Evaluation of germ-cell kinetics in infertile patients with proliferating cell nuclear antigen proliferating index

Li ZENG, Xiang-Tian KONG, Jin-Wei SU, Tong-Li XIA, Yan-Qun NA, Ying-Lu GUO

Institute of Urology, Peking University, Beijing 100034, China

Asian J Androl  2001 Mar; 3: 63-66

Keywords: male infertility; testis; kinetics; proliferating cell nuclear antigen

Abstract

Aim: To explore the usefulness of proliferating cell nuclear antigen proliferating index (PCNA PI) in the pathological diagnosis and treatment of male infertility. Methods: Testicular biopsy specimen obtained from 48 cases of male infertility and 2 normal controls were fixed and embedded. The sections were stained with anti-PCNA monoclonal antibodies or haematoxylin/eosin. Proliferating index (PI), expressed as the percentage of germ-cell nuclei positively stained with PCNA antibody, was assessed from more than 20 seminiferous tubules or 600 germ-cells. Results: The infertile patients  were divided into 4 groups: Group 1, normal spermatogenesis (14 cases); Group 2, hypospermatogenesis (16 cases); Group 3, germinal arrest (10 cases); Group 4, Sertoli cell only syndrome (8 cases). The PCNA PI of normal control testis was 86.5% (mean value). Group 3 had a significantly lower PCNA PI (29.8%) than normal testis; Group 1 and 2 had similar PIs (82.3% and 82.3%, respectively) as the control testis. PI of the negative control (Group 4) was 0 as no germ-cells were found. Conclusion: PCNA PI is useful for assessing germ-cell kinetics, especially for pathological diagnosis of germinal arrest which is difficult to differentiate by routine HE staining technique.  In germinal arrest, there is a significantly lowered PCNA PI, which is an indication of DNA synthesis deterioration, suggesting the use of therapies be different from those for hypospermatogenesis.

1 Introduction

2 Materials and methods

Testis biopsy specimen was obtained from 48 infertile and 2 normal men, aged 28.34.3 (20-35) years. Cases with chronic testitis or testicle tumors as well as other diseases were excluded. The testicular specimens were fixed in 10% neutral buffered formalin and embedded in paraffin.  Five m sections were cut and stained with either ordinary HE or immunohistochemical staining technique. For immunostaining, the sections were processed to block endogenous peroxidase activity and incubated with mouse anti-PCNA monoclonal antibody PC10 (Santa Cruz biotechnoligy, Inc, USA), diluted 1:100, followed by ordinary steps of SP staining.  The structure of seminiferous tubules, and the morphology, the number, the distribution and the proportion of different stage germ cells were observed on the HE stained sections. The mature sperm were also observed and counted.  In immunostained sections, the brown-stained nuclei of positively stained germ cells were investigated and counted. At least 20 transects of seminiferous tubules or 600 germ cells, including spermatogonia, primary spermatocytes, secondary spermatocytes, and spermatids were observed. Then the germ cell proliferating index, i.e., the percentage of positively stained germ cells, was calculated.

3 Results

The control sections with HE staining showed a normal testis structure with normal percentages of germ cells in different stages, and normal Sertoli and stromal cells in and around seminiferous tubules. The spermatogenisis of the control sections was also normal.  Normal control sections with PCNA immunohistochemical stain showed positively stained cells including spermatogones, spermatocytes and spermatids in the testis (Figure 1). No staining was found in sperm, stromal cells and most Sertoli cells.  Infertile cases with HE staining show different pathological changes. With the two staining techniques, the infertile cases can be divided into 4 groups (Table 1): (1) Normal group, 14 cases, accounted for about 29.2%. The average PI of this group was 82.4%.  In most cases, different stages of germ cells showed normal morphology and spermatogenesis, with approximately normal PI, whereas only 4 cases showed smaller numbers of mature sperm, with a mean PI of 82.2%. This condition might be called occult spermatogenetic stasis, which happened at the last phase of spermatogenesis, i.e., at the stage when sperm were detached from the Sertoli cells to become mature sperm. In these patients, the DNA synthesis and the cell kinetics of germ cells are usually normal. (2) Hypospermatogenesis group, 16 cases, accounted for about 33.3%. The average PI of this group was 82.3%. Most cases of this group showed a proportionate decrease in the numbers of all types of germ cells (Figure 2). The number of primary spermatocytes was either equal to or higher than that of the spermatogonia. The number of round spermatids was higher than that of primary spermatocytes, and the number of elongated spermatids was similar to that of spermatogonia. (3) Partial germinal arrest, 10 cases, accounted for 20.8%. Spermatogenic arrest was rare and most of the cases in our study belonged to spermatocytic arrest. The patients presented oligozoospermatism or azoospermatism. The average PI of this group was 29.8%. (4) Negative control (complete germinal arrest or Sertoli cell only syndrom), 8 cases, accounted for 16.7%. In this group, no mature sperm could be found in the biopsy specimen (Figure 3). The average PI of this group was 0. The seminiferous tubules were decreased in number and showed a slightly thickened tunica propria.  

Table 1. Classification of infertile cases by testis biopsy.

Figure 1. Immunohistochemical stain of PCNA in normal spermatogenic testis. Positively stained germ cells. (200)
Figure 2. Immunohistochemical stain of PCNA in hypospermatogenesis group. Positively stained germ cells were proportionally decreased. (200)
Figure 3. Immunohistochemical stain of PCNA in Sertoli cell only syndrom. Neither germ cell nor mature sperm were found. (200)

4 Discussion

In the 1940s, physicians started to perform testicular biopsy in the diagnosis of infertility^[8,9]. Later, under the impact of the development of morphometry, many semi-quantitative^[1] and quantitative^[10] studies were carried out with the biopsy specimen.  The greatest achievements of these studies were enhancement of the reversibility of lesions. The scoring of Johnsen^[2], the estimation of germ cell/Sertoli cell ratio for each germ cell type^[11], and the calculation of germ cell number per unit length of seminiferous tubules^[12] are reliable and useful methods. However, with any of these methods, the spermatogenic function cannot be evaluated. To the latter, more and more interest has been attached with the recent advancement in assisted reproduction technology.Germ cell kinetics has been assessed using estimation of DNA synthesis^[6,7]. There are 4 methods for investigating the cell kinetics and DNA synthesis: 1) tritiated thymidine in corporation followed by autoradiograpphy, 2) bromodeoxyuridine incorporation followed by anti-BrdU immunocytochemistry, 3) flow cytometric analysis, and 4) immunocytochemistry, using cell cycle specific monoclonal antibodies. Radiolabeling with tritiated thymidine has numerous drawbacks, including the requirement of fresh and viable tissue for thymidine incubation, problems with uniform uptake and potential alterations of cell kinetics in the tumor samples incubated in vitro, and the expense and dangers of handling radioactive substances. The use of BrdU and anti-BrdU antibodies eliminates some of these disadvantages, but still requires the use of fresh and viable tissue. Flow cytometrically determined kinetic analysis may use paraffin-embedded archival material, but its requirement for digested tissue sample will result in stripped nuclei with a significant loss of cell identification data.  Immunohistochemical methods can be done on a 5 m slide, and the same block can be kept for other uses. With the immunohistochemical method, the tissue architecture and cell-to-cell relationship can be preserved, and if necessary, it is also possible to perform double-staining with other cell-specific monoclonal antibodies to identify the proliferating cell on a single tissue section. Furthermore, through the use of image analysis system, more precise quantitative data can be obtained from anti-PCNA immunostained preparations. The study showed that DNA synthesis assessed by PCNA PI is extremely useful in the pathological diagnosis of infertility, especially for the differentiation of hypospermatogenesis from partial germinal arrest. 

References

[1] Meyer JM, Roos M, Rumpler Y. Statistical study of a semi-quantitative evaluation of testicular biopsies. Arch Androl 1988; 20: 71-4.
[2] Johnsen SG. Testicular biopsy score counta method for registration of spermatogenesis in human testis: normal values and results in 335 hypogonadal males.Hormones 1970; 1: 2-25.
[3] Fujisawa M, Yoshida S, Matsumoto O, Kamidono S. Deocyribonucleic acid polymerase activity in the testes of infertile men with varicocele. Fertil Steril 1988; 50: 795-800.
[4] Fujisawa M, Yoshida S, Matsumonto O, Komidono S. Topoisomerase I activity in the testes of infertile men with varicocele. Arch Androl 1988; 21: 45-50.
[5] Fujisawa M, Yoshida S, Matsumoto O, Kamidono S. Biochemical changes in varicocele testes. Arch Androl 1988; 22: 149-59.
[6] Celis JE, Celis A. Cell cycle dependent variations in the distribution of the nuclear protein cyclin proliferating cell nuclear antigen in cultured cells: Subdivision of S phase. Proc Natl Acad Sci USA 1985; 82: 3262-6.
[7] Jaskulski D, DeRiel JK, Mercer WE, Calabretta B, Baserga R. Inhibition of cellular proliferation by antisense oligodeoxy-nucelotides to PCNA/cyclin. Science 1988; 240: 1544-6. 
[8] Charny CW. Testicular biopsy, its value in male sterility. JAMA 1940; 115: 1429-32.
[9] Charny CW, Meranze DR. Testicular biopsy: Further studies in male infertility. Surg Gynecol Obstet 1942; 74: 836-42. 
[10] Zuckerman Z, Rodriguez-Rigau LJ, Weiss DB, Chowdhury AK, Smith KD, Steinberger E. Quantitative analysis of the seminiferous epithelium in human testicular biopsies, and the relation of spermatogenesis to sperm density. Fertil Steril 1978; 30: 448-55.
[11] Rowley MJ, Heller CG. Quantitation of the cells of seminiferous epithelium of human testis employing Sertoli cells as a constant. Z Zellforsch 1971; 115: 461-72.
[12] Steinberger E, Tjioe DY. A method for quantitative analysis of human seminiferous epithelium. Fertil Steril 1968; 19: 960-70.

home

Correspondence to: Dr. Li Zeng, Institute of Urology, Peking University, 1 Da Hong Lou Chang Street, Beijing 100034, China. 
Tel: +86-10-6617 1122 ext. 2579    Fax: +86-10-6617 6450 
e-mail: zengli@263.net
Received 2000-05-31                     Accepted 2001-02-28