1 Introduction

Apoptosis, an active cell suicide process characterized by a regulated degeneration of chromatin controlled by specific genes, is a completely different form of cell death in terms of morphology and biochemistry. Apoptosis is the principal mechanism of germ cell death during normal spermatogenesis and a decrease in the concentration of gonadotropins or testosterone (T) may further accelerate the apoptotic process^[1,2].    

2 Materials and methods

2.1 Subjects and treatments

Twenty healthy fertile Chinese men (Han race, all had signed appropriate papers to volunteer for the study), 25-45 years of age, participated in the Nanjing Center. TU (Zhejiang Xian Ju Pharmaceutical Corporation, Zhejiang, China) 1000 mg was given intramuscularly on admission, followed by 500 mg monthly for 3 months. The volunteers were requested to provide a semen sample before treatment and one sample every month for 3 months.

2.2 Semen examination

Semen samples were collected by masturbation with clean hands and containers. They were liquefied in a 37 water bath for 30 min. The sperm density and the concentration of the spermatogenic cell were examined under light microscope with Macro sperm counting chamber (Nanjing Yuancheng Co., Jiangsu, China).

2.3 Apoptotic cell observation

2.3.1 Preparation of slide

The semen sample was centrifuged at 1000 g for 5 min and the sediment washed with normal saline for three times. Centrifuge again (1000 g, 5 min) and the sediment was smeared on a glass slide. The slide was dipped in 95% ethanol for fixation for 10 min. For each semen sample, two slides were prepared.

2.3.2 Wright-Giemsa (WG) staining

Drop a few drops of the staining solution on the fixed slides and stain for 30-60 s. Then add an equal volume of sodium phosphate buffer (pH 6.9) and stand for 10 min. Examine under light microscope. The spermatogenic cells are distinguished as described elsewhere. Apoptotic cells are identified by the following morphological features: the presence of welldefined characteristic crescentic clumps of chromatin around apoptotic nuclei membranes; the chromatin materials are uniformly dense basophilic masses; the membrane of cytoplasm is shrunken or blebbing; the apoptotic bodies are round or oval eosinophilic cytoplasmic masses with or without basophilic nuclear material^[2,7,8]. The whole slide is observed and each type of apoptotic spermatogenic cell counted, then the apoptotic ratio is calculated separately.

2.3.3 TUNEL method^[2]

TUNEL is a method relying on the presence of fragmented DNA in apoptotic cells. Extensive DNA degradation is a characteristic event occurring in the early stages of apoptosis. Cleavage of the DNA may yield double-stranded, LMW DNA fragments (mono- and oligo-nucleosomes) as well as single stand breaks (nicks) in HMW-DNA. Those DNA strand breaks can be detected by enzymatic labeling of the free 3'-OH termini with modified nucleotides (XdUTP, X=biotin, DIG or fluorescein). In the present study fluorescein was used. TUNEL method employs terminal deoxynucleotidyl transferase as a labeling enzyme. The in situ cell death detection kit {horse-radish peroxidase (POD), Boehringer, Germany} is used to stain the slides. After fixation, samples are incubated in the blocking solution (0.3% H₂O₂ in methanol) for 30 min and then in 0.1% Triton X-100 in 0.1% sodium citrate solution for 2 min on ice before the addition of TUNEL reaction mixture. The reaction is allowed to proceed for 60 min. Wash with PBS. Then add converter-POD to the sample and incubate for 30 min. After substrate (DAB) reaction, the apoptotic spermatogenic cells are counted and the apoptotic ratio analyzed.

2.4 Data analysis

3 Results

One month after the use of TU, the sperm concentration and the number of castoff spermatogenic cells in semen started to decrease (P<0.01), and they remained low in the whole observation period; A gradual increase in the spermatogenic cells/sperm ratio indicated a continual increase in the relative number of castoff spermatogenic cells in the course of treatment (Table 1).

Table 1. Effects of  TU on concentrations of sperm and spermatogenic cells. n=20. means. ^cP<0.01, compared with the pretreatment levels (Bartlett's and Dunnett t tests).

References

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