{"id":49282,"date":"2023-06-30T11:34:35","date_gmt":"2023-06-30T11:34:35","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=49282"},"modified":"2023-07-11T05:52:06","modified_gmt":"2023-07-11T05:52:06","slug":"immunohistochemical-and-ultrastructural-evaluation-of-spermatogenic-alteration-by-p53-under-the-influence-of-bisphenol-a","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol16no2\/immunohistochemical-and-ultrastructural-evaluation-of-spermatogenic-alteration-by-p53-under-the-influence-of-bisphenol-a\/","title":{"rendered":"Immunohistochemical and ultrastructural evaluation of spermatogenic alteration by p53 under the influence of bisphenol-A."},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Cellular growth and\nproliferation are highly regulated processes. Among various factors, p53\nsuperfamily proteins play a significant role in cellular proliferation. In\ncases of abnormal stress or cellular damage, p53 signals repair machinery to\nmaintain genomic integrity. In cases where repair is not\npossible, it induces cell-cycle arrest and apoptosis.1<\/sup> According to many studies, p53 is silenced in most cancerous\ncells.2,3<\/sup> Apoptosis is an\nimportant phenomenon in cell growth and proliferation, in the absence of it,\ntumours may form in most cells. Hair follicles, skin cells, gastrointestinal\ncells, lymphocytes, germ cells, etc. require stringent control over\nproliferation and differentiation. Under slight variations in the cellular\nmicroenvironment, the role of p53 becomes extremely valuable in the regulation\nof damage destined to occur.<\/p>\n\n\n\n

Spermatogenesis is an ideal function to understand the\nimportance of p53. Mature sperm develop from the complex differentiation of\nspermatogonial cells, spermatocytes, and spermatids. However, before\nspermatogonia turn into spermatocytes, they follow a series of five subsequent\ndivisions, such as; A1, A2, A3, A4, and B 4<\/sup>. A study by Beumer et\nal.5 <\/sup>showed that differentiation of A2-B spermatogonia was\nrelatively more radioresistant than that of their parallel controls in p53\nknockout mice. Similarly, other studies also revealed a significant role for\np53 during the prophase of meiosis (Beumer et al.6<\/sup> leading to higher\nexpression of p53 in spermatocytes.7<\/sup><\/p>\n\n\n\n

Bisphenol A is a polymerizing agent used in plastics and plastic\nproducts. BPA is an endocrine disruptor that negatively interferes with the\nregulation of spermatogenesis.8<\/sup>\n<\/sup>There is\n ample\ninformation on BPA that it causes apoptotic damage in Sertoli cells 9-12 <\/sup>and\ngerm cells.13<\/sup> A study by Lloyd et\nal.14<\/sup> reported that BPA has a stimulatory effect on p53. BPA\nand p53 are also associated with each other by means of oxidative stress; previous studies have associated BPA with targeted oxidative\nstress in testicular tissue.15,16<\/sup> Interestingly, p53\nregulates antioxidant activities to ensure cell survival during low oxidative\nstress while promoting cell death during high oxidative stress.17<\/sup> Oxidative damages in\nsperms follow a typical pattern such as head tail separation, axonemal damage,\nand multiple morphological defects.18<\/sup> Based on earlier studies, the present study attempted to evaluate\nthe role of p53 at various stages of spermatogenesis in animals administered\nwith BPA. This study also investigated patterns in types of deformities\nassociated with oxidative stress in testicular sperm and related theoretically\nwith p53 expression.<\/p>\n\n\n\n

Materials and methods<\/strong><\/p>\n\n\n\n

Test material <\/strong><\/p>\n\n\n\n

Bisphenol A or 2,2-bis(4-hydroxyphenyl) propane (\u226599%) was\nmade commercially available from Sigma Aldrich, MO, USA.<\/p>\n\n\n\n

Test animals<\/strong><\/p>\n\n\n\n

Male albino rats (Rattus norvegicus<\/em>) were used in the\npresent study. These rats were selected based on their age (3 months) and\nweight (150\u2013200 g). All animals were maintained under the strict observation of\na veterinary expert. The university\u2019s departmental facility provided\npolypropylene cages (43\u00d727\u00d715 cm) and housing conditions where 12:12-h of\nlight: dark was ensured. Experiments carried out under the guidelines of the\nCommittee for the Purpose of Control and Supervision of Experiments and Animals.19<\/sup> Institutional Animal\nEthics Committee (IAEC)-approved experimental protocols were used and performed\nunder the procedures of the Indian National Science Academy (INSA), New Delhi,\nfor the care and use of animals.<\/p>\n\n\n\n

Experimental design<\/strong><\/p>\n\n\n\n

Animals were randomly divided into four groups, consisting of 10\nanimals each. Group I: vehicle-treated control; Group II: administered with 10\nmg BPA\/kg b.w. dissolved in olive oil; similarly, Group III: 50 mg BPA\/kg b.w.\ndissolved in olive oil; and Group IV: 100 mg BPA\/kg b.w. BPA was administered\nthrough oral gavage by dissolving it in olive oil in a 1:1 ratio. Accordingly,\nBPA was administered daily for 6 weeks.<\/p>\n\n\n\n

Detection and localization of Apoptotic marker <\/strong><\/p>\n\n\n\n

The immunohistochemistry of testis tissue was done by the\nAvidin-Biotin Complex (ABC) immunostaining method as described in the kit.\nFormalin-fixed, paraffin-embedded tissues were heated to expose antigenic\nresidue. A concentration of 2.5 \u03bcg\/ml (1:1000) of primary antibody was used for\nstaining, and the solution was incubated overnight at room temperature. After\nincubation with the primary antibody, slides were incubated with biotinylated\nsecondary antibodies, followed by horseradish peroxidase-streptavidin and\nchromogen.20<\/sup> Apoptotic profiles\nwere calculated based on set criteria in the focal plane.<\/p>\n\n\n\n

Scanning Electron Microscopy<\/strong><\/p>\n\n\n\n

Sperm collected from the cauda epididymis were washed twice with\nphosphate buffer (pH 7.0) and centrifuged at 1500 rpm for 15 minutes. Sperm\npellets were fixed in 2.5% glutaraldehyde for 30 minutes and washed three times\nin phosphate buffer followed by distilled water. A thin film of spermatozoa was\nsmeared on a clean glass slide, air dried, and mounted on an SEM stub with\nsilver paint. A coated sputter at 350 \u00c5was observed under ascanning\nelectron microscope.<\/p>\n\n\n\n

Results<\/strong><\/p>\n\n\n\n

Detection and localization of an apoptotic marker<\/strong><\/p>\n\n\n\n

Results indicated limited cellular expression of p53 in control (Group I) animals. The most stained areas were in and around Leydig cells. In Group II, the degree of p53 expression was higher, and these expressions were mostly localised in and around Leydig cells. Expression of p53 was also evident in seminiferous tubules, more specifically, in the lumen, but staining was not very strong. Group III’s histological slide indicated higher expression of p53 compared to control and Group II. Besides Leydig cells, the basal lamina also indicated high expression of p53. Group IV, notably, indicated lower expression of p53 in areas proximal to the basal lamina, compared to Group II. However, there were clear indications of higher expression in germ cells, which appeared to have fallen into the lumen. The lower expression at spermatogonial location was only due to a low cell count and high disorientation at cellular architecture (Figure 1A\u20131D).<\/p>\n\n\n\n\n\n
\"\"<\/td>\nFigure 1: Immunostaining of testicular tissues against p53 showed minimum number of positive spermatogonia
 in control (Group I) (A), likewise, slightly higher but limited to spermatogonial cells were found positive for p53
in Group II (B), <\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Scanning Electron Microscopy <\/strong><\/p>\n\n\n\n

SEM analysis of cauda epididymis spermatozoa was carried out to assess surface morphology. Control animals indicated normal morphology; sperm contained a perfect hook-shaped head with an uninterrupted surface and an intact acrosome (Figure 2A). Intact homogeneous plasma membrane throughout the head, middle piece, neck, and tail. Sperm midpiece width and sperm head width at the neck and head joints were normal. Treatment groups evidently showed various types of deformities in sperms (Figures 2B\u2013H). These include a flattened apical perforatorium, an irregular head cap segment, and a perifossal zone indicating abnormal appearance and damage to the connecting piece (Figure 2B). Dissolution of the dorsal acrosomal system with limited leakage of cellular fluid (Figure 2C). A combination of head and tail separation, an abnormal head, shrinkage in curvature, and a flattened perforatorium were apparent in BPA treated animals (Figure 2D). Abnormal rough surface on the head; cytoplasmic leakage at the acrosomal sheath; dysplasia of the fibrous sheath; and a damaged mid-piece were evident (Figures 2E\u2013F). Most BPA treated animals showed spermatozoons having coiled and looped tails (Figures 2G\u2013H).<\/p>\n\n\n\n\n\n
\"\"<\/td>\nFigure 2.(A-B): Control spermatozoon (A) shows intact plasma membrane (M), nucleus (N), acrosome (R),  perforatorium (P) and tail (T). BPA treated spermatozoa (B) showed flattened apical perforatorium and distorted head cap segment (HCS).<\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 2.(C-D): BPA treated spermatozoon (C) indicated dissolution of dorsal acrosomal system (AS) with partial leakage through dorsal plasma membrane. <\/strong><\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 2.(E-F): Treated animals indicated spermatozoa with damaged plasma membrane and formation of cytoplasmic droplets (Cd) in mid-piece (MP) and head. <\/strong><\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 2.G-H: BPA administered animals showed spermatozoon with coiled tail and free tail. There were number of sperms with bent tail and dag effects.<\/strong><\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

The tumour suppressor molecule p53 is also referred to as the\nguardian of cell cycle21 <\/sup>has specific roles in cellular development\nand proliferation. According to various studies, checkpoints such as; G1\/S and\nG2\/M are stringently controlled by p53.22-24<\/sup> Genetic instability through internal or external interferences\ncan upregulate expression of p53, leading to cell cycle arrest and eventually\napoptosis.25-26<\/sup> Expression of p53\ncould be dose-dependent, as an earlier study by Lloyd et al.14<\/sup>\nreported that an increase in the concentration of low-level BPA can inversely\naffect p53 expression. Similarly, another study by Dairkee et al.46<\/sup>\nreported that BPA induces aberrant expression of crucial p53 checkpoints. In\nresponse to high oxidative stress, prooxidative genes are upregulated by the\np53, causing an increased ROS level that promotes apoptosis in targeted cells17<\/sup>.\nBPA is well researched toxicant that has targeted induction of oxidative stress\nin testicular tissues.15, 27-28<\/sup> In the present study,\nmodulation of p53 expression in testicular tissues of BPA treated animals was\ninvestigated.<\/p>\n\n\n\n

The present study noted positive expression of p53 in the\ninterstitial spaces between seminiferous tubules in those animals treated with\nBPA. The p53 expression in Leydig cells was common in all dose groups and\nrelatively higher than control. A study by Inoue and Wada29 <\/sup>examined\nthe nuclear accumulation of p53 in testicular tumours of dogs and discovered\nthat Leydig cells were associated with a high level of p53 accumulation,\nwhereas, weak accumulation was evident in primary spermatocytes. There are many\nstudies that corroborate damages in Leydig cells under testicular oxidative\nstress 30<\/sup>. It appeared that under the influence of BPA, Leydig cells\nshowed a dose dependent increase in p53 expression. Similarly, BPA-administered\nanimals indicated higher p53 positive spermatogonia than control animals. It\nwas apparent that spermatogonia in animals administered with 50 mg\/kg b.w. of\nBPA were highly upregulated compared to animals administered with 10 mg\/kg b.w.\nand 100 mg\/kg b.w. BPA doses. Since most germ cells were severely disoriented\nin the 100 mg\/kg group and appeared to have fallen into the lumen, comparing\nthe spermatogonial expression of p53 was not parallel. Although a significant\nincrease in the expression of p53 was noted in the 50 mg\/kg groups compared to\nthe 10 mg\/kg group, It emerged that spermatogonia were more tolerant to\noxidative stress compared to Leydig cells. A study by Huang et al.31<\/sup> revealed\nthat spermatogonia were more tolerant to toxicants (Pb) than Leydig cells.\nOther studies also reported that spermatogonia are highly tolerant to oxidative\nstress.32-33<\/sup> It could be\nspeculated that spermatogonia may have a higher tolerance for BPA-induced\noxidative stress. However, at higher doses, expression of p53 was localised in\nvarious cells, including Leydig cells, spermatogonia, and spermatocytes.\nInterestingly, spermatocytes showed maximum tolerance against BPA-induced\noxidative stress and respective expression of p53. Nonetheless, animals\nadministered 100 mg\/kg BPA indicated relatively higher numbers of p53-positive\nspermatocytes. Although there is limited information on spermatocytes potential\ntolerability against reactive oxygen species (ROS), nevertheless, Agarwal et\nal.34<\/sup> reported that spermatogonia were more tolerant to\nROS-generated oxidative stress compared to primary spermatocytes. In contrast,\nthe present study indicated greater toleration in spermatocytes; a possible\nexplanation for this could be alternative action induced by oxidative stress\n(other than p53 activation) such as autophagy, which inhibits the progression\nof apoptosis.35<\/sup><\/p>\n\n\n\n

A SEM micrograph showed specific damages in the testicular\nspermatozoon, such as; flattened perforatorium area, indicating an acrosomal\ndefect. It is a part of the perinuclear theca, consisting of the post-acrosomal\nsheath, subacrosomal layer, and perforatorium.36-37<\/sup> The perforatorium is formed early during spermiogenesis, more\nspecifically around elongation phase.38<\/sup> <\/sup>This\nparticular part is not distinctly present in human sperm. Thus, damages found\nin this region of sperm indicate a significant alteration in morphosis during\nthe elongation phase of spermiogenesis. Spermiogenesis occurs quickly after\nmeiotic divisions. An alteration in the activity of p53 can impact\nspermiogenesis, leading to spermatids undergoing apoptosis.39<\/sup> A study by Sharma et\nal.40<\/sup> reviewed how oxidative insult can affect the epigenetic\nmechanisms of spermatogenesis. Perforatorium forms during the elongation phase\nof spermatogenesis, therefore, it is highly likely that defects occurred during\nthis stage, which is promoted by BPA-induced oxidative stress.47<\/sup> Although there is no\nclear evidence that oxidative stress may cause flattened perforatorium in\nspermatozoa, oxidative damage in sperm may lead to other head deformities. A\nreview by Lohiya et al.41<\/sup> reported that reactive oxygen species can\nvariably affect spermatozoa, leading to eventual loss of fertility. Authors\nclaimed that the plasma membrane of spermatozoa is highly responsive to\noxidative stress, leading to damage in the acrosome and absence of plasma\nmembrane in the mid-piece and tail. The present study also observed the\ndissolution of the plasma membrane and dorsal acrosomal system. Besides, loss\nof segmented columns and numeric aberration of the centriole in the neck are\nalso effects of oxidative stress.42<\/sup> <\/sup>This\nstudy indicated a similar pattern in the head and tail separation. It was\nnoticeable that both a flattened perforatorium and head-tail separation existed\nsimultaneously. Irregular expression of proteins leads to changes in the\npost-acrosomal sheath38<\/sup>. It is important to note that these\nalterations may have been carried out during spermiogenesis, which indicates\ninterference by BPA in spermatogenesis. Previous studies have indicated\nimpairment of spermatogenesis in BPA-exposed animals.13,43<\/sup> <\/sup>There\nis no direct impact of p53 on spermiogenesis, but it appears to have an impact\non overall spermatogenesis. The role of Sertoli cells during spermiogenesis is\nsuspected to have played an important role in deformities, however, the present\nstudy did not find a significant role for the p53 response in these cells. This\nstudy further suggests that apoptosis in Leydig cells may have been associated\nwith undernourished germ cells and subsequent deformities in sperm. This study\nalso observed typical coiling, bent tail, and dag effects indicative of\noxidative stress. Lipid peroxidation of the plasma membrane and cytoplasmic\ndroplets have a direct association.44<\/sup> Appearances of cytoplasmic droplets are indicative of failure\nto resist oxidative stress. There is another alternative for the presence of\ncytoplasmic residue: during spermiogenesis, a small amount of cytoplasmic\nresidue normally remains in sperm. These sperm could be defective even after\nmaturation and may impact fertility.45<\/sup><\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

Localization of p53 revealed differential expression in germ cells. It appeared that spermatocytes and spermatogonial cells were more tolerant to low-dose BPA-induced toxicity compared to other testicular cells. In conclusion, p53 positively impacted spermatogenesis under the influence of BPA, and in addition, it also impacted spermiogenesis. However, the present study was based on qualitative immunohistochemical observations thus further quantitative study on each phase of spermatogenesis would reveal more details on activities of p53 following BPA exposure. It appeared that p53 may have contributed to sperm deformities through specific metamorphic transitions from spermatocytes to spermatids. For further investigation of the role of Sertoli cells during spermiogenesis under targeted effects, BPA must be investigated in association with p53 and other apoptotic factors to understand specific sperm deformities.<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

There are no conflict of interest.<\/p>\n\n\n\n

Funding Sources<\/strong><\/p>\n\n\n\n

The authors are thankful to the Department of Science and\nTechnology (DST), New Delhi, for financial assistance provided by the DST\nINSPIRE FELLOWSHIP. The grant number is IF160160 and the Department of Zoology,\nUniversity of Rajasthan, for providing necessary facilities.
<\/p>\n\n\n\n

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