The Effects of Olive Leaf Extract on The Testis, Sperm Quality and Testicular Germ Cell Apoptosis in Male Rats Exposed to Busulfan

Abstract

Background: Busulfan (BU) has a destructive effect on the male reproductive system. The goal of this study was to assess the effects of olive leaf extract (OLE) as a source of antioxidants and phenolic compounds, on BU-induced damages in rat testes.

Materials and methods: In this experimental study, 40 male Wistar rats were randomly divided into 5 groups. The control group (CTL) received a single intraperitoneal (i.p.) injection of dimethyl sulfoxide (DMSO), followed by oral administration of distilled water for 5 weeks. In BU group, BU (10 mg/kg) was administrated i.p. once. In cotreatment groups, first, received BU (10 mg/kg, a single i.p. injection) then, OLE was administrated orally at different doses of 250 mg/kg (BU+OLE 250), 500 mg/kg (BU+OLE 500) and 750 mg/kg (BU+OLE 750), for 5 weeks. Next, blood and sperm samples were collected. The left testis was removed to investigate testicular parameters and apoptosis by using H and E and TUNEL staining, respectively. All data were analyzed by SPSS software and a P<0.05 was considered significant.

Results: There was a significant decline in sperm viability (P=0.017), number of primary spermatocyte (PS) (P=0.001) and Leydig cells (P=0.023) in the BU group versus the CTL group. OLE at three doses could repair these defects versus BU group. Increases in apoptotic spermatogonia cells (SG) due to BU were significantly reduced by OLE 250 and 500 mg/kg (P<0.01). A reduction in germinal epithelium height and an increase in apoptotic SG were observed in BU+OLE 750 group vs. other groups (P<0.01) and alkaline phosphatase (ALP) was at the highest level, also Aspartate aminotransferase (AST) increased markedly vs. CTL (P=0.024).

Conclusion: Oral administration of OLE at the doses of 250 and 500 mg/kg could be helpful in ameliorating BUinduced toxicity in rat testes, while OLE 750 mg/kg not only did not cause positive effects, but also could exacerbate the harmful effects.

Keywords: Apoptosis; Busulfan; Olive Extract; Spermatogenesis; Testicular Germ Cell.

Fig.1

The standard curves of phenols (gallic acid equivalents) and flavonoids (rutin equivalents) by drawing adsorption against concentration. A. Standard curves of rutin and B. Standard curve of gallic acid. The results have been resulted from triplicate experiments.

Fig.2

Light micrographs of rat testis (HA. Seminiferous tubules showing normal structure and active spermatogenesis in the control group, B. The most of spermatogonia and primary spermatocytes cells are destroyed in busulfan group, C. Photomicrographs of testis from rats that treated with 250 mg/kg, D. 500 mg/kg, and E. 750 mg/kg olive leaf extract (OLE) demonstrated that both 250 and 500 mg/kg OLE caused a normal and regular structure of seminiferous tubules and obvious improvement in spermatogenesis, while 750 mg/kg OLE reduced cell lines and height of seminiferous epithelium (.) and destroy interstitial space (*).

Fig.3

Immunohistochemical staining of the rat testis tissue in experimental groups. A. Light microscopy of TUNEL-stained rat testicular sections (×400 magnifications), a. Apoptotic cells are seen brown color. Apoptosis is extremely low in control testes, b. The most of testicular germ cells is undergoing apoptosis in busulfan testes, c. Although TUNEL-positive germ cells are still visible in testicular sections from rats that treated with olive leaf extract (OLE) at dose of 250 mg/kg, d. 500 mg/kg OLE caused a marked decrease in apoptotic testicular germ cells, e. High level of apoptotic cells was observed in testes of rats that treated with OLE at dose of 750 mg/kg and B. ×1000 magnification.

Fig.4

Effect of OLE treatment after busulfan exposureon percentage of apoptotic testicular germ cells. Significant differences (P<0.05) are indicated by ***; vs. all groups, *; vs. control and BU+OLE 500 groups, and #; vs. all groups except control. Values are expressed as % mean ± SEM. CTL; Control group, BU; Busulfan, PLE; Olive leaf extract, SG; Spermatogonia, and PS; Primary spermatocye cells.