:: Asian Journal of Andrology ::

Germ cell apoptosis induced by Ureaplasma urealyticum infection

Chen XU, Mei-Ge LU, Jing-Sheng FENG, Qiang-Su Guo, Yi-Fei WANG

Department of Histology & Embryology, Shanghai Second Medical University, Shanghai 200025, China

Asian J Androl 2001 Sep; 3: 199-204

Keywords: germ cells; apoptosis; Ureaplasma urealyticum; Fas/FasL; DNA electrophoresis

Abstract

Aim: To study the effect of Ureaplasma urealyticum (UU) infection on germ cell apoptosis of male rats. Methods: Male rats were infected artificially with UU serotype 8 (T₉₆₀). Morphological changes of germ cells in the seminiferous tubules and the lumen of the epididymides were observed under the light microscope. Fluorescence-conjugated polyclonal antibodies to Fas and Fas ligand (FasL) were used to localize Fas and FasL. TUNEL staining of germ cells and Sertoli cells was performed by the AKPase method. TUNEL-positive rate (% positive cells) and TUNEL -positive area (area occupied by stained cells) were analysed by KS400 Image Analysis System. The DNA laddering analysis was performed by agarose gels electrophoresis. Results: In those rats infected with UU: (1) Exfoliated germ cells were dramatically increased. Many multinucleated giant cells were found in the seminiferous tubules and the lumen of the epididymides. (2) The number of TUNEL-positive cells and the TUNEL-positive area were significantly increased. (3) The expression of Fas and FasL in germ cells and Sertoli cells was up-regulated. (4) Discrete bands of fragmented DNA were found in the testicular cells. Conclusion: In male rats, germ cell apoptosis was increased in UU infection.

1 Introduction

In the mammalian testis, overproliferation of early germ cells is tempered by selective apoptosis of their progeny^[1]. In addition, massive testicular germ cell loss is known to result from toxicant exposure^[2], depletion of growth factors^[3], changes of hormonal support^[4], heat stress^[5], radiation^[6], or treatment with chemotherapeutic compounds^[6]. In many of these situations, germ cells are known to undergo apoptosis. Ureaplasma urealyticum (UU) infection has been implicated in oligozoospermia^[^7] and male infertility[8]. However, the mechanisms of oligozoospermia and infertility caused by UU have not been elucidated. It has been demonstrated that UU can induce exfoliation of germ cells and formation of multinucleated giant cells in the seminiferous tubules^[8]. Fas (APO-1,CD) is a transmembrane receptor protein that belongs to the tumor necrosis factor/nerve growth factor receptor family. It contains a death domain and is capable of initiating apoptosis when stimulated by receptor cross-linking or binding to its ligand, FasL (CD 95L)^[9]. In the testis, the Fas system has been implicated in maintaining immune privilege. FasL-expressing Sertoli cells eliminate Fas-positive activated cells, providing general protection against graft-rejection in the testicular environment^[10]. In addition, it is reported that the Fas system is a key regulator of germ cell apoptosis in the testis^[11]. The present study was designed to investigate whether UU can induce germ cell apoptosis in male rats.

2 Materials and methods

2.1 Animal models

UU-free male Sprague-Dawley rats, weighing 200-250 g were used. UU serotype 8 (T₉₆₀) was kindly granted by Professor Janet A. Robertson. The method of inoculation was as follows. The inoculum used was a 16 h culture of the second passage which contained 10⁵ CCU/mL of UU. Following 2 % pentobarbital sodium (0.2 mL/100 g BW) anaesthesia, laparotomy was performed and the infected group was injected with 0.05 mL UU broth into the testes or epididymides (granulation formation was not seen). The possibility of anti-sperm antibody formation will be reported in another paper. The control group was injected with 0.05 mL of the vehicle.

2.2 FasL/Fas immunostaining and TUNEL

The animals were sacrificed at 48 and 72 h after initiating exposure to UU. For immunostaining of FasL and Fas, 20 m frozen sections of rat testes were prepared, fixed in acetone for 10 min at -20 , and rinsed in PBS for 20 min. Polyclonal antibodies to FasL and Fas, 0.5 g/mL in PBS (Santa Cruz Biotechnology, USA), were added to the sections and left for 1 h at room temperature. The primary antibody was detected using a FITC-conjugated goat-anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology, USA), 1 g/mL in PBS, for 45 min. The results were observed by the Laser Confocal Scanning Microscope (LSM-510, Zeiss, Germany).

For TUNEL staining, the method for frozen sections described by Lee et al^[12] with minor modification was employed. Briefly, testicular tissue was fixed in 10 % formaldehyde at 4 overnight, and then immersed in 25 % sucrose (w/v in water) at 4 overnight. The tissue was frozen at -80 after sinking to the bottom of the bottle. Ten m frozen sections were prepared and then immersed in 25 % (v/v) glacial acetic acid for 10 min. One g/mL proteinase K treatment was performed at 37 for 30 min, and then fixed in 4 % paraformaldehyde for 5 min. Sections were rinsed in succession with 0.1 mol/L Tris-HCL (pH 8.0), and double distilled water. Labelling solution (from In vitro cell Death Detection Kit AP, Roche, USA) was added to the sections at 37 and left for 60 min. Sections were rinsed with buffer (0.01 mol/L Tris-HCL and 0.02 mol/L EDTA) for 10 min to stop the reactions, and then rinsed with PBS buffer. The sections were treated with 10 % BSA at 37 for 30 min, followed by the addition of fluorescent IgG Fab fragments labelled by AKP. Sections were rinsed sequentially with PBS buffer, TSM1 and TSM2 in succession and were incubated with NBT and BICP at 25 for 60 min. For control sections, the procedure for TUNEL staining was the same, except for the removal of TdT from the labelling solution. Sections were observed under the light microscope.

To quantify the incidence of apoptosis at each time point, the TUNEL-positive rate and the TUNEL-positive area were analysed with the KS400 Image Analysis System (KS400, Zeiss, Germany). Briefly, five areas in each section were observed with the Image Analysis System. The size of each area was 512512. Data were analysed with SAS 6.12 software. Statistical assessment was made by Student's t-test and student-Newman-Keuls test and P<0.05 was set as significant.

2.3 DNA electrophoresis

For analysis of DNA fragmentation by agarose gels electrophoresis, total DNA was isolated from testicular cells. Briefly, testes were decapsulated. Tissue was homogenized in a lysis buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 7.4). The supernatant was digested with proteinase K (300 g/mL) at 54 for 4 h, andthen at 37 overnight. DNA was extracted with phenol and then with phenol:chloroform:isoamyl alcohol (25:24:1 by volume). DNA was precipitated overnight in -20 ethanol and centrifuged for 30 min at 10000 g. The pellet was resuspended in TE buffer. DNA was subsequently digested with DNase-free RNase (50 g/mL) at 37 for 60 min. DNA samples, of 190 g/mL per lane, were electrophoretically separated on 1.8 % agarose gel. Ethidium bromide was added to the electrophoresis buffer at a final concentration of 0.8 g/mL. DNA was visualized under a UV (302nm) transillu minalor and the gels were photographed with Laboratory Image and Analysis System (UVP Inc., USA).

2.4 Histological preparation

Histological exa mination was performed on testicular tissue and epididymal tissue of animal sacrificed 5 weeks after initiating exposure to UU. Testicular and epididymal tissue were fixed in Bouin's solution. All samples were embedded in paraffin, cut into 7 m sections and stained with haematoxylin and eosin. Samples were observed under an Olympus BH-2 microscope (Olympus, Japan).

3 Results

3.1 In situ TUNEL staining

Apoptosis in situ was assessed by the use of cellular TUNEL staining in frozen testicular cross sections. In control animals, there was a low incidence of apoptosis among the germ cells (Figure 1). After 24 h of UU exposure, an increased number of apoptotic cells was seen and plentiful TUNEL-stained cells were observed 48 and 72 h following UU exposure (Figure 2). Apoptosis in situ, determined by TUNEL staining, was quantified by a Laboratory Image Analysis System. It was shown that, in comparison with the control group, the TUNEL-positive rate, TUNEL-intensive positive rate, TUNEL-positive area and TUNEL-intensive positive area were increased dramatically in the UU-infected group (P<0.01)(Table 1). However, a significant difference in the incidence of TUNEL staining was found neither between different time points after UU exposure, nor between different inoculating approaches (e.g., in the testes or in the epididymides).

Figure 1. In situ TUNEL staining of testicular sections from control rat.Incidence of TUNEL-positive germ cells (arrows) was low. TUNEL stain, 600.
Figure 2. In situ TUNEL staining of testicular sections from rats observed 48 (A) and 72 h (B) following UU exposure. Compared with sections from control rat, an increased number of TUNEL-positive germ cells was seen (arrows). TUNEL stain, 600.
Table 1. TUNEL-positive incidence.

UU-infection
group

Control
group

P value

TUNEL-positive area

13304.89

445.47

0.01

TUNEL-intense positive area

3641.36

148.67

0.01

TUNEL-positive rate (%)

4.24

0.14

0.01

TUNEL-intense positive rate (%)

1.72

0.06

0.01

3.2 Fas/FasL immunostaining of testis

Immunohistochemistry of rat testicular cross-sections were performed to localize the FasL and Fas protein. In sections from untreated rats, FasL staining gave a little disperse fluorescence, characteristic of the location of Sertoli cells, whereas Fas staining was limited to germ cells. In sections from UU-infected rats, FasL staining was more intense and diffuse, and more germ cells were Faspositive (Figure 3).

Figure 3. Localization of FasL and Fas protein in rat testis by laser confocal scanning microscopy. A and B, FasL staining (arrowheads). C and D, Fas staining (arrows). Compared with sections from untreated rats (A and C), FasL staining was more intense and diffuse, and more germ cells were Fas positive in UU-infected testes (B and D). Immunofluorescence stain, 330.

3.3 Effects of UU-infection on apoptotic DNA fragmentation

The occurrence of apoptosis was defined by gel-fractionation analysis of DNA fragmentation. In testicular cells from untreated rats, no DNA fragmentation was found, which is consistent with the low incidence of apoptotic cells. In testicular cells from the UU-infected rats, however, discrete bands of fragmented DNA were demonstrated. These were high-molecular-weight DNA fragments in the range 400-800 bp (Figure 4).

Figure 4. Effects of UU-infection on apoptotic DNA fragmentation in rat testicular cells. The DNA samples, 190 g/mL per lane, were loaded on 1.8% agarose gel. Right lane contains molecular weight markers. Left lane represents testicular cells from UU-infected rat. Middle lane represents testicular cells from control rat. High-molecular-weight DNA fragments in the range 400-800 bp were found in UU-infected rat.

3.4 Morphological changes

In the UU-infected group, lots of multinucleated giant cells and groups of germ cells were found sloughed into the seminiferous tubular lumen and the epididymal duct lumen (Figures 5, 6). Many germ cells and multinucleated cells displayed signs of chromatin peripheral condensation (Figures 7, 8).

Figure 5. Testicular section from UU-infected rat stained by HE. Many multinucleated cells sloughing into the seminiferous tubule lumen (arrowheads). HE stain, 1000.
Figure 6. Epididymal section from UU-infected rat. An abundance of exfoliated germ cells were seen in the lumen of epididymis, among germ cells, multinucleated giant cells were seen (arrowheads) . HE stain, 1000.
Figure 7. Multinucleated cells in seminiferous tubule. Chromatin condensation and margination were seen (arrowheads) . HE stain, 1320.
Figure 8. Many germ cells sloughing into the seminiferous tubule. Chromatin condensation and margination were seen (arrows) . HE stain, 1000.

4 Discussion

In this study, we assessed the role of UU-infection in germ cell apoptosis in male rats. We localized FasL to Sertoli cells and Fas to germ cells in rat testis. The similar staining patterns of Fas-positive cells and TUNEL-positive cells suggested that germ cell apoptosis was mediated by Fas expression. After exposure to UU-infection, an upregulation of FasL and Fas was demonstrated, suggesting that the Fas-mediated pathway is involved in signalling germ cell apoptosis. The upregulation of Fas is well correlated with the increased incidence of germ cell apoptosis in all model systems tested, suggesting that Fas activation is a universal check point for germ cell viability in the testis. Apparently, multiple sources of cellular injury in the testis will lead to increased Fas expression and thus increased Fas may serve as a marker for cellular injury. This mechanism would ensure rapid elimination of the injured cells, which may be benefical for the remaining germ cells. On the other hand, if Sertoli cells are injured, their activity to up-regulate FasL is enhanced. As a result, the Fas-positive germ cells are eliminated^[12].

UU is one of the species of Mycoplasma. Induction of apoptosis in host cells by different species of Mycoplasma has been demonstrated by several other laboratories^[13-15]. It was reported that Mycoplasma nucleases were able to induce internucleosomal DNA degradation in cultured cells. Nuclei isolated from MCF-7 cells were used as a substrate to examine the time-dependent DNA degradation catalysed by the partially purified Mycoplasma endonucleases. After 1 h of incubation at 37 in the presence of Mycoplasma nucleases, most of the DNA was degraded into high molecular weight fragments and a small fraction into multiples of 200 bp^[16]. Earlier stages of nuclear DNA degradation have been described. It was shown that after proapoptotic treatments, DNA cleavage begins with the formation of high-molecular-weight DNA fragments in the range of 50-300 kb. These large fragments are normally degraded to nucleosomes and their oligomers. However, in some cases of apoptotic cell death only high-molecular-weight DNA fragments can be observed^[17-19]. Others morphological signs of apoptosis were observed at 5 weeks of inoculation by UU, for instance, sloughing of many multinucleated giant cells and groups of germ cells into the seminiferous tubular lumen and chromatin condensation and margination of the germ cells. When rats were exposed to 2,5-HD, one of the intensively studied environmental toxicant, which caused testicular injury in rats and the germ cells displayed apoptotic signs; the results are in consistent with the morphological findings of the present study. It gives an evidence for UU inducing germ cell apoptosis.

TUNEL staining of apoptotic cells and Fas/FasL staining for germ cells and Sertoli cells provides the earliest morphological signs of UU infection.

Acknowledgments

This study was supported by the National Natural Science Foundation ( 39870374) of China and Dawn Project Foundation of Shanghai ( 99SG42). We wish to thank Prof. Janet A. Robertson for kindly provided UU serotype 8 (T₉₆₀) and Drs. Shi-Xiao QIN and Jin-Mei WANG for their skillful assistance.

References

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Correspondence to: Prof. Chen XU, Department of Histology & Embryology, Shanghai Second Medical University, 280 Chongqing Road, Shanghai 200025, China.
Tel: +86-021-6384 6590-435 Fax: +86-021-5306 5329
E-mail: XCL203@pubilc3.sta.net.cn
Received 2001-07-09 Accepted 2001-08-24