1 Introduction

While it is generally accepted that the erectile response in mammals is regulated by androgens, the extent of involvement and the precise role of these steroids remains to be established. A number of neurotransmitters have been identified that may mediate erectile function, of which nitric oxide (NO) has been recently demonstrated as the principal mediator of penile erection^[1-3]. And NO is synthesized from L-arginine by nitric oxide synthase (NOS). Several research groups are actively investigating how androgens may affect the synthesis and action of these agents. Our previous research found that the NOS activity in the rat penis was significantly reduced by 70% after castration^[4]. It suggests that androgen deprivation may lead to the failure of NO-dependent penile erection.

2 Materials and methods

2.1 Materials

Adult male Sprague Dawley rats (Grade II, 150-200 g) were supplied by the Experimental Animal Center, Hubei Medical University. In Situ Cell Death Detection Kit was purchased from the Boehringer Mannheim (Germany), T undecanoate injection from the Xianju Pharmaceutical Co. LTD (Zhejiang, China), and low molecular weight DNA markers from the Promeger Corporation (USA).

2.2 Animal treatment

Thirty rats were randomized into 3 groups of 10 rats each. The Castration group underwent orchietomy under pentobarbital anesthesia. The T-supplementation group received T undecanoate (im, 13.7 mg/kg) at the time of castration. The Sham-operated controls only received sham-operation. One week after surgery, all animals were sacrificed and the corpora cavernosa harvested.

2.3 DNA fragment analysis

Fresh tissues were prepared and DNA was obtained by traditional phenol/chloroform extraction technique. It was then separated on a 1.5% garose gel (80 V) and stained with ethidium bromide. Forty minutes later, the gels were viewed under ultraviolet light for fragmentation of DNA and then photograph was taken.

2.4 In situ end labeling (ISEL) of DNA strand breaks in the nuclei

All specimens were fixed in 10 % formaldehyde neutral buffer solution (within 24 hours) and routine paraffin sections were prepared. According to the detailed procedures described in the kit introduction, ISEL was carried out. The main steps involved: 0.3% H₂O₂ inactivating endogenous peroxidaseprotein digestion by proteinase Kadd terminal deoxynucleotidyl transverse (TdT) and fluorescein-dUTPincubate for 60 min at 37analyze with fluorescence microscopyadd antifluorescein-POD (30 min, 37)stained with DAB substratecounterstained with hematoxylinobserve 10 random fields (400 magnification) for each corpus cavernosum with light microscopy and the apoptotic rate was calculated.

2.5 Statistical analysis

3 Results

3.1 DNA degradation in the erectile tissue after orchietomy

As illustrated in Figure 1, DNA extracted from the corpus cavernosum of controls and castrates with T-supplementation showed only a high molecular weight band. In contrast, DNA isolated from the simple castrates demonstrated distinct inter-nucleosomal DNA degradation resulting in a ladder pattern on the gel. This pattern is characteristic for apoptosis wherein endonuclease fragment DNA into multiple of 180 base pairs.

Figure 1.  DNA fragment analysis (Lanes from left to right represent DNA from Controls, T-supplementation, Simple castrates, and marker, respectively).

3.2 Apoptotic cells

The photomicrograph in Figure 2 shows the presence of apoptotic cell nuclei within the erectile tissue of simple castrates under fluorescence microscopy, with condensed, margined and fragmented cell nuclei, characteristic of the morphology of programmed cell death. Under light microscopy, both the nuclei of normal and abnormal cells were visualized because of hematoxylin counterstaining, as shown in Figure 3A; separate brown apoptotic cells dispersed among the blue normal cells. For the controls and the T-supplementation group, one or two apoptotic cells were occasionally observed. This is illustrated in Figure 3B.

Figure 2.  Apoptotic nuclei in erectile tissue of Simple Castrates observed under fluorescence microscopy (ISEL10 000).
Figure 3.  Apoptotic cells in erectile tissue of Simple Castrates (A) and Controls (B) under light microscopy (ISEL400).

3.3 Quantitative analysis of apoptosis

Apoptotic rate in the controls, the simple castrates and the T-supplementation group was 0.14%, 4.19% and 0.20%, respectively. Significant difference was found between the 3 groups by means of analysis of variance (P<0.05). When compared with each other, there was significant difference between the simple castrates and other 2 groups (P<0.01), while no statistical difference between the controls and the T-supplementation groups (P>0.05). Results suggest that apoptosis occurs in the corpus cavernosum when androgen is lacking; it can be prevented by T supplementation.

4 Discussion

Apoptosis, or programmed cell death, is a fundamental biological process that regulates tissue size and form. It is involved in the development and progress of many diseases^[7]. Ellisand Grayhack^[8] reported that 60% of potent men undergoing medical or surgical castration for metastatic prostate cancer became impotent, suggesting that factors other than androgen play important role in erectile function. The present study demonstrated the induction of apoptosis in erectile tissue by castration. Apoptosis is initiated by an increase in intracellular calcium and activation of endogenous endonucleases. Subsequent to it, fragmentation of nucleic DNA happens in the internucleosomal region in multiples of 180-200 base pairs and a ladder pattern appears after agarose gel electrophoresis. This is the most striking evidence for judging whether apoptosis occurs or not. In our experiments, DNA fragment analysis was carried out in all the 3 groups and only the simple castrates demonstrated a ladder pattern apoptosis.

The observation of morphological changes is another important approach to assay apoptosis. Under fluorescence microscopy, the characteristic condensed, margined and fragmented nuclei of apoptotic cells could only be found in the simple castrates.

ISEL technique was used for the quantitative analysis of apoptosis. The measure uses terminal deoxynucleotidyl transferase (TdT) to catalyze the attachment of fluorescein-dUTP to free 3'OH ends in genomic DNA. Then the positive cells and normal cells were counted and the mean apoptotic rate was calculated. In our experiment, only a small proportion of cells underwent apoptosis in the erectile tissue after one-week androgen depletion, which was out of our expectation. This may be due to the supposition that erectile tissue is so sensitive to androgen that a slight reduction of the blood hormone level will induce considerable amount of apoptotic cells which are readily degraded by phagocytes. Although androgen level decreased to the minimum one week after castration^[9], the peak of maximum apoptosis had passed. As a result, the apoptotic rate of the castrates is only 4.19%, but it is still much higher than that of the controls and the T-supplementation group.

This study raises some interesting questions. First, what is the mechanism responsible for apoptosis after androgen withdrawal? Mills et al^[10] indicated that in the rat the erectile response is regulated principally by the vascular smooth muscle, which controls the blood flow into the cavernosal sinuses. The smooth muscle contracts in response to an -agonist and relaxes in response to a drug which acts via NO. Furthermore, the relaxation of the smooth muscle may be under partial androgenic control. Thus, one possible answer to the above question is a change in penile blood flow after castration. Cavernosal ischemia might lead to cell death and subsequent erectile dysfunction. Another possible mechanism could be an increase in  the intracellular calcium, similar to an influx of calcium in the prostate after castration^[11,12]. A high Ca²⁺_in can activate endogenous endonuclease resulting in DNA degradation and subsequent apoptosis.

Second, which cell types are affected by castration. In situ end labeling identified apoptotic bodies within the erectile tissue, but the assay could not differentiate cell types. Further study on this problem is worthwhile.

In one study, rats were castrated and received T supplementation 2 weeks later. DNA synthesis occurred as early as 48 hours after the replenishment^[13]. We indicated that apoptosis could be prevented by T supplementation at the time of surgery. These studies suggest that administration of testosterone to castrated rats may inhibit the apoptotic cascade.

References

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