Experimental animals
Male ICR mice were purchased from Shanghai Experimental Animal Center and kept in the Experimental Animal Building of Shanghai Institute of Family Planning Science (Clean grade, license No.: SCXY(Shanghai)2018-0017).
Reagents and antibodies
Cyclophosphamide (8H257F, Baxter International Inc, Illinois, USA). anti-TRA98 ( ab82527,Abcam, Cambridge, MA,USA). anti- GATA4 ( ab84593, Abcam, Cambridge, MA,USA). anti-3β-HSD (sc-515120, Sant Cruz, Oregon, USA ). anti-Ki67 (AF1738, Beyotime, Shanghai, China). TUNEL kit (C1086, Beyotime, Shanghai, China). LIVE/DEAD Sperm Viability kit (Thermo Fisher Scientific, Waltham, MA). PSA-FITC (012M4037V, Sigma, St. Louis, MO,USA).
Preparation of Sheng Jing Decoction
Sheng Jing Decoction was combined with Rehmannia glutinosa (Gaertn.) DC., Astragalus membranaceus (Fisch.) Bunge, Pseudostellaria heterophylla (Miq.) Pax, Dipsacus acaulis (A.Rich.) Napper, Lycium arenicolum Miers, Astragalus complanatus Bunge and Gleditsia sinensis Lam.. All herbs pieces were purchased from Shanghai Wanshicheng TCM CO., Ltd, and made into SJD by TCM Pharmacy of Seventh People's Hospital of Shanghai University of TCM. Rehmannia glutinosa (Gaertn.) DC., Astragalus membranaceus (Fisch.) Bunge, Dipsacus acaulis (A.Rich.) Napper, Lycium arenicolum Miers, Astragalus complanatus Bunge and Gleditsia sinensis Lam. can be checked in “The Plant List” (www.theplantlist.org) and Pseudostellaria heterophylla (Miq.) Pax is available in MPNS (http://mpns.kew.org) The details of herbs can be checked in Supplemental Table 1.
Experimental design
The oligozoospermia model mice were constructed by cyclophosphamide inducing. Cyclophosphamide was dissolved in PBS and injected intraperitoneally (ip) to the mice (60mg/kg), once in a day for a period of 5 days. High (33g/kg), medium (16.5 g/kg) and low (8.25 g/kg) doses of SJD were given orally to mice, once in a day for a period of 35 days. The medium dose is converted from the therapeutic dose of human. Body weights were measured every week. Testes were quickly dissected out and weighed. One half of testes were collected and stored at -70 ◦C for real-time RT-PCR analysis, and the other half of testes were fixed in Bouin’s fixative (0.2% picric acid/2% (v/v) formaldehyde in PBS) for histological evaluation.
Sperm motility and count
The epididymis was placed in 1ml normal saline, and then a deep hole was cut in each caudate nucleus with micro scissors. Sperm was allowed to release for 5 minutes in an incubator with a temperature of 34°C and 5% CO2. The sperm suspension was incubated in CO2 incubator for 30 min and a drop of sperm suspension was uniformly smeared on clean glass slide. Sperm motility and count were analyzed by CASA system and bright-field microscopy (BX43 Olympus, Tokyo, Japan).
Immunohistochemistry
Immunohistochemistry was performed according to standard procedures. The primary antibodies anti-TRA98 (1: 200), anti- GATA4 (1: 200), anti-3β-HSD (1: 100) and anti- Ki67(1: 100), ABC Kit (Vector, USA) and DAB (ZSGB-bio, China) were used for staining. The results were obtained by fluorescence microscope (BX51 Olympus, Tokyo, Japan) and Color digital camera (DFC425 Leica, Wetzlar, Germany).
Real-time RT-PCR analysis
Total RNA was prepared using TRIzol (Sigma, USA) and was reversely transcribed into cDNA with the QuantiNova Reverse Transcription Kit (Qiagen, Dusseldorf, Germany). Real-time quantitative PCR was performed using a CFX96TM Real-time system (Bio-Rad, USA) and SYBR Premix Ex Taq (TakaRa, Dalian, China) according to the manufacturer’s instruction. Raw data were normalized to the internal GAPDH and presented as relative expression level calculated by 2△△Ct method. Primers pairs were as following: Lin28a:5'-GGTGGTGTGTTCTGTATTGGGA-3' and 5'-AGTTGTAGCACCTGTCTCCCTTTG-3'. Kit: 5'-GGGCTAGCCAGAGACATCAG-3' and 5'-AGGAGAAGAGCTCCCAGAGG-3'. Sohlh2: 5'-TCTCAGCCACATCAC AGAGG-3' and 5'-GGGGACGCGAGTCTTATACA-3'. Stra8: 5'-ACCCTG GTAGGGCTCTTCAA-3' and 5'-GACCTCCTCTAAGCTGTTGGG-3'.
JC-1 staining
JC-1 staining kit was purchased from Beyotime (Shanghai, China), Equal volume sperm and JC-1 staining working fluid were mixed. Incubated at 37℃ with 5%CO2 for 30min. Then the sperm were washed twice with JC-1 staining buffer. The results were observed under fluorescence microscope (BX51 Olympus, Tokyo, Japan).
Sperm plasma membrane integrity test
The Sperm (1×10 /mL) suspension was added syBR-14 and PI dye kit(Thermo Fisher Scientific, Waltham, MA) successively. Mixed and incubated in dark for 5 min. After washing, stained sperm were observed by fluorescence microscope (BX51 Olympus, Tokyo, Japan).
Statistics and data analyses
Data are expressed as the mean ± SEM, and statistical evaluation was performed using the Student’s t-test for independent groups. Values of p < 0.05 were considered statistically significant.