Effect of bilateral testicular resection on thymocyte and its microenvironment in aged mice

Xi-Yun WEI, Jin-Kun ZHANG, Jun LI, Su-Biao CHEN

Department of Anatomy, Histology and Embryology, Shantou University Medical College, Shantou  515031, China

Asian J Androl  2001 Dec; 3: 271-275

Keywords:   testis; orchiectomy; thymocyte; microenvironment; mice

Abstract

Aim:  To observe the changes in thymocyte and its microenvironment in aged mice after bilateral testicular resection. Methods: In male old mice, at the 25th day after testicular resection, the peripheral blood and thymus were collected. Blood and thymus suspension smears were prepared for quantitative histochemistry and immunohistochemistry study under light and electron microscopes. Results: In testes resected mice the size and the weight of thymus were markedly increased. The demarcation between cortex and medulla was clear. The cortex was thickened and the cell density was increased. The ratio of cortex/medulla stereometry was increased. The total cell count, thymocyte count, the percentage of acid -naphthyl acetate esterase (ANAE) positive thymocytes, nonlymphocytes and the rosette formation of macrophages and thymocytes were all increased. The thymocytes surrounded closely to the light thymic epithelial cells, dendritic cells or macrophages. The lymphocytes, particularly the ANAE positive lymphocytes of peripheral blood were increased. Conclusion: After bilateral testicular resection, the thymus of aged male mice showed morphological regeneration and the thymocytes and its microenvironment appeared to be definitely improved. It is suggested that testicular resection may improve immune function.

1 Introduction

In prostatic cancer, bilateral testicular resection may prolong the 5-year survival rate to 90.5%^[1] and additional active immunotherapy with dendritic cell induced cytotoxic lymphocyte (CTL) has resulted in better therapeutic outcome^[2,3]. Previous research showed that normal thymus degeneration was delayed after gonad resection in young mice. In this report, the changes in thymocyte and its microenvironment induced by bilateral testicular resection were studied in aged mice under light and electron microscopes.

2 Materials and methods

2.1 Animals and treatment

Forty ICR mice, 15 months old, were randomly divided into the control and experimental groups of 20 animals each. The experimental mice were subjected to bilateral testicular resection under ether anesthesia. At the 25th day after operation, the peripheral blood, the thymus and the spleen were obtained immediately after sacrificing the animals. The thymus was divided to three pieces after weighing.

2.2 Specimen preparation  

2.2.1 Smears for histochemsitry and immunohistochemistry staining

One piece of the thymus was used for the routine preparation of cell suspension, which was fixed in polyaldehyde-glutaraldehyde after cell counting. It was then centrifuged and the sediment cells were used for the preparation of smears. Histochemistry staining showing acid phosphatase (AcP, Gomori modification), alkaline phosphatase (AkP, Gomori modification), adenosine triphosphatase (ATP, Glick method), ANAE (Mueller modification) and periodic acid Schiff (PAS) reaction was performed. Immunohistochemistry staining was also done to detect S-100 protein according to the avidin-biotin-peroxidase complex (ABC) method. Smears were incubated with the primary antibody (1:100, 12 h, rabbit anti-cow S-100 protein, DAKO product), followed by biotin-labeled secondary antibody (1:200, sheep anti-rabbit IgG, ABC kit, DAKO product). After washing, ABC (1:100, ABC kit) was applied for 30 min. Brown staining was done by 5 min treatment of diaminobenzidine (DAB). Counterstaining was performed with hematoxylin. In the negative controls, the primary antiserum was omitted. In addition, the peripheral white blood cell was also counted. The blood smear was stained with ANAE.  

2.2.2 Specimens for light microscopy

One piece of the thymus was prepared according to the routine histological method (paraffin section and hematoxylin and eosin HE] staining) for light microscopy.

2.2.3 Specimens of electron microscopy

The third piece of the thymus was prefixed in 2.5% glutaraldehyde and postfixed in 1% osimate for electron microscopy. The tissues were infiltrated and embedded in Epon 618. Ultra-thin sections were prepared with LKB-8800-3 microtome, stained with uranyl acetate and lead citrate and were observed under Hitachi H-600 transmission electron microscope.

2.3 Quantitative detection

2.3.1 Morphometry of cortex/medulla

HE sections of the biggest area in the series sections were selected for detecting metrical point P and PC of cortex and medulla under square metrical board. The Delless method was used to calculate the area ratio for the cortex and medulla (An=P/PC).

2.3.2 Quantitative detection of thymocytes in cortex and medulla

The cortex and medulla thymocytes were counted using a micrometer.

2.3.3 Quantitative detection of nonlymphocyte and rosette

Under the light microscopy, a total 2000 cells, 200 ANAE staining thymocytes and 200 macrophages were counted and the percentages the thymic nonlymphocytes, ANAE positive thymocytes, and the rosette formations between the ANAE positive thymocytes and the macrophages were calculated. A big rosette is a rosette when macrophages bind with over five thymocytes, a small rosette; when bind with 3-5 thymocytes, a negative rosette; when bind with 1-2 thymocytes. In addition, the percentage of ANAE positive lymphocytes was calculated by counting 200 lymphocytes.

2.3.4 Data processing

The significance of difference was analyzed with the t-test and P<0.05 was set as significant.

3 Results  

3.1 General morphology of thymus

Compared with the control group, the size and weight of thymus in the experimental animals were significantly increased and the total cell count was also significantly increased (Table 1). Under the light microscope, there was connective tissue hyperplasia in the thymus of the control group and some were replaced with  adipose tissue (Figure 1); the cortex became thinner and the demarcation between the cortex and the medulla was unclear. In the experimental group the cortex was thicker and the demarcation between the cortex and the medulla was clear (Figure 2). The ratio of cortex/medulla morphometry increased significantly (Table 1).

Figure 1.    The control group, thymus parenchyma partly replaced by fatty tissue (HE staining, 400)
Figure 2.    The experimental group, thymus cortex thickened, clear demarcation between cortex and medulla (HE staining, 400)

3.2 Thymocytes

In the experimental group, the density of thymocytes and the percentage of ANAE ositive thymocytes were increased significantly, which was more clearly observed in the cortex than in the medulla (Table 1). Under the electron microscope, in the control group there were clusters of degenerating thymocytes with little cytoplasm and swollen nuclei; the nucleoli were distinct, which looked like nude nuclei. The experimental group did not show these signs (Figures 3,4).

Figure 3.    The control group, degenerate thymocytes look like nude nuclei (7000)
Figure 4.    The experimental group, thymocytes normal (10000)

3.3 Thymic nonlymphocytes

The percentage of nonlymphocytes in the experimental group was significantly higher than that of control group (Table 1).

Table 1.   Thymic changes in aged mice after testicular resection.

3.3.1 Epithelial cells

Under the electron microscope, the epithelial cells in the experimental group were similar in morphology to the control group, but with a higher cell density. There were also more cell bodies and processes of bright epithelial cells, that were binding with the thymocytes, forming rosettes (Figure 5).

Figure 5.    The experimental group, rosette formation between epithelial cell and thymocytes (5000)

3.3.2 Macrophages

With histochemistry staining, the macrophages showed similar results with regard to AkP(-), S-100 protein(-), AcP(+), PAS(+), ATP(+) and ANAE(+) both in the control and experimental groups. Only ANAE positive strength was distinctly higher in the experimental than in the control group. Under light and electron microscopes, the macrophage-thymocyte rosette formation rate was higher in the experimental than in the control groups (Table 1, Figure 6).

Figure 6.    The experimental group, rosette formation between macrophage and thymocytes (7000)

3.3.3 Proplasmacytes and plasmacytes

In the control group under the electron microscope, the proplasmacytes and plasmacytes in the medulla were fewer in number and solitarily distributed, while in the experimental animals, they were far more in number and were distributed in clusters with swollen rough endoplasmic reticulum (RER) and floccules deposited in the RER antrum; Some mature plasmacytes can also be seen.

3.3.4 Dendritic cells

S-100 protein staining showed that the dendritic cells showed brown particles and irregular shape dendrites. Dendritic cells were more popular in the experimental than in the control group. Dendritic cell-thymoctyes rosettes can also be seen.

3.3.5 Other findings

The body weight, the spleen weight and the total peripheral white cell count had no significant difference between the experimental and the control group. The percentage of peripheral blood ANAE(+) lymphocytes was higher in the experimental  (65.45%6.17%) than in control group (34.13%7.93%).

4 Discussion

The results of the present paper demonstrate that the degenerating thymus of the aged mice may be regenerated after bilateral testicular resection. Similar result has been shown in rats^[4].

Thymus is the site for T cell development. The microenvironment of thymocytes is composed of epithelial cells, macrophages, dendritic cells, etc. The epithelial cells stimulate T cell maturation through the secretion of thymosin and thymopoietin. Recently, new evidences showed that the bright epithelial cells could produce neuropeptides, such as oxytocin, neurophysin and vasopressin to the microenvironment. Macrophages secret IL-1 to moderate thymoctye proliferation and differentiation.  Dendritic cells negatively select the thymocytes by expression of the major histocompatibility complex -(MHC)-I and MHC-II. Only thymocytes adapted to self MHC molecule can survive (2%) and continue to mature; thymic epithelial cells induce T cell clone deletion by apoptosis^[5]. Macrophage-thymocyte rosette and dendritic cell-thymocyte rosette formation may be important evidence indicating that macrophages and dendritic cells play an important role in thymocyte differentiation, functional maturation, processing of dead thymocytes and negative election^[6]. In the present paper, an increase in the epithelial cells, light epithelial cells, macrophages, dendritic cells, rosette formation, thymocytes, peripheral lymphocytes and ANAE (T cell marker) positive cells all points to an improvement of thymus function and microenvironment.

In the experimental group, proplasmacytes and plasmacytes are found in the medulla of thymus, suggesting that both humoral and cellular immune reaction may take place in thymus.

Testicular resection decreased the level of angrogens and inhibited the metastasis of prostatic cancer^[7]. Specific active immune therapy has obtained rapid progress in recent years^[8,9]. As far as prostate carcinoma is concerned, three prostate-associated antigens, namely prostate-specific antigen (PSA), prostatic-acid phosphatase (PAP), and prostate-specific membrane antigen (PSMA), are widely used in clinical practice. These antigens, binding human leukocyte antigen (HLA)-A2 may activate specific T lymphocytes. Prostate carcinoma vaccine, i.e., dendritic cell pulsed with PSMA and its associated peptide, has recently been developed and beneficial effect has been obtained in the treatment of stage I and II prostatic cancer patients^[10,11]. Our experiment confirmed that testicular resection reverses thymus degeneration and improves the immune function, which may be of help as an adjuvant in the immune therapy for cancer.

References

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Correspondence to:  Prof. Xi-Yun WEI, Department of Anatomy, Histology and Embryology, Shantou University Medical College, Shantou 515031, China
Tel:+86-754-853 7504, Fax:+86-754-855 7562 
E-mail: junkunzh@163.net
Received 2001-07-24    Accepted 2001-12-03