Effect of Gosha-Jinki-Gan on Levels of Specific mRNA Transcripts in Mouse Testes after Busulfan Treatment

Abstract

With the increase in survival rates of cancer patients in recent years, infertility caused by anticancer treatments has become a significant concern for cancer survivors. Some studies have suggested that Sertoli cells play a key role in mediating testicular immunology in busulfan-induced aspermatogenesis. We recently demonstrated that Gosha-jinki-gan (TJ107), a traditional Japanese medicine, can completely recover injured spermatogenesis in mice 60 days after busulfan injection. In the present study, we sought to examine the levels of mRNA transcripts encoding markers of 25 Sertoli cell-specific products and 10 markers of germ cell differentiation. Our results demonstrated that only supplementation of TJ107 at day 60 after busulfan injection could significantly recover the increase in five mRNA species (Amh, Clu, Shbg, Testin, and Il1a) and the decrease in four mRNA species (Aqp8, CST9, Wnt5a, and Tjp1) in response to Busulfan (BSF) at day 120, with the increase of all examined spermatogenic markers.

Keywords: anticancer treatment; aspermatogenesis; oriental medicine; specific mRNA transcript.

Figure 1

Testicular histology and the levels of three mRNA transcripts encoding markers of germ cell differentiation in each group at day 120 (n = 5): (A) testes sections show morphology in groups I–V. Intact seminiferous tubules showing normal germinal epithelium from the spermatogonia to spermatozoa are observed in groups I and II. Atrophic seminiferous tubules with azoospermia are observed in group III. The presence of both atrophic and intact seminiferous tubules with spermatogenesis was observed in the group IV mice. Normal-appearing seminiferous tubules are observed in the group V mice (bar = 50 µm). (B) Expression was measured by real-time RT PCR, and the results are expressed relative to the internal control GAPDH. Data show expression of the spermatogonial marker Stra8, the spermatocyte marker Spo11, and the spermatid marker Tnp1. The results are expressed as the mean values ± standard deviation of five mice in each group, and the y-axis shows relative mRNA intensity. (C) Expression was measured by real-time RT PCR, and the results are expressed relative to the internal control GAPDH. Data show expression of the premeiotic markers (cKit, Gfra1, and Vasa), and the meiotic and postmeiotic markers (Boll, Crem, Prm1, and Acrosin). The results are expressed as the mean values ± standard deviation of five mice in each group, and the y-axis shows relative mRNA intensity. * p < 0.05 vs. group I; ^# p < 0.05 vs. group III.

Figure 2

Effect of TL107 on the levels of 25 mRNA transcripts encoding markers of Sertoli cell-specific products in each group at day 120: expression was measured by real-time RT PCR, and the results are expressed relative to the internal control GAPDH. The results are expressed as the mean values ± standard deviation of five mice in each group, and the y-axis shows relative mRNA intensity. Transcripts showing a significant increase in levels after BSF treatment compared to control values are grouped in (A); transcripts showing a significant decrease in levels after BSF treatment compared to control values are grouped in (B). Transcripts that showed no change in levels after BSF treatment are grouped in (C). * p < 0.05 vs. group I; ^# p < 0.05 vs. group III.

Figure 3

Testicular histology and levels of three mRNA transcripts encoding markers of germ cell differentiation in groups I, III, and IV at day 60 (n = 5): (A) seminiferous tubules showing normal spermatogenesis from spermatogonia to spermatozoa are observed in the group I mice. Both atrophic and intact seminiferous tubules with spermatogenesis were observed in the group III and group IV mice (bar = 40 µm). (B,C) Expression was measured by real-time RT PCR, and the results are expressed relative to the internal control GAPDH. (B) Data show expression of the spermatogonial marker Stra8, the spermatocyte marker Spo11, and the spermatid marker Tnp1. (C) Data show expression of the premeiotic markers (cKit, Gfra1, and Vasa), and the meiotic and postmeiotic markers (Boll, Crem, Prm1, and Acrosin). The results are expressed as the mean values ± standard deviation of five mice in each group, and the y-axis shows relative mRNA intensity. * p < 0.05 vs. group I. (D) Testicular weights and epididymal spermatozoa count in groups I, III, and IV. Data are presented as mean ± standard deviation. ^a p < 0.05 vs. group I; ^b p < 0.05 vs. group III.

Figure 4

Effect of TL107 on the expression levels of 25 mRNA transcripts encoding markers of Sertoli cell-specific products in groups I, III, and IV mice at day 60: expression was measured by real-time RT PCR, and the results are expressed relative to the internal control GAPDH. The results are expressed as the mean values ± standard deviation of five mice in each group, and the y-axis shows relative mRNA intensity. Transcripts showing a significant increase in levels after BSF treatment compared to control values are grouped in (A); transcripts showing a significant decreased in levels after BSF treatment compared to control values are grouped in (B); and transcripts showing no change in levels after BSF treatment are grouped in (C). * p < 0.05 vs. group I; ^# p < 0.05 vs. group III.