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Volume 20, Issue 2 (February 2022)
IJRM 2022, 20(2): 101-110 |
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Amad S N, Ahmad K R A, Usma U A, Fiza F M, Ali R, Amir-ud-din U, et al . 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. IJRM 2022; 20 (2) :101-110
URL: http://ijrm.ir/article-1-2002-en.html
URL: http://ijrm.ir/article-1-2002-en.html
Syeda Nadia Amad1 
, Khawaja Raees Ahmad Ahmad * 2, Usma Abdullah Usma3 , Fiza Malik Fiza4 , Rabiyah Ali4 , Umara Amir-ud-din4 , Muhammad Ali Kanwal4 , Iram Inayat4
, Khawaja Raees Ahmad Ahmad * 2, Usma Abdullah Usma3 , Fiza Malik Fiza4 , Rabiyah Ali4 , Umara Amir-ud-din4 , Muhammad Ali Kanwal4 , Iram Inayat4
1- Government Associate College for Women Bhera, District Sargodha, Pakistan.
2- Department of Zoology, University of Sargodha, District Sargodha, Pakistan. , k.r.ahmad@gmail.com
3- Government Degree College for Women Mianwali, Sargodha, Pakistan.
4- Department of Zoology, University of Sargodha, District Sargodha, Pakistan.
2- Department of Zoology, University of Sargodha, District Sargodha, Pakistan. , k.r.ahmad@gmail.com
3- Government Degree College for Women Mianwali, Sargodha, Pakistan.
4- Department of Zoology, University of Sargodha, District Sargodha, Pakistan.
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1. Introduction
CCl4 is also called carbona, benzinoform and methane tetrachloride. Humans are exposed to CCl4 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).
Reactive oxygen species produced by CCl4 exposure include trichloromethyl free radicals (CCl3) and trichloroperoxyl radicals (CCl3O2), which are highly reactive and increase lipid peroxidation (3). The radicals damage the membranes of testicular cells and lead to testicular toxicity. Biological exposure to CCl4 due to its extensive environmental and occupational applications has led to various fertility issues like impaired spermatogenesis, hormonal changes, and degenerations of various differentiating spermatozoa (4). CCl4 mainly targets the polyunsaturated fatty acids in the sperm membrane, causing lipid peroxidation and increased free radical concentration (5).
Antioxidant agents are chelating agents that scavenge free radicals and save cell membranes and eventually tissues from damage (6). The recent trends of research indicate the use of various medicinal fruit pulp extracts (FPEs) like turmeric, Nepeta paulsenii, ginger and cinnamon for the mitigation of noxious chemical agents like CCl4 on the body organs, especially the testes (7-9). Basella alba contains certain distinct phytosterols like kaempferol and beta-sitosterol that are capable of reducing stress-related immuno-cellular responses (10). Likewise, Grewia asiatica and Ficus carica contain phytosterols, e.g campesterol and stigmasterol (11, 12), while Solanum nigrum contains various potent non-steroidal phytochemicals like solasonine, gallic acid and rutin (13).
In the present study, the bio-ameliorative potentials of the above-mentioned FPEs were compared against CCl4 induced testicular histopathologies in adult albino mice. The novelty of this work was the use of commonly available wild medicinal plant extracts and the comparison of their ameliorative potential for testes. This is the first study to the best of our knowledge that has simultaneously addressed the testicular histopathologies of CCl4 exposure and their mitigations by the use of the four medicinal FPEs of Basella alba, Solanum nigrum, Ficus carica and Grewia asiatica.
2. Materials and Methods
This experimental study was conducted for the exploration of the pathological effects of CCl4 (a product of Riedal-de Haen, Germany, batch number 32215) and their ameliorations through various medicinal fruit extracts in albino laboratory mice.
2.1. Animal conditions and dose groups
Sixty 7-8-wk-old, male Swiss Webster albino mice (Mus musculus) of 28-30 g weight were kept in the animal house of the Department of Zoology, University of Sargodha under standard housing conditions. Animals were divided into six groups of 10 mice each.
a) Vehicle control (Vc) group: Received 0.1 mL corn oil (single dose) by gavage on day one, followed by free normal drinking water on days 2-6.
b) CCl4 (C) group: Received single dose of 0.1 mL of 0.2 mL/kg CCl4 solution in corn oil (containing 0.006 mL CCl4 for the 30 gr animal weight) on day one by gavage, followed by free normal drinking water for days 2-6.
c) CCl4+Basella alba (CBa) group: Initial dose treatment was the same as in the C group on day one, followed by 0.1 mL freshly obtained FPE of Basella alba for days 2-6 every 12 hr.
d) CCl4+Solanum nigrum (CSn) group: Initial dose treatment was the same as in the C group on day one, followed by 0.1 mL freshly obtained FPE of Solanum nigrum for days 2-6 every 12 hr.
e) CCl4+Ficus carica (CFc) group: Initial dose treatment was the same as in the C group on day one, followed by 0.1 mL freshly obtained FPE of Ficus carica for days 2-6 every 12 hr.
f) CCl4+Grewia asiatica (CGa) group: Initial dose treatment the was same as in the C group on day one, followed by 0.1 mL freshly obtained FPE of Grewia asiatica for days 2-6 every 12 hr.
2.2. 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 (14-17). The freshly thawed FPEs were provided to the animals of the relevant groups.
2.3. Preparation of CCl4 solution
The laboratory grade CCl4 was dissolved in corn oil (15:85 v/v respectively) to prepare the stock solution. This stock CCl4 solution was further diluted (6:94 v/v) to prepare the required strength of 0.006 mL CCl4/0.1 mL (single dose volume for each animal in the relevant experimental groups).
2.4. 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.
2.5. Digital histometry of testis and testicular smear
Digital histometry of testicular sections was conducted in CorelDRAW 11 under calibrated scales (cm: µ). Digital images (x400 magnification) of randomly selected sections of testes and testicular smears from each group were used for this purpose. The mean measurements of spermatogonia along the basement membrane µ-1, mean cross-sectional area (CSA) of the sperm head, mean tail length of sperm and mean number of whirls of spermatogonia/seminiferous tubules were estimated. The CSA of the sperm head was calculated using the formula CSA = (length × width)/4*π.
2.6. Ethical considerations
All procedures of this study were approved by the ethical committee of the Department of Zoology, University of Sargodha, Sargodha, Pakistan. (Code: SU/Zol/2665).
2.7. Statistical analysis
For the histometric and micrometric analyses, mean ± standard error of the mean and mean CSA were calculated. The data were further analyzed using the Statistical Package for the Social Sciences (SPSS.13) software, a brand of International Business Machines Corporation (IBM), SPSS Statistics, New York, USA. Two-way ANOVA followed by Tukey’s Multiple Range Test were used. The mean values at p < 0.05 were considered as significant.
3. Results
3.1. 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 in concentric 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). In the CBa slides, rehabilitative ultrastructural architectural arrangements were observed as shown by the partial rehabilitation of whirls of spermatogenic cells - an indication of the restoration of spermatogenesis. Healthy interstitial tissue was also observed with many regenerated Leydig cells (Figure 1C). In the CSn group slides, the size of the seminiferous tubules was much reduced as compared to the Vc group. The shape of the seminiferous tubules was also variable showing polygonal irregular margins. The spermatogonia, primary spermatocytes, secondary spermatocytes and spermatids were not present in proper alignment. This situation seemed more deteriorated than the arrangement of spermatogonia and spermatocytes in the C group. Moreover, all seminiferous tubules were devoid of differentiating spermatids and spermatozoa. The interstitial tissue showed necrosis of the Leydig cells (Figure 1D). The obvious ameliorative and regenerative indications were observed in the CGa group such as that the CSA of the seminiferous tubular sections were much enlarged. The lumen of the seminiferous tubules was occupied with an enormous number of mature spermatozoa. Regeneration of interstitial tissue was observed from the pericytes resulting in the rehabilitation of the Leydig cells (Figure 1E). The best regenerative signs were seen in the CFc group, including rounded and much enlarged seminiferous tubular sections as compared to the C group. However, small aggregates of spermatozoa were present in the central luminal spaces of the seminiferous tubules. The interstitial tissues seemed more rehabilitated than those of the C group (Figure 1F).
3.2. 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).
3.3. Histometric results of testes
The micrometric data of the testicular sections also indicated a significantly lower (p < 0.001) mean number of spermatogonia along the length (1 µ) of the basement membrane in the C group, compared with the rest of the five groups. The mean CSA of the sperm heads was significantly higher in the CSn and C groups than in the other four groups, and the mean tail length of the sperm was significantly lower in the C and CSn groups than in the rest of the four groups. Likewise, the mean number of whirls of spermatogonia per seminiferous tubule was significantly lower in the C group than in the other five groups (Table II).
3.4. Testicular smear results
The testicular smears in the Vc group showed a large number of spermatozoa with normal parakeet beak-shaped heads and a long straight tail. Some spermatogenic cells and partially formed spermatozoa at various stages of spermiogenesis were also seen along with hormone-secreting interstitial Leydig cells (Figure 2A). In the CCl4 group, maturing and mature spermatozoa were rarely observed; however, various spermatids with a small rudimentary tail or no tail were frequently observed. Clumps of interstitial cells were rarely seen (Figure 2B). In the CBa group slides, many maturing and mature spermatozoa, spermatocytes, spermatogonia, spermatids and some aggregations of interstitial cells were observed. Nevertheless, many of these spermatogenic and interstitial cells were also found showing intracellular vacuolations, indicating signs of intracellular toxicity and necrosis (Figure 2C). In the CSn group slides, the situation seemed to be worse in terms of a large number of necrotizing spermatids and other spermatogenic cells with no matured or maturing spermatozoa (Figure 2D). In the CGa group slides, spermatids were frequently observed. Some maturing spermatozoa were also found while the other spermatogenic cells like spermatogonia and spermatocytes were also observed. The frequency of occurrence of spermatogonia seemed even better than in the CBa group (Figure 2E). In the CFc group, although all spermatogenic cells (spermatogonia, spermatocytes, maturing spermatids and maturing and matured spermatozoa) and a few clumps of interstitial cells were present, some spermatogenic cells also showed signs of apoptosis (cytoplasmic vacuolations) (Figure 2F).
4. Discussion
In contrast to testicular sections in the Vc group (Figure 1A) the CCl4 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, followed by the CGa and CFc groups (Figures 1C, 1E and 1F, respectively). However, in the CSn group, worse conditions were observed with extreme shrinkage of the tubular sections of the CCl4 exposure damage, and no signs of recovery (Figure 1D). The prominent signs of rehabilitation of steroidogenesis and spermatogenesis in the Cba, CGa and CFc groups were the at least partially regenerated interstitial tissue and the realigned sperm-producing cells within the tubules. This included re-arrangement of whirls of spermatogonia along the basement membrane, followed by the inner whirls of spermatogenic cells at various stages of spermiogenesis (Figures 1C, 1E and 1F, respectively). These rehabilitative activities were likely partially attributable to the unique blends of phytosterols like sitosterol, stigmasterol and campesterol found in the FPEs of G. asiatica, B. alba and F. carica (10-13) that mimic the role of male sex hormones and thus logically contribute towards stress alleviation, by promoting the anabolic activities in the testis, and thereby assisting the restoration and rehabilitation of spermatogenesis. The process of testicular histoarchitectural rehabilitation may involve the activation of dormant pericytes present at the margins of the seminiferous tubules by the androgenic phytosterols present in these FPEs. The elicited pericytes may rehabilitate the functional Leydig cells through proliferation (18). Also, mitotic reactivation of the scanty spermatogonial cells that escaped CCl4 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 CCl4 and CSn groups (Table II) that must have been a consequence of halted spermiogenesis owing to severe damage to the Sertoli cells after CCl4 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 CCl4 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 CCl4 exposure (22). Nevertheless, the direct CCl4 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 CCl4 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).
5. 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 CCl4 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 CCl4, 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.
Acknowledgements
We thank the University of Sargodha, Sargodha, Pakistan for permission to conduct this study and for the provision of appropriate funding, and we thank the lab and animal house facilities.
Conflict of Interest
The authors declare that there is no conflict of interest.
Full-Text: (300 Views)
1. Introduction
CCl4 is also called carbona, benzinoform and methane tetrachloride. Humans are exposed to CCl4 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).
Reactive oxygen species produced by CCl4 exposure include trichloromethyl free radicals (CCl3) and trichloroperoxyl radicals (CCl3O2), which are highly reactive and increase lipid peroxidation (3). The radicals damage the membranes of testicular cells and lead to testicular toxicity. Biological exposure to CCl4 due to its extensive environmental and occupational applications has led to various fertility issues like impaired spermatogenesis, hormonal changes, and degenerations of various differentiating spermatozoa (4). CCl4 mainly targets the polyunsaturated fatty acids in the sperm membrane, causing lipid peroxidation and increased free radical concentration (5).
Antioxidant agents are chelating agents that scavenge free radicals and save cell membranes and eventually tissues from damage (6). The recent trends of research indicate the use of various medicinal fruit pulp extracts (FPEs) like turmeric, Nepeta paulsenii, ginger and cinnamon for the mitigation of noxious chemical agents like CCl4 on the body organs, especially the testes (7-9). Basella alba contains certain distinct phytosterols like kaempferol and beta-sitosterol that are capable of reducing stress-related immuno-cellular responses (10). Likewise, Grewia asiatica and Ficus carica contain phytosterols, e.g campesterol and stigmasterol (11, 12), while Solanum nigrum contains various potent non-steroidal phytochemicals like solasonine, gallic acid and rutin (13).
In the present study, the bio-ameliorative potentials of the above-mentioned FPEs were compared against CCl4 induced testicular histopathologies in adult albino mice. The novelty of this work was the use of commonly available wild medicinal plant extracts and the comparison of their ameliorative potential for testes. This is the first study to the best of our knowledge that has simultaneously addressed the testicular histopathologies of CCl4 exposure and their mitigations by the use of the four medicinal FPEs of Basella alba, Solanum nigrum, Ficus carica and Grewia asiatica.
2. Materials and Methods
This experimental study was conducted for the exploration of the pathological effects of CCl4 (a product of Riedal-de Haen, Germany, batch number 32215) and their ameliorations through various medicinal fruit extracts in albino laboratory mice.
2.1. Animal conditions and dose groups
Sixty 7-8-wk-old, male Swiss Webster albino mice (Mus musculus) of 28-30 g weight were kept in the animal house of the Department of Zoology, University of Sargodha under standard housing conditions. Animals were divided into six groups of 10 mice each.
a) Vehicle control (Vc) group: Received 0.1 mL corn oil (single dose) by gavage on day one, followed by free normal drinking water on days 2-6.
b) CCl4 (C) group: Received single dose of 0.1 mL of 0.2 mL/kg CCl4 solution in corn oil (containing 0.006 mL CCl4 for the 30 gr animal weight) on day one by gavage, followed by free normal drinking water for days 2-6.
c) CCl4+Basella alba (CBa) group: Initial dose treatment was the same as in the C group on day one, followed by 0.1 mL freshly obtained FPE of Basella alba for days 2-6 every 12 hr.
d) CCl4+Solanum nigrum (CSn) group: Initial dose treatment was the same as in the C group on day one, followed by 0.1 mL freshly obtained FPE of Solanum nigrum for days 2-6 every 12 hr.
e) CCl4+Ficus carica (CFc) group: Initial dose treatment was the same as in the C group on day one, followed by 0.1 mL freshly obtained FPE of Ficus carica for days 2-6 every 12 hr.
f) CCl4+Grewia asiatica (CGa) group: Initial dose treatment the was same as in the C group on day one, followed by 0.1 mL freshly obtained FPE of Grewia asiatica for days 2-6 every 12 hr.
2.2. 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 (14-17). The freshly thawed FPEs were provided to the animals of the relevant groups.
2.3. Preparation of CCl4 solution
The laboratory grade CCl4 was dissolved in corn oil (15:85 v/v respectively) to prepare the stock solution. This stock CCl4 solution was further diluted (6:94 v/v) to prepare the required strength of 0.006 mL CCl4/0.1 mL (single dose volume for each animal in the relevant experimental groups).
2.4. 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.
2.5. Digital histometry of testis and testicular smear
Digital histometry of testicular sections was conducted in CorelDRAW 11 under calibrated scales (cm: µ). Digital images (x400 magnification) of randomly selected sections of testes and testicular smears from each group were used for this purpose. The mean measurements of spermatogonia along the basement membrane µ-1, mean cross-sectional area (CSA) of the sperm head, mean tail length of sperm and mean number of whirls of spermatogonia/seminiferous tubules were estimated. The CSA of the sperm head was calculated using the formula CSA = (length × width)/4*π.
2.6. Ethical considerations
All procedures of this study were approved by the ethical committee of the Department of Zoology, University of Sargodha, Sargodha, Pakistan. (Code: SU/Zol/2665).
2.7. Statistical analysis
For the histometric and micrometric analyses, mean ± standard error of the mean and mean CSA were calculated. The data were further analyzed using the Statistical Package for the Social Sciences (SPSS.13) software, a brand of International Business Machines Corporation (IBM), SPSS Statistics, New York, USA. Two-way ANOVA followed by Tukey’s Multiple Range Test were used. The mean values at p < 0.05 were considered as significant.
3. Results
3.1. 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 in concentric 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). In the CBa slides, rehabilitative ultrastructural architectural arrangements were observed as shown by the partial rehabilitation of whirls of spermatogenic cells - an indication of the restoration of spermatogenesis. Healthy interstitial tissue was also observed with many regenerated Leydig cells (Figure 1C). In the CSn group slides, the size of the seminiferous tubules was much reduced as compared to the Vc group. The shape of the seminiferous tubules was also variable showing polygonal irregular margins. The spermatogonia, primary spermatocytes, secondary spermatocytes and spermatids were not present in proper alignment. This situation seemed more deteriorated than the arrangement of spermatogonia and spermatocytes in the C group. Moreover, all seminiferous tubules were devoid of differentiating spermatids and spermatozoa. The interstitial tissue showed necrosis of the Leydig cells (Figure 1D). The obvious ameliorative and regenerative indications were observed in the CGa group such as that the CSA of the seminiferous tubular sections were much enlarged. The lumen of the seminiferous tubules was occupied with an enormous number of mature spermatozoa. Regeneration of interstitial tissue was observed from the pericytes resulting in the rehabilitation of the Leydig cells (Figure 1E). The best regenerative signs were seen in the CFc group, including rounded and much enlarged seminiferous tubular sections as compared to the C group. However, small aggregates of spermatozoa were present in the central luminal spaces of the seminiferous tubules. The interstitial tissues seemed more rehabilitated than those of the C group (Figure 1F).
3.2. 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).
3.3. Histometric results of testes
The micrometric data of the testicular sections also indicated a significantly lower (p < 0.001) mean number of spermatogonia along the length (1 µ) of the basement membrane in the C group, compared with the rest of the five groups. The mean CSA of the sperm heads was significantly higher in the CSn and C groups than in the other four groups, and the mean tail length of the sperm was significantly lower in the C and CSn groups than in the rest of the four groups. Likewise, the mean number of whirls of spermatogonia per seminiferous tubule was significantly lower in the C group than in the other five groups (Table II).
3.4. Testicular smear results
The testicular smears in the Vc group showed a large number of spermatozoa with normal parakeet beak-shaped heads and a long straight tail. Some spermatogenic cells and partially formed spermatozoa at various stages of spermiogenesis were also seen along with hormone-secreting interstitial Leydig cells (Figure 2A). In the CCl4 group, maturing and mature spermatozoa were rarely observed; however, various spermatids with a small rudimentary tail or no tail were frequently observed. Clumps of interstitial cells were rarely seen (Figure 2B). In the CBa group slides, many maturing and mature spermatozoa, spermatocytes, spermatogonia, spermatids and some aggregations of interstitial cells were observed. Nevertheless, many of these spermatogenic and interstitial cells were also found showing intracellular vacuolations, indicating signs of intracellular toxicity and necrosis (Figure 2C). In the CSn group slides, the situation seemed to be worse in terms of a large number of necrotizing spermatids and other spermatogenic cells with no matured or maturing spermatozoa (Figure 2D). In the CGa group slides, spermatids were frequently observed. Some maturing spermatozoa were also found while the other spermatogenic cells like spermatogonia and spermatocytes were also observed. The frequency of occurrence of spermatogonia seemed even better than in the CBa group (Figure 2E). In the CFc group, although all spermatogenic cells (spermatogonia, spermatocytes, maturing spermatids and maturing and matured spermatozoa) and a few clumps of interstitial cells were present, some spermatogenic cells also showed signs of apoptosis (cytoplasmic vacuolations) (Figure 2F).
4. Discussion
In contrast to testicular sections in the Vc group (Figure 1A) the CCl4 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, followed by the CGa and CFc groups (Figures 1C, 1E and 1F, respectively). However, in the CSn group, worse conditions were observed with extreme shrinkage of the tubular sections of the CCl4 exposure damage, and no signs of recovery (Figure 1D). The prominent signs of rehabilitation of steroidogenesis and spermatogenesis in the Cba, CGa and CFc groups were the at least partially regenerated interstitial tissue and the realigned sperm-producing cells within the tubules. This included re-arrangement of whirls of spermatogonia along the basement membrane, followed by the inner whirls of spermatogenic cells at various stages of spermiogenesis (Figures 1C, 1E and 1F, respectively). These rehabilitative activities were likely partially attributable to the unique blends of phytosterols like sitosterol, stigmasterol and campesterol found in the FPEs of G. asiatica, B. alba and F. carica (10-13) that mimic the role of male sex hormones and thus logically contribute towards stress alleviation, by promoting the anabolic activities in the testis, and thereby assisting the restoration and rehabilitation of spermatogenesis. The process of testicular histoarchitectural rehabilitation may involve the activation of dormant pericytes present at the margins of the seminiferous tubules by the androgenic phytosterols present in these FPEs. The elicited pericytes may rehabilitate the functional Leydig cells through proliferation (18). Also, mitotic reactivation of the scanty spermatogonial cells that escaped CCl4 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 CCl4 and CSn groups (Table II) that must have been a consequence of halted spermiogenesis owing to severe damage to the Sertoli cells after CCl4 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 CCl4 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 CCl4 exposure (22). Nevertheless, the direct CCl4 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 CCl4 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).
5. 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 CCl4 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 CCl4, 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.
Acknowledgements
We thank the University of Sargodha, Sargodha, Pakistan for permission to conduct this study and for the provision of appropriate funding, and we thank the lab and animal house facilities.
Conflict of Interest
The authors declare that there is no conflict of interest.
Type of Study: Original Article |
Subject:
Reproductive Biology
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