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Testicular histology in streptozotocin-induced diabetes.

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THE ANATOMICAL RECORD 214:378-382 (1986)
Testicular Histology in Streptozotocin-Induced
Diabetes
JUDY E. ANDERSON AND JAMES A. THLIVERIS
Department of Anatomy, University of Manitoba, Winnipeg, Manitoba, R3E OW3, Canada
ABSTRACT
The use of the streptozotocin rat model for diabetes has been questioned by the appearance of extrapancreatic cytotoxicity, notably renal and hepatic.
In this study the model was made specific to diabetic, drug-induced, and starvation
effects on parameters of testicular histology. Formulation of orthogonal contrast
expressions permitted the statistical separation of these influences. Tubules from
moderately diabetic animals showed frequent thinning, and premature desquamation of pachytene spermatocytes and early spermatids from the germinal epithelium.
Results showed that only diabetes significantly decreased seminiferous tubule diameter and increased testicular blood vessel numbers. In addition, significant alteration from the control pattern of tubule stage distribution was noted, particularly at
stages IX-XI. Due to the inclusion of a drugtreated but nondiabetic group, streptozotocin itself was shown to have no significant effect on these parameters.
The streptozotocin rat model for diabetes is being
widely used despite questions of extrapancreatic cytotoxicity [induction of renal tumors (Horton et al., 1977)
and hepatic injury (Viatteters et al., 1978)]. General
testicular histology (Oksanen, 1975) showed wide individual variation in seminiferous tubule diameter and an
overall decrease in tubule size. A large variation in the
spermatogenesis process was also seen between animals
in the diabetic group, which increased with the duration
of diabetes. Seminiferous tubules first showed spermatocyte and early spermatid sloughing, progressing to
early spermatocyte sloughing and then to complete atrophy. These observations were not correlated with body
weight or blood glucose at the time of death. Moreover,
no reference was made to severity of diabetes or to any
monitoring of metabolic parameters which might have
shown correlation to the testicular alteration, and no
quantification of the staging was made.
A morphometric study of testes in streptozotocin-induced diabetes (Rossi and Aeschlimann, 1982) showed a
significant reduction in tubule cross-sectional area after
8 months. Extremely high individual differences within
diabetic groups were noted, but a constant (20-25%)
“total or subtotal block of spermatogenesis” at spermiocyte stages I1 and I11 was seen in small tubules in all
diabetic groups, regardless of the duration of the condition. In neither study by Oksanen (1975) or Rossi and
Aeschlimann (1982) was any direct toxicity of the diabetogenic agent determined.
found in both diabetic and non-diabetic drug-treated animals 7 weeks after injection of streptozotocin (Thliveris
et al., 1984). This led to the present work, which is part
of a larger investigation into Leydig cell ultrastructure
and steroidogenesis in streptozotocin-induced diabetes.
Experimental design controlled for any effects of the
drug itself by inclusion of both the animal group which
failed to become hyperglycemic with the drug treatment, and a group treated with insulin to correct the
0 1986 ALAN R. LISS, INC
diabetes. The influence of protein-calorie-malnutrition
on testicular endocrine structure and function was controlled for by addition of a food-restricted group of animals to the study. In a n attempt to reduce individual
variations in diabetic animals, less of the diabetogenic
agent was administered to the rats than in previous
work, and an intermediate duration of diabetes in adult
rats was used.
MATERIALS AND METHODS
Diabetes was induced in 16 adult male (250-300 g)
Long Evans rats with a single intravenous injection of
40 m g k g streptozotocin (Sigma) in cold citrate buffer
(pH 4.5). Hyperglycemia (blood glucose a t 3 days > 300
mg/dl) and glycosuria resulted within 3 days in ten
animals, whereas the remaining six maintained normoglycemia. The former group was randomly subdivided
into a n untreated diabetic (n = 51, and an insulin-treated
diabetic (n = 5) group (3 IU subcutaneous Connaught
ultralente insulin daily to achieve near-normal glycemia). A control group of six citrate-injected rats was
age-matched to the diabetic group. Animals were fed
Wayne F6 rodent blox and kept for three months with
water and food ad libitum. A fifth group of six animals
was age- and weight-matched to the diabetic group by
titrating food restriction over 3 months.
Testes were perfused with glutaraldehyde (1%)
in Sorenson’s buffer (Anderson et al., 1983), dissected away
from epididymus, and weighed. Decapsulated and longitudinally bisected organ halves were postfixed in
Bouin’s fluid, embedded in paraffin, sectioned, and
stained with hernatoxylin and eosin. Coded slides were
examined separately for histopathology, tubule diameter, and blood vessel number.
Received November 16, 1984, accepted October 30,1985.
TESTICULAR HISTOLOGY IN DIABETES
Analysis of Testicular
379
by Kruskal-Wallis nonparametric analysis of variance
within each stage class. In all cases a probability of less
than 0.05 was accepted for rejection of the null
hypothesis.
Histology
RESULTS
0
tubule
diameter
Fig. 1. Analysis of testicular histology. Tubule diameter: Smallest
measure of 20 patent tubules ( 5 0 ‘~p
ro’ection)
in each of five areas.
J in
. each
Blood vessel count: Number per mm ( X 100)
of five areas.
For determination of testis tubule diameter, slides
were projected a t 50 x and measurements were systematically made from five preset, area-weighted regions of
the organ section (Weibel, 1979). In each region, the
smallest diameter for each of 20 patent, nearly round
tubule cross sections was measured (Fig. 1).Mean and
standard error of the mean were derived from these 100
tubule diameter measurements per animal.
To count blood vessels, slides were viewed at lOOx,
and five preset, area-weighted fields (1 mm2) of crosssectioned tubules were scanned through a n ocular grid
(Zeiss). All vessel profiles in the outlined area were
counted, disregarding those intersected by the “forbidden line” (Weibel, 1979) (Fig. 1). This was adjusted to a
number per tubule, and the five vessel counts per animal were averaged.
Frequency distributions of the stages of spermatogenesis (Leblond and Clermont, 1952) were made by scanning the sections from pole to pole of the organ, and
staging the first 100 tubules viewed per animal.
Score data (tubule diameter and blood vessel numbers)
were analysed by Tukey’s method for multiple mean
comparisons and by analysis of variance. Algebraic
expressions for the separate effects of diabetes, streptozotocin, and starvation were formulated (Snedecor and
Cochran, 1980)for orthogonal contrasts of means:
Ldm = 1/4(C + STV f Sdm + 1 f S n o t dm) - Sdm
LS 1/2(sdm + I + Snot dm) - c
L s w = 1/3(C + Sdm + I + Sno, dm) - STV
where C = citrate control, STV = semistarved, Sdm =
streptozotocin diabetic, S b + I = streptozotocin diabetic
and insulin treated, and Snotdm = streptozotocin injected but not diabetic.
Frequency distributions were analyzed by chi-square
contingency tables for between-group independence, and
Testicular histology of the citrate-injected control, the
nondiabetic drug-treated group, and the insulin-treated
diabetic group showed smoothly rounded seminiferous
tubules lined by germinal epithelium at various stages
of spermatogenesis. A thin, regular basal lamina separated the tubule epithelium from the interstitum, composed of Leydig cells and blood vessels in loose array
between tubule profiles (Fig. 2A). In testes from diabetic
animals there were some degenerating pachytene nuclei
and frequent sloughed pachytene spermatocytes in the
tubule lumen, resulting in a thinned germinal epithelium. This was most apparent at stages IX-XI1 and IIIV where the heads of maturing spermatids were unusually close to the basal lamina, and the spermatocytes
(and early spermatids) did not form regular “columns”
between their flagella (Fig. 2B). The basal lamina in the
diabetic group appeared irregular in thickness, and
lacked the smoothly rounded profile seen in the control
groups, but precise measures of this component must
await the higher magnification available with electron
microscopy. Tubules from semistarved animals occasionally had prematurely sloughed spermatids in the early
stages of acrosomal cap formation. Testis weights in the
diabetic animals tended to be smaller than in the other
groups, but the difference was not significant whether
expressed as single or total organ weight or as total
testicular weight per 100 g of body weight.
Tubule diameter and blood vessel data are listed in
Table 1. For the five groups analysed by analysis of
variance and Tukey’s procedure, there was a significant
difference in tubule diameter between the diabetic and
the citrate-injected control groups ( P < .05), and between the diabetic and nondiabetic streptozotocin-injected groups ( P < ,011. Chi-square analysis of
histograms constructed from all diameter measurements in each group (100 per animal) showed a significant (x2 = 438.9, df = 36, P < .001) shift to smaller
tubule diameters in untreated diabetic animals (Fig. 3).
Blood vessel number per tubule showed no significant
difference between any two groups by Tukey’s procedure.
The linear, orthogonal contast expressions compared
means on the basis of a chosen factor-namely, the separate effects of diabetes, streptozotocin, and starvation.
Diabetes had the only significant effect on either variable: decreasing tubule diameter (mm x 50) by 1.52 -t
0.8 ( P < .001), and increasing blood vessel number per
tubule by 1.4 -t 1.1 ( P < .02). These are approximately
- 10% and +30% changes, respectively. Streptozotocin
toxicity, as an influence on either parameter, was
excluded.
The frequency distribution of seminiferous tubules a t
stages I, I1 and 111, IV-VI, VII and VIII, IX-XI, XI1 and
XIII, and XIV are given in Table 2. Chi-square analysis
of the data showed significant (x2 = 60.2, df = 24, P <
.001) dependence of the distribution on the animal treatment groups. Within each treatment group there were
significant individual variations in stage frequency distributions by chi-square analysis only in the untreated
J.E. ANDERSON AND J.A. THLIVERIS
380
Fig. 2. Testicular histology. (A)Control testis (H & El. Note germinal
epithelium lining seminiferous tubules in various stages of spermatogenesis, and cleared blood vessels in the interstitium ( ~ 8 0 )$3)
. Testis
from streptozotocin-diabetic animal (H & E). Note disrupted and
thinned germinal epithelium, sloughing of spermatocytes, and decreased tubule diameter ( ~ 8 0 ) .
TABLE 1. Tubule diameter and number of blood vessels per tubule in testes of diabetic
and control groups of rats
Condition
(n)
Citrate, control'
(6)
Streptozotocin, diabetic2
(5)
Streptozotocin, not diabetic'
(4)
Streptozotocin, diabetic + insulin'
(5)
Tubule
diameter (mm @ 50 x)
(X
k SEMI
Blood vessels
per tubule
( x k SEMI
11.48 + 0.20"
9.85 f 0.36*$
3.4 f 0.4
4.9 k 0.7
11.79 f 0.704
3.6 f 0.7
11.19 f 0.19
3.6 f 0.3
11.02 i- 0.19
3.2 & 0.1
Semistarved'
(6)
'Blood glucose 120.7 1.6 mgidl after 3 months.
'Blood glucose > 400 mg/dl after 3 months.
*Significant at P < .05 level.
$Significant at P < .01 level.
diabetic ( P < .005) and semistarved ( P < .01) groups.
Together these analyses suggest that streptozotocin-induced diabetes may alter the spermatogenesis process,
that insulin correction of diabetes does not appear to
entirely prevent that alteration, and that the wide individual variation in testicular histology seen by previous
authors is likely due to a combination of the diabetic
state and the semistarvation it includes.
Analysis of the variance between frequencies of each
stage of individual animals, with the nonparametric
Kruskal-Wallis test, showed significant ( P = .008) variation only at stages IX-XI. This may indicate the initial
site of a block in spermatogenesis, and account for the
sloughing of pachytene spermatocytes into the tubule
lumen from these stages, and for the epithelial thinning
seen in stages IX-XI1 and 11-IV.
DISCUSSION
The data examined here reveal that both a reduction
in testicle tubule diameter and a n increase in number
381
Control
l:::
0
8
10
14
12
16
Y,
-1
V
oc
Lu
cLu
Strep- Injected
I. j
80
z o
9
n
LLI
4
z
Y,
LLI
a
2
2
8
Yt
0
8
10
12
14
16
Strep- Injected
Nondiabetic
6
8
10
14
12
16
-
Insulin Treated
80
23
l 60
OI
Z
6
80
&
Lu
Diabetic
n= 500
Ilt
Semistarved
80
0
6
8
10
12
TUBULE DIAMETER
14
16
(mm x 5 0 )
Fig 3. Tubule diameter for each treatment group Note shift toward
smaller tubule diameters in untreated diabetic animals Arrow =
group mean; n = sample size.
of blood vessels distributed per tubule were present in
the diabetic state. While alteration of the former parameter is confirmatory evidence, the augmented blood vessel density, not an artifact of decreased testicular size in
this work, may be a n important addition to the understanding of diabetic pathology. Reduced fertility in diabetes (Paz and Homonnai, 1979) may involve vascular
proliferation and consequently increased testicular temperature, which has been shown to affect both spermatogenesis (Jegou et al., 1984; Bedrak et al., 1980) and
Sertoli cell function (Damber and Bergh, 1980; Bergh et
al., 1984) in experimentally cryptorchid rats. In rats
treated only with heat for 4-7 weeks, a “novel endocrine
balance” (Bedrak and Chap, 1984) was acquired where
a decreased number of Leydig cell hCG receptors with a
higher affinity for hCG was combined with a post-receptor defect to result in a decreased capacity to synthesize
testosterone. Increased temperature has also been shown
to raise capillary permeability (Sharpe, 1984), as also
occurs in diabetes (Parving et al., 1983), influencing
testosterone availability to target cells in both interstitial and tubular compartments of the testis.
Although moderate, the changes in tubule diameter
(-10%) and blood vessel density (+30%) in adult animals with diabetes of relatively short duration induced
with a small dose of streptozotocin, and in the absence
of a significant decrease in testicular weight, suggest
that such alterations occur early in the course of the
disease. They may relate to the altered response to testosterone or its accessibility to target tissue seen in
adult male Wistar rats after 30 days of diabetes (Jackson
and Hutson, 1984).
The altered frequency distribution of the stages of
spermatogenesis, significant at stages IX-XI, with premature release of pachytene spermatocytes into the lumen and a thinned germinal epithelium, appears to
correspond to changes in testicular histology seen with
short-term exposure to heat. It also coincides with the
tubule stage exhibiting maximal numbers of nuclear
androgen receptors (Isomaa et al., 1985). One complete
cycle of spermatogenesis in the Long Evans strain of rat
takes 48 days (Rich and de Kretser, 1983); thus significant disruption of the process a t one particular stage
after 3 months of the onset of diabetes suggests that
such disruption has recently begun or that it begins
early in the disease.
TABLE 2: Frequency distribution of seminiferous tubules by stage
Stages
I
1
11, I11 IV-VI
2,3
4-6
VII, VIII
7, 8
IX-XI
9-11
XII, XI11 XIV
12, 13
14 Total
Condition (n)
Citrate, control (6)
Streptozotocin
diabetic ( 5 )
Streptozotocin
not diabetic (4)
Streptozotocin,
diabetic & insdin (5)
Semistarved (6)
‘Raw frequency.
22l
24
17
66
81
39
191
117
83
185
159
132
54
53
69
64
45
39
18
21
21
600
500
400
22
71
129
149
64
43
22
500
37
92
142
152
100
47
30
600
382
J.E. ANDERSON A1VD J.A. THLIVERIS
Comparison of the stage frequency distribution of control animals in this study with those of previous authors
(Leblond and Clermont, 1952; Clermont and Morgentaler, 1955) shows major differences between any two
control distributions. Whether these are attributable to
strain differences or to inconsistencies in staging tubules between observers, it serves to emphasize the caution with which inferences from histopathology of a
tissue as complex as the testis must be made. Comparison between stage distributions in other models and
durations of diabetes would strengthen understanding
of the processes involved in the disruption of the normal
tubule histology and possibly its timing.
The distinction between diabetic and streptozotocininduced alterations in a model is not a trivial one. A
recent study on the effects of alloxan and alloxan-induced diabetes on the kidney (Evan et al., 1984) stressed
this concern. Close examination of the drug-induced diabetic model suggested two separate contributions to
renal pathology: one by diabetes and a second by the
diabetogenic agent. Thus, nondiabetic, drug-injected animals should not be discarded, as they serve as crucial
internal controls.
ACKNOWLEDGMENTS
The technical assistance of D. Jones, C. Rempel and R.
Simpson is gratefully acknowledged. J.E. Anderson is
the recipient of a Manitoba Health Research Council
Studentship.
LITERATURE CITED
Anderson, J.E., J.A. Thliveris, and S.B. Penner (1983) Effect of fixation
on the ultrastructural localization of A53P-hydroxysteroiddehydrogenase in adrenocortical and Leydig cells of the rat. Acta Histochem., 73/79-86.
Bedrak, E., and Z. Chap (1984) Activity of LH receptor, LH-stimulated
cyclic AMP and testosterone production in the Leydig cell of heatacclimatized rats. J. Endocrinol., 102:167-173.
Bedrak, E., Z. Chap and K. Fried (1980) Factors for consideration in
the interpretation of the adverse effects of elevated environmental
temperatures on reproduction in the male rat. Int. J. Biometerol.,
24W: 117- 128.
Bergh, A., J.-E. Damber, and M. Ritzen (1984) Early signs of Sertoli
and Leydig cell dysfunction in the abdominal tetes of immature
unilaterally cryptorchid rats. Int. J. Androl., 7r398-408.
Clermont, Y.,
and Morgentaler (1955) Quantitative study of spermatogenesis in the hypophysectomized rat. Endocrinology 57:369-382.
Damber, J.-E., and A. Bergh (1980) Decreased testicular response to
acute LH-stimulation and increased intratesticular concentration
of oestradiol-17 in the abdominal testes in cryptorchid rats. Acta
Endocrinol. 95416-421.
Evan, A..P., S.A. Mong, B.A. Connors, G.R. Aronoff, and F.C. Luft
(1984) The effect of alloxan, and alloxan-induced diabetes on the
kidney. Anat. Rec., 208:33-47.
Horton, L., C. Fox, B. Corrin, and P.H. Sonksen (1977) Streptozotocininduced renal tumors in rats. Br. J. Cancer, 36~692-699.
Huhtaniemi, I., A. Bergh, H. Nikula, and J.-E. Damber (1984) Differences in the regulation of steroidogenesis and tropic hormone receptors between the scrota1 and abdominal testes of unilaterally
cryptorchid rats. Endocrinology, 115:550-555.
Isomaa, V., M. Parvinen, O.A. Janne, and C.W. Bardin (1985) Nuclear
androgen receptors in different stages of the seminiferous epithelial cycle and the interstitial tissue of rat testis. Endocrinology,
116(11:132-1347.
Jackson. F.L.. and J.C. Hutson (1984)Altered resDonses to androgen
- in
diabetic male rats. Diabetes, 33:819-824.
Jeqou, B., A.O. Laws, and D.M. de Kretser (1984)Changes in testicular
function induced by short-term exposure of the rat testis to heat:
Further evidence for interaction of germ cells, Sertoli cells and
Leydig cells. Int. J. Androl., 7244-257.
Leblond. C.P.. and Y. Clermont (1952) Definition of the stages
of the
"
~~~cycle of the seminiferous epithelium of the rat. Ann.-N.Y. Acad.
Sci.. 55548-573.
Oksanen, A. (1975) Testicular lesions of streptozotocin diabetic rats.
Horm. Res. 6t138-144.
Parving, H-H., G.C. Viberti, H. Keen, J.S. Christiansen. and N.A.
Lassen (1983) Hemodyuamic actors in the genesis of diabetic microangiopathy. Metabolism, 32~943-949.
Paz, G., and Z.T. Homonnai (1979) Development of diabetic-infertility
induced by streptozotocin in the male rat. Int. J. Androl., 2182192.
Rich, K.A., and D.M. de Kretser (1983) Spermatogenesis and the Sertoli cell. In: The Pituitary and Testis, Clinical and Experimental
Studies. D.M. de Kretser, H.G. Burger,and B. Hudson, eds. Springer-Verlag, Berlin, Heidelberg, pp. 84-105.
Rossi, G.L. and M. Aeschlimann (1982) Morphometric studies of pituitary glands and testes in rats with streptozotocin-induced diabetes.
Andrologia, 14(6);532-542.
Sharpe, R.M. (1984) Intratesticular factors controlling testicular function. Biol. Reprod., 30:29-49.
Snedecor, G.W., and W.G. Cochran (1980) Statistical Methods, ed 7.
Iowa State University Press, p. 224.
Thliveris, J.A., G.F. Paz, E. Rempel, and C. Faiman (1984) An extrapancreatic direct effect of streptozotocin in the hypothalamo-hypophyseal-testicular axis in the rat. Anat. Am., 157:213-219.
Viatteters B., E. Barrat, V. Lassmann, J. Guidon, E. Alomare, C.
Simon, and P.H. Vague (1978) Polykystose hepatique chez le rat
renda diabetique par la streptozotocine et treite par greffe d'ilots
de Langerhans: Role de la stretozotocine. An Anat. Pathol. (Paris)
23:351-360.
Weibel, E.R. (1979) Stereological Methods, Volume 1. Academic Press,
Chpts. 3 and 4.
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