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Studies on apoptosis of spermatogenic cells in normal fertile men treated with supraphysiological doses of testosterone undecanoate                      

Yi-Feng GE1, Yu-Feng HUANG1, Gui-Yuan ZHANG2, Xing-Hai WANG3, Jian-Ping XU1   

1Laboratory of Reproduction and Genetics, General Hospital of Nanjing Command, PLA, Nanjing 210002, China 
2National Research Institute for Family Planning, Beijing 100018, China 
3Jiangsu Family Planning Research Institute, Nanjing 210029, China

Asian J Androl  1999 Sep; 1: 155-158

Keywords: anti-spermatogenic effect; testosterone undecanoate; apoptosis; germ cells; semen
Aim: To study the anti-spermatogenic mechanism of supra-physiological doses of testosterone undecanoate (TU). Methods: Twenty fertile adult men received four intramuscular injections of TU at monthly intervals, 1000 mg upon admission and 500 mg for the subsequent injections. The apoptotic germ cells in the semen were studied under light microscope with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and Wright-Giemsa staining methods. Results: After treatment, the sperm density and the number of spermatogenic cells in the semen were significantly decreased (P<0.01), while the apoptotic ratios of spermatocytes and spermatids increased significantly (P<0.01) as compared with the pretreatment levels. Apoptosis was found to be augmented in the whole series of castoff spermatogenic cells. Conclusion: Besides its suppressive effect on spermatogenesis through a negative feed-back mechanism, TU enhances apoptosis of spermatogenic cells, which may be an additional mechanism of its anti-spermatogenic activity.

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].    

From 1985 to 1995, the World Health Organization (WHO) has organized multi-center studies on the male contraceptive effect of testosterone enanthate (TE), a long-acting T preparation, at 16 centers in 10 countries. Results indicated that TE could safely induce reversible azoospermia or severe oligospermia in men[3]. However, a dosing regime of weekly intramuscular injection made this preparation unacceptable for practical application. Testosterone undecanoate (TU), another long-acting T preparation with a longer half-life and persistent time than those of TE, has been preliminarily shown to suppress spermatogenesis in male volunteers[4-6]. Large scale multi-center studies on TU have also been launched to confirm its anti-spermatogenic efficacy. TU requires a much lower injection frequency. If TU could be confirmed to be similarly efficacious, it will be superior to TE as a male contraceptive method. The present paper is a part of this TU project aiming at the investigation of the apoptotic phenomenon in the castoff spermatogenic cells in the semen.

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

Data were presented as means. Statistical analysis was performed using Bartlett's, Dunnett, Rank sum and SNK tests. Significance of differences was setat P<0.05.

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).


Concentrations (1109/L)

Spermatogenic cell/
Sperm ratio


spermatogenic cell





Day 30




Day 60




Day 90




The apoptotic ratio, the ratio of the number of a particular type of apoptotic spermatogenic cell to the total number of that type of cell, is an expression of the degree or rate of apoptosis.

It can be seen from Table 2 that the apoptotic ratios of spermatocyte and spermatid were significantly increased on Day 30 as compared with the pretreatment level (P<0.01); The ratios on Day 60 and 90 were further increased to values significantly higher than those on Day 30 (P<0.01). Data indicated a gradual enhancement of apoptosis in the course of TU administration. Apoptosis was found to be augmented in the whole series of castoff spermatogenic cells, however, spermatogonia were too few to be accurately counted.

Table 2. Effects of TU on apoptotic ratios of spermatogenic cells. means. cP<0.01, compared with the pretreatment levels (Rank sum and Dunnett t tests). fP<0.01, compared with the Day 30 values (Rank sum and SNK tests).



Apoptotic ratio (%)



WG stain


WG stain








Day 30






Day 60






Day 90






4 Discussion

Recent experimental studies indicated that exogenous interference in spermatogenesis, no matter accomplished by whatever means, such as FSH neutralizatiion[9], LH deprivation[10], GnRH antagonists[11], or selective intoxication of Leydig cells[12], may induce apoptosis in different stages of spermatogenic cells. Only in case of Leydig cell intoxication, was apoptosis enhanced in all stages of spermatogenic cells.

The present paper showed that after TU treatment, the apoptotic spermatogenic cells in the semen were gradually increased, indicating a progressive enhancement of apoptosis in the course of treatment. Furthermore, apoptosis was found to be augmented in the whole series of castoff spermatogenic cells, an occurrence that bears a similarity with the effect of Leydig cell intoxication. It is well known that large doses of exogenous T suppresses endogenous T secretion through a negative feed back mechanism with a resultant deprivation of the intratesticular T, while in Leydig cell intoxication, the T production is arrested and the intratesticular T is deprived. It can be seen that the two measures reach the same consequence of intratesticular T deprivation. However, supraphysiological doses of TU suppress GnRH, LH and FSH secretion as well, which may also contribute to the enhancement of apoptosis.

The ratio of castoff spermatogenic cells to spermatozoa was increased gradually from the pretreatment value of 0.027 up to 0.226 on Day 90. It may imply a gradual exacerbation of spermatogenic arrest with a relative enhancement of spermatogenic cells exfoliation and/or a decreased spermatozoa formation.

In conclusion, TU could induce apoptotic changes in the spermatogenic cells in the semen, and apoptosis may be an additional anti-spermatogenic mechanism of TU action.


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Correspondence to Dr. Yi-Feng GE.
Tel: +81-25-482 6808     Fax: +81-25-480 3061
Received 1999-04-16     Accepted 1999-09-16