Protective effect of grape seed extract against cadmium-induced testicular dysfunction

Abstract

Cadmium (Cd) is the most prevalent toxic metal present in livestock feed; therefore, the present study aimed to examine the ameliorative effects of grape seed extract (GSE) on cadmium chloride (CdCl2)‑induced testicular dysfunction of Wistar rats. Male adult Wistar rats (40 rats; n=10/group) were divided into four equal groups. Group one was used as a control, and was given ad libitum access to food and water. Groups 2‑4 were treated with CdCl2 [5 mg/kg body weight (BW)], GSE (400 mg/kg BW, orally), and GSE plus CdCl2, respectively. Blood and testicular tissues were collected and assayed for biochemical and histopathological changes, respectively. Testicular genes were expressed using semi‑quantitative RT‑PCR analysis. The results of the present study demonstrated that there was a decrease in serum testosterone levels following CdCl2 toxicity, which were normalized after GSE co-administration. Furthermore, CdCl2 significantly increased the serum levels of malondialdehyde, and decreased levels of antioxidants. At the histopathological level, the testes of the CdCl2 group exhibited congestion, edema in the interstitial blood vessels, irregular arrangement of the epithelial lining of the seminiferous tubules, and degeneration and sloughing of the spermatogenic cells, which accumulated in the center of the seminiferous tubules. Such pathological alterations were ameliorated following treatment with GSE in the CdCl2 plus GSE group. The immunohistochemical expression of B‑cell lymphoma 2‑associated X protein was high in the CdCl2 group, and low in the control and GSE groups. Co‑treatment with GSE and CdCl2 exhibited ameliorative effects on the immunoreactivity of B‑cell lymphoma 2‑associated X protein. CdCl2 toxicity induced a significant downregulation in the mRNA expression levels of cytochrome P450 cholesterol side‑chain cleavage enzyme, cytochrome P450 17A1, 3β‑hydroxysteroid dehydrogenase (3β‑HSD), 17β‑HSD, androgen receptor, steroidogenic acute regulatory protein, and follicle‑stimulating hormone receptor. GSE administration exhibited a stimulatory effect on steroidogenesis‑associated enzymes, and co‑treatment with GSE and CdCl2 normalized and upregulated the mRNA expression levels of these examined genes. This study concluded that GSE has beneficial protective effects against the deleterious effects of CdCl2 on the testis.

Figure 1

(A) Alterations in glutathione S-transferase (GST) and superoxide dismutase (SOD) expression in the testes of rats treated with cadmium chloride (CdCl₂) and grape seed extract (GSE). Experimental groups were administered water as a control (CNT), CdCl₂ (Cd), GSE, or CdCl₂ plus GSE (Cd+GSE). (B) Densitometric analysis of genes from 5 rats per treatment group. Data are presented as the mean ± standard error of the mean. ^*P<0.05 vs. the control group; ^#P<0.05 vs. the Cd group. ^$P<0.05 vs. the GSE group. G3PDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 2

Photomicrographs of the testes. (A) In the control group, numerous seminiferous tubules (st), spermatogonia (sg), spermatocytes (sc) and spermatozoa (z) were detected. Interstitial connective tissue and Leydig cells (L) were observed [hematoxylin & eosin (H&E); magnification, ×10). (B) In the grape seed extract (GSE) group activated spermatocytes and spermatozoa (z) were detected (H&E; magnification, ×40). (C) In the cadmium chloride (CdCl₂) group, edema (o) and sloughing of spermatogonia (s) were detected (H&E; magnification, ×40). (D) Accumulation of the sloughed cells was detected in the center of the seminiferous tubules (arrow) (H&E; magnification, ×40). (E) In the co-treated group (GSE and CdCl₂), the seminiferous tubules were characterized by congestion (C) and slight edema (o) (H&E; magnification, ×10).

Figure 3

Photomicrograph of the immunoreactivity of B-cell lymphoma 2-associated X protein (Bax) in rat testes. (A) Few Bax-positive cells were immunostained in the control group (arrows; magnification, ×40). (B) Weak Bax immunostaining was detected in the grape seed extract (GSE) group (arrows; magnification, ×40). (C) Strong Bax immunostaining was detected in the cadmium chloride (CdCl₂) group (arrows; magnification, ×40). (D) Few Bax-positive cells were immunostained in the co-treated group (GSE and CdCl₂) (arrows; magnification, ×10).

Figure 4

Photomicrograph of the immunoreactivity of Ki-67 in the rat testes. (A) Strong Ki-67-positive immunostaining was detected in the spermatogenic cells of the control group (arrows; magnification, ×40). (B) Ki-67-positive immunostaining was detected in the grape seed extract (GSE) group (arrows; magnification, ×40). (C) Ki-67 immunostaining was very weak in the cadmium chloride (CdCl₂) group (arrows; magnification, ×40). (D) Ki-67-positive immunostaining was detected following co-treatment with GSE and CdCl₂ (arrows; magnification, ×10).

Figure 5

Semi-quantitative reverse transcription-polymerase chain reaction analysis of the alterations in the expression of steriodogenesis-associated genes in the testes of rats treated with cadmium chloride (CdCl₂) and grape seed extract (GSE) for 3 months. (A) mRNA expression levels of steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), cytochrome P450 17A1 (P450c17) and follicle-stimulating hormone receptor (FSHR). (B) mRNA expression levels of 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD) and androgen receptor (AR). (C and D) Densitometric analysis of gene expression levels. Experimental groups were administered water as a control (CNT), CdCl₂ (Cd), GSE, or CdCl₂ plus GSE (Cd+GSE). Densitometric analysis was carried out for 5 rats per treatment group. Data are presented as the mean ± standard error of the mean. ^*P<0.05 vs. the control group; ^#P<0.05 vs. the Cd and control groups; ^$P<0.05 vs. the Cd group. G3PDH, glyceraldehyde 3-phosphate dehydrogenase.