histometric investigations of the effect of cyclosporin a on the testicular tissue of rats

EXPERIMENTAL AND MOLECULAR PATHOLOGY 49, 185-195 (1988)

Histometric Investigations of the Effect of Cyclosporin A on the Testicular Tissue of Rats’

CHRIS CHEN, MICHAEL H. SCOTT, PAUL L. WOLF, A. R. MOOSSA, AND SUN LEE

Departments of Surgery and Pathology School of Medicine at The University of California, San Diego, California 92103; and San Diego Microsurgical Institute, San Diego, California

Received February 17, 1988; and in revised form March 29, 1988

The effect of cyclosporin A (GA) on rat testes was assessed in Lewis rats which were given 10 mgikg of CsA im per day. The rats were divided into 13 subgroups, and 1 subgroup was sacrificed each month. The rat testes were measured and examined for histometrical and morphological changes in comparison to controls. Histometrical analysis included testicular cross-sectional surface area, tubular density, tubular diameter, and the amounts of testicular germinal epithelium, lumen, and interstitial tissue. In the parameters examined, there were no overall differences between CsA-treated animals and controls. CsA does not affect rat testicular tissue. 8 1988 Academic press, Inc.

INTRODUCTION

Cyclosporin A (CsA) is a fungal metabolite first isolated in 1972 in the Sandoz Research Laboratories in Basel, Switzerland. This new drug has been shown to have powerful immunosuppressive properties (1) by its selective action on the T-lymphocytes via inhibiting the membrane-cytoplasmic signal required for the formation of lymphokine mRNA transcripts (2).

CsA has been used extensively throughout the world for immunosuppression in experimental and clinical transplantation of various organs and as well as in some diseases of immunological etiology (1). Especially in the human sector, clinicians have reported successful treatment and rehabilitation of diseases with CsA. Since many patients being treated with CsA are young and in their child-bearing ages, any treatment must take into consideration potential long-term side effects (3). And in the rehabilitation of their patients, clinicians will inevitably need to answer questions regarding fertility and reproduction which may be important determi- nants of the quality of life of patients and their families (4).

Little is known, however, of the effects of CsA on the gonads in humans or in animals. The rat is an excellent model of human testicular histology and morphology (5) which are related to testicular dysfunction (4, 6, 7). The aim of this study is to quantitatively compare the testes and the testicular tissues of CsA-treated versus untreated rats by histometrical analysis.

MATERIALS AND METHODS

Animals

Male Lewis rats were housed individually in wire suspension cages in a con- trolled environment (1ight:dark cycle 12: 12,72”F, at 45% humidity) and fed Purina Rat Chow and tap water ad libitum.

’ Supported in part by NIH Grant 5-ROI-AM3312502.

185 0014-4800/88 $3.00 Copyright 0 1988 by Academic Press, Inc. All tights of reproduction in any form reserved.

186

Experimental Protocol

CHEN ET AL.

Age-matched male Lewis rats were randomly assigned to one of two groups: Group 1 (n = 27), the controls which received no further treatment; and Group 2 (n = 33), the treatment group which received intramuscularly 10 mg/kg/day CsA (iv preparation, Sandoz). CsA levels were evaluated monthly with the Sandoz RIA Kit.

Starting from the third month of life, one to live rats from both groups were sacrificed each month for the next I1 months (thus the rats in Group 2 were treated with CsA for a maximum of 13 months), The rats were further subdivided into four groups corresponding to 3-month intervals, except for the fourth and last group which was comprised of rats of 2 months.

Preparation of Tissue for Microscopy

Following sacrifice of the rat, the testicles were removed and fixed in Bouins solution for 72 hr. Then they were sectioned. Tissue dehydration, clearance, and infiltration were performed automatically using the “autotechnican,” and the samples were then embedded in parafIin. The sections were cut at 4 pm and stained using hematoxylin-eosin and the HOPA technique (hemalum, orange- G-solution, phosphomolybdic acid, and aniline blue) (8). A random coded number was assigned to each slide so that the following histometric and morphologic studies would be blind studies.

Morphometric Analysis

Testicular Cross-sectional area. The diameters of sectioned and fixed testes were measured using a point to point micrometer. From these measurements, the maximum cross-sectional surface areas of the testicles were calculated.

Tubdar density. At a power of 100x magnification (Leitz, Wetzlar micro- scope), the number of tubules per field (low-power field or LPF) were counted in five randomly picked areas of each testicle. Only areas that consisted entirely of cross-sectioned tubules were counted. A mean was then calculated for each rat.

Tubular size. In each testicular section, 20 approximately circular cross- sectioned seminiferous tubules were randomly selected. The tubules were measured using a Nikon graduated reticle at a magnification of 100x. Mean tubular diameters were then calculated.

Histometric Determinations

Testicular composition was measured using an adaptation of the point-counting technique of Henning (8). We used a Nikon 5-mm reticle. The ocular has a net- work of 121 points which were arranged in the area to be examined by microscopy. Using the method described by Schoffling et al. (8), the tissue element under a grid point was described as a “hit. ” The sum of the hits of each tissue component were then related to the total number of hits, thus giving a percentage composition. In each rat testicle, 20 randomly selected fields of tubules at an objective magnification of 100: 1 were counted, thus giving a total of 2420 hits for each section.

In our test, we differentiated between germinal epithelium, lumen, and interstitial tissue. The various developmental stages of spermatogenesis, including Sertoli cells were considered as germinal epithelium. Those tissue components

EFFECT OF CYCLOSPORIN A ON RAT TESTES 187

which were not attached to the germinal epithelium but were scattered throughout the center of a tubule were considered lumen. Interstitial tissue included connective tissue, Leydig cells, and tunica propria, as well as the separation which was occasionally caused by shrinkage of tissue during preliminary treatment.

For each section, an assessment of tubular architecture was made with the prominent cell types being noted.

Statistical Analysis

The mean values in comparative series of animals were compared. All results were expressed as means 2 1 SD. The significance of differences between means was determined using the Mann-Whitney confidence interval and test. Signifi- cance was not rejected at (Y (P) less than or equal to 0.05.

RESULTS

Group l-Control Rats

The 27 rats in this group did not develop any complications throughout the 13 months. Although the mean tubular cross-sectional areas overall decreased with age (Table I), the differences between the subgroups were insignificant. We found small but insignificant decreases in the mean percentages of tubular germinal epithelium and in the mean tubular diameters. There were also small but insignificant increases in the mean percentages of interstitial tissue and in the tubular densities. The mean percentages of lumen did not change significantly with age.

TABLE I Results of Histometric Determination in Untreated and &A-Treated Rat Testicular Tissue

Study parameter

Months of study

Untreated (Mean 2 SD)

Testicular CISA

Mean No. tubules per LPF

Percentage germinal epithelium

Percentage interstitium

Percentage lumen

Mean tubular diameter

3-5 63.14 2 22.59 6-8 60.30 +- 29.20 9-11 35.86 -c 14.17

12-13 43.00 2 13.73 3-5 14.43 + 4.20 6-8 18.43 2 2.30 9-11 21.00 ? 6.61

12-13 21.83 2 3.87 3-5 71.29 -t 6.10 6-8 72.51 t 5.65 9-11 65.14 2 9.41

12-13 61.83 ” 5.04 3-5 15.43 k 7.28 6-8 15.14 “_ 6.54 9-11 22.14 * 12.59

12-13 22.67 +- 4.68 3-5 13.00 2 2.77 6-8 12.57 k 2.15 9-11 12.86 ” 3.24

12-13 15.17 2 2.19 3-5 286 k 33.2 6-8 274 2 19.1 9-11 267 ” 19.6

12-13 269 ” 39.7

CsA-treated (Mean 2 SD)

53.00 k 25.28 65.14 * 24.28 49.20 k 30.40 35.50 ‘- 3.54 23.78 r+_ 10.78 21.86 k 5.98 23.00 + 3.16 20.50 “_ 3.54 69.10 2 2.81 66.86 * 5.73 70.40 t 5.81 70.00 2 1.00 14.50 !I 4.28 12.00 -c 5.54 10.20 k 3.56 10.50 ” 0.71 16.20 ” 3.79 21.00 ? 1.16 19.60 2 3.21 19.00 2 1.41

255 ?I 24.2 258 + 15.8 256 k 18.8 265 2 6.0

(l$

188 CHEN ET AL.

FIG. 1. (A) Histological section of testis of 13-month-old rat which was a control animal not treated with cyclosporin A. The number of seminiferous tubules and the germinal epithelium are normal. The interstitial connective tissue and Leydig cells are normal. H & E Stain, X 150. (B) Section of histologic testis of the same animal depicted in (A). This higher magnification of a seminiferous tubule demon- strates that spermatogenesis is normal with normal progression of formation of normal spermatozoa. The sertoli cells are normal as is the lumen size. A small number of Leydig cells in the interstitium are normal. H & E stain, x600.


secfronal area ,n CsA-

treated rats YS age

Months

FIG. 2. Comparison of mean testicular cross-sectional area in untreated and CsA-treated rats vs age in months.

Morphologically, the tubular germinal tissues appeared normal and unchanged with normal spermatogenic cells throughout the 13 months (Figs. 1A and 1B).

Group 2-CsA-Treated Rats

Twenty-nine percent of the rats in this group died during the 13 months. The majority of the deaths were believed to be due to pulmonary infections.

As compared to the controls, there was also an overall, but insignificant, decrease of the mean testicular cross-sectional areas with age (Table I, Fig. 2). The mean tubular densities in Group 2 did not show any changes with age. Except for the 3- to Smonth subgroups which had a standard deviation of k10.78 in the treatment subgroup, there were no significant differences between the corresponding control and treatment subgroups (Fig. 3). Both the tubular mean percentages of germinal epithelium and interstitial tissue did not have any significant changes with age. Except for the 9- to 11-month and the 12- to 13-month interstitial tissue subgroups, there were no significant differences between the corresponding control and treatment subgroups in both the germinal epithelium and interstitial tissue subgroups. The difference observed at O-11 and 12-13 months in the CsA-treated group in the interstitial tissue was due to a shrinkage of the

25

20

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2 3

2

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10 k h

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I i , //, //,

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9-11 II

Q Cl ,, , :: ,f, i/i 45 12-13

Mea” number 01 tubules/LPF I” CsA-

FIG. 3. Comparison of mean number of tubules/LPF in untreated and CsA-treated rats vs age in months.

190

Mean % germma, e,,,fheC”m ,n CsA- treated mt.3 “S age

Months

FIG. 4. Comparison of mean percentage of germinal epithelium in untreated vs GA-treated rats vs age in months.

connective tissue and lymphatic components possibly due to the cyclosporin A. The Leydig cells were not affected. The 9- to 11-month subgroup in Group 1 had a standard deviation of ? 12.59 (Figs. 4 and 5). There were no significant changes with age in the mean percentages of lumen in the treatment group. In comparison between the control and treatment groups, the differences in the 6- to 8-month and the 9- to 11-month subgroups were significant. There were no significant differences between the corresponding 3- to 5-month and 12- to 13-month subgroups (Fig, 6). Finally, there were no significant differences in the mean tubular diameters between the control and the corresponding CsA-treated subgroups (Fig. 7).

Morphologically, in comparison to each corresponding age subgroup, we did not find any changes (Fig. 8).

DISCUSSION

CsA is a very potent immunosuppressive agent, and its use in bone marrow and organ transplantation will likely continue to increase, especially as we continue to learn about its side effects and the management of complications. Currently, the use of CsA has been mainly limited by its nephrotoxic properties which have been well-documented in laboratory animals as well as in the clinic. In rats treated with

3-5 6-6 9-11

,I

; 5 :. k 1 I 3

Months

FIG. 5. Comparison of mean percentage of interstitial tissue in untreated and CsA-treated rats vs age in months.


Months

FIG. 6. Comparison of mean percentage of lumen in untreated and GA-treated rats vs age in months.

CsA, workers have noted focal tubular cell vacuolation (P-16), tubular inclusion bodies, microcalcification (17, 18), elevated blood urea nitrogen (19, 20), and elevated serum creatinine (20).

CsA nephrotoxicity in man is very similar to that in the rat, but is associated with more severe functional changes (18). In man, it is well-documented that CsA causes injury to proximal tubules with diffuse interstitial fibrosis, vascular changes, atrophy, vacuolation, and intracellular calcification (18, 21, 22). Func- tionally, CsA nephrotoxicity includes reduced creatinine clearance with elevation of serum creatinine (21,.23), disproportionate increases in blood urea nitrogen, hyperkalemia (22), and decreased glomerular filtration rate (15, 24).

Hepatic toxicity has also been reported, although the findings have varied in different studies (25). Cyclosporine has been noted to cause severe degranulation and hydropic degeneration of islet Q cells in rats (26).

Due to the selective effect on T cells, CsA may impair resistance to Epstein- Barr virus-induced B cell proliferation (22). Patients using CsA are susceptible to other types of infections, such as those caused by Pneumocystis carinii (28, 29) and herpesvirus (30), and to lymphoproliferative disorders accompanied by dys- gammaglobinemia (13, 16, 31-34).

3-5 6-6 9-11 12-13

Mean tubular drameter m untreated rats “S. age

Mean tubular diameter I” CsA-trea,ed rars YS age

Months

FIG. 7. Comparison of mean tubular diameter in untreated and CsA-treated rats vs age in months.

192 CHEN ET AL.

FIG. 8. Section of histologic testis of 13-month-old rat which was treated with cyclosporin A. The seminiferous tubule is normal with normal spermatogenesis. There is normal progression of formation of spermatozoa. The sertoli cells are normal as is the lumen. H & E stain, X600.

In pregnant rats, doses of CsA up to 15 to 17 mg/kg/day are asociated with normal fertility. However at higher doses, embryo-lethal effects and retarded development are present (13). At CsA dosage of 25 mg/kg/day, there are necrosis of decidual and placental tissue (37), decrease in viable litter size, and arrest of fetal development (38, 39).

Fetuses of transplant patients using conventional immunosuppressants are sub- ject to substantial hazards. One study of infants born to female renal homograft recipients has noted 49% prematurity, 30% with one or more serious complications, and 9% with congenital anomalies (40, 41).

To look at the effect of CsA on the male gonads, we can use the rat as a model. The germinal epithelium and the mean tubular diameter are probably the most specific histological indicators of testicular damage (8, 42-44). They are also in- dependent of the interstitium and lumen. In aging or diabetic rats and humans (5, 6, 45) as well as in uremic patients (7), atrophy of the germinal epithelium and decreased tubular diameter (6) have been well documented. In our study, the tubular diameter and the testicular germinal tissue of control versus CsA-treated rats do not differ, both histometrically and morphologically.

In recent studies, the interstitial compartment is also reported to be abnormal (5, 6). We did not find any overall differences in CsA versus control groups, except for the 9- to 11-month and the 12- to 13-month groups. In the CsA-treated animals the decrease in the interstitial tissue was due to a shrinkage of the connective tissue and lymphatic components possibly due to the cyclosporin A. The Leydig cells were not affected.

We did not observe any overall differences in the mean tubular density between


the two groups. The P value of the 3- to 5-month subgroup is likely due to the same reason given above for the interstitial compartment.

The mean percentage of testicular lumen is the least reliable of all the parameters that we have examined. It is dependent on interstitial supporting structures which prevent collapse of the germinal epithelium; therefore, lumen size is dependent on supporting structures and collapsing germinal epithelium. Thus, there are inconsistencies in the mean lumen sizes of both the control and treatment groups resulting in low P values in the 6- to &month and the 9- to 11-month subgroups.

Finally, tubular cross-sectional surface areas ahove been noted to be smaller in diabetic rats as compared to normal rats (44). In man, mean testicular volumes, which are related to tubular cross-sectional surface areas, are noted to be decreased in patients with uremia undergoing hemodialysis (4). In our study, there are no significant differences between control and CsA-treated groups, although in both groups there is a general decrease in mean cross-sectional surface areas with age.

The relationship between testicular morphology and function has been examined by several authors. Singhal et al. suggest that the various changes in testicular histology in diabetic men are associated with abnormalities only and are not responsible for the appearance of impotency. They believe libido and potency are hormone dependent (45). Handelsman ef al., however, have noted that human testicular volume is inversely correlated to both basal and stimulated LH and FSH levels. They postulate that serum FSH is a prognostic indicator to the return of fertility since fertile men in their study have the lowest serum FSH (4). This correlation is further supported by a number of authors who have recorded marked improvement in gonadal function including fertility (40) and spermatogenesis (35) following successful renal transplantation. Another study has also reported 50 male renal transplant recipients who have fathered 67 infants (40). An increase in testosterone is also reported after transplantation (35). Furthermore, following transplantation, there are increases in mean testicular volumes (16) and reversal of hypogonadism and germinal cell aplasia (7, 46). In our laboratories in San Diego, this reversal of testicular lesions has also been observed in diabetic rats following pancreas transplantations (47).

In conclusion, the effect of CsA on the human gonad is not known, although several normal pregnancies in bone marrow (3) and renal transplant (41) women recipients on CsA have been reported. The effect of CsA on the male gonads is even more limited. From previous studies, we know that although high doses of CsA can cause fetotoxicity in rats, doses under 15-17 mg/kg/day are associated with normal fertility without fetotoxicity in rats (13). In rat testes, we can state from our study that CsA at 10 mg/kg/day for at least up to 13 months does not cause gonadal toxicity.

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histometric investigations of the effect of cyclosporin a on the testicular tissue of rats

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