Effect of Formaldehyde and Curcumin on Histomorphological Indices, Gene Expression Associated with Ovarian Follicular Development, and Total Antioxidant to Oxidant Levels in Wistar Rats

The present experimental study was undertaken to investigate the effect of formaldehyde (FA) and curcumin (CUR) on histomorphological features, antioxidant potential, and messenger ribonucleic acid (mRNA) levels of genes related to follicular development in FA-exposed rats. 24 Wistar female rats were divided into four study groups and given intraperitoneal injections of FA (10 mg/kg) (N = 6), FA (10 mg/kg) + CUR (100 mg/kg) (N = 6), sham (N = 6), and control (N = 6) for 14 days. Ovarian follicular histology, the related gene expression, blood factors, and anti/oxidation potentials were assessed using ovarian tissue and serum, respectively. The klotho was significantly overexpressed in the FA group compared with controls and shams. Contradictory, the factor in germ line alpha was significantly down-regulated in FA and FA + CUR groups compared to shams and controls. A significant decline was seen in the number of ovarian follicles in the FA group, independent of the developmental stage. Regarding the comparison of the FA + CUR group to other groups, a significant change was seen in the number of secondary, graafian, and atretic follicles. The FA group demonstrated significantly lower hemoglobin, red blood cell count, hematocrit, and mean corpuscular hemoglobin concentration than controls. The activity of glutathione peroxidase increased significantly in the FA group than in the controls. Despite the deleterious effects of FA on histological and molecular aspects of rat ovarian follicles, CUR does not appear to have a protective effect against the hazardous effects of this chemical. However, CUR in some cases has positive effects such as reducing follicular destruction and interstitial edema.


Introduction
Formaldehyde (FA), a pervasive environmental pollutant, has long been discussed concerning the environmental policy. Te human body or any animal can be exposed to organic solvents such as FA in many situations, including at work, during cleaning, and during excessive use. Te three main ways that this chemical is absorbed are through the digestive system, skin, and respiratory system. It is detrimental to various organs in addition to being a potent respiratory system infammatory trigger [1]. Exposure to FA in the air for a long time has been associated with several reproductive symptoms, including irregular menstruation, genital infections, abortions, low birth weight, and female birth abnormalities [2]. In investigations on animals, exposure to FA harmed the ovarian histology and oocyte structure in female rats and resulted in oocyte apoptosis [3]. According to the previous studies, FA exposure caused deoxyribonucleic acid (DNA) damage to the oocyte as well as apoptosis in the uterus and ovary [4].
Curcumin (CUR) is the main polyphenol in turmeric (curry powder) [5]. Recently, herbal medical compounds have been considered to be investigated for possible treatments. It has been demonstrated that CUR is benefcial to the gonads, testis, and ovaries due to its antioxidant, anticancer, antiapoptotic, and anti-infammatory properties through multiple mechanisms, such as its efects on the expression profle and the associated signaling [6][7][8]. By preventing the generation of reactive oxygen species (ROS), CUR can lessen the harm FA causes to the parameters of the sperm and testicular structure [9]. CUR is also a powerful antioxidant that protects cells from damage caused by oxidative stress and biochemical changes [10]. CUR can also decrease oxidative stress in patients with polycystic ovary syndrome due to its anti-infammatory properties [11,12].
Tere are a variety of reports on the potential efects of FA on oogenesis [13], structure and function of the ovary [14], and also benefcial impacts of CUR on premature ovarian failure [15], oocyte apoptosis [12], and polycystic ovary syndrome [16]. However, to the best of our knowledge, this is the frst study using a diferent methodology than others to investigate the efect of either FA or CUR and both together on histomorphological indices, expression of genes associated with ovarian follicle development, and antioxidant potential in the animal model.

Experimental Design.
All animal experiments in this study were approved by the Animal Ethics Committee of Yazd University of Sciences (IR.SSU. MEDI-CINE.REC.1400.121). 24 Female Wistar rats, 8 weeks old, weighing 150-200 gr, were obtained from the animal house of Yazd Institute of Reproductive research, Yazd. Iran. All experiments were performed under standard laboratory conditions. Tey received water and food while undergoing a separate 12-hour light/dark acclimatization period. All rats were randomly and equally assigned into four experimental groups as follows: (1) Te FA group including the rats was intraperitoneally injected with 10 mg/kg FA daily for 14 days [17]. (2) Te CUR + FA group including the rats was intraperitoneally injected with 100 mg/kg CUR and 10 mg/kg of FA daily for 14 days [4,18]. (3) Te sham group including rats was intraperitoneally injected with dimethyl sulfoxide (DMSO) solvent daily for 14 days, and (4) the control group included rats that received no injection and were kept in normal conditions.

Administration of FA and CUR and Sample Collections.
All Wistar rats in FA and FA + CUR groups were intraperitoneally injected by FA (Sigma Lowis ST, Mo) at a dose of 10 mg/kg. 100 mg/kg of CUR (368 MW. a Da, 94% of purity, Sigma. Louise, MO, USA) was intraperitoneally injected into six rats of FA + CUR group along with FA. 10% DMSO, to resolve the CUR for absorption, was used for sham interventions. After 14 days of injections, all rat ovaries were removed during a midline laparotomy following anesthetization with xylazine and ketamine (100 mg/kg and 10 mg/kg, respectively). Due to obtaining the ovaries, each animal's abdomen was opened. Te ovaries were weighed and then collected for further investigation. A sterile syringe was used to obtain blood samples from the heart. All serums were then separated from blood cells by centrifugation at 1500 × g for 15 minutes.

Relative Gene Expression Assessments.
Genes expression assessments associated with folliculogenesis (klotho (KI), follicle-stimulating hormone receptor (FSHR), growth differentiation factor-9 (GDF-9), factor in germ line alpha (FIGLA), B-cell lymphoma 2 (BCL-2), and BCL-2-like protein 4 (BAX)) were studied by quantitative real-time-polymerase chain reaction (qRT-PCR). Using a total RNA extraction kit (Parstous Co. Iran), total ribonucleic acid (RNA) was extracted from ovarian tissue while adhering to the manufacturer's instructions. Following that, RNA concentration was determined by using spectrophotometry and set to amounts of 10 ng/ml. Te complementary deoxyribonucleic acid (cDNA) synthesis reaction was carried out as follows: 10 min at 25°C, 60 min at 47°C, and 5 min of striking at 85°C to stop the reaction. Te cDNA was then used for qRT-PCR using SYBR Green RT-PCR Master Mix and the one-step Applied Biosystems real-time thermocycler. As it is shown in Table 1, the β-ACTIN gene was considered as the reference gene. Each PCR run was repeated three times. Te RT-PCR was performed as follows: frst denaturation phase at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 56-60°C (based on the optimal melting temperature set for each primer) for 20 s, and 72°C for 30 s. To perform PCR, a 20 µl reaction mix comprising 1 µl of cDNA was used, with 1 µl of each forward and reverse primer, 12.5 µl of master mix, and 5.5 µl of diethyl pyro carbonate (DEPC) treated water. To standardize the results by eliminating variations in mRNA and cDNA quantity and quality, the β-ACTIN transcript was used. Te level of gene expression between the treatment groups was quantifed using the 2 −∆∆Ct method [19].

Ovary Tissue Preparation.
Te prepared ovaries were embedded in cylinder-shaped parafn blocks and divided into sections using isotropic uniform random (IUR) sections. For this purpose, the parafn block of the ovary was positioned on the φ-clock at random (Figure 1(a)).
Te parafn block was then put on the θ-clock, and the division was performed along with the chosen score ( Figure 1(a)) [20].
Afterward, 5 and 20 μm sections were cut with a microtome, mounted on slides, and stained with hematoxylin and eosin (H&E).

2.4.2.
Determining the Ovarian Size. Te volumes of the ovary were assessed by Cavalieri's principle [20]. For this purpose, 8-12 sections per rat were chosen by systematic random sampling. Te frst section was chosen randomly, and the subsequent sections were gained at the same intervals. Te living fgures were subjected to the stereology probes by using the point-counting software at the magnifcation of 30×. Te volumes of the ovary were measured using the consequent formula: " P" was the whole number of points superimposing on the ovarian pictures, a/p was the area related to each point, and "T" is the distance between the chosen sections.

Determining the Volumes of the Cortex, Medulla, and
Corpus Luteum. Te volumes of the cortex, medulla, and corpus luteum were estimated using sections with 5 µm thickness. Te borders of the cortex, medulla, and corpus luteum were identifed in each ovary segment (Figure 1(b)). Te volume density of the cortex, medulla, and corpus luteum was calculated using the "point-counting approach," and the following formula after the 386.66× fnal magnifcation stereology probes were applied to the live fgure ( Figure 1(c)) [20,21]:  Vv(structure) � P(structure) P(total) . (2) Te complete points that struck the selected structure in this formula, " P" (cortex or medulla or corpus luteum)" and " P (total ovary section)," were the ovary sections, respectively. To calculate the volumes of the cortex, medulla, and corpus luteum, the ovarian thickness was multiplied by "V (ovary)": V (structure) � Vv (structure) × V (ovary).

Estimating the Number of Follicles.
Te follicular number was determined using the optical dissector technique. An Eclipse microscope (E200, Nikon, Tokyo, Japan) with a high numerical aperture (NA � 1.30) × 40 oil immersion objective was housed in the optical dissector, along with the electronic microcator (MT12, Heidenhain, Traunreut, Germany), for calculating Z-axis movement.
Te counting frame is a three-dimensional stereological probe that is used in conjunction with a stereology software system to count the number of follicles (Stereolite, SUMS, Shiraz, Iran). At a fnal magnifcation of ×386, the unbiased counting frame was placed on the live picture ovary (Figure 1(d)). Te guard zone is the area above and below each portion of the ovary. To prevent tissue artifacts, these locations were utilized from cutting through the ovarian sections in this location during tissue processing. Te frst 3.5 μm of the guard region above or below the follicle was not counted. Te "height" of the dissector, in this case, 10 μm, determined the separation between the above and below guard zones.
Te follicles were detected, and the nucleus was visible in the greatest focus of the counting frame, was positioned wholly or partially there, and avoided making contact with the banned line of choice (Figure 1(d)) [20,21]. Te following algorithm is used to calculate the follicles' numerical density (NV): where "ΣQ-" represents the number of diferent follicles located within the height of the dissector, "ΣP" represents all counting frames in the microscopic felds, "h" represents the height of the dissector, "a/f" represents the region of the counting frame, "t" represents the calculation of the section compactness by the microcator, and "BA" represents the block precession of the microtome. Te density of the ovarian section was measured throughout the microscope viewing area using IUR from each section of the ovary. Te numerical density (Nv) was multiplied by V (ovary) to approximate the total number of follicles.

Te Classifcation of the Ovarian Follicles.
Te following classifcation was performed to identify each ovarian follicle during the cell count: primordial follicle (the oocyte was enclosed by a single layer of fat granulosa cells). Te primary follicle (the oocyte was enclosed by a layer of cubic granulosa cells). Te secondary or antral follicle (the oocyte was enveloped by two or three layers of cubic granulosa cells with a space between the cells). Te mature or graafan follicle (the oocyte surrounded by more than 4 layers of granulosa cells with C-shaped spaces and cumulus oophorus). Te atretic follicle (the oocyte contained apoptotic granulosa cells detachment, oocyte autolysis, and the degeneration of the zona pellucida [20].

Measurement of Total Antioxidant, Oxidant Capacity, and Blood
Factors. Blood samples were collected from all groups at the end of the 14 days of administration. Blood was drawn after anesthesia and kept in tubes containing ethylene diamine tetra acetic acid as the anticoagulant for 10 min at room temperature. Te sample tubes were then centrifuged at 3500 rpm for 10 minutes. 1.5 ml of isolated blood serum was placed in microtubes. White blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin [22], mean corpuscular hemoglobin concentration (MCHC), platelets (PLT), and lymphocytes (LYM) and neutrophils were quantitatively assessed. Te serum levels of glutathione peroxidase-1 (GPx), total antioxidant capacity (TAC), and total oxidant status (TOS) were determined by applying the associated enzyme-linked immunosorbent assay kits (ELISA) (Navand Salamat Co. IRAN) according to the manufacturer's guidelines.

Statistical Analysis.
We used GraphPad Prism software version 9 to present graphs. Data analyses were undertaken by using IBM SPSS mean ± standard deviation version 25.00 which was used to represent the obtained data. Te data's normality was measured using the Kolmogorov-Smirnov test. Data were compared with the one-way ANOVA (Tukey test). Te Mann-Whitney test was utilized for comparison among the two groups, and the Kruskal-Wallis test was utilized for comparison between all groups. To compare the ratios between the groups, the chisquared test was used. Te p values <0.05 were regarded as signifcantly diferent.

Histomorphological and Stereological Analysis.
Although an 18% decrease was seen in ovarian weight in FA + CUR and FA groups compared to control and sham groups, there were no notable changes in either group. FA + CUR and FA groups did not difer signifcantly in terms of the volumes of the ovarian cortex, medulla, and corpus luteum (Table 3). In comparison to the controls and shams, the mean number of primordial follicles was signifcantly reduced in FA + CUR and FA groups (p < 0.0001). However, no considerable diference was found between FA + CUR and FA groups (p � 0.92). A signifcant decline in the number of primary follicles was observed in FA + CUR and FA groups compared to controls and shams (p < 0.0001). No considerable diference was found between FA + CUR and FA groups (p � 0.61). Tere was a signifcant decrease in the mean number of secondary follicles among FA + CUR and FA groups compared to controls and shams (p < 0.0001). A signifcant diference was found in FA + CUR and FA groups (p � 0.03). Te mean number of graafan follicles in FA + CUR and FA groups was signifcantly lower than in shams and controls (p < 0.0001). Te results showed that FA + CUR and FA are signifcantly diferent from each other in terms of graafan follicles (p � 0.009). In comparison to other groups, the greatest number of atretic follicles belonged to the FA group ( Figure 2).

Ovarian Morphology.
A group of follicles at diferent stages (primordial, primary, secondary, and mature) were seen in the ovary of the control and sham groups (Figures 3(a) and 3(b)). However, in the FA group, an increase in atretic follicles was observed along with an increase in edema in the medulla and vascular hyperemia (Figure 3(c)). In the FA + CUR group, follicular destruction is reduced and interstitial edema is less visible (Figure 3(d)).

Quantifcation Efects on Blood Parameters.
In this study, the FA group showed signifcantly lower concentrations of RBC, HGB, MCHC, and HCT than controls (p � 0.03, 0.02, 0.04, and 0.04, respectively). Among the study groups, a signifcant change in neutrophil counts was seen (p � 0.03). Te most counts of neutrophils were observed between the FA + CUR group and controls (p � 0.08). Tere was no noticeable diference in any variable between the other study groups (p ≥ 0.05).

Oxidant and Antioxidant Status.
Although TAC levels were higher in FA + CUR and FA groups compared to shams and controls, it did not lead to a statistically signifcant diference (p > 0.05). Although there was no remarkable diference between any of the research groups, the GPx level in the FA group was signifcantly greater than in the controls (p � 0.005). Te TOS level did not remarkably change between the study group (p > 0.05). As seen in Table 4, the FA + CUR group represented the highest TAC/TOS ratio.   Te data were represented by the mean ± SD. Two groups were compared using the post hoc (Tukey) test, and all groups were compared using the one-way ANOVA test. SD: standard deviation.  Figure 2: A comparison of the number of ovarian follicles among study groups. Each column presents the mean number ± SD of primordial (a), primary (b), secondary (c), graafan (d), and atretic follicles (e) in study groups. P < 0.05 were considered statistically signifcant. FA: formaldehyde, CUR: curcumin, and SD: standard deviation. * p � 0.02, * * p � 0.009, and * * * p < 0.0001. We used GraphPad Prism software version 9 to present graphs. Two groups were analyzed using the post hoc (Tukey) test, and all groups were analyzed using the one-way ANOVA test. In this experiment, the efects of FA and CUR on histomorphological features, antioxidant potential, and mRNA levels of genes related to follicular development in FAexposed rats were investigated. FA environmental exposure occurs mostly by inhalation. However, a lot of earlier animal experiments involved indirect exposure techniques such as intraperitoneal and subcutaneous injections [3,23]. In the current study, an intraperitoneal injection was to ensure the same intensity and efect of exposure to FA and further treatment with CUR for associated rats and to minimize the error of the study design. One of the treatment techniques to decrease the destructive efects of FA is the use of efective and safe antioxidants.
Te fndings of this investigation demonstrated that the FA group had higher levels of KL expression. Tis gene is a protective factor against oxidative stress damage, so its upregulation in the FA group indicates the external induction of oxidative stress. Similarly, several studies have reported that KL can attenuate oxidative damage and apoptosis [24,25].
Our results showed a signifcant down-regulation of FIGLA in FA + CUR and FA groups compared to controls. Following RNA level analysis, it was revealed that FIGLA was primarily expressed in mice gonads and that the ovary had a substantially higher abundance of its transcript than the testis. FIGLA was primarily expressed in the oocytes of primordial follicles in the ovary, but as the follicle grew, its expression reduced [26]. Despite having normal embryonic gonadal development, female mice lacking FIGLA are unable to generate primordial follicles after birth, leading to huge oocyte loss and infertility [27]. Previous research has shown that the lack of FIGLA greatly slows down the meiotic process, damages DNA, and causes oocyte death [28]. Taken together, due to the important role of FIGLA in folliculogenesis, its low expression in FA + CUR and FA groups could indicate the negative efect of FA on molecular pathways related to folliculogenesis.
Apoptosis is caused by the extracellular factors that control the BCL-2 and BAX genes [29]. Our fndings in the number of follicles as well as apoptotic gene expression declared that apoptosis was more pronounced in the groups receiving FA. Te fndings of FA and CUR on apoptosis-related genes in the ovaries revealed that the ratio of BAX to BCL-2 was higher in the FA group in comparison with the other groups. However, it seems neither FA nor CUR could not regulate the apoptotic pathway dependent on the BCL-2 family. Tese results may be due to the dose and duration that were used in this study. Terefore, it seems that further investigations are needed with diferent methodologies and other apoptotic-related genes.
Disruption of folliculogenesis, which is shown by a reduction in the number of distinct types of follicles in the ovarian parenchyma and interstitial edema, may consider one of the main adverse consequences of the FA injection. FA is one of several chemical solvents that could damage the ovaries and disrupt follicular growth by impairing corpus luteum function [13].
Rats in the FA group had follicular destruction as well as interstitial edema. Tis experiment indicates that FA caused most of the follicles to enter the atresia stage. A probable cause of follicular atresia could be an insufcient defense against ROS [30]. Additionally, ROS in the ovary causes alterations in ovarian blood fow and the death of granulosa [31,32]. On the other hand, the information gathered from the results on the ovary, cortex, and medulla's total volume supported the fndings of the investigation of the number of follicles, and we observed a decrease in the volume of the ovary and its subgroups (this decrease in volume was due to follicular destruction), although this decrease in volume was not signifcant between the groups. Tese results showed that FA interferes with folliculogenesis, negatively afects the ovarian structure, and leads to oxidative stress. Kareem et al. in 2014 reported similar fndings. Based on their results, the number of follicles in the ovaries of rats exposed to FA by inhalation was reduced [13].
Coadministration of CUR with FA compensated for follicular destruction and decreased interstitial edema. According to ovarian structure results, CUR can reduce oxidative stress damage caused by FA to some extent, and this efect was seen in the reduction of atresia follicles in the FA + CUR group. Te decline in follicular destruction after injection of CUR at a concentration of 100 mg/kg in rats may be due to its antioxidant and anti-infammatory properties [33,34]. Based on the literature, the efects of CUR on FA-induced ovarian damage have not yet been investigated. Oxidative stress occurs when the oxidative balance is disturbed [33,34]. Te balance of oxidants and antioxidants is essential for the normal biological function of cells and tissues. Te ratio of TAC/TOS was higher in the FA + CUR group compared with other groups. Similarly, Wang et al. in 2017 found that CUR can eliminate free radicals and improve antioxidant status in mouse ovaries [35].

Conclusion
Despite the deleterious efects of FA on histological and molecular aspects of rat ovarian follicles, CUR does not appear to have a protective efect against the hazardous efects of this chemical, at least at the dose and timing used in this study. But in some cases, it has positive efects such as reducing atretic follicles, and increase in secondary and graafan follicles was also observed. According to our data, it could be suggested to work with a diferent dosage of FA and CUR, a longer duration as well as alternative methods of the intervention such as the gavage.

Data Availability
Te data that support the fndings of the study are available from the corresponding author on request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.