• Users Online: 89
  • Home
  • Print this page
  • Email this page
Home About us Editorial board Search Ahead of print Current issue Archives Submit article Instructions Subscribe Contacts Login 

 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 45  |  Issue : 2  |  Page : 79-91

Histological and immunohistochemical study on the effect of gibberellic acid on the seminiferous tubules of testis of adult albino rat and the possible protective role of grape seeds proanthocyanidin extract


Department of Histology, Faculty of Medicine, Tanta University, Tanta, Egypt

Date of Submission08-Nov-2016
Date of Acceptance12-Feb-2017
Date of Web Publication13-Oct-2017

Correspondence Address:
Eman M El-Beltagi
Department of Histology, Faculty of Medicine, Tanta University, Tanta
Egypt
Login to access the Email id


DOI: 10.4103/tmj.tmj_38_16

Rights and Permissions
  Abstract 


Background
Gibberellic acid (GA) is widely used in Egypt and other countries to increase the growth of many fruits and vegetables. GA can induce disturbances in the testicular enzymes and hormones. Recent studies have revealed that grape seed proanthocyanidin extract (GSPE) has a potential protective effect in male reproductive disorders.
Aim
This work aimed to study the effect of GA on the seminiferous tubules of testis of adult albino rat and to evaluate the possible protective role of GSPE.
Materials and methods
Forty adult male albino rats were divided into four groups: control group, group II treated with GSPE (100 mg/kg once daily for 4 weeks), group III treated with GA (20 mg/kg once daily for 4 weeks), and group IV concomitantly treated with GSPE and GA with the same dose and duration of previous groups. Specimens from the testes were processed for light and electron microscopy. Immunohistochemical study was carried out using antibodies against Bcl-2.
Results
Specimens from GA-treated animals showed disturbance in the normal architecture with obvious structural changes. Some spermatogenic cells appeared disorganized with wide intercellular spaces, and others were exfoliated into the lumen of the tubules. Many spermatogonia showed vacuolated cytoplasm and deeply stained nuclei. Ultrastructurally, the cytoplasm of Sertoli cells showed markedly dilated smooth endoplasmic reticulum. Some primary spermatocytes appeared with vacuolated cytoplasm and dilated perinuclear cisternae. The early spermatids appeared with electron-dense bodies, concentric lamellar formations, and irregular outlined nuclei. Abnormally shaped spermatozoal heads were detected. The immunohistochemical study showed a highly significant decrease in Bcl-2 immunoreaction. In contrast, minimal changes were observed in rats treated concomitantly with both GSPE and GA, with a nonsignificant change in the immunoreaction.
Conclusion
GA induced structural changes of the seminiferous tubules of the rat testis that could be minimized by the coadministration of GSPE.

Keywords: Bcl-2, gibberellic acid, grape seed proanthocyanidin extract, seminiferous tubules


How to cite this article:
El-Beltagi EM, Elwan WM, Mohsen El-Bakry NA, Salah EF. Histological and immunohistochemical study on the effect of gibberellic acid on the seminiferous tubules of testis of adult albino rat and the possible protective role of grape seeds proanthocyanidin extract. Tanta Med J 2017;45:79-91

How to cite this URL:
El-Beltagi EM, Elwan WM, Mohsen El-Bakry NA, Salah EF. Histological and immunohistochemical study on the effect of gibberellic acid on the seminiferous tubules of testis of adult albino rat and the possible protective role of grape seeds proanthocyanidin extract. Tanta Med J [serial online] 2017 [cited 2017 Dec 14];45:79-91. Available from: http://www.tdj.eg.net/text.asp?2017/45/2/79/216689




  Introduction Top


Plant growth regulators are endogenous plant hormones that are used in agriculture to enhance and control the production of a wide variety of crops [1]. Gibberellic acid (GA) is a plant growth regulator that is widely used to increase the growth of many fruits and vegetables by increasing cell division [2], promoting stem elongation, flowering, and fruit development [3]. Some studies demonstrated that GA was associated with oxidative stress and cellular damage in soft organs particularly the breast, lung, kidney, heart, stomach, and spleen [4].

Medicinal plants have played an important role in maintaining human health and improving quality of life for thousands of years [5]. Recent trends prefer the use of these plants in treating and controlling different diseases, as they have several advantages such as having fewer side effects, better patient tolerance, being safe, and less expensive [6].

Proanthocyanidin is a natural antioxidant extracted from grape seeds that has been reported to have a broad spectrum of biological, pharmacological, and therapeutic activities against free radicals and oxidative stress [7]. It has been suggested that intake of grape seed proanthocyanidin extract (GSPE) may protect multiple organs such as liver, kidney, and bone from a variety of toxic materials [8]. Besides its antioxidant activity, it has been described as an antimicrobial, anticancer, anti-inflammatory, and antifatigue agent [9]with cardioprotective properties [10]. In addition, recent studies have demonstrated that GSPE has a potential protective effect in various male reproductive disorders [11].

The aim of this work was to study the histological changes that might occur in the seminiferous tubules of the rat testis after GA exposure and to evaluate the possible protective effect of GSPE.


  Materials and methods Top


Materials

Chemicals

GA was obtained from El-Gomhoria Company for Trading Pharmaceutical Chemicals and Medical Appliances (Tanta, Egypt), in the form of vials. Each vial contained one gm of GA powder. The experimental dose of GA is 20 mg/kg/day [12].

GSPE was obtained from Armal GNC (Riyadh, KSA), in the form of a package containing 60 capsules, 50 mg each. The dose of GSPE is 100 mg/kg/day corresponding to the human dose [13],[14].

Animals

The present study was carried out on 40 adult male albino rats, with an average weight of 160–200 g each. The rats after being grouped were housed in suitable clean properly ventilated cages at room temperature (22–25°C) and in a photoperiod of 14 h light/10 h dark. The animals were fed on similar commercial laboratory diets and water. They were acclimatized to their environment for one week before starting the experiment, which was approved by the local ethics committee.

The animals were divided into four equal groups (10 animals each):

Group I (control group): Rats of this group were subdivided into two equal subgroups − subgroup Ia received no treatment and subgroup Ib received 0.5 ml/100 g distilled water orally by a gastric tube once daily for 4 weeks.

Group II (GSPE-treated group): Rats of this group were administered 100 mg/kg GSPE orally by a gastric tube once daily for 4 weeks.

Group III (GA-treated group): Rats of this group were administered 20 mg/kg GA orally by a gastric tube once daily for 4 weeks.

Group IV (GSPE-treated and GA-treated group): Rats of this group were concomitantly administered both GSPE and GA at the same doses and duration as in groups II and III, respectively.

After 24 h from the last administration, the animals were anesthetized by an intraperitoneal injection of sodium thiopental (30 mg/kg) [15],[16]. The testes were dissected and specimens from the testes were obtained and processed for histological and immunohistochemical examination.

Methods

Histological examination

The testicular specimens were immediately fixed in Bouin’s solution, dehydrated, cleared, and embedded in paraffin. Sections of 5 μm thickness were stained with hematoxylin and eosin [17],[18].

Immunohistochemical examination

Sections of 5 µm thickness were dewaxed, rehydrated, and washed with PBS. The sections were then incubated overnight in a humid chamber with the primary antibody (rabbit monoclonal Bcl-2 antibody)) Sigma Aldrich, Cairo, Egypt) diluted in PBS at 4°C. Washing in PBS buffer and coincubation with biotinylated secondary antibody for one hour at room temperature were carried out. Streptavidin peroxidase was then added for 10 min and rinsed again three times in PBS. Immunoreactivity was visualized using 3,3′-diaminobenzidine-hydrogen peroxide as a chromogen. Sections were counterstained with Mayer’s hematoxylin. The negative control sections were prepared by excluding the primary antibodies [19],[20].

Electron microscopic examination

The specimens were fixed by immersion in 2.5% phosphate-buffered glutaraldehyde, processed, and embedded in epoxy resin by routine protocol. Semithin sections (1 µm thick) were obtained and stained with 1% toluidine blue and examined by light microscopy. Ultrathin sections (80–90 nm) were then stained with uranyl acetate and lead citrate to be examined by JEOL electron microscope (JEOL, Egypt) at 80 kV in EM Unit, Faculty of Medicine, Tanta University [21],[22].

Morphometric study

Image analysis system (Leica Qwin 500C Image Analyzer Computer System; Leica Imaging System Ltd, Cambridge, England) at Central Research Lab. was used to measure the color intensity of Bcl-2 immunoreactivity (in diaminobenzidine-stained sections) in all groups. Ten different nonoverlapping randomly selected fields at a magnification of 400 were examined for each slide.

Statistical analysis

The data obtained were analyzed using SPSS software (version 13; SPSS Inc., Chicago, Illinois, USA), and then compared by one-way analysis of variance test followed by Tukey’s test to compare different groups with the control group. The results were expressed as mean±SD. The differences were considered statistically significant if probability value was P-value less than 0.05 and highly significant if P-value less than 0.001.


  Results Top


Histological results

H&E-stained sections

Group I (control group): Sections from the testes of both control subgroups (Ia and Ib) showed the normal histological structure. The seminiferous tubules appeared rounded or oval. They were lined by a complex stratified epithelium showing different stages of spermatogenesis ([Figure 1]).
Figure 1 Photomicrographs of testis section of the control group. (a) Apparently normal oval to rounded seminiferous tubules (T) are seen. Spermatozoa (Sz) are filling the lumina of the tubules. The interstitial tissue (IT) can be seen in between the tubules. Hematoxylin and eosin, ×100. (b) A seminiferous tubule lined with different types of spermatogenic cells including spermatogonia (Sg), primary spermatocytes (Sp), spermatids (Sd), and spermatozoa (Sz). Groups of interstitial cells of Leydig (L) are seen in between the seminiferous tubules. Hematoxylin and eosin, ×400

Click here to view


Group II (GSPE-treated group): Sections from the testes of this group showed similar results as the control group.

Group III (GA-treated group): Sections from the testes of this group showed disturbance in the normal architecture with evidence of structural changes. Some of the seminiferous tubules were widely separated from each other and the basement membrane was focally separated from the overlying germinal epithelium. The interstitial spaces revealed deposition of homogeneous acidophilic material ([Figure 2]). Some spermatogenic cells appeared disorganized with wide intercellular spaces and others with deeply stained nuclei were exfoliated into the lumen of the tubules. Many spermatogonia revealed vacuolated cytoplasm and deeply stained nuclei ([Figure 3]).
Figure 2 Photomicrographs of a rat testis of group III. (a) The seminiferous tubules are widely separated from each other (*). The basement membrane is focally separated from the overlying germinal epithelium (arrows). Hematoxylin and eosin, ×100. (b) The interstitial spaces contain homogeneous acidophilic substance (*). Hematoxylin and eosin, ×100

Click here to view
Figure 3 Photomicrographs of a rat testis of group III. (a) The spermatogenic cells showing wide intercellular spaces (*) and the lumen of the tubule is filled with desquamated cells (arrows). Hematoxylin and eosin, ×400. (b) The spermatogonia show highly vacuolated cytoplasm (arrowheads). Hematoxylin and eosin, ×400

Click here to view


Group IV (GSPE-treated and GA-treated group): Sections from the testes of this group showed partial preservation of the normal structure of the seminiferous tubules. Most of them were more or less resembling the normal structure. Some spermatogenic cells showed mild cytoplasmic vacuolation ([Figure 4]).
Figure 4 Photomicrographs of a rat testis of group IV. (a) The seminiferous tubules (T) are apparently normal with interstitial cells of Leydig (L) in between. Their lumina are seen full of spermatozoa (Sz). Hematoxylin and eosin, ×100. (b) The cytoplasm of some spermatogenic cells showing mild vacuolation (arrowhead). Hematoxylin and eosin, ×400

Click here to view


Toluidine-blue-stained sections

Group I (control group)

Sections from the testes of both control subgroups (Ia and Ib) showed different stages of spermatogenesis at the basal and the adluminal compartment of the seminiferous tubules ([Figure 5]).
Figure 5 Photomicrographs of a semithin section of control rat testis showing parts of seminiferous tubules lined by Sertoli cell (St) and different spermatogenic cells. (a) Type A dark spermatogonium (SgAd), type A pale spermatogonium (SgAp), early rounded spermatids (Sd), and late spermatids (Sdl) are illustrated. Toluidine blue, ×1000. (b) Type B spermatogonium (SgB), primary spermatocytes (Sp), and early rounded spermatids (Sd) are seen. Bm, basement membrane; My, myoid cell. Toluidine blue, ×1000

Click here to view


Group II (grape seed proanthocyanidin extract-treated group)

Sections from the testes of this group revealed similar results as the control group.

Group III (gibberellic acid-treated group)

Sections from this group showed spermatogonia with irregular outlined nuclei, whereas the nuclei of early spermatids illustrated disrupted membranes ([Figure 6]).
Figure 6 Photomicrographs of a semithin section of a rat testis of group III. (a) Many spermatogonia with irregular nuclei (arrowheads) and vacuolated cytoplasm (V) are seen. Notice the irregular basement membrane. Toluidine blue, ×1000. (b) Some early rounded spermatids showing disrupted nuclear membranes (arrowheads). Toluidine blue, ×1000

Click here to view


Group IV (grape seed proanthocyanidin extract-treated and gibberellic acid-treated group)

Sections from the testes of this group showed mild cytoplasmic vacuolation of few spermatogenic cells ([Figure 7]).
Figure 7 A photomicrograph of a rat testis of group IV. The cytoplasm of some spermatogenic cells shows mild vacuolation (arrowheads). Toluidine blue, ×1000

Click here to view


Immunohistochemical results

Examination of Bcl-2-immunostained sections obtained from testes of both control group and GSPE-treated group revealed strong positive cytoplasmic reaction of the cells lining the seminiferous tubules ([Figure 8]a). While the sections obtained from GA-treated group showed weak reaction ([Figure 8]b), on the other hand strong positive cytoplasmic immunoreaction was detected on examination of sections obtained from rats treated with both GSPE and GA ([Figure 8]c).
Figure 8 (a) A photomicrograph of a rat testis of control group showing strong Bcl-2-positive cytoplasmic immunoreaction in the cells of the seminiferous tubule (arrows). Bcl-2 immunostaining, ×1000. (b) A photomicrograph of a rat testis of group III showing weak positive cytoplasmic reaction in the cells lining the seminiferous tubule (arrow). Bcl-2 immunostaining, ×1000. (c) A photomicrograph of a rat testis of group IV showing strong positive cytoplasmic immunoreaction for Bcl-2 in the cells of the seminiferous tubule (arrow). Bcl-2 immunostaining, ×1000

Click here to view


Electron microscopic results

Group I (control group)

Electron microscopic examination of the ultrathin sections of the testes from the control group showed the basal compartment of the seminiferous tubules formed of cells resting on the basement membrane formed of  Sertoli cells More Details and spermatogonia. Sertoli cell appeared as a large pyramidal cell having large indented nucleus with prominent nucleolus ([Figure 9]). Type A dark spermatogonia appeared with oval nuclei containing peripheral clumps of heterochromatin ([Figure 10]) and type B spermatogonia appeared with almost rounded nuclei ([Figure 11]). The adluminal compartment showed primary spermatocytes, spermatids, and spermatozoa. The primary spermatocytes revealed rounded to oval nuclei ([Figure 12]); secondary spermatocytes were rarely seen as they have short life. The early rounded spermatids were identified by their characteristic acrosomal caps ([Figure 13]). The spermatozoa appeared with their characteristic heads and tails ([Figure 14] and [Figure 15]).
Figure 9 An electron micrograph of a control rat testis showing a Sertoli cell having a large indented nucleus (N) with a prominent nucleolus (Nu). Multiple mitochondria (M), smooth endoplasmic reticulum (S), and electron-dense bodies (E) are seen in the cytoplasm. BL, basal lamina. Magnification, ×3000

Click here to view
Figure 10 An electron micrograph of a control rat testis showing type A dark spermatogonium (SgAd) having an oval nucleus (N) with peripheral clumps of heterochromatin (arrowheads). BL, basal lamina. Magnification, ×3000

Click here to view
Figure 11 An electron micrograph of a control rat testis showing type B spermatogonium (SgB) having rounded nucleus (N). BL, basal lamina. Magnification, ×2500

Click here to view
Figure 12 An electron micrograph of a control rat testis showing a primary spermatocyte with a large rounded to oval nucleus (N) containing fine chromatin. The cytoplasm shows multiple rounded to oval mitochondria (M) with widened cristae. Magnification, ×2500

Click here to view
Figure 13 An electron micrograph of a control rat testis showing an early rounded spermatid at the cap phase. The acrosomal cap (Ac) extends over the anterior pole of the nucleus (N) that appears rounded with fine granular chromatin. The cytoplasm shows mitochondria (M) with widened cristae arranged at the periphery of the cell. Magnification, ×3000

Click here to view
Figure 14 An electron micrograph of a control rat testis showing a spermatozoal head with an electron-dense nucleus (N) surrounded from inside to outside by (1) nuclear membrane, (2) subacrosomal space, (3) inner acrosomal membrane, (4) acrosomal material, and (5) outer acrosomal membrane. Magnification, ×8000

Click here to view
Figure 15 An electron micrograph of a control rat testis showing transverse sections of spermatozoal tails. The middle piece (Mp) shows (1) axoneme, (2) nine outer dense fibers, (3) circumferentially arranged mitochondria, and (4) flagellar membrane. The principle piece (Pp) shows (1) axoneme, (2) nine outer dense fibers, (3) ventral and dorsal columns, (4) circumferential ribs, and (5) flagellar membrane. The end piece (Ep) shows (1) axoneme and (2) flagellar membrane. Magnification, ×8000

Click here to view


Group II (grape seed proanthocyanidin extract-treated group)

Examination of ultrathin sections from the testes of animals of this group showed similar results as the control group.

Group III (gibberellic acid-treated group)

Examination of ultrathin sections from the testes of this group revealed some ultrastructural alterations. The cytoplasm of Sertoli cells showed markedly dilated smooth endoplasmic reticulum (SER) ([Figure 16]). Some type A dark spermatogonia showed rarified cytoplasm and irregular outlined nuclei ([Figure 17]). The cytoplasm of some type B spermatogonia appeared vacuolated and their nuclei showed dilated perinuclear cisternae and vacuoles ([Figure 18]). Some primary spermatocytes revealed vacuolated cytoplasm and dilated perinuclear cisternae ([Figure 19]). The early rounded spermatids appeared with vacuolated cytoplasm, electron-dense bodies, concentric lamellar formations, and irregular shaped nuclei ([Figure 20]). The spermatozoal heads revealed abnormal shapes ([Figure 21]). The middle pieces of the tails were encountered with an excess retained vacuolated cytoplasm ([Figure 22]).
Figure 16 An electron micrograph of a rat testis of group III showing a Sertoli cell with dilated smooth endoplasmic reticulum (S). Notice the large lipid droplet (L). BL, basal lamina; My, myoid cell. Magnification, ×1500

Click here to view
Figure 17 An electron micrograph of a rat testis of group III showing type A dark spermatogonium (SgAd) with areas of rarified cytoplasm (*) and an irregular outlined nucleus (N). Notice the irregular basal lamina (BL). Magnification, ×2500

Click here to view
Figure 18 An electron micrograph of a rat testis of group III showing type B spermatogonium (SgB) with vacuolated cytoplasm (V). The nucleus (N) appears with vacuoles (arrowheads) and dilated perinuclear cisternae (arrows). Notice the wide intercellular space (*) and the irregular basal lamina (BL). My; myoid cell. Magnification, ×2500

Click here to view
Figure 19 An electron micrograph of a rat testis of group III showing a primary spermatocyte with cytoplasmic vacuoles (V) and the nucleus (N) shows dilated perinuclear cisternae (arrowheads). Magnification, ×2000

Click here to view
Figure 20 An electron micrograph of a rat testis of group III showing early rounded spermatids at the cap phase. The cytoplasm appears with concentric lamellar body (arrow), electron-dense bodies (arrowheads), and vacuoles (V). Their nuclei (N) appear to have irregular shapes. Magnification, ×1500

Click here to view
Figure 21 An electron micrograph of a rat testis of group III showing the spermatozoal heads with abnormal shapes (arrows). Magnification, ×3000

Click here to view
Figure 22 An electron micrograph of a rat testis of group III showing middle pieces (Mp) of spermatozoal tails having irregular outlines and an excess retained cytoplasm (arrows) with a vacuole (V). Magnification, ×8000

Click here to view


Group IV (grape seed proanthocyanidin extract-treated and gibberellic acid-treated group)

Examination of ultrathin sections of the testes obtained from this group revealed partial preservation of the ultrastructure of both Sertoli cells ([Figure 23]) and spermatogenic cells ([Figure 25],[Figure 27],[Figure 28]). On the other hand, few spermatogonia showed discontinuous nuclear membranes ([Figure 24]) and some early rounded spermatids showed few heterogeneous electron-dense bodies ([Figure 26]).
Figure 23 An electron micrograph of a rat testis of group IV showing an apparently normal Sertoli cell having a large indented nucleus (N) with two prominent nucleoli (Nu), mitochondria (M), and electron-dense body (E). BL, basal lamina; My, Myoid cell. Magnification, ×3000

Click here to view
Figure 24 An electron micrograph of a rat testis of group IV showing type A dark spermatogonium (SgAd) having an oval nucleus (N) with interrupted nuclear membrane (arrowhead). BL, basal lamina. Magnification, ×3000

Click here to view
Figure 25 An electron micrograph of a rat testis of group IV showing a primary spermatocyte with rounded nucleus (N). The cytoplasm shows multiple oval to rounded mitochondria (M). Magnification, ×4000

Click here to view
Figure 26 An electron micrograph of a rat testis of group IV showing an early spermatid at the cap phase. The acrosomal cap (Ac) extends over the anterior pole of the nucleus (N) that appears rounded with fine chromatin. The cytoplasm shows mitochondria (M) that are arranged at the periphery of the cell. Few heterogeneous electron-dense bodies can be seen (arrows). Magnification, ×3000

Click here to view
Figure 27 An electron micrograph of a rat testis of group IV showing an apparently normal spermatozoal head with an electron-dense nucleus (N). Magnification, ×5000

Click here to view
Figure 28 An electron micrograph of a rat testis of group IV showing apparently normal transverse sections of the spermatozoal tails. Mp, middle piece; Pp, principle piece; Ep, end piece. Magnification, ×4000

Click here to view


Morphometric results and statistical analysis

The color intensity of Bcl-2-positive immunoreaction in the GA-treated group (group III) showed a highly significant decrease compared with the control group, whereas GSPE-treated and GA-treated group (group IV) showed a nonsignificant change compared with the control group ([Table 1] and Figure 29).
Table 1 The mean color intensity of Bcl-2 immunoreaction in different studied groups

Click here to view
Figure 29 The mean color intensity of Bcl-2 immunoreaction in different studied groups

Click here to view



  Discussion Top


GA is widely used as a plant growth regulator in many countries. However, it has potential hazardous effects on human health and male reproductive functions [1],[23],[24]. Recent studies proposed GSPE to be beneficial in combating various male reproductive disorders [25]. Therefore, the present work was designed to investigate the effect of exposure to GA on the histological structure of the seminiferous tubules of testis of adult albino rats and to evaluate the possible protective role of GSPE.

Widening of the intertubular spaces was observed in this study, and this could be attributed to the deposition of homogeneous acidophilic material in most of the interstitial spaces, which was described as hyaline material [26]. This hyaline material can be attributed to the excess lymphatic exudates oozing from degenerated lymphatic vessels, as well as the increase in vascular permeability that results from accumulation of free radicals and reactive oxygen species (ROS) [27],[28].

Soliman et al. [29] reported that GA administration is associated with the generation of ROS that interact with the tissues resulting in numerous pathological changes. Chen et al. [30] added that the ROS could damage every major cellular component, including membranes, lipids, carbohydrates, and DNA. Other investigators reported that GA administration can cause peroxidation of polyunsaturated fatty acids, which leads to an oxidative destruction of cellular membranes and organelles, which in turn increases the production of toxic free radicals ending in cell death [31],[32].

Widening of the intercellular spaces in the present study may be explained by the disruption of tight junctions of blood–testis barrier, upon exposure to the ROS, leading to entry of excess water and toxic agents between the spermatogenic cells, with consequent widening of the intercellular spaces [33]. In addition, the loss of cell cohesiveness may be attributed to destruction of the cellular processes of Sertoli cells that fill the spaces between the germ cells leading to exfoliation of the spermatogenic cells into the lumen of the seminiferous tubules [34].

As regards the vacuolated cytoplasm of spermatogenic cells, this could be attributed to lipid peroxidation with consequent damage to the cell membrane caused by GA, as well as membranes of the cell organelles with subsequent increase in their permeability [35]. Some authors reported that the clear vacuoles within the cytoplasm represent distended and pinched-off segments of the endoplasmic reticulum. They added that the cellular swelling may occur as a result of failure of energy-dependent ion pumps in the plasma membrane, leading to an inability to maintain ionic and fluid homeostasis, and they referred this pattern of nonlethal injury to be hydropic change or vacuolar degeneration [36].

The present results demonstrated pyknotic nuclei in some spermatogonia. Kroemer et al. [37] attributed the nuclear pyknosis to be a feature of apoptosis. On the other hand, Kumar et al. [36] reported that pyknosis is a pattern of nuclear changes related to cell necrosis and characterized by nuclear shrinkage with increased basophilia as its DNA condenses into a solid shrunken mass.

The dilatation of SER that was observed in the cytoplasm of some Sertoli cells may reflect the accumulation of water inside it as a result of lipid peroxidation of the SER membranes [38]. The nuclei of some spermatogonia and primary spermatocytes showed dilated perinuclear cisternae. These changes could be a result of lipid peroxidation of the nuclear membranes [39]. The results also showed some nuclei of spermatogonia containing vacuoles. These nuclear vacuoles may be considered as an indicator of genotoxicity [40],[41].

In addition, some concentric membranous lamellae were observed in the cytoplasm of early spermatids. Such structures were described by Castejón [42] as myelin figures. He stated that myelin figures are concentric membranous lamellar formations that have been related to drug administration, anoxic–ischemic conditions, wide variety of pathological processes, and potent inducers of apoptotic cell death. Some heterogeneous electron-dense bodies were observed in the cytoplasm of early rounded spermatids. This finding may be attributed to the autophagy of damaged cytoplasmic debris [43]. These heterogeneous electron-dense bodies were proven to be secondary lysosomes [44].

Some spermatozoal heads appeared with abnormal shapes. These abnormalities could be due to the disruption of spermatogenesis with consequent deterioration of motility and content of spermatozoa, as well as morphological abnormalities [45]. Moreover, it has been demonstrated that the spermatozoa are particularly susceptible to oxidative damage, as their cell membranes contain high percentage of polyunsaturated fatty acids and their cytoplasm contains low concentrations of scavenging enzymes [46],[47].

The middle pieces of spermatozoal tails illustrated an excess retained cytoplasm, which can be attributed to the spermiogenesis arrest and to the interruption of cytoplasmic extrusion. Previous studies have reported that once the spermatogenesis process was exposed to considerable degeneration, the cytoplasmic extrusion mechanisms will not act under the normal conditions and the released spermatozoa carry excess residual cytoplasm and will be considered as immature and functionally defective spermatozoa [48],[49],[50].

Immunohistochemically, the present study demonstrated that GA induced apoptosis in the spermatogenic cells, as indicated by the highly significant decrease in the expression of Bcl-2 protein. Al-Azemi et al. [51] stated that Bcl-2 protein is an antiapoptotic marker. It has been reported that GA can initiate a series of events that end in programmed cell death (apoptosis). The ROS are key elements in this programmed cell death. The amounts and activities of ROS-scavenging enzymes, including catalase, ascorbate peroxidase, and superoxide dismutase, are strongly downregulated with GA. These data imply that GA-exposed cells lose their ability to scavenge ROS, and this loss ultimately results in oxidative damage and cell apoptosis [52].

The present work demonstrated that GSPE coadministration minimized the structural changes of seminiferous tubules induced by GA as evidenced by the histological findings. This partial preservation could be attributed to the ability of GSPE to overcome the oxidative stress and upregulate the endogenous antioxidant defense system [53]. In addition, GSPE contains polyphenolic compounds that have powerful free-radical-scavenging effect [54]. In addition, GSPE was found to be beneficial in ameliorating the degree of apoptotic cell death. This antiapoptotic effect of GSPE could be probably mediated through the upregulation of Bcl-2 protein expression as observed from the immunohistochemical results of the current work. This was in agreement with the results of other authors who have documented the antiapoptotic effect of GSPE against cisplatin-induced testicular apoptosis in rats and they explained this antiapoptotic activity of GSPE to be through upregulation of Bcl-2 protein expression [55].


  Conclusion Top


On the basis of the histological and immunohistochemical findings obtained from this study, it could be concluded that GA caused marked alterations in the structure of the seminiferous tubules of the rat testis that may be minimized by the coadministration of GSPE.

Acknowledgements

The authors thank all the technicians in the Department of Histology and Electron Microscope Unit, Faculty of Medicine, Tanta University, Tanta, Egypt, for their continuous help throughout this work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Ashikari M, Sakakibara H, Lin S, Yamamoto T, Takashi T, Nishimura A et al. Cytokinin oxidase regulates rice grain production. Science 2005; 309:741–745.  Back to cited text no. 1
    
2.
Silverstone AL, Sun T. Gibberellins and the green revolution. Trends Plant Sci 2000; 5:1–2.  Back to cited text no. 2
    
3.
Arous S, Boussaid M, Marrakchi M. Plant regeneration from zygotic embryo hypocotyls of Tunisian chili (Capsicum annuum L.). J Appl Hortic 2001; 3:17–22.  Back to cited text no. 3
    
4.
Celik I, Tuluce Y. Effects of indoleacetic acid and kinetin on lipid peroxidation and antioxidant defense in various tissues of rats. Pestic Biochem Physiol 2006; 84:49–54.  Back to cited text no. 4
    
5.
Monobe M, Ogino A, Ema K, Tokuda Y, Yamamoto MM. A crude extract from immature green tea (Camellia sinensis) leaves promotes Toll-like receptor 7-mediated interferon-a production in human macrophage-like cells. Cytotechnology 2012; 64:145–148.  Back to cited text no. 5
    
6.
Tabassum N, Hamdani M. Plants used to treat skin diseases. Pharmacogn Rev 2014; 8:52–60.  Back to cited text no. 6
    
7.
Bagchi D, Bagchi M, Stohs SJ, Ray SD, Sen CK, Pruess HG. Cellular protection with proanthocyanidins derived from grape seeds. Ann N Y Acad Sci 2002; 957:260–270.  Back to cited text no. 7
    
8.
Yilmaz Y, Toledo RT. Health aspects of functional grape seed constituents. Trends Food Sci Tech 2004; 15:422–433.  Back to cited text no. 8
    
9.
Shan Y, Ye X, Xin H. Effect of the grape seed proanthocyanidin extract on the free radical and energy metabolism indicators during the movement. Sci Res Essays 2010; 5:148–153.  Back to cited text no. 9
    
10.
Serrano J, Puupponen-Pimia R, Dauer A, Aura AM, Saura-Calixto F. Tannins: current knowledge of food sources, intake, bioavailability and biological effects. Mol Nutr Food Res 2009; 53:310–329.  Back to cited text no. 10
    
11.
Bayatli F, Akkus D, Kilic E, Saraymen R, Sonmez MF. The protective effects of grape seed extract on MDA, AOPP, apoptosis and eNOS expression in testicular torsion: an experimental study. World J Urol 2013; 31:615–622.  Back to cited text no. 11
    
12.
Erin N, Afacan B, Erosy Y, Ercan F, Balci MK. Gibberellic acid, a plant growth regulator, increases mast cell recruitment and alters substance P levels. Toxicology 2008; 254:75–81.  Back to cited text no. 12
    
13.
Karthikeyan K, Bai BR, Devaraj SN. Cardioprotective effect of grape seed proanthocyanidins on isoproterenol-induced myocardial injury in rats. Int J Cardiol 2007; 115:326–333.  Back to cited text no. 13
    
14.
Attia SM, Helal GK, Abd-Ellah MF, Mansour AM, El-sayed EM. The effects of oral grape seed extract on cisplatin-induced cytogenotoxicity in mice. Saudi Pharma J 2008; 16:161–167.  Back to cited text no. 14
    
15.
Karthikeyan M, Arunakaran J, Balasubramanian K. The effects of prolactin and corticosterone on insulin binding to rat Leydig cells. Reprod Biol 2009; 9:189–194.  Back to cited text no. 15
    
16.
Kushawaha S, Malpani A, Aswar UM, Bodhankar SL, Malpani A, Shivakumar S. Effect of different anesthetic agents on cardiovascular parameters in male Wistar rats. Res J Pharma Bio Chem Sci 2011; 2:685.  Back to cited text no. 16
    
17.
Wulff S, Hafer L. Guide to special stains. 1st ed. Carpinteria, CA: DakoCytomation; 2004.  Back to cited text no. 17
    
18.
Suvarna SK, Layton C, Bancroft JD. Bancroft’s Theory and practice of histological techniques. 7th ed. Philadelphia, PA: Churchill Livingstone, Elsevier; 2013.  Back to cited text no. 18
    
19.
Bancroft JD, Gamble M. Theory and practice of histological techniques. 6th ed. Philadelphia, PA: Churchill Livingstone, Elsevier; 2008.  Back to cited text no. 19
    
20.
Sharma R, Gandhi E. Localization of interleukin-2 in goat ovary. IOSR J Pharma 2012; 2:7–11.  Back to cited text no. 20
    
21.
Bozzola JJ, Russell LD. Electron Microscopy: principles and techniques for biologists. 2nd ed. Boston, MA, Toronto, London, Singapore: Jones and Bartlett Publishers; 1999.  Back to cited text no. 21
    
22.
Kuo J. Electron microscopy: methods and protocols. 2nd ed. Totowa, NJ: Humana Press Inc.; 2007.  Back to cited text no. 22
    
23.
Aksglaede L, Juul A, Leffers H, Skakkebaek NE, Anderson AM. The sensitivity of the child to sex steroids: possible impact of exogenous estrogens. Hum Reprod Update 2006; 12:341–349.  Back to cited text no. 23
    
24.
Chaari-Rkhis A, Maalej M, Messaoud SO, Drira N. In vitro vegetative growth and flowering of olive tree in response to GA3 treatment. Afr J Biotechnol 2006; 5:2097–2302.  Back to cited text no. 24
    
25.
Zhao YM, Gao LP, Zhang HL, Guo JX, Guo PP. Grape seed proanthocyanidin extract prevents DDP-induced testicular toxicity in rats. Food Funct 2014; 5:605–611.  Back to cited text no. 25
    
26.
El-Sherif NM, El-Mehi AE. Effect of semicarbazide on the testis of juvenile male albino rat. J Interdiscipl Histopathol 2015; 3:9–18.  Back to cited text no. 26
    
27.
Salama N, Bergh A, Damber JE. The changes in testicular vascular permeability during progression of the experimental varicocele. Eur Urol 2003; 43:84–91.  Back to cited text no. 27
    
28.
Ravikumar S, Srikumar K. Metabolic dysregulation and inhibition of spermatogenesis by gibberellic acid in rat testicular cells. J Environ Biol 2005; 26:567–569.  Back to cited text no. 28
    
29.
Soliman HA, Mantawy MM, Hassan HM. Biochemical and molecular profiles of gibberellic acid exposed albino rats. J Am Sci 2010; 6:224–229.  Back to cited text no. 29
    
30.
Chen Z, Jiang H, Wan Y, Bi C, Yuan Y. H2O2-induced secretion of tumor necrosis factor-α evokes apoptosis of cardiac myocytes through reactive oxygen species-dependent activation of p38 MAPK. Cytotechnology 2012; 64:65–73.  Back to cited text no. 30
    
31.
Celik I, Ozbek H, Tuluce Y. Effects of subchronic treatment of some plant growth regulators on serum enzyme levels in rats. Turk J Biol 2002; 26:73–76.  Back to cited text no. 31
    
32.
Troudi A, Ben Amara I, Soudani N, Samet AM, Zeghal N. Oxidative stress induced by gibberellic acid on kidney tissue of female rats and their progeny: biochemical and histopathological studies. J Physiol Biochem 2011; 67:307–316.  Back to cited text no. 32
    
33.
Mohamed D, Saber A, Omar A, Soliman A. Effect of cadmium on the testes of adult albino rats and the ameliorating effect of zinc and vitamin E. Br J Sci 2014; 11:72–95.  Back to cited text no. 33
    
34.
Sugandhy O, Pannerdoss S, Suryavathi V. Toxic influence of mercuric chloride on antioxidant system in the testis and epididymis of albino rats. The 10th International Conference on Mercury as a Global Pollutant Halifax, Canada 2011; 29: pp. 45–100.  Back to cited text no. 34
    
35.
Sakr SA, Okdah YA, El-Abd SF. Gibberellin A3-induced histological and histochemical alterations in the liver of albino rats. Sci Asia 2003; 29:327–331.  Back to cited text no. 35
    
36.
Kumar V, Abbas AK, Aster JC. Robbins basic pathology. 9th ed. Philadelphia, PA: Saunders, Elsevier; 2013.  Back to cited text no. 36
    
37.
Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES, Baehrecke EH et al. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ 2009; 16:3–11.  Back to cited text no. 37
    
38.
Elharoun H, Bashandy MA. Testicular toxic effect of Di-N-Butyl phthalate on adult male albino rat and the possible protective role of Vitamin C & E (ultrastructural, histological and histochemical study). J Am Sci 2014; 10:147–157.  Back to cited text no. 38
    
39.
Kumar V, Abbas AK, Fausto N. Robbins and cotran pathologic basis of disease. 7th ed. Philadelphia, PA: Saunders, Elsevier; 2005.  Back to cited text no. 39
    
40.
Ergene S, Cavas T, Celik A, Koleli N, Kaya F, Karahan A. Monitoring of nuclear abnormalities in peripheral erythrocytes of three fish species from the Goksu Delta (Turkey): genotoxic damage in relation to water pollution. Ecotoxicology 2007; 16:385–391.  Back to cited text no. 40
    
41.
Strunjak-Perovic I, Coz-Rakovac R, Topicpopovic N, Jadan M. Seasonality of nuclear abnormalities in gilthead sea bream Sparus aurata (L.) erythrocytes. Fish Physiol Biochem 2009; 35:287–291.  Back to cited text no. 41
    
42.
Castejón OJ. Electron microscopy of myelin figures in normal and pathological tissues. A review. Acta Microsc 2008; 17:13–19.  Back to cited text no. 42
    
43.
Yang JL, Chen HC. Serum metabolic enzyme activities and hepatocyte ultrastructure of common carp after gallium exposure. Zool Stud 2003; 42:455–461.  Back to cited text no. 43
    
44.
Kample P, Kulkarni S, Bhiwgade DA. Ultrastructural and antioxidant studies of etoposide treated kidney of rat. J Cancer Sci Ther 2013; 5:137–141.  Back to cited text no. 44
    
45.
Garcia-Leston J, Mendez J, Pasaro E, Laffon B. Genotoxic effects of lead: an updated review. Environ Int 2010; 36:623–636.  Back to cited text no. 45
    
46.
Pace BM, Lawrence DA, Behr JM, Parson JP, Dias AJ. Neonatal lead exposure changes quality of sperm and number of macrophages in testes of BALB/c mice. Toxicology 2005; 210:247–256.  Back to cited text no. 46
    
47.
Hosseinchi M, Soltanalinejad F, Najafi G, Roshangar L. Effect of gibberellic acid on the quality of sperm and in vitro fertilization outcome in adult male rats. Vet Res Forum 2013; 4:259–264.  Back to cited text no. 47
    
48.
Sun JG, Jurisicova A, Casper RF. Detection of deoxyribonucleic acid fragmentation in human sperm: Correlation with fertilization in vitro. Biol Reprod 1997; 56:602–607.  Back to cited text no. 48
    
49.
Malekinejad H, Mirzakhani N, Razi M, Cheraghi H, Alizadeh A, Dardmeh F. Protective effects of melatonin and Glycyrrhiza glabra extract on ochratoxin A-induced damages on testes in mature rats. Hum Exp Toxicol 2011; 30:110–123.  Back to cited text no. 49
    
50.
Rengan A, Agarwal A, Linde M, Plessis SS. An investigation of excess residual cytoplasm in human spermatozoa and its distinction from the cytoplasmic droplet. Reprod Biol Endocrinol 2012; 10:92.  Back to cited text no. 50
    
51.
Al-Azemi M, Omu FE, Kehinde EO, Anim JT, Oriowo MA, Omu AE. Lithium protects against toxic effects of cadmium in the rat testes. J Assist Reprod Genet 2010; 27:469–476.  Back to cited text no. 51
    
52.
Fath A, Bethke PC, Jones RL. Enzymes that scavenge reactive oxygen species are down-regulated prior to gibberellic acid-induced programmed cell death in barley aleurone. Plant Physiol 2001; 126:156–166.  Back to cited text no. 52
    
53.
Hassan HA, Al-Rawi MM. Grape seeds proanthocyanidin extract as a hepatic-reno-protective agent against gibberellic acid induced oxidative stress and cellular alterations. Cytotechnology 2013; 65:567–576.  Back to cited text no. 53
    
54.
Nada SA, Gowife AM, El-Denshary ES, Salama AA, Khalil MG, Ahmed KA. Protective effect of grape seed extract and/or silymarin against thioacetamide-induced hepatic fibrosis in rats. J Liver 2015; 4:178.  Back to cited text no. 54
    
55.
Zhao Y, Zhang H, Gao L. Anti-apoptotic effect of grape seed proanthocyanidin extract on cisplatin-induced apoptosis in rat testis. Food Sci Technol Res 2015; 21:805–811.  Back to cited text no. 55
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16], [Figure 17], [Figure 18], [Figure 19], [Figure 20], [Figure 21], [Figure 22], [Figure 23], [Figure 24], [Figure 25], [Figure 26], [Figure 27], [Figure 28], [Figure 29]
 
 
    Tables

  [Table 1]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Materials and me...
Results
Discussion
Conclusion
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed96    
    Printed0    
    Emailed0    
    PDF Downloaded28    
    Comments [Add]    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]