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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 Asian J Androl 1999 Sep; 1: 155-158 Keywords:
AbstractAim: 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].
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).
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).
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.
References [1]
Ge YF, Huang YF. Apoptosis
of male germ cells. J Clin Lab Sci (China)
1998; 16: 254-6. Correspondence
to Dr. Yi-Feng GE.
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