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Responsiveness of testis morphology to chemotherapy in childhood leukemia.

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THE ANATOMICAL RECORD 209:491-500 (1984)
Responsiveness of Testis Morphology to Chemotherapy
in Childhood Leukemia
GIULIANA ANELLI, GIUSI COZZI, EMILIA FRANCHI, MARIELLA
PARLAVECCHIA, FRANC0 CHIARA, GIUSEPPE MASERA, AND
MARINA CAMATINI
Department of Biology, Comparative Anatomy (G.A., G.C., E.F., M.P., M.C.),
2nd Obstetrical-Gynaecobgical Clinic (RC.), and Clinic of Pediatry (G.M.),
University of Milano, 20133 Milano, Italy
ABSTRACT
The appearance of seminiferous tubules and interstitial cells
of children, aged 2.5 to 13 years, affected by acute lymphoblastic leukemia was
analyzed in sections. The testicular biopsies were performed a t the end of
therapy (vincristine, prednisone, L-asparaginase, 6-mercaptopurine, intrathecal methotrexate), which was affected for the same period and a t the same
doses. Three age groups were considered (I, 2.5 to 5 years; 11, 6 to 9 years; 111,
12 to 13 years). Age groups I and I1 presented damage of some tubules (2535%) and areas of degeneration. Histometric analysis performed for A type
spermatogonial population gave a mean value corresponding to controls in age
group I and a mean value significantly lower with respect to controls in age
group 11. Moreover, age group I1 presented a lack of increase in tubular cross
section. These results suggest that there is a vulnerability both of whole
tubules and of some areas of Sertoli cells and germ cells to cytotoxic-induced
damage. Leydig cells appear to be the cells least sensitive to drugs, and
hormonal data indicate that the hypothalamic pituitary function appears to be
intact, despite chemotherapy. Long-term prospective studies of reproductive
function in children receiving cancer chemotherapy are needed to determine
the magnitude and duration of damage resulting from therapeutic treatment.
We have previously demonstrated (Camatini et al., 1982) that inter-Sertoli junction
formation in prepuberal boys who received
antileukemic agents was similar to that of
control children of a similar age (Camatini et
al., 1981). Our observations have now been
extended to the appearance of seminiferous
epithelium and interstitial tissues in the
same patients. Relatively few studies have
been performed on the effect of chemotherapeutic drugs on development of testis in children, whereas there are recent data on the
histologic and ultrastructural appearance of
human prepuberal testis in normal individuals (Vilar et al., 1970; Hadziselimovic, 1977;
Nistal et al, 1982; Muller and Skakkebaek,
1983), so that a comparison of the data reported here with those in the literature is
possible. Unfortunately, limited information
about the spermatogenic cycle is available in
young (Hadziselimovic, 1977) and even adult
humans (Rowley et al., 19711, and the stem
cell has not been unequivocally identified.
0 1984 ALAN R. LISS, INC.
Type A spermatogonia includes three spermatogonial types: A long (Al), A pale (Ap),
and A dark (Ad) (Rowley et al., 1971), each of
which may be a candidate for stem cell in
adults. In children only Ap and Ad spermatogonial types have been described in control
testes (Hadziselimovic, 1977). Thus, type A
spermatogonial population includes the stem
cell, and the histometric count of this spermatogonial population in the developmental
stages of the testis may furnish some indication on the cytotoxicity of drugs on these
cells.,Obviously, there is no way to predict if
the damage caused by the treatment will
persist and result in a n increased cell loss
during spermatogenesis.
The high incidence of testicular relapse in the treatment of
childhood acute leukemia, after cessation of therapy, has
prompted the requirement of bilateral testicular biopsies prior
to discontinuation of therapy. Children have been biopsied following the DEW policy on the protection of human subjects and
after informed consent.
Received May 20, 1983; accepted February 21, 1984.
492
G. ANELLI ET AL.
TABLE 1. Characteristics of 24 patients with childhood leukemia
Group
No. of
patients
I
I1
111'
8
12
A
B
C
D
Age at
biopsy (years)
Range
Mean
4.50
6.75
2.5-5
6-9
12
12
12
12
No, with
testicular
infiltrate
1
1
Mean
hormone value
(mIU/ml)
_____
FSH
LH
2.03
2.95
2.26
3.46
+
+
+-
'Patient A, prepuheral. Following hone marrow relapse, he underwent testicular irradiation (Fig.
11). Patient B, pubertal. Patient C, pubertal. Patient D, pubertal. He received a more aggressive
chemotherapy (ara-C and cyclophosphamide) (Fig. 10).
Since there is a n improved prognosis for
children treated for acute lymphoblastic leukemia, attention must be paid to the quality
of life of the survivors (Shalet, 1980),and the
knowledge of gonadal morphology following
chemotherapy represents a n important aspect, which must be taken into consideration.
Controversial results (Berry et al., 1972; Rapola et al., 1973; Lendon et al., 1978) are
related to the variability of drugs administered and their doses and combinations.
Moreover, differences in the sensitivity of the
prepuberal, pubertal, and adult testis to alkylating agent chemotherapy have been ren
ported, and these results are conflicting
(Lendon et al., 1978; Schilsky et al., 1980).
The results presented here are significant,
since they consider only prepuberal patients
who have been treated with the same combination of chemotherapeutic agents, for the
same period and at the same doses. Evidence
is presented that the combined chemotherapy used resulted in damage to 20% of seminiferous tubules in the first age group
considered (2.5 to 5 years) and 35% in the
second age group (6 to 9 years). The unaltered tubules of the second group presented
a reduction in number of type A spermatogonia with respect to control testis. Leydig
cells' morphology and endocrinologic data
were not apparently affected by the drugs.
MATERIALS AND METHODS
Twenty-four bilateral wedge testicular
biopsies (Rowley and Heller, 1966) of children aged between 2 and 12 years affected
by acute lymphoblastic leukemia were considered (Table 1). Testicular biopsies were
performed, under general anaesthesia and
following parents' consent, after 30 months
of therapy to recognize the presence of lymphoblastic cells in the interstitial tissue.
Twenty-three patients received standard chemotherapy (vincristine, prednisone, L-aspar-
I age
group
I I age
g roll P
Fig. 1. Histogram showing mean spermatogonia
number (n) per tubular cross section in group I (range
2.5-5, mean age 4.5 years) and group I1 (range 6-9,
mean age 6.75 years), P < 0.08.
Fig. 2. Area of preserved tubules in a 5-year-old testis. Spermatogonia (G) and Sertoli cells (S) are easy to
distinguish. The appearance of tubules and interstitial
tissue correspond to a control testis of the same age.
x200.
Fig. 3. Area of damaged tubules in a 9-year-old testis.
Extensive vacuolized regions are evident (arrows); Sertoli cells (S)present irregularly shaped condensed nuclei;
germinal elements are not distinguishable. x 580.
CHEMOTHERAPY ON TESTIS LEUKEMIA
493
494
G. ANELLI ET AL.
aginase, 6-mercaptopurine) and intrathecal
methotrexate and cranial irradiation (1,8002,400 rads) for the prevention of central nervous system leukemia. One patient (D, Table
1) received a more aggressive chemotherapy
(arabinofuranosyl-cytosine [ara-C] and cyclophosphamide [CP]) after bone marrow relapse. Five of the 24 patients (Table 1)showed
diffuse testicular infiltrate. One of these infiltrated patients (A, Table 11, following bone
marrow relapse, underwent testicular radiation and had a second testicular biopsy.
For ultrastructural analysis, samples of
testicular tissue were fixed in a mixture of
glutaraldehyde-paraformaldehyde in 0.2 M
cacodylate buffer, pH 7.4 (It0 and Karnovsky,
1968), and postfixed in 1% 0 ~ 0 4 .Tissues
were stained en bloc with aqueous 2% uranyl
acetate, dehydrated in ethanol, and embedded in Epon-Araldite. Thin sections stained
with lead citrate were examined with a Philips 400 electron microscope. One-micronthick sections were stained with methylene
blue and saphranine for light microscope and
histometric analysis and were photographed
with a Leitz Dialux 20 microscope. The values of FSH and LH (Table 1)were measured
by radioimmunoassay (Weintraub et al.,
1973).
type spermatogonia number per tubular cross
section was calculated for each patient and
for the entire group, counting a mean number of 20 undamaged tubules for each patient
in group I and a mean number of 30 undamaged tubules for each patient in group 11.
RESULTS
Tubules with gonia and Sertoli cells, which
appeared a s shown in Figure 2, were considered undamaged, since their histological
characteristics corresponded to control tubules. Tubules, such as those in Figure 3, were
considered damaged on the basis of disappearance of the spermatogonial population,
extensive vacuolated areas (Fig. 3, arrows),
and the appearance of the Sertoli cells, whose
fine structure is presented in Figure 6. The
ultrastructure of the damaged tubules confirmed that both germ cells and Sertoli cells
were affected (Fig. 8). The contour of Sertoli
cells appeared modified as a consequence of
retractions where plasma membranes were
juxtaposed (Fig. 8, arrows); the nucleus presented evident chromatin condensation.
These aspects are indicative of dysfunction of
Sertoli cells. Germ cells, surrounded by such
Sertoli cells, were altered too, showing a n
unusual vacuolization in the cytoplasm (Fig.
8).
Histometric Study
About 20% of the total number of tubules
The analysis of the general appearance of counted presented the above-mentioned morseminiferous tubules was performed before phology in age group I and about 35%in age
the histometric count. Tubules presenting group 11.
disappearance of spermatogonial population,
The mean diameter of undamaged tubules
vacuolated Sertoli cells, and altered contour in group I corresponded to that of the same
were considered damaged. We considered age age group reported in the literature for congroup I (mean age 4.5, range 2.5-5 years) trol testis (65 pm, according to Hadziseliand age group I1 (mean age 6.75, range 6-9 movic 119771and Nistal et al. [1982].In group
years), since the diameter of seminiferous 11, the mean diameter of normal undamaged
tubules, spermatogonia count, and Sertoli tubules corresponded to that of control testis
cell appearance are very similar within each
group in the analysis of control testis (Hadziselimovic, 1977; Furuya et al., 1980). Age
Fig. 4. Tubules in a 9-year-old testis. The diameter of
group I11 is not presented in the diagram
(Fig. 11, since the appearance of seminiferous the tubules is not increased with respect to the first
Gonia (arrows) are less numerous than in group
tubules varies greatly during late prepuberal group.
I. x300.
age; thus, it was not considered as a homoFig. 5. Aspect of tubules in a n 8-year-oldtestis. Many
geneous group. In age group I 20% and in
age group I1 35% damaged tubules were Sertoli cells ( S )present an irregular profile, a condensed
nucleus, and vacuolized areas. In such tubules spermaobserved.
togonia cannot be recognized. x220.
The number of transversely sectioned unFig. 6. Ultrastructure of a portion of a damaged tubdamaged seminiferous tubules per semithin
section and the number of A type spermato- ule in a 9-year-old testis. Degenerated Sertoli cells have
their limiting pIasma membrane; the nuclei (N)
gonial population per transverse tubular sec- lost
present indentations, chromatin condensation, and irtion were calculated using a calibrated ocular regular profiles. Degenerated mitochondria (arrows) and
micrometer with a x40 objective. Mean A vacuoles are present in the cytoplasmic area. ~8,800.
CHEMOTHERAPY ON TESTIS LEUKEMIA
495
496
G. ANELLI ET AL.
only in the case of the 6- to 7-year-old patients. The 9-year-old patients did not present the normal increase in cross-tubular
diameter (Fig. 4).
In group I, no relevant alteration in the
interstitial tissue was observed. Fibroblasts,
which represent the most common cell type,
were normal, and scattered groups of precursors of Leydig cells, mainly surrounding the
tubular wall, were present (Fig. 2).
In group 11, peritubular fibrosis and thickening of the basal membrane were frequent:
Leydig cells presented some characteristic
features of adult cells. Also in group 111, Leydig cells were recognizable even when a
heavy infiltrate was present (Figs. 9,121. Postradiation biopsy (Fig. 11)showed a striking
interstitial and tubular fibrosis. Irreversibly
damaged tubules in which cell types could
not be recognized appeared in consequence of
testis radiation. Moreover, a complete loss of
germ cells (Fig. 10) was observed in another
patient of this group treated with ara-C and
CP therapy. Leydig cells were still visible.
Germ Cells
It was previously demonstrated that from
birth to puberty Ap, Ad, and B spermatogonia as well a s primary spermatocytes can
be discerned (Hadziselimovic, 1977). The A
type spermatogonial population includes the
stem cells, and the histometric analysis was
conducted on this type of gonia (Fig. 11, which
could be easily recognized by their size and
shape (Fig. 2).
In group I (Fig. 2) the mean value (3.5 gonia
per tubule) obtained is in agreement with
that reported in the literaturefor control testis of the same age group (Hadziselimovic,
1977).
A type spermatogonia count carried out on
group I1 (Fig. 4) showed that the mean spermatogonia number per tubule (1.6 gonia) was
markedly decreased in comparison with that
of group I (Fig. 2).
Group I11 showed different morphologic
patterns (Table 1).Two pubertal patients (B
and C, Table 1) presented leukemic infiltrates (Figs. 9, 12) at the end of therapy and
displayed tubules with normal diameter, a
high number of gonia, and active spermatogenesis. One patient (A, Table 1) was still
prepuberal; he underwent testicular radiation and presented completely damaged seminiferous tubules (Fig. 11).The last one was
pubertal (D, Table l), but only Sertoli cells
were present in tubules (Fig. 10). This patient had been treated with ara-C and CP.
Sertoli Cells
Seminiferous tubules of testes from groups
I and I1 showed immature Sertoli cells. These
presented a pseudostratified disposition and,
in unaffected tubules, could be easily identified among germ cells (Figs. 2, 4). Their normal ultrastructural features are presented in
Figure 7. The nucleus had a small nucleolus
and dispersed chromatin except for a peripherally placed chromatin band. The cytoplasm
was characterized by small mitochondria,
abundant endoplasmic reticulum cisternae,
and vesicles.
The appearance of Sertoli cells was completely modified in damaged tubules (Fig. 3)
and also in scattered areas of some preserved
tubules (Fig. 5). Thick sections evidenced irregularly shaped, intensely stained nuclei,
surrounded by vacuolized regions (Fig. 3).
The contour of these cells could not be recognized. At ultrastructural level the nucleus of
Sertoli cells presented numerous indentations and irregular masses of condensed
chromatin; the cytoplasm was filled with a
large number of vacuoles and altered mitochondria, which lost their internal structure
(Fig. 6).
DISCUSSION
There are many difficulties in performing
human studies concerning the effect of combined chemotherapeutic drugs on fertility,
and scanty and controversial are reports concerning damage on gonadal function of
treated children (Rapola et al., 1973; Lendon
et al., 1978). The action of single tested drugs
on the seminiferous tubules of adult rodents
(Lu and Meistrich, 1979) is reported in literature. The most apparent effect of Vinca alkaloids (such as vincristine [VCR] and
vinblastine [VLB]) on testicular cells was a
partial killing of type B spermatogonia, and
these drugs apparently produced less damage than did ara-C and CP.
Despite these results on adult rodents, it is
well known that the reproductive function of
Fig. 7. Aspect of normal, immature Sertoli cells (S) in
a 5-year-old testis, showing round-shaped nuclei (N) with
dispersed chromatin. The cytoplasm is characterized by
the usual components. Sertoli-Sertoli contacts (arrows)
are well preserved. X3,500.
Fig. 8. Ultrastructure of a portion of a tubule in which
Sertoli cells (S) present a beginning of a degenerative
process: altered contacts (arrows) and nuclei (N) with
partially condensed chromatin. The germ cell (G) presents an extensive vacuolization in the cytoplasm.
~4,700.
CHEMOTHERAPY ON TESTIS LEUKEMIA
497
498
G. ANELLI ET AL.
CHEMOTHERAPY ON TESTIS LEUKEMIA
man is seriously affected by VLB Wilar, 1975)
and CP (Pairley et al., 1972).
The results herein reported on the effect of
a combined therapy on testicular morphology of children suggest that there is evident
damage which involves less than 35%of the
observed tubules. The reason why tubules
display different sensitivity to drugs is unknown: drug penetration would be the same
for all tubules, since during this period no
compartmentalization of seminiferous tubules exists. Moreover, it is impossible to establish if the combined therapy has a
preferential effect upon stem cells or whether
it is secondary to a n effect upon Sertoli cells.
A progressive increase in the number of A
type spermatogonia per transverse tubular
section has been reported in 4- to 10-year-old
control testis (Hadziselimovic, 1977; Miiller
and Skakkebaek, 1983).In group 11, the number of type A spermatogonia appears reduced
in comparison with control testis of the same
age. This result may be related with some
spermatogonial degeneration or to a mitotic
arrest in the existing spermatogonial population. Thus, age group I1 appears the most
affected.
In age group 111, two patients presented
some preserved tubules with spermatogenesis, suggesting that the cytotoxic effect of
drugs did not prevent the development of
progeny of some stem cells.
Leydig cells appear to be the cells least
sensitive to drugs in prepuberal testes studied. Their morphology correlates well with
the gonadotropins mean values (Table 1)
which are in agreement with the data of the
Fig. 9. Seminiferous tubules in a 13-year-oldpubertal
boy. Active spermatogenesis is evident; leukemic cells
(arrows) are present in the interstitial tissue among Leydig cells. X420.
Fig. 10. Seminiferous tubules of a pubertal 13-yearold boy who received ara-C therapy. The loss of germ
cells is evident, and only Sertoli cells occupy the tubules.
The irregular profile of tubules and thickening of the
basement membrane (arrows) evidence the damage of
this therapy. X420.
Fig. 11. Appearance of tubules, following testis radiation, in a 12-year-old testis. It is possible to recognize
only the profile of the tubules, which are completely
damaged. A Leydig cell (L)is morphologically identifiable in the interstitial tissue. X420.
Fig. 12. Leydig cells (L) in a pubertal testis with
lymphoblasts. Among blast cells (arrows) a Leydig cell
presents the typical morphology, with numerous lipid
inclusions. x 1,280.
499
literature (Fischer and Aur, 1982). Thus, despite chemotherapy and radiation, the hypothalamic pituitary function appears intact in
acute lymphoblastic leukemia.
The results presented demonstrate that the
combined chemotherapy used appears to be
considerably less destructive than other therapies, such as CP, ara-C (Pairley et al., 1972;
Buchanan et al., 1975) (Fig. lo), and radiation (Fig. 11).It can be foreseen that suspension of the therapy, in cases with favorable
prognosis, will result in reestablishment of
development conditions, at least in apparently undamaged tubules.
ACKNOWLEDGMENTS
This research was supported by MPI 1982.
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