0%	0%
Estrus	0%	0%	0%	0%	0%
Metestrus	16.7%	0%	33.3%	33.3%	16.7%
Diestrus	83.3%	100%	66.7%	66.7%	83.3%

Fig. 1.

Photomicrograph of vaginal smears at different stages of estrous cycle. (A) Proestrus, with numerous round nucleated epithelial cells (NEC). (B) Estrus, clumps of cornified epithelial cell (CEC). (C) Metestrus, with round nucleated epithelial cells, cornified epithelial cells and a high number of leukocytes. (D) Diestrus, with numerous leukocytes (Lkc). (10X magnification)

PYR effects on the morphology of ovaries

PYR effects on the morphology of ovaries were inspected through histology. The microphotographs were analyzed for any alterations to tissue integrity, follicular structure, and stages of follicular development. The ovarian histoarchitecture in the control group appeared normal, with ovarian follicles at various developmental stages. There were few empty spaces, and follicular cell dispersion was not observed (Fig. 2a). The ovarian surface epithelium (called Basal membrane) was well intact. In the ovarian cross sections of G1 rats, increased optical spaces were seen, accompanied by the distortion of basal membrane, thus, causing follicular cell dispersion and damage to the tissue integrity of ovaries (Fig. 2b). In G1 most of the follicles were in the primary and secondary phases of folliculogenesis; in addition, previously formed corpora lutea were degenerating (rupturing). Mostly, there were secondary follicles, a few newly formed corpora lutea, and a few atrial follicles (Figs. 2c and d).

Fig. 2.

Photomicrograph (10X) of rat ovary of control group. (A and B) The H&E cross section shows the previously formed corpus luteum (PCL), newly formed corpus luteum (NCL), antral follicle (Tertiary follicle, ANF), primordial follicle (PMF), ovarian bursa (OB), well intact basal membrane (BM), Atretic follicle (AF), secondary follicle (SF), oocyte (yellow arrow), undistorted granulosa cells (red arrow), ovarian blood vessels (blue arrow). Microphotographs of G1 ovary, treated with 62 mg/kg PYR. (C and D) shows the distortions in granulosa cells of follicles (GCD), large water-filled cysts (Cyst), lots of disruption in the basal membrane (BMD), also the degeneration of previously formed corpus luteum (CLD), and many optical empty spaces (yellow arrows). Microphotographs of H&E sections of G2 rats’ ovaries treated with 124 mg/kg. (E and F) shows various optical space (OES) caused due to the degeneration of basal membrane (BMD), the abundance of previously formed corpus luteum (PCL) and newly formed corpus luteum (NCL), and secondary follicles (SF) with degenerating granulosa cells layer. Microphotographs of H&E cross sections of ovaries from G3 treated with 186 mg/kg PYR. (G and H) shows the rupturing follicles (RF) with degenerating granulosa cells (DGC), previously and newly formed corpus luteum (PCL, NCL), a cyst filled with water (Cyst), a secondary follicle (SF), and some empty spaces (yellow arrows)

The H&E sections of G2 ovaries showed tormented basal membranes, leading to increased empty spaces and disturbed tissue compaction. The microphotographs showed large numbers of previously and newly formed corpora lutea. Many of the seen follicles were degenerating and a few were found to be in the secondary stage of folliculogenesis (Fig. 2). To substantiate these observations, a blinded histomorphometric analysis was performed, and the results are summarized in Table 6. The analysis revealed a significant, dose-dependent decline in the number of healthy antral follicles and a concurrent increase in the number of atretic follicles in the PYR-treated groups. Furthermore, the incidence of cystic structures and the area occupied by empty spaces were significantly higher in the G2 and G3 groups compared to the control. The number of corpora lutea showed a non-significant increasing trend, possibly reflecting anovulation and the persistence of cyclic structures.

Table 6.

Histomorphometric analysis of ovarian follicles and structures in rats treated with PYR

Group	Healthy Antral Follicles	Atretic Follicles	Corpora Lutea	Cysts	Empty Space Area (%)
Control	8.5 ± 0.8a	1.2 ± 0.4a	6.8 ± 0.6	0.0 ± 0.0a	2.1 ± 0.5a
G1 (62 mg/kg)	5.8 ± 0.7a, b	3.5 ± 0.6b	7.5 ± 0.7	0.3 ± 0.2a	5.8 ± 1.1a, b
G2 (124 mg/kg)	3.3 ± 0.5b, c	6.2 ± 0.8c	8.2 ± 0.9	1.5 ± 0.3b	11.4 ± 1.8b
G3 (186 mg/kg)	1.7 ± 0.4c	8.8 ± 0.9d	8.7 ± 0.8	2.8 ± 0.4c	18.9 ± 2.3c

Values represent Mean ± SEM per ovarian section. Different superscript letters (a, b, c) within a column denote significant differences (p < 0.05) as determined by one-way ANOVA followed by Tukey’s test

Discussion

Repeated and inappropriate exposure to synthetic pesticides may be associated with a range of health issues in humans, including cancer, endocrine disruption, immunosuppression, and reproductive dysfunction [25]. PYR is one of the most extensively used pesticides in the world and is well known for its ability as an embryogenesis inhibitor in insects [13]. It accumulates in the environment, causing harmful effects on non-target organisms such as plants, fish, amphibians, birds, and mammals. These effects include growth retardation, hormonal imbalance, impaired reproduction, and neurodevelopmental toxicity [40, 41].

The reprotoxic effects observed in this study, including hormonal imbalance, lipid profile disruption, and severe ovarian histopathological damage, exhibited a clear dose-dependent relationship. It is important to contextualize these experimental doses with potential real-world exposure. The doses used here (62–186 mg/kg/day) are substantially higher than the Acceptable Daily Intake (ADI) for humans (0.1 mg/kg/day) set by regulatory bodies. These high doses are standard in toxicological hazard identification studies to elucidate a compound’s intrinsic toxic potential and mechanism of action under conditions of exaggerated exposure. The significant damage observed at these levels confirms the potent reprotoxicity of PYR, which warrants caution and further investigation into the effects of long-term, low-dose environmental exposure on reproductive health.

Oral administration of PYR was performed in the current study, because of its use in drinking water for mosquito control and on different crops for controlling insect pests, leading to its oral exposure through food and water [18, 23, 27]. In toxicological studies, body weight, organ weight, and blood biochemical profiles are essential indicators of organ toxicity. We noticed a non-significant reduction in the average body weight and BMI of the treated groups. A previous study on male Swiss albino mice indicated that the oral administration of PYR caused a significant drop in the body weights in treatment groups [18]. Similarly, an investigation on pregnant female mice authenticated that the oral administration of PYR (30, 100, 300, 1000 mg/kg) caused a decrease in the pup’s body weights in a dose-dependent manner [27]. In contrast to the studies mentioned above, the non-significant changes observed in the current investigations may be attributed to the use of a rat model rather than a mouse model. A reduction in ovarian weight was noted following the administration of PYR, with the extent of this decrease correlating with the dosage administered. In the current study, we noticed that oral administration of PYR instigated a significant rise in absolute weights of the uterus, kidney, liver, and heart as compared to control. Similarly, a previous study on Sprague Dawley rats indicated that six months of oral PYR administration increased heart, liver, and kidney weights and decreased ovarian weight compared to controls [42]. In a recent study, the toxic effects of PYR in Labeo rohita were thoroughly examined. The findings unambiguously demonstrated that PYR administration resulted in a significant increase in the weights of the liver, kidney, brain, and gills [26]. Research suggested that the increased weight might be due to oxidative stress, mitochondrial dysfunction, impaired glucose and lipid metabolism, and disruption of metabolic regulation in pathways related to liver function, thyroid function, and adipose tissue metabolism [43, 44].

The assessment of key general metabolites associated with energy metabolism, such as proteins, lipids and glucose, is a direct approach, compliant with a rapid response for any treatment [45]. These biomolecules are crucial for the proper functioning of an organism, and deviations in their concentrations could be attributed to irregular metabolism induced by the presence of a contaminating agent [46]. The present study revealed that PYR exposure leads to a decrease in blood glucose levels with passing days. In PYR-treated groups, rats showed significantly low blood glucose levels on the 28th day of the experiment. In a previous study, the blood biochemistry results of female rats after 26 weeks of treatment with PYR displayed a significant decrease in blood glucose levels [42]. We have recorded altered lipid profiles and biochemical parameters in PRY treatment groups compared to the control group. This might be due to PYR’s adverse effects on the blood cells, the liver, and in combination with various enzyme-inhibitory, metabolic, and transcriptional events acting at cellular and molecular levels [47, 48]. Total plasma proteins represent the total concentrations of proteins in plasma e.g. albumins, globulins etc. Plasma proteins are an important component of blood that perform a wide range of functions in the human body. They help to maintain fluid balance, transport various substances in the blood, play a crucial role in the immune system, and are involved in blood clotting. The present study showed a non-significant increase in total plasma proteins in PYR-treated groups as compared to the control group. A study reported that PYR exposure caused an increase in total protein concentration in female and male Sprague Dawley rats [42]. In another research elevation in total protein in hemolymph of silkworm larvae was reported because of pyriproxyfen residue in the body [49]. In the current study, it was found that the PYR administration leads to an increase in total cholesterol concentration and plasma triglycerides levels as compared to the control group. Also, there was a significant decrease in HDL concentrations in the PYR-treated groups as compared to the control. A study done on silkworm larvae showed that PYR had elevating effects on hemolymph cholesterol levels [49]. Koyama and coworkers revealed that PYR administration in Sprague Dawley rats results in an elevation in cholesterol and TGL concentrations in the plasma [42]. Another study performed on Labeo rohita, showed that PYR exposure leads to increased concentration of Cholesterol and TGL [26]. Additionally, there is no prior record related to PYR exposure and HDL concentration.

The sub-acute oral administration of different doses of PYR was found to be directly affecting the reproductive cycle (estrous cycle) in adult female Sprague Dawley rats. PYR treatment led to shortened proestrus, prolonged metestrus, and lengthened diestrus phase in treated rats. Previous data regarding the effects of PYR on estrous cyclicity is lacking, but in studies performed on pregnant female mice, it was found to be causing a reduction in litter size and the number of live births as compared to the control [27]. Previous observation depicted that the fetal indices of Wistar rats treated with different doses of PYR (100, 300, and 500 mg/kg) resulted in a significant decrease in several fetuses in the treated groups. This might be due to the PYR interference with the female hormonal function, which then leads to negative effects on the reproductive system through disruption of the hormonal balance necessary for proper functioning. The hormonal function may be disrupted in many more ways through pesticide exposure [50].

For studies of reproductive toxicity, the measurement of sex hormones has been deliberated as one of the most functional and integrative points [51]. PYR may cause reproductive and endocrine problems by interfering with the HPG axis. Studies have shown that male and female zebrafish when separately exposed to various doses of PYR for 21 days, experienced suppressed estradiol levels in female zebrafish [28]. In contrast to the previous work of Manabe et al. 2006, a mixture of two pesticides prothiofos/pyriproxyfen increased estradiol levels by using MtT/Se cell proliferation assay [52]. In the present findings, all the treated groups showed a reduction in plasma estradiol levels as compared to the control. The decrease in estradiol level might be due to PYR inhibiting aromatase enzyme production so the expression of the CYP19a gene might be decreased which may affect female reproduction [53, 54]. Similarly, a reduction was observed in the concentration of plasma progesterone level as compared to control. Our results are consistent with Naito and colleagues, who observed a reduction in progesterone levels in adult virgin rats exposed to various doses of another insecticide, chlorpyrifos [55]. The decrease in progesterone concentration might be due to the PYR effect on the steroidogenic pathway by inhibiting the expression of Cytochrome P450 proteins and Steroidogenic Acute Regulatory Protein genes [56]. In the current study, a prominent decrease in cortisol concentrations was also detected in all the treated groups compared to the control group. Gusso et al. 2020 also observed similar effects. Exposure to different doses of PYR (0.125, 0.675, and 1.75 mg/l) in zebrafish resulted in a reduction in cortisol levels in the treated group compared to the control [57]. Low cortisol levels serve as an indicator of potential underlying diseases, such as primary adrenal insufficiency, or may reflect conditions that directly impair the function of the adrenal glands, resulting in reduced cortisol production. However, the underlying reason is unknown.

Moreover, the deteriorating effects of PYR on the ovarian histoarchitecture of rats were noticed in the current study. The chronic ovarian administration had led to basal membrane distortion, cyst formation, granulosa cell degeneration, increased optical empty spaces, increased corpus luteum formation, and follicular dispersion. Although there are no previous studies related to the effects of PYR on ovarian histology, there are findings of its effects on testicular histology in a study done on mice, in which the PYR administration (1200, 600, 320, 200, 100, 40, 20, 0 mg/kg) for 28 successive days caused shrinkage of seminiferous tubules, vacuolization in seminiferous tubules, and reduction in lumen diameter [25]. Another study performed on adult zebrafish reported the negative effects of PYR exposure on ovary histology [28]. In another case, a group of researchers performed experiments on Christmas Island red crabs and reported that PYR had negative effects on the ovarian histology of red crabs [58]. These studies on various animal models indicate the potentially toxic effects of PYX exposure on female reproduction and support our findings.

Conclusion

From the current findings it is concluded that exposure of adult female rats to PYR for 28 consecutive days causes a reduction in blood glucose levels, increases the total plasma protein content and cortisol levels, disrupts estrous cyclicity, reduces the plasma estradiol, progesterone levels, affects ovarian histoarchitecture. This suggests that an alarming increase in the usage of PYR in agricultural and industrial sectors is contributing to its bioaccumulation in the environment could affect all life forms. Therefore, a controlled use of PYR is advised to limit its potential hazardous effects on the ecosystem.

Study limitations

While this study provides valuable insights into the reprotoxic effects of Pyriproxyfen (PYR) in adult female Sprague Dawley rats, several limitations must be acknowledged. The main limitation is the narrow dose range (62, 124, and 186 mg/kg) and short experimental duration, so future studies should include a broader spectrum of doses and chronic exposure to provide a more comprehensive toxicity profile and chronic effects of PYR exposure on reproductive health. The study focused primarily on biochemical and histopathological parameters. A deeper molecular investigation, including gene expression studies on ovarian and endocrine pathways, would provide mechanistic insights into PYR toxicity. Moreover, our study includes the Single-Species model, results may not fully translate to other animal models or humans. Comparative studies with other species would strengthen the findings. PYR exposure in this study was oral, reflecting one of the common exposure routes in real-life scenarios. However, dermal and inhalation exposure, which are also significant in agricultural and environmental contexts, were not explored. Factors such as stress, diet composition, and hormonal cycles may have introduced variability in the results. Although efforts were made to maintain controlled conditions, some external influences may have affected the outcomes. The histopathological evaluation was limited to ovaries. Examining additional reproductive organs, such as the fallopian tubes and uterus, could provide a more holistic view of PYR’s reproductive toxicity. Furthermore, the study was focused on biochemical and histopathological endpoints. A molecular investigation into the expression of key genes involved in steroidogenesis (e.g., CYP19, StAR) and folliculogenesis would provide crucial mechanistic insights into how PYR disrupts ovarian function. Such studies are a logical and necessary next step.

Future directions

To address the existing limitations, future research should prioritize the examination of chronic exposure effects across a broader range of doses. Additionally, it is essential to integrate molecular and genetic analyses to elucidate the mechanism of action of PYR. The investigation of alternative exposure routes, such as dermal and inhalation pathways, should also be conducted. Moreover, assessing the behavioral and neurological impacts of PYR exposure is imperative. By systematically addressing these areas, future studies will be better positioned to offer a comprehensive risk assessment of PYR exposure and its potential implications for reproductive health.

Abbreviations

PYR

Pyriproxyfen

EDCs

Endocrine-disrupting chemicals

BMI

Body Mass Index

H&E

Hematoxylin and eosin stain

PBS

Phosphate Buffered saline

RF

Rupturing follicles

DGC

Degenerating granulosa cells

CL

Corpus luteum

SF

Secondary follicle

Author contributions

Conceptualization, methodology and experimentation done by JA. Data curation and writing—original draft preparation done by SA, MD, JF, HA and SJ; writing—review and editing done by TA, SR, FMH, DA and HS; visualization, supervision and funding acquisition done by SJ and SR. All authors have read and agreed to the published version of the manuscript.

Funding

The authors extend their appreciation to the Researchers Supporting project number (ORFFT-2025-07-6), King Saud University, Riyadh Saudi Arabia and Department of Zoology Quaid-i-Azam University, Islamabad for funding this project. The funding body has no role in study design.

Data availability

Raw data will be available from the corresponding author on request.

Declarations

Ethical approval and consent to participate

The study protocol (BAS#345A) was approved by the ethics committee of animal studies, at Quaid-i-Azam University, Islamabad, Pakistan. The study is reported per ARRIVE guidelines [30]. All methods were carried out following relevant guidelines and regulations.

Competing interests

The authors declare no competing interests.