INTRODUCTION:

Lead is a high-density metal commonly used in the manufacturing industry for producing various products, such as battery, pipe, gasoline, paint, cosmetics, and children's toys¹. The use of lead in various daily products causes an environmental hazard. Eventually, humans are exposed to lead, and it can damage several organs, including ovary as reproductive organ².

Lead exposure may cause infertility, miscarriage, preeclampsia, gestational hypertension, and preterm birth. Cellular death is an active mechanism that requires energy and does not involve inflammation. This phenomenon is also known as cellular apoptosis, a process that requires caspase-3 protein to execute³.

In the ovary, caspase-3 is expressed inside the shrinking granulosa cells [atresia] by the presence of both oxidative stress agents and reproductive hormones⁴

The presence of lead manipulates female reproductive hormones by decreasing FSH level, which affects estradiol and progesterone levels down as well⁵. FSH [Follicle Stimulating Hormone] is essential for follicle maturation, especially at the gonadotropin-dependent phase in which the follicles develop further⁶. The diameter of follicles is determined at this stage. If the process does not work correctly, the follicles will shrink [atresia]. The diameter of follicles itself relates to the amount of granulosa cell and the oocyte's ability to mature and be fertilized^7,8. Follicle atresia is a mechanism that usually occurs in any stage of follicle maturation⁹. Atresia process in antral follicles can occur due to a decrease of estradiol level¹⁰.

This research aims to observe the extent of damage that oral-administered lead inflicts on female rats' ovaries by assessing the expression of caspase-3 as an indicator of ongoing apoptosis of antral granulosa cells and measuring the diameter of tertiary follicles and the amount of follicle atresia.

MATERIALS AND METHODS:

In this research, the trial animals used were 24 female Wistar rats [Rattus Norvegicus] aged 8 to 10 weeks, weighed 100 to 200 grams, obtained in a healthy condition. One millilitre of lead acetate was given orally to samples each day for 30 days. Twenty-four female rats were classified into 4 different groups. Group 1 is not administered with any lead acetate. Groups 2, 3, and 4 are administered with 30 ppm, 100 ppm, and 300 ppm of lead acetate.

The rats were examined for their oestrous cycle the day after the last day of lead acetate administration. The dissection was undergone during the pro-oestrous phase. After that, ovaries were obtained, weighed, and stored inside a 10 % formalin buffer container. The staining was conducted in the Laboratory of Anatomical Pathology, Faculty of Medicine, Universitas Brawijaya. Ovaries, previously stored in formalin buffers, are paraffinized, coloured using Hematoxylin stain, Eosin 1% for 10-15 minutes, and dehydrated using Alcohol 70%, 80%, and 96% for 3 minutes each. The preparations are then drenched in Xylol for 60 minutes, mounted using Entellan, covered using cover-glasses, and left to dry for observation. Each slide of preparation is observed under Dot Slide Olympus XC microscope using 400x magnifying power.

The expression of caspase-3 was assessed through immunohistochemistry using BIOSS's Caspase-3 Polyclonal Antibody [bs-0081R]. This method was conducted in the Laboratory of Biomedicine, Faculty of Medicine, Universitas Brawijaya. The preparation slides were deparaffinized by washing them with Xylol three times for 3 minutes each then were dehydrated by soaking them in an absolute ethanol solution [2x10 minutes], ethanol 90% [1x5 minutes], ethanol 80% [1x5 minutes], ethanol 70% [1x5 minutes], and distilled water [3x5 minutes]. Furthermore, the slides undergo an antigen retrieval by heating them in a 95°C water bath for 20 minutes, then chilled and PBS-washed [3x5 minutes]. Before doing immunostaining, the preparation should be undergone endogenous peroxide blocking using Peroxide 3% in methanol [20 minutes], incubated in a room temperature [15 minutes], PBS-washed [1x5 minutes and 2x2 minutes]. After that, unspecific protein blocking was conducted using background sniper, and then the slides were once again incubated and PBS-washed as in previous steps. Finally, the primary antibody incubation should be performed overnight under 4°C. On the second day, the preparation slides were PBS-washed [1x5 minutes and 2x2 minutes] then performed upon them with secondary antibody incubation for 60 minutes, PBS-washing, SA-HRP incubation for 40 minutes, PBS-washing, and distilled water rinsing for 3-4 times, chromagen DAB application for 3 minutes, and distilled water rinsing [3x5 minutes]. The clean preparation slides were finally counter-stained using Meyer's incubation for one minute. Three drops of double-distilled water are applied upon the slides, and they are re-incubated for 5 minutes. After the final rinsing using distilled water, the preparation slides were ready to be mounted and covered with glass.

To identify tertiary follicles, we found a specific characteristic of the follicle that was four layers of granulosa cells circling the follicles. An antrum also must present on the HE-stained ovarian tissue when it is scanned using an Olympus microscope supported by the Dot Slide software application. The scan was then registered into the Image-J software application to measure the diameter of tertiary follicles. The diameter of each follicle was summed and mean-calculated. Follicle atresia was identified from HE-stained ovarian tissue and scanned using the Olympus microscope. The method used for identifying follicle atresia is observing the follicles in 10 high-powered fields using Osman's criteria [1985]. The data recorded from the experiment was statistically analyzed using SPPS for Windows version 20. The expression of caspase-3 and follicle atresia were analyzed using the ANOVA test, followed by LSD. The diameter of tertiary follicles was analyzed using the Kruskal-Wallis test, followed by Mann-Whitney test. The significance level in this research was set by 0.05.

RESULTS:

The Expression of Caspase-3 in Antral Granulosa Cells:

The brown colour in granulosa cells characterizes the expression of caspase-3. K: normal expression of caspase-3. P1 [30 ppm]: enhanced expression of caspase-3 compared to K. P2 [100 ppm]: further enhancement of caspase-3 expression. P3 [300 ppm]: the most enhanced caspase-3 among the groups.

Figure 1: Expression of Caspase-3 in Antral Granulosa Cells. The expression of caspase-3 was observed using immunohistochemistry under the 400x magnifying power of a microscope supported with Olympus digital camera

Table 1: The effect of oral applied lead acetate on the expression of caspase-3 in antral granulosa cells

Caspase-3	*Mean ± SD*	*p-value*
Control	32.97 ± 11.68	0.007
30 ppm of lead acetate	41.17 ± 15.18
100 ppm of lead acetate	45.34 ± 14. 55
300 ppm of lead acetate	61.68 ± 8.41*

Data shown as mean ± SD. *: P< 0.05.

ANOVA test shows that the oral administration of lead acetate significantly enhances the expression of caspase-3 in antral granulosa cells [p-value of 0.007]. Follow-up LSD test shows that the enhancement of caspase-3 is significant in the 100 ppm and 300 ppm groups. Figure 2 shows the average enhancement of caspase-3 expression.

Figure 2: Histogram for Caspase-3 Expression of Caspase-3 in Antral Granulosa Cells

P1: 30 ppm of lead acetate; P2: 100 ppm of lead acetate; P3: 300 ppm of lead acetate.

The measurement of diameter of antral follicles using scan from Olympus microscope with 400x magnifying power in ImageJ software application.

K: normal follicle diameter. P1 [30 ppm]: smaller diameter compared to K. P2 [100 ppm]: smaller diameter compared to K and P1. P3 [300 ppm]: the smallest diameter among the groups.

Figure 3: Ovarian Tertiary Follicles

Table 4: The effect of orally-administered lead acetate to the diameter of tertiary follicle

Diameter of tertiary follicles	Mean ± SD	*p-value*
Control	237.484 ± 45.81	0.002
P1	218.825 ± 8.45
P2	194.497 ± 23.04*
P3	158.267 ± 23.037*

Data shown as mean ± SD. *: P< 0.05.

Kruskal-Wallis test shows that lead acetate's oral administration significantly shrinks the diameter of tertiary follicles in experiment groups [p-value of 0.002]. Follow-up Mann-Whitney tests showed that the diameter shrinks significantly between the 100 ppm and 300 ppm groups. Figure 4 shows an average decrease in diameter.

Figure 4: Histogram for Average diameter of tertiary follicles

P1 = 30 ppm of lead acetate; P = 2 100 ppm of lead acetate; P3 = 300 ppm of lead acetate.

Follicle Atresia:

Figure 5: Image of follicle atresia viewed from from Olympus microscope under 400x magnifying power.

K: follicle atresia from control group. P1, P2, P3: image of follilce atresia taken from ovaries of rats receiving lead acetate of 30 ppm, 100 ppm, and 300 ppm respectively.

Table 5: The effect of orally-administered lead acetate to the amount of follicle atresia

Follicle Atresia	Mean ± SD	*p-value*
Control	2.67 ± 1.21	0.037
P1	2.83 ± 2.48
P2	2.00 ± 0.89
P3	5.17 ± 2.22^*

Data shown as mean ± SD. *: P< 0.05

The ANOVA test results in the significant increase of follicle atresia from experiment group [p-value of 0.037]. Follow-up LSD tests showed that the number of follicle atresia significantly increases in 100 ppm and 300 ppm groups. Figure 6 shows a graphic of the average increase for follicle atresia.

Figure 6: Histogram for Average Number of Follicle Atresia

P1: 30 ppm of lead acetate; P2: 100 ppm of lead acetate; P3: 300 ppm of lead acetate.

DISCUSSION:

The expression of caspase-3 is significantly enhanced in the 300 ppm group. Whereas 30 ppm and 100 ppm groups both show caspase-3 enhancement yet insignificantly. This phenomenon is presumably due to low dose and short duration of lead administration, affecting only little on the change of FSH, estradiol, progesterone, and oxidative stress level in ovaries. Oxidative stress acts as the lead agent causing cellular damage. Lead bonds with sulfhydryl group from Glutathione, a natural substance inside the human body acting as an antioxidant, making it nonworking. Low-level GSH will lead to cellular apoptosis^3,11. The enhancement of caspase-3 expression proves the apoptosis of antral granulosa cells due to lead exposure. This result favours the previous findings stating that lead acetate exposure enhances caspase-3 expression¹². Caspase-3 is the principal prosecutor that initiates cellular apoptosis, characterized by DNA-fragmentation inside the nucleus^13–15. The enhancement of caspase-3 in this research reciprocates apoptosis index from other similar research¹². This enhancement can be triggered by various apoptosis pathway^15,16, one of which is by destroying cellular mitochondria¹⁷. Lead acetate itself becomes the agent that directly inflicts damage on mitochondria, enhancing caspase-3 expression.

Other than that, histopathologic changes also occur so that the diameter of tertiary follicle shrinks and the amount of follicle atresia increases. Follicle growth itself is highly dependent towards FSH level. Lead has been proven to lower FSH, estradiol, and progesterone level^5,18. The low FSH level recorded from the experiment causes the diameter shrinkage of the follicles. The shrinkage eventually leads to follicle atresia. Follicle atresia is an apoptosis mechanism that occurs explicitly in the ovary⁹. Furthermore, the low FSH level also lowers the level of GSH in the ovary¹⁹.

The damage to the ovary caused by orally-administered lead acetate can be assessed by enhancing caspase-3 expression in antral granulosa cells, the diameter shrinkage of tertiary follicles and the increase of follicle atresia. These phenomena are triggered by the presence of oxidative stress in the ovary and the low level of FSH.

CONCLUSION:

It found that significant rat body weight loss and ovary weight loss from week 0 to week 4 of an experiment in the 30 ppm, 100 ppm, and 300 ppm groups. These results prove that rats suffer from growth restriction, possibly due to anaemia, nausea, vomiting, and epigastric pain caused by lead poisoning.

CONFLICT OF INTEREST:

The authors declared there were no competing interests in the study.

ETHICAL APPROVAL:

This research was declared an ethical pass test by the Ethics Committee of Faculty of Brawijaya, Malang, East Java Indonesia with number 261/EC/KEPK/S2/06/2016.

FUNDING:

The research funded by the authors.

AVAILABILITY OF DATA AND MATERIAL:

Data will be available on request.

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Received on 09.01.2021 Modified on 14.02.2021

Research J. Pharm.and Tech 2021; 14(11):6007-6011.

DOI: 10.52711/0974-360X.2021.01044