Basella alba, Grewia asiatica, Solanum nigrum and Ficus carica fruit extracts ameliorate the testicular histopathology induced by CCl4 exposure in albino mice: An experimental study

Abstract Background Carbon tetrachloride (CCl 4 ) is extensively used in various industries and induces oxidative stress in humans. Unfortunately, it is one of the neglected potent male reproductive toxicants. Objective The present research reports the testicular histopathology of CCl 4 and ameliorations by four medicinal fruit pulp extracts (FPEs) in mice. Materials and Methods Sixty male albino mice were divided into six groups (10/group) as per the following: 1. Vehicle control (Vc); 2. CCl 4 (C); 3-6. CCl 4 +Basella alba (CBa), CCl 4 +Solanum nigrum (CSn), CCl 4 +Ficus carica (CFc) and CCl 4 +Grewia asiatica (CGa). Except for the control group, CCl 4 (0.1 mL of 0.2 mL kg -1 ) was given to the animals in corn oil. The four plant extracts (0.1 mL each) were respectively given to the relevant FPE group animals for the next five consecutive days, while the animals in the Vc and CCl 4 groups received water instead of FPE. Results The CCl 4 exposure led to various histometric and histological alterations (loss of interstitial tissue and various dislodged tailless spermatids with enlarged heads) that were recovered in all except Solanum nigrum FPE mice post-treatment. The micrometric data of testicular sections also indicated significant decline in the number of spermatogonia, while the cross-sectional area of the sperm heads remained significantly higher in the CSn and C groups. Conclusion Except for Solanum nigrum,the three FPEs, especially Ficus carica,showed rehabilitative properties against CCl 4 exposure-related modifications in testicular histopathologies.


Introduction
CCl 4 is also called carbona, benzinoform and methane tetrachloride. Humans are exposed to CCl 4 through inhalation, dermal contact and ingestion. It is used in the production of chlorofluorocarbon refrigerants, foam-blowing agents, paints, plastics and as a solvent in metal cleaning and in fumigants (1). It damages body organs like the liver, kidney and testis (2). on the body organs, especially the testes (7)(8)(9).

Materials and Methods
This experimental study was conducted for the exploration of the pathological effects of CCl 4 (a product of Riedal-de Haen, Germany, batch number 32215) and their ameliorations through various medicinal fruit extracts in albino laboratory mice.

Fruit extract preparation
Fresh fruits of Grewia asiatica and Ficus carica were obtained from the market whereas the Basella alba and Solanum nigrum fruits were collected from the suburbs of Sargodha city. All fruits were thoroughly washed, air dried, and crushed gently. Finally, the FPEs of all mentioned plants were prepared by following the lab protocol (14)(15)(16)(17). The freshly thawed FPEs were provided to the animals of the relevant groups.

Animal dissections, organ recovery and histological studies
Animals were sacrificed through cervical dislocation on day seven. Testes were recovered and their general appearance and weight were recorded. One testis from each animal in all the groups was randomly selected for smear preparation while the other was processed for microtomy and hematoxylin and eosin staining. The smears and serial sections were finally observed for various landmarks of toxicological and ameliorative activities.

Statistical analysis
For the histometric and micrometric analyses, mean ± standard error of the mean and mean CSA were calculated. The data were further The mean values at p < 0.05 were considered as significant.

Histological results of testes
The histological results of the testes showed that in the Vc group, rounded seminiferous tubules delimitated externally with the basement membrane were well visualized. In each tubular section, the spermatogonia, primary spermatocytes, spermatids and spermatozoa were found arranged concentrically peripheral to central whirls. Healthy and normal interstitial tissue was found filling the spaces among the tubular sections ( Figure 1A). Contrary to these, the C group sections showed irregularly shaped seminiferous tubules.
The internal arrangement of the spermatogenic cells was also disrupted. These cells were found rather scattered and poorly differentiated. Clumps of spermatogonial cells at peripheral margins of the tubular sections were also seen ( Figure 1B). The interstitial tissues seemed more rehabilitated than those of the C group ( Figure   1F).

Mean weight parameters for animals and organs (testes)
The mean group body weights in all five experimental groups were significantly lower than the Vc group on day seven; however, the maximum decrease was recorded in the CSn and C groups.
The mean testicular weight was also significantly lower in the C and CSn groups than in the rest of the four groups (Table I).
International Journal of Reproductive BioMedicine Medicinal fruits ameliorate testicular pathology of CCl 4

Histometric results of testes
The  (Table II).

Testicular smear results
The

Discussion
In contrast to testicular sections in the Vc group ( Figure 1A) the CCl 4 group showed severely damaged interstitial tissue and delocalized spermatogenic cells. The interstitial tissue showed severe damage as reflected by the cellular obliterations leaving wide empty spaces containing small masses of the androgenic hormone secreting Leydig cells ( Figure 1B). These histoarchitectural damages were logical consequences of lipid peroxidation in the cell membrane and the membranous organelles leading to cellular necrosis in the actively metabolizing interstitial and Sertoli cells ( Figure 2B).
The most appreciable rehabilitation from testicular pathologies was seen in the CBa group, FPEs. The elicited pericytes may rehabilitate the functional Leydig cells through proliferation (18). Also, mitotic reactivation of the scanty spermatogonial cells that escaped CCl 4 toxicity may have been involved in the restoration of spermatogenesis (19). This idea is further supported as significantly fewer spermatogonia were found per µ along the basement membrane in the C group than in that of the Vc, CBa, CFc and CGa groups. The best rehabilitative effect on the number of spermatogonia was observed in the CFc group, which perhaps could be ascribed to the large battery of unique phytosterols (12).
The increased number of spermatogonia in the CSn group was probably partly due to the extreme shrinkage of the seminiferous tubules observed because of the anti-steroidogenic effects of the solasonine in S. nigrum (Table II), which may have halted the surviving spermatogonia and their role in the process of spermatogenesis (20).
The histometric parameters of the developing sperms indicated gross alterations in the head and tail sizes in the CCl 4 and CSn groups (Table II) that must have been a consequence of halted spermiogenesis owing to severe damage to the Sertoli cells after CCl 4 exposure; this damage may have been worse in the CSn group due to its solasonine (21).
Rehabilitation of the spermatic tissue involved differentiation of primary spermatogonial cells and partial rehabilitation of the stressed Sertoli cells in the plant extracts of the CGa, CBa and CFc groups. In the CCl 4 treated group, cellular damage was indicated by frequently observed loss of maturing spermatozoa and necrotizing spermatogenic cells. This was a logical consequence of an acute nutritional deficiency in the spermatogenic cells because of their general dislodgement from the narcotized nurturing Sertoli cells on account of direct CCl 4 exposure (22). Nevertheless, the direct CCl 4 exposure might also have contributed to the oxidative damage to the plasma membrane of the spermatogenic cells and maturing spermatozoa (5). Additionally, this exposure may have also interfered with the process of mitosis and meiosis in the spermatogenic cells (23,24). The further negative consequences observed in the CSn group indicated that the S. nigrum FPE further enhanced CCl 4 toxicity, which may have been attributable to solasonine selectively damaging the process of steroidogenesis (25,26). Among the other three groups treated with the FPEs, the F. carica group showed the best rehabilitation of spermatogonial cell populations which can likely be attributed to its phytosterols. Thus, the F. carica treatment might have led to the fastest alleviation of oxidative stress, helping in rapid rehabilitation of the interstitial tissue and further resulting in natural androgen secretion, ultimately causing almost complete rehabilitation of the processes of meiosis and spermiogenesis (Figure 3).

Conclusion
Out of the four plant extracts used in this study, unlike Solanum nigrum, Ficus carica, Grewia asiatica and Basella alba showed good capacity for testicular histoarchitectural rehabilitation after CCl 4 exposure, an indication of the importance of phytosterols and natural antioxidants. This study demonstrated the importance of ethnomedicinal compounds against the testicular toxicological damage of CCl 4 , an environmental chemical with multiple occupational exposure risks. The findings indicated a need for further investigation into the antioxidant ingredients of these plants and their biochemical approach.