World J Mens Health.  2015 Apr;33(1):20-29. 10.5534/wjmh.2015.33.1.20.

Protective Effect of Administered Rolipram against Radiation-Induced Testicular Injury in Mice

• Lee W
• Son Y
• Jang H
• Bae MJ
• Kim J
• Kang D
• Kim JS

• KMID: 2320736
• DOI: http://doi.org/10.5534/wjmh.2015.33.1.20

Abstract

PURPOSE
Pelvic irradiation for the treatment of cancer can affect normal cells, such as the rapidly proliferating spermatogenic cells of the testis, leading to infertility, a common post-irradiation problem. The present study investigated the radioprotective effect of rolipram, a specific phosphodiesterase type-IV inhibitor known to increase the expression and phosphorylation of the cyclic adenosine monophosphate response element-binding protein (CREB), a key factor for spermatogenesis, with the testicular system against pelvic irradiation.
MATERIALS AND METHODS
Male C57BL/6 mice were treated with pelvic irradiation (2 Gy) and rolipram, alone or in combination, and were sacrificed at 12 hours and 35 days after irradiation.
RESULTS
Rolipram protected germ cells from radiation-induced apoptosis at 12 hours after irradiation and significantly increased testis weight compared with irradiation controls at 35 days. Rolipram also ameliorated radiation-induced testicular morphological changes, such as changes in seminiferous tubular diameter and epithelial height. Additionally, seminiferous tubule repopulation and stem cell survival indices were higher in the rolipram-treated group than in the radiation group. Moreover, rolipram treatment counteracted the radiation-mediated decrease in the sperm count and mobility in the epididymis.
CONCLUSIONS
These protective effects of rolipram treatment prior to irradiation may be mediated by the increase in pCREB levels at 12 hours post-irradiation and the attenuated decrease in pCREB levels in the testis at 35 days post-irradiation in the rolipram-treated group. These findings suggest that activation of CREB signaling by rolipram treatment ameliorates the detrimental effects of acute irradiation on testicular dysfunction and the related male reproductive functions in mice.

Keyword

Cyclic AMP response element-binding protein; Radiation; Rolipram; Testis

MeSH Terms

Adenosine Monophosphate
Animals
Apoptosis
Cyclic AMP Response Element-Binding Protein
Epididymis
Germ Cells
Humans
Infertility
Male
Mice*
Phosphorylation
Rolipram*
Seminiferous Tubules
Sperm Count
Spermatogenesis
Stem Cells
Testis
Adenosine Monophosphate
Cyclic AMP Response Element-Binding Protein
Rolipram

Figure

• 

  Fig. 1 Protective effect of rolipram from apoptotic cell death in seminiferous tubules following pelvic irrdiation (IR) (2Gy). (A) Representative images (×400) showing apoptotic cells stained with transferase dUTP-biotin nick end labeling (TUNEL) in irradiation testis with or without rolipram treatment at 12 hours post-irradiation. Apotosis was easily recognized by the presence of entire apoptotic bodiesstained by peroxidase. Irradiation increased the expression of the apoptotic nuclei in the germ cells of the mice. Most of the apoptotic cells were in the spermatogonia and primary spermatocytes. Rolipram treatment before irradiation decreased the TUNEL-positive cells compared with the vehicle-treated mice. (B) The graph depicts the number of apoptotic cells per seminiferous tubule in the testis section, as observed using the TUNEL method. Values are reported as the mean±standard deviation of five mice in each group (*p<0.05 as compared to the irradiation control group).

• 

  Fig. 2 Protective effect of rolipram on the body and testis weight 35 days following pelvic irrdiation (IR) (2Gy). Graphs showing body weight (A), testis weight (B), and testis per body weight (C). Values are reported as the mean±standard deviation of five mice in each group (*p<0.05 as compared with the irradiation control group).

• 

  Fig. 3 Protective effect of rolipram on radiation-induced histopathological changes in the testis 35 days following pelvic irrdiation (IR) (2Gy). (A) Representative images (×400) showing H&E and Ki-67-stained testis sections from the irradiation control and rolipram+irradiation groups. Graphs showing the histological features of epithelial height (B) and seminiferous diameter (C). Ki-67 positive cells were detected primarily in the lower layer of the seminiferous tubule, and the immunopositive cells correlated with the tubule germinal cells. (D) Repopulation index (RI) was calculated to show the survival of spermatogonial stem cells. (E) The stem cell survival index (SSI) was calculated using the following equation: SSI=-[lne (1-RI÷100)]. Irradiation reduced SSI and increased RI, but rolipram attenuated these changes. Values are reported as the mean±standard deviation of five mice in each group (*p<0.05 as compared with the irradiation control group).

• 

  Fig. 4 Protective effect of rolipram on sperm characteristics in the testis 35 days following pelvic irrdiation (IR) (2Gy). Graphs showing the number of sperm per cauda epididymis (A), and sperm mobility (B). Values are reported as the mean±standard deviation of five mice in each group (*p<0.05 as compared with the irradiation control group).

• 

  Fig. 5 Effect of rolipram on phosphorylated CREB (pCREB) and total (tCREB) expression in the testis after pelvic irradiation (IR) (2 Gy). (A) Western blot analysis of pCREB and tCREB expression after 12 hours and 35 days following pelvic irrdiation (2 Gy). Values are reported as the mean±standard deviation of five mice in each group (*p<0.05 as compared with the irradiation control group). (B) Representative images (×100) showing pCREB-stained testis sections from the sham control, rolipram+sham control, irradiation control, and rolipram+irradiation groups 35 days after irradiation. OR: odds ratio.

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