effects of pancreaticoduodenal allografts on diabetic testicular lesions in the rat

Journal of Clinical Laboratory Analysis 2:7-15 (1988)

Effects of Pancreaticoduodenal Allografts on Diabetic Testicular Lesions in the Rat

Chris Chen, Michael H. Scott, Paul L. Wolf, A.R. Moossa, Parviz Haghighi, and Sun Lee

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

San Diego, California

Abnormal testicular histology is common in untreated diabetic animals and men. Reversal of testicular lesions with return of function and fertility are seen in controlled diabetes. We investigated the effects of pancreaticoduodenal (PD) allografts on testicular lesions of alloxan-induced diabetic rats. For periods of l to 15 months, 190 diabetic Lewis rats were allowed to remain diabetic before receiving PD transplants and Cyclosporin A (CsA); 190 other rats were divided into 4 control groups: normal rats with and without CsA, and diabetic rats with and without CsA. Following transplantation, transplanted rats and corresponding controls were sacrificed at 3- month intervals. Testicles were morphom- etrically examined. CsA did not affect rat testicular histology. All successfully transplanted rats (n = 97) had complete reversal of hyperglycemia. In the first 6 months following transplantation, the testicular lesions appear to worsen; thereafter, the tes-

ticular sizes, tubular densities, and percentages of germinal tissue converge toward nondiabetic levels. This convergence was more marked in the later transplantation groups (2 9 months of diabetes before receiving transplantation) than in the earlier groups (< 9 months). Overall, these results suggest that PD transplantation may partially reverse testicular lesions. Al- though pancreas transplantation may initially worsen diabetic testicular lesions, after 6 months, improvement of the lesions can be expected. Thus, an important result from this study relating to the clinical laboratory is that good control of the blood glucose in diabetics causes reversal of diabetic testicular lesions with return of function and fertility. In addition, Cyclosporin levels should be monitored in clinical laboratories in transplant patients to prevent toxic injury to the kidney and liver. How- ever, Cyclosporin did not damage the testis in our study.

Key words: alloxan diabetes, animal, diabetes mellitus, experimental, histology, homologous transplantation, pancreas, seminiferous epithelium, seminiferous tubules, testis, transplantation

INTRODUCTllON

Pancreas transplantation is being used with increasing fre- quency as a treatment for a small proportion of the type-1 insulin-dependent diabetic population. The rationale for this treatment rests on the hypothesis that the microangiopathic and other lesions associated with diabetes mellitus are sec- ondary to disordered metabolism (1). Thus, theoretically, perfect control of carbohydrate metabolism should prevent the development or halt the progression of these lesions. Previous work has shown this to be the case with some diabetic renal (2,3), ophthalmic (4), and nervous lesions (5) in rats successfully treated with pancreatic tissue transplants.

Testicular changes (6,7,8) and reproductive dysfunction (9,lO) are frequent findings in both animal models of diabetes mellitus and in diabetic patients. Although all the causes of infertility in diabetics cannot be attributed to the testicular lesions, they do represent a major problem. Preg-

0 1988 Alan R. Liss, Inc.

nancy after reversal of hyperglycemia has been reported in animals after pancreas transplantation (1 1). However, except for fetal pancreas transplants (12), previous studies in- vestigating the effects of pancreatic tissue, either islets (3) or whole organ (2,4,7) transplantation, on diabetic lesions have utilized isografts. This does not reproduce the real-life clinical allograft situation where immunosuppressive therapy can cause multiorgan damage (13). Hence, to realistically observe the effects of pancreas grafting on diabetic gonadal lesions, allografting with Cyclosporin A (CsA) immuno- suppression is needed. The aim of this study, therefore, is to assess the effects of pancreaticoduodenal transplantation on the early and established testicular lesions of alloxan diabetic rats.

Received May 15, 1987; accepted July 16, 1987.

8 Chen et al.

MATERIALS AND METHODS

Animals

All transplant recipients and controls were male Lewis rats, with male Brown Norway rats as donors. The animals were housed individually in wire suspension cages in a controlled environment (1ight:dark cycle 12:12, 72°F at 45% humidity) and fed Purina rat chow and tap water ad libitum. Experimental diabetes was induced in all potential recipients and 50% of controls in the ninth week of life by penile vein injection of alloxan (42 mg/Kg body weight) solution. Blood glucose levels were monitored on postalloxan days 3, 7, and 14 using a Beckman glucose analyzer 11. Rats were considered diabetic if all 3 levels were greater than 350 mg/dl. Blood was drawn from amputated tail tips.

Transplant Technique

The grafting technique used in this study was first devel- oped in 1971 by Sun Lee (14) and recently updated in our laboratory (15). The pancreaticoduodenal (PD) graft was harvested from the donor rat through a midline abdominal incision. The duodenum and pancreas were separated from the colon and greater omentum. Then the left gastric artery was divided, and the spleen was excised after ligation and division of its vascular pedicle. The duodenum was ligated just beyond the pylorus and divided. The bile duct and hepatic artery were ligated and divided close to the liver. The aorta was isolated from above the celiac artery to below the superior mesenteric artery, and all of its branches except the celiac and superior mesenteric arteries were ligated and divided. The portal vein was divided at the level of the duo- denal-jejunal junction. The aorta was cross-clamped, and the graft was perfused with iced 0.85% saline by injection through the aorta. Then the aorta was divided to produce an aortic segment with attached celiac and superior mesenteric arteries supplying the graft. The portal vein was divided at the liver hilum; the duodenum was divided at its distal third; and the entire pancreas with attached duodenum, aortic segment, and portal vein was excised and placed in iced saline.

The pancreaticoduodenal graft was transplanted to the re- cipient rat through a midline abdominal incision. The recip- ient aorta and inferior vena cava below the level of the left renal vein were cleaned and occluded with a partial occlu- sion clamp. End-to-side vascular anastomoses were performed using continuous 9-0 nylon sutures between the graft aortic segment and the host aorta, and between the graft portal vein and the host inferior vena cava. The open distal end of the graft duodenum was anastomosed end-to-side to the third portion of the host duodenum using a continuous 7-0 silk suture. Transplants were considered successful if the blood sugar fell to less than 170 mg/dl within 12 hours. Re- jection was taken as the first of 2 consecutive days when the blood sugar was greater than 200 mg/dl.

To prevent rejection, Cyclosporin A (I.V. preparation Sandoz) was given intramuscularly at a dosage of 10 mg/Kg/ day for the life of the animal. This dosage was previously shown by us to be the minimum amount necessary to prevent pancreaticoduodenal rejection in this combination of rats.

Experimental Design

Age-matched, nondiabetic male Lewis rats were randomly assigned to 1 of 2 groups: group A (n = 40), which received no further treatment, and group B (n = 5 3 , which received 10 mg/Kg/ddy of CsA. Similarly, there were 2 diabetic animal control groups: group C being nontreated diabetics (n = 50) and group D being diabetic rats receiving 10 mg/Kg/day of CsA (n = 45). Group E consisted of 190 rats that were allowed to remain diabetic for periods of 1, 3, 9, 12, and 15 months before receiving pancreaticoduodenal transplants.

All control rats were weighed and bled for glucose eval- uation. Transplanted rats were weighed and bled daily for 1 week postoperatively, weekly for 6 weeks, and then monthly until sacrifice. CsA levels were evaluated monthly in rele- vant rats (Sandoz RIA Kit).

Control and transplanted rats were sacrificed at 3-month intervals. Following sacrifice of the rat, the left testicle was removed and fixed in Bouins solution for 72 hours. Then they were sectioned longitudinally. Tissue dehydration, clearance, and infiltration were performed automatically using the “autotechnician,” and the samples were embedded in paraffin. The sections were cut at 4 microns and stained using Hematoxylin-eosin and the HOPA technique (hem- alum, orange-G-solution, phosphomolybdic acid, and ani- line blue) (16). A random coded number was assigned to each slide so that the following histometric and morphologic studies would be blind studies.

Morphometric Analysis

Testicular size

When the testicle was removed from the Bouins fixative and sectioned longitudinally through its point of maximum width, the mean testicular diameter and length were measured using a point-to-point micrometer. From these mea- surements, the mean testicular volume or the mean testicular size was calculated.

Tubular size

In each testicle section, 20 approximately circular cross- sectioned seminiferous tubules were randomly selected. The tubules were measured using a Nikon graduated reticle at a magnification of 100 X . The mean tubular cross-sectional surface areas (USA) were then calculated.

Pancreatic Allografts on Rat Testes 9

Fig. 1. a. Section of 9-month-old rat testis demonstrating normal testicular tissue. HOPA staining. b. Higher magnification.

Tubular density made, with the prominent cell types being noted.

At a power of 100 x magnification (Leitz, Wetzlar micro- scope), the number of tubules per field (2.61 mm2) were Statistical Analysis counted in 5 randomly picked areas of each testicle. Only The mean values in comparative series of animals were areas that consisted entirely of cross-sectioned tubules were compared. All results were expressed as the mean counted. A figure was thus calculated for the mean tubular dard deviation. The significance of differences between density (number of tubules/mm2). means was determined using the Mann-Whitney confidence

interval and test. Significance was not rejected at alpha (P) less than or equal to 0.05. Testicular cellular composition

RESULTS Testicular composition was measured using an adaptation of the point counting technique described by Hennig (17) and later employeld extensively by Schoffling (61, using a Nikon Group A. Untreated Nondiabetic Rats

For each section, an assessment of tubular architecture was

5mm reticle. This ocular had a network of 121 points, which were arranged in the area to be examined by microscopy. Using the method described by Federlin (18), the tissue ele- ment under a grid point was described as a “hit.” The sum of the “hits” of each tissue component was then related to the total number of “hits,” thus giving a percentage composition. The mean percentage of germinal epithelium was determined. The mean percentage of lumen (% lumen or lumen size) was calculated to be 100 % multiplied by the mean proportion of lumen divided by the sum of lumen and germinal epithelium. In1 each rat testicle, 20 randomly selected fields of tubules at an objective magnification of 1OO:l were counted, thus giving a total of 2,420 “hits” for each section.

In our test, we differentiated between germinal epithe-

All rats remained healthy throughout the study, with normal growth patterns and blood glucose levels (15 1 f 7 mg/ dl). Testicular sizes remained constant throughout the adult life-span of the rats. There were slight, insignificant increases in tubular densities and lumen sizes, and an insignificant decrease in tubular C/SAs and mean percentages of germinal epithelium (Table I). Although our calculations did not indicate any significant changes in the examined parameters over time, morphologically some of the tubules of rats greater than 9 months old appeared to have had increased tubular densities and decreased tubular diameters. Some of the older tubules even appeared to have had collapsed (Figs. 1 a, b, 2).

Group C. Untreated Diabetic Rats lium, lumen, and interstitial tissue. The various develop- mental stages of spermatogenesis, including Sertoli cells, were considered as germinal epithelium. Those tissue com- During the course of the study, 20% (10/50) of the dia- ponents that were not attached to the germinal epithelium but betic rats died; the death rate was constant throughout this were scattered throughout the center of a tubule were con- period, approximately 1 to 1.5% per month. Diabetic rats sidered as lumen. Interstitial tissue included connective tis- were significantly smaller (p < 0.05) than corresponding sue, Leydig cells, tunica propria, as well as the separation normal controls. The blood sugar of the diabetic rats was 576 that was occasionally caused by shrinkage of tissue during & 70, which was significantly greater than group A rats. preliminary treatment. Germinal epithelial architecture remained normal for the first

10 Chen et al.

Fig. 2. Section of 12-month-old normal rat demonstrating that some tubules have collapsed with reduced amounts of germinal epithclium and spermatogenic cells.

7-9 months, but after 9 months of diabetic induction, significant changes were seen both morphologically and histo- metrically. By 12 months, there was a depopulation of germinal epithelial cells with very little evidence of spermatogenesis in any tubules. Many tubules were lined with Sertoli cells and degenerating germinal epithelium (Fig. 3). In the histometric studies, as compared to each previous month interval, the mean tubular C/SAs significantly decreased (p < 0.03) by 26%, whereas the mean densities significantly increased (p < 0.04) by 36%, progressing at 15 months to 42% and 52%, respectively. Testicular sizes and the percentage of germinal epithelium remained normal for the first 9 months. After this time, however, there was a rapid decrease in testicular sizes. Over the 15 months of study, the diabetic testicular sizes and percentage of germinal epithelium significantly decreased (p < 0.05 and p = 0.008, respectively). The percentage of lumen was the only parameter that had an insignificant increase over the 15 months of study (p = 0.1, Table 1).

Groups B and D. Cyclosporin A-Treated Rats

During the study, 18% (10/55) and 49% (22/45) of diabetic CsA-treated rats died. The majority of the deaths were believed to be due to pulmonary infections.

The presence of CsA generally made no significant differ- ence to either the diabetic or nondiabetic testicular state; that is, group B was no different from group A, and, similarly, group C was no different from group D (Figs. lb, 4, 5a,b, 6). The exceptions were in the percentage of lumen at 4-6 and 7-9 months of the healthy groups (groups A,B, Table 1).

Group E. Transplanted Rats

Testicle data was obtained from 51% (97/190) of the transplanted rats. Data could not be obtained from the re-

Fig. 3. Section of an alloxan-induced diabetic rat at 12 months. Most tubules show loss of germinal epithelium with only a thin layer of spermatogonia.

Fig. 4. normal testicular tissue.

Section of rat trcated with CsA for 9 months demonstrating

maining 93 rats because of perioperative deaths. The fasting blood glucose levels of the successfully transplanted rats fall permanently from a preoperative mean of 536 f 62 mg/dl to a mean of 143 mg/dl, irrespective of the duration of diabetes at the time of transplantation.

Testicular volume (Fig 7.): Following transplantation in rats with early diabetes (1- and 3-month durations), there was a rapid decrease in testicular size (volume) for 6 months. The loss in size was much greater than that seen in diabetic rats of corresponding ages. The rate of decrease in size was equivalent to that seen in the more established diabetic rats of 9-12 months diabetes duration. Following the rapid loss of testicle size in these rats, there was a slow recovery to a size greater than that seen in correspondingly aged diabetic rats, but still not back to that seen in normal rats. Rats transplanted at 6- and 9-month diabetic durations displayed the same rapid decrease in testicular size over the ensuing 6 months. This pattern corresponded to that seen in diabetic


Fig. 5. a. Section of an alloxan-induccd diabetic rat at 9 months demonstrating normal testicular tissue. b. Higher magnification.

rats of the same age. After this rapid loss, there was a sub- stantially larger and slightly significant (p = 0.05) recovery in testicular size as compared to corresponding diabetic rats (410 + 42 after 9 months transplantation vs. 223 -t- 100 in diabetic controls). This increase in size was still smaller than normal. In rats with established diabetes of 12 and 15 months following transplantation, there was a rapid recovery of testicular size (590 141 and 575 f 163, respectively; p = 0.05) to near the normal range.

Tubular C/S,4 (Fig. 8): Following PD transplantation, all groups of rats showed an early decrease in mean tubular cross-sectional area. After a further 3-6 months, all groups had a recovery in C/SA to better than that seen in equally aged diabetic rats. This recovery, however, was statistically insignificant as; tubular C/SA in rats with transplantation after 15 months diabetes had standard deviations of + 1,000 square-microns. Morphologically in each microscopic field, it was obvious that amidst collapsed tubules, some tubules had significant increases in ClSAs. This was seen in the 6-15 months transplantation groups (Figs. 3,7).

Tubular density (Fig. 9): Return to a less than diabetic tubular density was seen in all groups of rats. This took longer in the short diabetic duration transplants (1- and 3-month groups) than in the others, and only the diabetic transplants after 9 months or greater had a significant return to near nondiabetic levels, although the nondiabetic controls were consistently lower than the transplant groups.

Percentage of lumen (Fig. 10): Rats transplanted at 1, 3, and 6 months of diabetes all immediately demonstrated a return of lumen size to normal, nondiabetic sizes. The rats transplanted after a longer diabetic duration (9, 12, and 15 months) had a significant shrinking of tubular lumen to a degree much greater than that seen in normal rats.

Percentage of germinal epithelium and testicular architecture. Rats transplanted at 1-6 months did not amear to have

trols (Fig. 11). In the later (9-15 months) transplantation rats, there were insignificant increases of percentage of germinal epithelium. Of interest is the transplantation after 15 months diabetes group (E15), which approached nondiabetic levels of germinal epithelium. This, however, was insignificant with S.D. = 20. But generally the transplanted groups had lower percentages of germinal lumen as compared to both diabetic and nondiabetic control groups. Morphologi- cally, the tubules in the early transplantation rats showed normal populations of spermatogenic elements. Whereas there was considerable depletion of these cells in many tubules of later transplantation groups as compared to the nondiabetic control groups, some of the tubules appeared to have some return of germinal epithelium. The later pancreas transplantation groups, however, still had less normal germinal epithelium as compared to the diabetic control groups (Figs. 3,12). All groups of transplanted rats showed some increases in interstitial tissue. This was marked in the longer duration diabetic groups (i.e., > 9 months of diabetes before transplantation) with marked Leydig cell hyperplasia. The testicular architecture in these groups often became very deranged with a major degree of tubular distortion and collapse.

DISCUSSION

Observations of testicular pathology have been reported in both diabetic men and animals. Of the animals studied, the rat has been most extensively investigated (19). It is an ex- cellent model to observe human testicular histology and morphology (20), which are related to testicular dysfunction (2 1,22,23).

The testicular lesions seen in our alloxan-induced diabetic rats are in accordance with the majority of publications on I.

any differences-as compared to corresponding diabetic con- this issue. Histologically, the most obvious alteration is the

12 Chenetal.

TABLE 1. Results of Histometric Determination in Testicular Tissue of Controls

Tubular Months Testicular CISA Tubular of volume ( ~ 1 0 ~ density % Germinal study Group (mm3) micron') (#/mm') % Lumen epithelium

1-3 A 832 f 86 65.0 f 15 5.52 f 1 15.4 f 3 71.3 f 6 B 795 f 88 55.7 f 8.5 7.01 f 1 14.4 f 4 72.4 f 6 C 753 f 65 47.8 f 11 7.13 f 2 18.2 f 5 65.1 f 3 D - 46.0 f 7.0 7.95 f 1 17.2 f 3 76.4 f 10 P [A-B] > 0.5 > 0.1 > 0.1 > 0.5 > 0.5 P [C-D] > 0.1 > 0.5 > 0.5 > 0.5 > 0.1 P [A-C] > 0.05 > 0.1 > 0.05 > 0.05 > 0.05

4-6 A 812 f 210 59.4 f 8.7 7.02 f 0.8 14.9 f 2 72.6 f 6 B 704 f 280 50.4 f 7.7 8.58 f 4 17.3 f 4 66.5 f 4 C 782 f 130 48.9 f 6.9 7.89 f 3 19.9 f 2 64.8 f 5 D - 40.6 f 11 8.21 f 1 22.1 f 5 62.6 f 10 P [A-B] > 0.5 > 0.5 > 0.1 < 0.05 > 0.05 P [C-D] = I > 0.1 > 0.1 > 0.1 > 0.5 P [A-C] > 0.05 > 0.05 > 0.05 > 0.05 > 0.05

7-9 A 746 f 180 56.3 f 8.3 8.05 f 3 16.4 f 2 65.2 f 9 B 796 f 400 53.7 f 11 8.66 f 2 15.7 f 4 71.4 f 6 C 752 f 260 46.1 f 11 8.28 f 3 18.1 f 6 65.0 f 5 D - 41.8 f 14 10.73 f 3 18.2 f 5 70.0 k 8 P [A-B] > 0.05 > 0.5 > 0.5 < 0.05 > 0.1 P [C-D] > 0.5 > 0.5 > 0.1 > 0.5 > 0.1 P [A-CI > 0.05 > 0.05 > 0.05 > 0.05 > 0.05

10-12 A 821 f 300 58.3 f 16 8.01 f 1 17.1 f 4 63.0 f 6 70.2 f 1 19.3 f 2 B 585 f 110 53.2 f 2.6 8.05 f 1

C 519 f 180 34.0 f 14 12.95 f 5 20.1 f 6 59.6 f 10 D - 27.9 f 3.2 12.03 f 2 26.7 f 4 59.2 f 3 P [A-B] > 0.5 > 0.5 = I > 0.1 > 0.05 P [C-D] > 0.5 > 0.5 = I > 0.05 > 0.5 P [A-C] > 0.05 < 0.01 < 0.05 > 0.05 > 0.05

13-15 A 672 i. 250 54.6 f 13 7.62 f 1 18.2 f 5 62.0 16 C 223 f 100 26.6 f 20 17.16 f 3 23.6 f 3 56.3 f 8 D - 24.2 f 9.3 10.59 f 3 19.6 f 9 61.5 f 2 P [C-D] = 1 = I > 0.5 > 0.5 > 0.1 P [A-C] < 0.05 < 0.05 < 0.05 > 0.05 > 0.05

A, Nondiabetic rats. B, Nondiabetic rats + CsA. C, Diabetic rats. D, Diabetic rats + CsA

900 r

S 300-

c 200 - m 2 loo/ , , , , , , , I .--.-a €15 Jrsnsp lantamn atlei

15 rnonln diabelel

1-3 4-6 7-9 10-12 13-15 16-18 19-21 22-24 'I T'ansp'anlaf'on

2 loo/ , , , , , , , I .--.-a €15 Jrsnsp lantamn atlei

15 rnonln diabelel

1-3 4-6 7-9 10-12 13-15 16-18 19-21 22-24 'I T'ansp'anlaf'on

Months

Fig. 6. strating essentially normal testicular tissue.

Section of a 9-month-old diabetic rat treated with CsA demon- Fig. 7. age of rats in months.

Effect of pancreas transplantation on mean testicular volume vs.


l:L, I 1 I ' J

1-3 4-6 7-9 10-12 13-15 16-16 19-21 22-24

A. Non-diabetic controls

---.I2 Diabelic C O ~ I ~ O I S

--4 €1 Transplanfation m e , 1 month diabetes

.-.-A E3 Transplantalron afler 3 monrhs diabeles

Transplanfallan alter 6 monfhs diabeles

--+ €6

.-.+ E9 TraOSplanlallOn alter 9 monlhs diabetes

Tiansplanlalroo al le i 12 monrhs 01 diabeles

Transplantalron alfer 15 monlh diabeles

--4 El2

.--.I El5

\I Traosplanlalion

Months

8. Effect of pancreas transplantation on mean tubular cross-sectional (c/s) area vs. age of rats in months.

--+ €6 Traorp lan lamn atlei 6 rnomho diabeles

.- +€9 Trarlsplarildlion atier 9 months diabetes

---a El2

II 1-3 4-6 7-9 10-12 13-15 16-18 19-21 22-24

Months

Transplanfatlo" aller 12m0ofhs 01didbele~ E l 5 Traosplanlatron aller 15 muolh diabsle3 TrmsoIaolaIion

Fig. 10 Effect of pancreas transplantation on mean percentage of lumen/ tubule vs. age of rats in months.

01 1 1 1 ' 1 ' I ' 1-3 4-6 7-9 10-12 13-15 16-18 19-21 22-24

4 A

- c

---A €1

Nm-diabefic conlrots

Diabelrc control^

Tramplanlalion aller 1 monlh drabeles

'-'* €3 TranSplanlallOn alfer 3 mOnlhSdiabeles --+ €6 Transplantalion aller 6 monlhs diabeles .-.* €9 Transplanlalion a m , 9 m o n I h ~ diabeles

--a €12 TranSplanldflO" m e r 12 r n m l h s 01 drabelm

.--.4 €15 TrarlSplanlal,"" after 15 monlh diabeleS

II T,a"splanIahon

Months

Fig. 9. Effect of pancreas transplantation on mean tubular density vs. age of rats in months.

rransplantalron ahei 15 months didbelei :: 1-3 4-6 7-9 10-12 13-15 16-18 19-21 22-24 I1 TrenlplaOlallO"

Months

Fig. 11. Effect of pancreas transplantation on mean percentage of germinal epithelium vs. age of rats in months.

arrest of spermatogenesis, which is well-documented in both humans (24) and rats (7,8,20,25). Our findings of germinal epithelium deterioration with distorted tubules lacking prod- ucts of spermatogenesis (such as mature spermatozoa and spermatids), but sparing Sertoli cells in diabetic rats, are supported by other authors (6,7,20,26). These lesions occur at a much younger age in diabetic as compared to nondiabetic rats. Similar findings have been reported in human diabetics as well as in other animal models. Although atrophic testes of diabetic animals are histologically similar to those with senile atrophy, the exact etiology and pathogenesis of these lesions remain to be elucidated (20).

Reduction in tubular size is also well-documented in diabetic rat testicles (7,s). Our finding of significantly smaller tubular cross-sectional surface areas at greater than 9 months Of diabetes is by Rossi et (27). These authors also have repofied a decrease in the Percentage of large and

Fig. 12. Section of testicular tissue of a 12-month-old diabetic rat 3 months after pancreas transplantation. Most tubules show only a thin layer of stxrmatoponia. which has collaDsed into luminal area. A tubule (arrow)

I

an increase in the percentage of small tubular cross-sectional shows some return of spermatogenic elements.

14 Chen et al.

surfaces (8). This finding is reflected in our determinations of mean tubular density, which is also increased after 9 months. The increase in density is due to the smaller size of tubules, as well as the loss of supporting structures in the in- terstitium, thereby enabling us to count more tubules per microscopic field.

Schoffling has reported increases in luminal size of tubules in diabetic rats (6). Our studies, however, do not reveal any significant changes in diabetic versus nondiabetic age-matched testicles. The mean percentage of testicular lumen is dependent on interstitial supporting structures that prevent collapse of the germinal epithelium; therefore, lumen size is dependent on supporting structures and collaps- ing germinal epithelium and probably is not as reliable as the other parameters.

The eventual decrease in testicular size has been noted by most investigators; however, in at least one study of chemi- cally (streptozotocin) induced diabetic rats, a loss of testicle weight was noted within 2 weeks of the onset of diabetes (7). This may represent the differences in measuring testicular size (volume in our study and weight in the other) or the method of diabetic production.

In all of the parameters studied, CsA generally does not significantly affect the diabetic and nondiabetic testicles with the exception of significance in 2 subgroups of groups A and B (Table 1, Figs. lb, 4, 5b, 6). As previously mentioned, lumen size is dependent on surrounding testicular structures. Thus, there are inconsistencies resulting in low P values in the subgroups of 4-6 and 7-9 months. But overall, CsA does not appear to affect testicular histology.

Reversal of testicular lesions and return of function (28) and fertility (8) are well-documented in renal transplant patients. Reversal of hypogonadism and germinal aplasia (23,29) and marked improvement in gonadal function, including fertility (8), spermatogenesis, and an increase in testosterone (28), have followed successful renal transplantation. Although some authors have reported low apparent fertility rates (22) after transplantation, reports of fertility after renal transplantation are nevertheless encouraging. One study has reported 50 male renal transplant recipients who have fathered 67 infants. However, thus far, there has been no report of fertility in men after receiving liver or pancreas transplants (30).

In our pancreas transplantation groups of 9 months or greater, there is a return of tubular density to near but less than nondiabetic levels. Tubular cross-sectional surface area and percentage of germinal epithelium are insignificantly increased posttransplantation. These insignificances may be caused by our large standard deviations reflecting large in- dividual variations. In regard to testicular volume, some authors have noted a decrease in testicular volumes of diabetic men during uremia and hemodialysis and a subsequent increase after renal transplantation (22). This supports our finding of increase in testicular size in rats with transplan-

tation after 9 or more months of diabetes. Thus, most of our parameters reveal that transplantation causes a reversal of testicular lesions in longer duration diabetic rats, but the reversal is not complete.

From our studies, it seems that once the diabetic testicular lesions develop, pancreas transplantation has the effect of arresting or even partially reversing some of them. There- fore, from morphometric analysis it seems that pancreatic transplantation is of benefit to established testicular diabetic lesions. However, when the architecture of the long-duration diabetic transplanted rats is assessed, it is obvious that there is severe testicular damage to the extent of virtually no normal spermatogenesis. This is not seen in rats with a short duration of diabetes before receiving transplantations. It is likely that the long-term effect of diabetes results in irrevers- ible damage to tubular morphology.

In diabetic rats with early pancreatic transplantation, testicular volume and tubular C/SA decrease and tubular density increases almost immediately after transplantation. The rate of change in early transplanted rats appears to parallel that in controls after 9 months of diabetes. The reason may relate to the development of testicular lesions caused by diabetes mellitus or operative stress and healing after the transplant procedure. We have certainly observed that transplantation initially causes a worsening of the testicular lesions in diabetic rats. Perhaps there may be a critical rate for the development of testicular damage in this type of animal model.

Six months following transplantation, all of the transplanted groups have a convergence in all of the measured parameters toward nondiabetic levels. It has been established that the beta cells of Langerhans can function almost immediately after pancreas transplantation to restore glucose levels to normal in diabetic rats. It may take at least 6 months to identify histological changes in the testicles. It would now be important that we follow these rats for a longer duration of time to determine the effect of age on rat testes.

The effects of pancreatic transplantation are complicated. Even though it has previously been shown that administer- ing insulin to diabetic rats to normalize blood glucose levels is capable of preventing the development of testicular weight loss in the short term (over 30 days ) (31,32), it would appear from our results that pancreatic transplantation in newly diabetic rats does not completely prevent the development of diabetic testicular lesions, although the long-term result is an improvement over age-matched diabetic rats.

Thus, an important result from this study relating to the clinical laboratory is that good control of the blood glucose in diabetics causes reversal of diabetic testicular lesions with return of function and fertility. In addition, Cyclosporin levels should be monitored in clinical laboratories in transplant patients to prevent toxic injury to the kidney and liver. How- ever, Cyclosporin did not damage the testis in our study.


16. Tonutti E: Zur histophysiologie der leydigschen zwischenzellen des rattenhodens. Z Zellforsch 32:495, 1943.

17. Hennig A: Das Problem der Kernmessung; eine Zusammenfassung und Erweiterung der mikroskopischen Messtechnik. Mikroskopie, Wien 12(5-6): 174, 1957.

18. Federlin C, Schoffling K, Neubronner P, Pfeiffer EF: Histometrische Untersuchungen am Hodengewebe des Diabetikers mit Keimdriisen- unter funktion. Diubetologiu 1:85, 1965.

19. Foglia VG, Rosner JM, Ramos M, Lema BE: Sexual disturbances in the male diabetic rat. Horm Res 1:72, 1969.

20. Wright JR, Yates AJ, Sharma HM, Shim C, Tigher RL, Thibert P: Testicular atrophy in the spontaneously diabetic BB Wistar rat. Am J Puthol 108:72, 1982.

21. Cameron DF, Murray FT, Drylie DD: Interstitial compartment pathology and spermatogenic disruption in testes from impotent diabctic men. Anur Rec 213(1):53, 1985.

22. Handelsman DJ, Ralec VL, Tiller DJ, Horvath JS, Turtle JR: Testic- ular function after renal transplantation. Clin Endocrinol 14(5):527, 1981.

23. Lim VS, Fang VS: Gonadal dysfunction in uremic men. A study of the hypothalamic-pituitary-testicular axis before and after renal transplantation. Am J Med 58:655, 1975.

24. Singhal KC, Rastogi GK, Aikat BK, Chhuttani PN: Testicular histology in diabetics with impotence. Indiun J Puthol Bucferiol 12(4): 145, 1969.

25. Murray FT, Cameron DF, Orth J: Gonadal dysfunction in the spontaneously diabetic BB rat. Metabolism 32(7 Suppl.): 141, 1983.

26. Rosenmann E, Yanko L, Cohen AM: Comparative study of the pathology of sucrose-induced diabetes in the rat and adult-onset diabetes in man. Israel J Med 2(7):753, 1975.

27. Rossi GL, Bestetti G: Morphological changes in the hypothalamic- hypophyseal-gonadal axis of male rats after twelve months of streptozotocin-induced diabetes. Diubetologiu 21:476, 1981.

28. Handelsman DJ, McDowell IFW, Caterson ID, Tiller DJ, Hall BM, Turtle JR: Testicular function after renal transplantation: Comparison of Cyclosporin A with azathioprine and prednisone combination re- gimes. Clin Nephrol22(3): 144, 1984.

29. Morley JE, Distiller LA, Unterhalter S, Myers JB, Rabkin R, Katz M: Effects of renal transplantation on pituitary gonadal function. Metabolism 27:781, 1978.

30. Penn I, Makowski EL, Harris P: Parenthood following renal and hepatic transplantation. Transplanlation 30(6):397, 1980.

31. Chatterjee A: Prevention of gonadal degeneration in male diabetic rats by the use of insulin. Endokrinologie 50: 11, 1966.

32. Hutson JC, Stocco DM, Campbell GT, Wagoner J: Sertoli cell function in diabetic, insulin-treated diabetic, and semi-starved rats. Diu- betes 32:112, 1983.

ACKNOWLEDGMENTS

This work was supported in part by NIH grant 5-ROI- AM33125-02.

REFERENCES

I . Tchobroutsky G : Relationship of diabetic control to development of microvascular complications. Diubefologiu 15: 143, 1978.

2. Bell R, Fernandez Cruz L, Brimm J, Sayers H, Lee S, Orloff M: Prevention by whole pancreas transplantation of glomerular mem- brane thickening in alloxan diabetes. Surgery 88:31, 1980.

3. Federlin K, Bretzel R: The effect of islet transplantation on complications in experimental diabetes of the rat. World.! Surg 8: 169, 1984.

4. Worthen D, Lee S, Nakaji N, Sayers H, Orloff M: Effect of whole pancreas transplantation on eye lesions of alloxan diabetes. Surg Forum 27:44, 1976.

5. Macedo A, Skivlocki W, Thompson K, Bell R, Lee S , Sayers H, Orloff M: Morphometric electron microscopy study of the effect of whole pancreas transplantation on alloxan diabetic neuropathy. Surg Forum 32:379, 1981.

6. Schoffling K, Federlin K, Schmidtt W, Pfeiffer EF: Histometric investigations on the testicular tissue of rats with alloxan diabetes and Chinese hamsters with spontaneous diabetes. Acfu Endocrinol54:335, 1967.

7. Okasanen A: Testicular lesions of streptozotocin rats. Hormone Res 6:138, 1975.

8. Rossi GL, Aeschlirnann M: Morphometric studies of pituitary glands and testes in rats with streptozotocin-induced diabetes. Andrologiu 14532, 1982.

9. Rodriguez-Rigau L: Diabetes and male reproductivc function. J An- drology 1:105, 1980.

10. Kolodny R: Sexual dysfunction in diabetic men. Diabetes 23:306, 1974.

11. Brown J, Heininger D, Kuret J, Mullen Y: Normal response to pregnancy in rats cured of streptozotocin diabetes by transplantation of one fctal pancreas. Diubefologiu 22(4):273, 1982.

12. Tuch BE, Shed AG, Ng AB, Turtle JR: Transplantation of cultured human fetal pancreas into insulin-dependent diabetic humans. Truns- plunnt Proc 18(2):260, 1986.

13. Cohen DJ, Loertscher R, Rubin MF, Tihey NL, Carpenter CB, Strom TB: Cyclosporine: A new immunosuppressive agent in organ transplantation. Ann Intern Med 101:667, 1984.

14. Lee S, Tung K, Koopmans H, Chandler J, Orloff M: Pancreaticod- uodenal transplantation in the rat. Trunspluntution 13:421, 1972.

IS. Lee S, Scott MH: Pancreaticoduodenal transplantation in the rat-an update. Trunspluntution 1986 (in preparation).

effects of pancreaticoduodenal allografts on diabetic testicular lesions in the rat

Documents