Korean J Androl.  2011 Apr;29(1):33-42. 10.5534/kja.2011.29.1.33.

The Changes of Testis and the Effects of Anthocyanin on Spermatogenesis in Rat Induced Varicocele

• Jang H
• Kim SJ
• Yuk SM
• Han DS
• Ha US
• Hong SH
• Lee JY
• Hwang TK
• Hwang SY
• Kim SW

• KMID: 2298624
• DOI: http://doi.org/10.5534/kja.2011.29.1.33

Abstract

PURPOSE
Varicocele is known as a main cause of primary male infertility and it supposed to be associated with oxidative stress. Anthocyanin is known as a natural plant pigment and novel antioxidant. This study was designed to investigate the effects of anthocyanin on a rat model of varicocele.
MATERIALS AND METHODS
Twenty four male rats, induced varicocele by partial obstruction of left renal vein, were divided into four experimental groups: the group induced varicocele for four weeks without anthocyanin, the group received anthocyanin (80 mg/kg) right after varicocele induction, group induced varicocele for eight weeks without anthocyanin, and the group received anthocyanin (80 mg/kg) after four weeks observation following varicocele induction. After anthocyanin treatment, testes from the rats in all groups were removed, weighed, and subjected to histological examination. Apoptosis in the testes was measured by the TUNEL assay. And the oxidative stress was evaluated by measurement of 8-hydroxy-2'-deoxyguanosine (8-OHdG).
RESULTS
Induction of varicocele led to decreasing left testis weight, decreasing spermatogenic cell density significantly (p<0.05). Also it led to increasing apoptotic body counts and increasing concentration of 8-OHdG significantly (p<0.05). However administration of anthocyanin right after varicocele induction prevent this change meaningfully (p<0.05). In group received anthocyanin after four weeks observation following varicocele induction, interestingly, there was no significant difference in testis weight, spermatogenic cell density, apoptotic body count and concentration of 8-OHdG compared to group induced varicocele for eight weeks without anthocyanin administration.
CONCLUSIONS
These results suggest that anthocyanin is effective in decreasing the oxidative stress of testis in rat induced varicocele and may be effective in making a healthy sperm in patient of varicocele in early stage. However in patient under way in advanced stage, it is supposed that the anthocyanin cannot help having a protective effect from oxidative stress narrowly unless the condition of oxidative stress by varicocele is corrected. Further studies are needed to better understand the mechanisms and actions of anthocyanin and varicocele, and these studies may lead to the clinical application of anthocyanin in preventing male infertility by varicocele.

Keyword

Anthocyanin; Antioxidant; Apoptosis; Varicocele; Infertility

MeSH Terms

Animals
Anthocyanins
Apoptosis
Cell Count
Deoxyguanosine
Humans
In Situ Nick-End Labeling
Infertility
Infertility, Male
Male
Oxidative Stress
Plants
Rats
Renal Veins
Spermatogenesis
Spermatozoa
Testis
Varicocele
Anthocyanins
Deoxyguanosine

Figure

• 

  Fig. 1. (A) It shows ligation of proximal renal vein with plastic probe. Black arrow indicates plastic probe and white arrow indicates the engorgement of left gonadal vein and distal renal vein. (B) It shows partial obstruction of proximal renal vein after removal of plastic probe. Gray arrow indicates engorged left gonadal vein.

• 

  Fig. 2. Histopathologic findings of left testis (H&E stain). The thickness of germinal cell layer (B) is more increased than that (A) statistically (×400). (C) and (D) necrosis of germinal cells was observed and the thickness of germinal cell layer was more decreased than that (B). And there was no difference in spermatogenic cell density between (C) and (D) statistically (×400). (A) Group induced varicocele for 4 weeks without anthocyanin administration. (B) Group received oral dose of anthocyanin (80 mg/kg) for 4 weeks right after varicocele induction. (C) Group induced varicocele for 8 weeks without anthocyanin administration. (D) Group received oral dose of anthocyanin (80 mg/kg) for 4 weeks after 4 weeks observation following varicocele induction.

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  Fig. 3. Mean weight of testes in each groups. Left testis weight of group received anthocyanin right after varicocele induction is significantly higher than group induced varicoele for 4 weeks without anthocyanin administration statistically (p＜0.05). But there was no difference between group induced varicocele for 8 weeks without anthocyanin and group received anthocyanin after 4 weeks observation following varicocele induction statistically (p＞0.05). ∗Anthocyanin 80 mg/kg: group received oral dose of anthocyanin (80 mg/kg) right after varicocele induction. ∗∗Anthocyanin 80 mg/kg: group received oral dose of anthocyanin (80 mg/kg) after 4 weeks observation following varicocele induction.

• 

  Fig. 4. Spermatogenic cell density (germinal cell layer thickness/ diameter of seminiferous tubule). Spermatogenic cell density of group received anthocyanin right after varicocele induction is significantly higher than group induced varicocele varicocele for 4 weeks without anthocyanin administration statistically (p ＜0.05). But there was no difference between group induced varicocele for 8 weeks without anthocyanin administration and group received anthocyanin after 4 weeks observation following varicocele induction statistically (p＞0.05). ∗Anthocyanin 80 mg/kg: group received oral dose of anthocyanin (80 mg/kg) right after varicocele induction. ∗∗Anthocyanin 80 mg/kg: group received oral dose of anthocyanin (80 mg/kg) after 4 weeks observation following varicocele induction.

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  Fig. 5. Apoptotic bodies in TUNEL stain of left testis. Positive TUNEL stain cell called apoptotic body was showed in dark brown or black color in TUNEL stain (×200). (A) Counts of apoptotic bodies was more increased than that (B). (C) and (D) apoptotic bodies were more increased than that (A). But there was no difference in counts of apoptotic bodies between (C) and (D) statistically (×200). (A) Group induced varicocele for 4 weeks without anthocyanin administration. (B) Group received oral dose of anthocyanin (80 mg/kg) for 4 weeks right after varicocele induction. (C) Group induced varicocele for 8 weeks without anthocyanin administration. (D) Group received oral dose of anthocyanin (80 mg/kg) for 4 weeks after 4 weeks observation following varicocele induction.

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  Fig. 6. Numbers of apoptotic bodies. In group received anthocyanin right after varicocele induction, count of apoptotic bodies is significantly lower than that of group induced varicocele for 4 weeks without anthocyanin administration statistically (p＜0.05). But there was no difference between group induced varicocele for 8 weeks without anthocyanin. administration and group received anthocyanin after 4 weeks observation following varicocele induction statistically (p＞ 0.05). ∗Anthocyanin 80 mg/kg: group received oral dose of anthocyanin (80 mg/kg) right after varicocele induction. ∗∗Anthocyanin 80 mg/kg: group received oral dose of anthocyanin (80 mg/kg) after 4 weeks observation following varicocele induction

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  Fig. 7. Concentration of 8-OHdG (8-hydroxy-2'-deoxyguanosine). In group received anthocyanin right after varicocele induction, concentration of 8-OHdG is significantly lower than that of group induced varicocele for 4 weeks without anthocyanin administration statistically (p＜0.05). But there was no difference between group induced varicocele for 8 weeks without anthocyanin administration and group received anthocyanin after 4 weeks observation following varicocele induction statistically (p＞0.05). ∗Anthocyanin 80 mg/kg: group received oral dose of anthocyanin (80 mg/kg) right after varicocele induction. ∗∗Anthocyanin 80 mg/kg: group received oral dose of anthocyanin (80 mg/kg) after 4 weeks observation following varicocele induction

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