Studies on the integration of hepatitis B virus DNA sequence in human sperm chromosomes

Jian-Min HUANG^1,*, Tian-Hua HUANG¹, Huan-Ying QIU², Xiao-Wu FANG¹, Tian-Gang ZHUANG¹, Jie-Wen QIU³

Asian J Androl 2002 Sep; 4: 209-212 

Keywords: hepatitis B virus; spermatozoa; human chromosomes; fluorescence in situ hybridization; virus integration; vertical disease transmission

Abstract

Aim: To study the integration of hepatitis B virus (HBV) DNA into sperm chromosomes in hepatitis B patients and the features of its integration. Methods: Sperm chromosomes of 14 subjects (5 healthy controls and 9 HB patients, including 1 acute hepatitis B, 2 chronic active hepatitis B, 4 chronic persistent hepatitis B, 2 HBsAg chronic carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free hamster oocytes and human spermatozoa. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes. Results: Specific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis B. In 9 (9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots and the others 2 to 4 signals. The fluorescence intensity showed significant difference among the signal spots. The distribution of signal sites among chromosomes seems to be random. Conclusion: HBV could integrate into human sperm chromosomes. Results suggest that the possibility of vertical transmission of HBV via the germ line to the next generation is present.

Hepatitis B virus (HBV) infection is a serious public health problem worldwide, especially in the Far-East Asia. In HBV infection, HBV can be found in the saliva, vaginal secretion, semen and tissues other than the liver and blood [1-3]. It has been documented that HBV DNA could integrate not only into the host hepatocytes but also the spermatozoa [4-7]. However, the characteristic features of HBV DNA integration into sperm chromosomes are poorly understood. The present work was designed to study the integration of HBV DNA into sperm chromosomes in hepatitis B patients and the features of the integration. The genetic significance of the results is discussed.

2 Materials and methods

2.1 Subjects

Nine men with HBV infection, including 1 acute hepatitis, 6 chronic hepatitis (2 chronic active, 4 chronic persistent) and 2 HBsAg chronic carriers with no clinical symptoms were recruited for the study. Five healthy men served as the controls. All the subjects were 22 to 38 (mean 27) years of age. The status of the markers of HBV infection of these subjects is listed in Table 1.

Table 1. Markers of HBV infection and results of FISH analysis. *Hepatitis patients: No.1: acute hepatitis; No.2, 3: chronic active hepatitis; No.4-7: chronic persistent hepatitis; No.8, 9: chronic HBsAg carriers.

The technique of interspecific in vitro fertilization of zona-free hamster oocytes was used to prepare the sperm chromosomes. For the procedures of the techni-que, including the treatment of semen samples, supero-vulation, egg processing, insemination, postinsemination culture and preparation of chromosome slides, please refer to previous literature [8,9]. Slides were placed at 70 for fluorescence in situ hybridization (FISH).

The recombinant plasmid, pHBV-1, containing the whole length 3.2kb HBV genomic DNA, was taken to be amplified, extracted and purified according to routine method. The whole length 3.2kb HBV DNA probe with its vector (altogether 6.2kb) was labeled with biotin-14-dATP by nick translation (GIBCOBRL No. 18247-015). The unincorporated nucleotides were separated by the cold ethanol precipitation method.

2.3.2 In situ hybridization

Sperm chromosome slides were treated with RNase (Sigma) 100 mg/mL for 60 min at 37, pepsin (Sigma) 50 mg/mL in 0.01 N HCl for 10 min at 37, and 1 % polyformaldehyde in PBS for 10 min at room temperature in succession. Chromosomes were denatured at 75 for 4 min in 70 % formamide in 2XSSC. In situ hybridization with the denatured DNA probe was performed with a modification of the procedure described in our previous paper [10]. Briefly, 10 mL hybridization buffer (50 % deionized formamide, 10 % dextran sulfate and 2XSSC) containing 40 ng/mL biotin-labeled HBV DNA probe and 500 ng/mL sheared salmon sperm DNA was placed on the slide. A coverslip (1818 mm) was applied and sealed with rubber cement. The slides were then incubated overnight in a humidified chamber at 37.

Post-hybridization washes were performed accod-ing to Korenberg and Chen [11], first in 40 % forma-mide 2XSSC for 10 min at 40, then twice in 2XSSC for 5 min each at room temperature. Hybridization signals were detected with FITC-conjugated avidin and amplified with goat biotinylated anti-avidin antibody, followed by another layer of FITC-avidin (both avidin and anti-avidin, Vector Laboratories, 5 mg/mL in 4XSSC with 0.2 % Tween 20). To increase the intensity of the hybridization signal, a second round of amplification was applied by the sandwich method as above. In order to reduce the nonspecific binding, slides were preincubated in 4XSSC with 15 % nonfat dry milk for 10 min at 37 prior to each step. Chromosomes were counterstained with propidium iodide (PI, Sigma) and 4,6-diamidino-2-phenylindole (DAPI, Sigma), 2 mg/mL each in PBS/glycerol (1:9,v/v) containing 0.2 % (1,4)-diazobicyclo-(2,2,2)octane (DABCO, Sigma) as an anti-fade agent. Photo-graghs were taken under a fluorescence microscope (BH-2, Olympus) with the G excitation filter, EY-455 help excitation filter and Y-475 barrier filter, using Fuji ASA 400 color film.

The slides were viewed with a fluorescence microscope . About 50 sperm metaphases per subject and more than 200 hamster oocyte metaphases were observed. Photograghs were taken as above.

3 Results

In situ hybridization studies indicated that the sperm metaphase of one chronic persistent hepatitis patient (subject 6) presented positive signals; spermatozoa of all other subjects and all hamster oocytes did not have FISH signal (Table 1). In subject 6, of the 42 chromosome complements observed, 9 showed clearly twin yellow spots on some chromosomes. In 9 sperm metaphase spreads with positive signals, one had 5 signals distributed at different chromosomes and the rest had 2 to 4 spots at various chromosomes. The intensity of the signals presented distinct difference among the spots. In different spermatozoa, the positive signals detected did not show identical chromosome distribution. (Figure 1).

Figure 1. Detection of HBV DNA sequences in sperm chromosomes by FISH with biotinylated whole length HBV DNA probe: (A) a well separated chromosome plate of subject 6 (chronic persistent hepatitis) with three fluorescent signals at different chromosomes, (B) a poorly separated chromosome plate of the same patient with five fluorescent signals.

In our study the HBV DNA sequence integration into sperm chromosomes was directly visualized, suggesting that HBV infection could be inherited through germ cells carrying altered genetic constituent resulting from the insertion of virus DNA. This is the first attempt so far available in the literature to clarify the characteristics of HBV-sperm integration and the influence of the integration on human sperm chromosomes.

FISH onto sperm chromosomes by specific HBV DNA as probe showed positive signals in a HBV patient. The results suggested that HBV could penetrate blood-testes barrier, enter male germ cells and integrate into their genome. So the possibility of vertical transmission of HBV via the germ cells was further demonstrated [6,7]. Our preliminary observations showed that HBV integration into sperm chromosomes presented the feature of multi-site integration and that there seems to be no predilection site for the integration. These results are similar with previous reports about HBV-related hepatocellular carcinoma either in HCC-derived cell lines or in liver tumor samples [4,5]. It is possible that relocation of viral sequences and multi-site integration could occur via genetic recombination during HBV insertion to male reproductive cells, but the genetic effect of initial HBV insertion into various spermatogenic stages must vary greatly. If the insertion occurs at the stem cells, their daughter cells will all carry the integrated form of viral DNA and the effects will be long continued.

We were surprised at the high intensity of the FISH signals in the patients upon using single small DNA probe.The reasonable explanations for this result include: (1) the HBV strand invasion may be multiple copied, (2) the HBV sequence generates duplications in situ after invasion into the host genome, (3) in the processes of sperm production, unequal cross-over repeatedly occurs at the HBV insertion site or (4) HBV insertion into the DNA of stem cells leads to DNA rearrange-ment.

Through preliminary sperm chromosome aberration analysis, it was shown that the subject with HBV integration had a relatively high incidence of aberration, including gap, acentric fragment, deletion, triradial, ring chromosome, dicentric chromosome, pulverization and numerical anomaly. This correlation implied that viral DNA integration to germ cell significantly increased the instability of genome, that frequently occur in host hepatocytes or hepatocellular carcinoma cell lines with HBV DNA integration [4,5]. HBV integration into spermatogenic cells may cause inheritable effects, producing infected fetuses, modifying genetic constituent and inducing mutations [12].

It is concluded that HBV may be integrated into the human sperm chromosomes and the possibility of vertical transmission of HBV via the germ line to the next generation is present.

We thank Professor Qin Da-Nian, Department of Physiology, Shantou University Medical College for reviewing the manuscript.

The Project was supported by grants from the Natural Science Foundation of Guangdong Province, China (Grant No. 940567) and the National Natural Science Foundation of China (Grant No. 39970374).

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Correspondence to: Dr. Tian-Hua Huang, Department of Cell Biology and Medical Genetics, Shantou University Medical College, Shantou 515031, China.
Tel: +86-754-890 0497 Fax. +86-754-855 7562
E-mail: thhuang@stu.edu.cn
* Now in: Allergy and Inflammation Institute, Shantou University Medical College, Shantou 515031, China.
Tel: +86-754-890 0380 Fax. +86-754-890 0192
E-mail: jmhuang@stu.edu.cn
Received 2002-05-10      Accepted 2002-06-28