Histological and Ultrastructural Studies on the Protective Effects of Chlorella vulgaris on Healthy Testis against Toxicity of The Therapeutic Regimen of Cisplatin

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Chlorella vulgaris is commonly utilized as a functional food or a magical supplement for people because of its abundant component.Its advantageous impacts on human health have been proven (Vartak et al., 2022).
We thus focused on investigating the induced testis injury by cisplatin on healthy tissue along with the potential use of Chlorella vulgaris as an ameliorative agent to overcome these injuries.To achieve such an objective, the male sex hormone and testicular tissue of rats assessed by performing histopathological and ultrastructure examinations.

MATERIALS AND METHODS Experimental Groups and Design of Work:
The animals were placed into four groups, each with five rats, after a one-week acclimation period.Saline (NaCl) was administered intraperitoneally to the rats in the control group.Cisplatin group: For three months, the animals received 134 mg/kg of cisplatin intraperitoneally, which is the same amount as was utilized in the human treatment procedure.Chlorella group: According to Zainul Azlan et al. (2020), the animals got 150 mg/kg of chlorella orally every day for three months.Cisplatin/Chlorella group: Over the course of three months, the rats received 134 mg/kg of cisplatin intraperitoneally once per week and 150 mg/kg of chlorella orally daily.

Blood and Testes Sampling:
Under anesthesia with (100 mg/Bwt) thiopental sodium, blood samples were taken from the medial retro-orbital venous plexus of rats using capillary tubes (Micro Hematocrit Capillaries) (Abdi-Azar and Maleki, 2014).Following the blood sample, the animals were dislocated to cause death, and the testis was removed for histology and ultrastructure analysis.

Male Sex Hormone:
According to Dada and Nduka (1980), testosterone was found in the blood serum using Elisa kits (MyBioSource company) (catalog no.MBS580035).

Histological Preparation:
The testis of each animal was rapidly removed when they were slaughtered, rinsed with normal saline, and preserved in 10% neutral formaldehyde.The samples were dehydrated, cleaned in terpineol for two days, rinsed in benzene for ten minutes, and then embedded in three different layers of pure paraffin wax.A microtome was used to cut sections of around 5 microns thickness, which were then placed on spotless glass slides for histological preparation.Hematoxylin and Eosin (H&E) were used to stain a piece of the tissue.The slides were then mounted with DPX and cleaned with xylene (Fischer et al., 2008).

Electron Microscopy Analysis:
Under a dissecting microscope, little testicular fragments were removed and sliced in the presence of 2% glutaraldehyde.Specimens were placed in a solution containing 2% glutaraldehyde fixative and 0.1 M sodium cacodylate buffer for 24 hours.They were then rinsed in 0.1 M phosphate buffer at 4 °C and post-fixed with 1% osmium tetraoxide.The tissues were then injected with resin following dehydration in a progressive series of ethyl alcohol.On the ultramicrotome (Leica Ultracut UCT, Heidelberger, Germany), portions of tissue-containing blocks were cut into semi-thin layers.The transmission electron microscope was used to analyze ultrathin slices that had been stained with lead citrate and uranyl acetate (Sato et al., 2008).

Statistical Analysis of Data:
The testosterone data was expressed in mean ± standard error (SE) and analyzed using SPSS 11.0 for Windows.The statistical significance of differences when p-value less than 0.05 was evaluated by using a one-way analysis of variance (ANOVA) followed by post-hoc Duncan to make the comparison between means.

Effect of Cisplatin and Chlorella Supplement on The Testosterone of Rats:
For the control and various treated groups, serum testosterone values are estimated.In terms of data compared to the control group, the chlorella and chlorella/cisplatin groups both significantly outperformed the cisplatin group.

Histological Examination:
Rats treated with chlorella and control rats with examined testicles both displayed normal histological structures Figure 2(a&b).The tunica albuginea, a capsule of connective tissue, appeared to surround the testis.Seminiferous tubules with uniform contours that resembled spherical or oval shapes made up the testicular parenchyma.There is fragile loose connective tissue and Leydig cells in the interstitial spaces between the tubules.
A basement membrane surrounding myoid cells and lined with stratified spermatogenic cells and supporting Sertoli cells encloses the seminiferous tubules.The seminiferous tubules are typically organized in several series of spermatogenic layers and spermatozoa in control and chlorellatreated rats.In the lumen of every seminiferous tubule, spermatogonia continued to develop into adult spermatozoa.Rat testes that had received cisplatin treatment Seminiferous tubules in Figure 2 (c) were severely degenerating and atrophic, and the lumen of the tubules included spermatozoa debris and hypocellular spermatogenic cells.Moreover, the lumen of the majority of tubules lacked free mature spermatozoa.Figure 2(d) illustrates the typical appearance of the germinal epithelium and the spermatogenic cells with the regular arrangement of the seminiferous tubules in rats that had been given cisplatin and chlorella treatment.

Ultrastructure Examination:
Ultrastructure examination of the testis sections of the control and chlorella groups Figure 3 (a&b) showed that the seminiferous tubule appeared to be surrounded by a basement membrane and myoid cell.The basement membrane was regular and of uniform pattern; the myoid cell was slender, smooth musclelike, and was present outside the basement membrane within the basal lamina.The spermatogonia were rounded and rested on the basement membrane containing rounded euchromatic nuclei and prominent nucleoli, and many mitochondria were observed scattered within the cytoplasm.The 1ry spermatocytes appeared to be large cells with large euchromatic nuclei.Spermatids appeared with large euchromatic nuclei and its characteristic acrosomal vesicle; many sperms or fully formed spermatozoa were also detected embedded within the Sertoli cell, which appeared as a long dark cell with an irregular outline.Sections of mature sperm revealed the normal structure of middle pieces of sperms, which were formed of a central axoneme surrounded by a fibrous sheath, then a mitochondrial sheath, and covered by a plasma membrane.The end piece of the tail was formed of axoneme only.Sertoli cells, with their cytoplasm, extend from the basal lamina to the lumen of the seminiferous tubule and envelop the adjacent germinal elements.The nucleus is enfolded and sometimes has a very irregular shape; the nucleoplasm is homogenous, and the cytoplasm contains abundant endoplasmic reticulum, ovoid Golgi apparatus, and spherical or cylinder-shaped mitochondria.
Ultrastructure examination of the testis sections of the cisplatin group Figure 3 (c) showed that the seminiferous tubule appeared to be surrounded by a thin basement membrane and degenerated myoid cell.The cytoplasm is vacuolated and disrupted, and some of their cells appear ruptured; despite that, the spermatids were recognized by the characteristic acrosomal vesicle surrounded by a widened intracellular bridge.Sertoli cells, with their cytoplasm, the chromatin clumped in their margin, and some of their membranes are ruptured.Ultrastructure examination of the testis sections of the chlorella/cisplatin group Figure 3 (d) showed a normal structure like the control group.S3) with its large euchromatic nuclei and its characteristic acrosomal vesicle (AV).The spermatogonia (SGL) were rounded and rested on the basement membrane, containing rounded euchromatic nuclei within the cytoplasm.Sertoli cells (ST), with their cytoplasm, extend from the basal lamina to the lumen of the seminiferous tubule and envelop the adjacent germinal elements.The nucleus is enfolded and sometimes has a very irregular shape; the nucleoplasm is homogenous, and the cytoplasm contains abundant endoplasmic reticulum ovoid Golgi apparatus, dense chromatin bodies (CH), lipid droplets (L) and spherical or cylindershaped mitochondria.(c) treated group with cisplatin showed cytoplasm is vacuolated (V) and disrupted, and some of the cells appeared ruptured; despite that, the spermatids (S3) were recognized by the characteristic acrosomal vesicle (AV) surrounded with a widened intracellular bridge.(d) treated group with chlorella in combination with cisplatin showed normal spermatids (S3) were recognized by the characteristic acrosomal vesicle (AV).The cytoplasm contains lipofusion granules (CE).

DISCUSSION
This study used histopathological analysis and cellular examination of testes tissues to examine the potential ameliorative effects of Chlorella vulgaris supplement against the therapeutic regime of cisplatin druginduced toxicity on the testis of healthy rats.Both healthy spermatogenesis and t he preservation of seminiferous tumbles' proper structure depend on testosterone (Sharpe et al., 1992).Chlorella treatment effectively reversed the drop in testosterone levels caused by cisplatin, which followed the preceding investigations of Silici et al. (2009) and Osama et al. (2019).As one of the potential causes, cisplatin-induced significant damage to Leydig and Sertoli cells due to the increased formation of free radicals could account for this substantial decrease in the hormone level (Ilbey et al., 2009).Additionally, numerous studies suggested that cisplatin may affect testosterone levels by impairing the expression of the luteinizing hormone (LH) receptor and cholesterol mobilization to mitochondrial cytochrome P450, thereby obstructing the initial stages of testosterone production (Essawy et al., 2017).
Chlorella's anti-oxidative function, which guards against OSinduced Leydig cell damage, may account for its favorable impact on testosterone levels (Abdel-khalek et al., 2023).Rats exposed to pollutants or toxins in the laboratory have been evaluated for health using histopathological and ultrastructural alterations as indicators (Jiang et al., 2021).Cisplatin has a toxic reproductive effect (Abdel-Wahab et al., 2020).The present study's testicular injury in cisplatin-treated rats served as a warning sign of the drug's negative effects.In the testis of rats given cisplatin treatment, there were fewer Leydig cells, fewer spermatogenic cells lining seminiferous tubules, and fewer spermatogenic cells themselves.In the lumen of the seminiferous tubules, spermatozoa debris was seen.These changes in the histology of the testis, highly dividing cells, may be due to the fact that cisplatin has been thought to be a powerful inhibitor that prevents cell division (Bunch and Eastman, 1996) or commonly linked to testicular OS induced by cisplatin (Moradi et al., 2021).These findings are in agreement with Bushra and Bastwrous (2022), that observed that during cisplatin treatment, it was seen that the number of spermatogenic cells decreased and the epithelial height of the seminiferous tubules decreased.As opposed to the cisplatin group, rats treated with chlorella plus cisplatin shield the seminiferous tubules from deterioration.In the ultrastructural examination, the testis of treated rats with cisplatin showed evidence of increased intercellular spaces between the various components of the seminiferous tubules, which is indicative of tubular edema (Krishna et al., 2018).In this present study, one of the early and most common morphological features of damage to the testis is vacuolations.These might have formed due to the autophagosomes formed for the phagocytosis of necrotic germ cells by Sertoli cells (Wang and Han, 2019).Another reason for the vacuolization may be due to the swelling and coalescence of intracellular membrane-bound organelles like the ER (Krishna et al., 2018).Spermatogenic cells and Sertoli cells degenerated in the current investigation.This could be a result of ROS, which was detected in the measured parameter, directly harming either these spermatogenic cells or Sertoli cells.Sertoli cells are crucial to the process of spermatogenesis (Alves et al., 2013).Sertoli cells produce lactate, which is important for the survival of germ cells (Boussouar and Benahmed, 2004).Any injury to Sertoli cells, as found in the present study, would affect the nutrition and sustenance of spermatogenic cells (Li et al., 2009).Additionally, as seen in this work, the separation of Sertoli cells from spermatogenic cells as a result of intercellular edema would hinder the passage of nutrients from Sertoli cells to the spermatogenic cells (El Shafai et al., 2011).

Conclusions
The current study showed that the equivalent therapeutic regime and dose of cisplatin caused adverse changes to the healthy testes of rats.Cisplatin-induced histological and ultrastructural changes of testes.Moreover, Chlorella protects the testis from the damage induced by cisplatin.

Fig. 1 :
Fig. 1: Effect of Cisplatin and Chlorella supplement treatment on testosterone.Data were expressed as means ± SEM, n=5.Data were statically analyzed using One-way ANOVA followed by Duncan multiple comparisons test.Different letters showed data from the different rows, which is statistically significant, P≤0.05.

Fig. 2 :
Fig.2: Photomicrographs of the testis of (a) control rats and (b) treated rats with chlorella showed normal seminiferous tubules (ST) with different spermatogenic stages.(c) treated rats with cisplatin showed atrophic seminiferous tubules (AT) devoid of the spermatogenic stage, and free mature spermatozoa were absent in the lumen of most tubules.(d) treated rats with chlorella and cisplatin showed normal seminiferous tubules (ST) with different spermatogenic stages.(H & E×200).

Fig. 3 :
Fig.3: An electron micrograph of the testis from (a) the control group showed normal ultrastructure of Spermatids (S3) with its large euchromatic nuclei and its characteristic acrosomal vesicle (AV).(b) treated group with chlorella showed normal ultrastructure of Spermatids (S3) with its large euchromatic nuclei and its characteristic acrosomal vesicle (AV).The spermatogonia (SGL) were rounded and rested on the basement membrane, containing rounded euchromatic nuclei within the cytoplasm.Sertoli cells (ST), with their cytoplasm, extend from the basal lamina to the lumen of the seminiferous tubule and envelop the adjacent germinal elements.The nucleus is enfolded and sometimes has a very irregular shape; the nucleoplasm is homogenous, and the cytoplasm contains abundant endoplasmic reticulum ovoid Golgi apparatus, dense chromatin bodies (CH), lipid droplets (L) and spherical or cylindershaped mitochondria.(c) treated group with cisplatin showed cytoplasm is vacuolated (V) and disrupted, and some of the cells appeared ruptured; despite that, the spermatids (S3) were recognized by the characteristic acrosomal vesicle (AV) surrounded with a widened intracellular bridge.(d) treated group with chlorella in combination with cisplatin showed normal spermatids (S3) were recognized by the characteristic acrosomal vesicle (AV).The cytoplasm contains lipofusion granules (CE).