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THE ANATOMICAL RECORD 247:379–387 (1997)
Effects of Carbendazim (Methyl 2-Benzimidazole Carbamate; MBC)
on Meiotic Spermatocytes and Subsequent Spermiogenesis
in the Rat Testis
of Veterinary Anatomy, Faculty of Agriculture, Miyazaki University,
Miyazaki, Japan
2Department of Veterinary Biosciences, University of Illinois, Urbana, Illinois
Background: Benzimidazole fungicide, carbendazim, is
known to adversely affect Sertoli cells by disrupting microtubules, which
induces sloughing of elongate spermatids in a stage-specific manner. This
study determines the direct effects on dividing germ cells and the subsequent effects on spermiogenesis.
Methods: Carbendazim was administered orally to male rats (100 mg/kg),
and their testes were processed for histological evaluation at various
post-treatment intervals up to day 20.0.
Results: The sloughing of elongate spermatids was observed as reported
previously. In addition to this Sertoli cell lesion, necrosis of dividing
spermatocytes in stage XIV was observed at 8 hours post-treatment. At day
1.5, empty spaces of missing step 1 spermatids were seen in stage I. At days
4.5 and 7.5, normal round spermatids were missing, but large round
spermatids (megaspermatids) and binucleate spermatids were common.
The megaspermatid nucleus was approximately 33% larger in diameter
than normal round spermatids. At day 10.5, megasteps 10–12 spermatids,
binucleate spermatids, and three to four different steps of spermatids
coexisting in the same tubule section were present in stages X–XII. In
addition, abnormally shaped elongating spermatids were observed having distorted heads and nuclear invagination containing microtubules. At
day 20.0, empty spaces of missing diplotene spermatocytes were seen in
stage XIII.
Conclusions: The present observations show that carbendazim has
rapid direct effects on meiotic spermatocytes and latent effects on spermatids, leading to morphological abnormalities and failure of spermiogenesis. These effects are found independent of occlusions in the efferent
ductules. Anat. Rec. 247:379–387, 1997. r 1997 Wiley-Liss, Inc.
Key words: carbendazim; rat; spermatocytes; spermatids; spermiogenesis; testis
Studies of the effects of benzimidazole fungicides,
benomyl and carbendazim, on the testis have shown
that these agents cause cleavage of the apical cytoplasmic processes of Sertoli cells and sloughing of immature
germ cells (Parvinen and Kormano, 1974; Hess et al.,
1991; Nakai and Hess, 1994). The proposed mechanism
contributing to the sloughing is deformation of Sertoli
cells, mainly due to disruption of its microtubules
(Nakai and Hess, 1994; Nakai et al., 1995). When the
animals are exposed to relatively high doses of benomyl
or carbendazim, the efferent ductules become irreversibly occluded with the sloughed materials from the
seminiferous epithelium, which subsequently leads to
seminiferous tubular atrophy and male infertility in
the rat (Carter et al., 1987; Hess et al., 1991; Nakai et
al., 1992; 1993).
The present study was designed to determine the
effects, unrelated to occlusions of the efferent ductules
by sloughed germ cells, of carbendazim on the seminiferous epithelium. The design incorporates the exposure
of animals to a low dose of carbendazim. This experimental design is important for determining the prognosis of
carbendazim-induced male infertility, as there is a
possibility of recovery of the seminiferous epithelium
that has undergone sloughing, except when efferent
ductules are occluded. The data show that recovery
Received 20 May 1996; accepted 9 September 1996.
*Correspondence to: Masaaki Nakai, Department of Veterinary
Anatomy, Faculty of Agriculture, Miyazaki University, Miyazaki 88921, Japan.
from massive sloughing is possible if the efferent ductules are intact; however, abnormal spermatids could be
produced in the recovering seminiferous epithelium
due to the extended effects of carbendazim.
Animals and Experimental Design
A total of 43 male Sprague-Dawley rats (90–100 days
of age) were used. They were housed two or three per
cage with a 12-hour alternating light-dark cycle and
were allowed free access to diet and water. Five or six
animals were assigned to individual sampling intervals. Carbendazim suspended in corn oil was administered to the animals by a single oral gavage. The known
minimum effective dose of carbendazim is 50 mg/kg,
but changes in the seminiferous epithelium with this
dose are subtle (Nakai et al., 1992). Therefore, the dose
of 100 mg/kg was used in the present study.
At selected time intervals after treatment with carbendazim, the animals were deeply anesthetized with
an intraperitoneal injection of pentobarbital (1 ml/
animal), and the testes and epididymides were fixed
with 4% glutaraldehyde in 0.1 M cacodylate buffer,
using a vascular perfusion technique (Hess and Moore,
1993), at 8 hours, and again at 1.5, 4.5, 7.5, 10.5, and
20.0 days post-treatment. As controls, three animals
each were assigned to days 7.5 and 20.0, and one
animal each to the remaining intervals. Corn oil alone
was given to control animals, and their testes were
processed using the same technique as used for the
treatment group.
Histological Methods
Testicular tissue blocks were embedded in JB-4 plastic resin (Polyscience, Inc., PA). Sections were cut with
glass knives at 2.5 µm, and stained with periodic
acid-Schiff reaction (PAS) and hematoxylin. Caput epididymides, excised away from the testes, were embedded in paraffin. Caput epididymides, containing the
efferent ductules and the initial segment, were serially
sectioned at 4 mm. Every 10th section was mounted on
a glass slide and stained with hematoxylin and eosin.
Patency of the efferent ductules was confirmed by the
presence of intact ductules and sperm in the epididymal
ducts. Testes were used if there were no associated
efferent ductal occlusions, or when only minor ductal
occlusions were observed and the associated seminiferous tubules exhibited no expansion in diameter. A total
of 64 out of 86 testes, including controls, were utilized to
evaluate the testicular damages. When particular cell
types were missing or showed abnormalities in the
course of observation, we predicted the original cell
type affected by treatment using a software program
named STAGES (version 1.0, Vanguard Productions,
Inc., IL). In the STAGES program, it is possible to
backtrack in time to the original cell type that the
observed affected cell would have been at the time of
Testes collected at day 10.5 were further processed
for transmission electron microscopy, because various
abnormalities in spermatid head morphology were observed by light microscopy. Tissue blocks were rinsed in
0.1 M cacodylate buffer overnight. They were post-fixed
in 1% osmium tetroxide containing 1.25% potassium
ferrocyanide (Russell and Burguet, 1977), dehydrated
in graded ethanol series, and embedded in Quetol 812.
Thick sections for light microscopy were stained with
toluidine blue. Ultrathin sections were stained with
uranyl acetate and lead citrate and observed by a
Hitachi H-800MU transmission electron microscope.
Quantitative Analysis
To determine whether to include a testis in which a
few efferent ductules were occluded, the mean diameter
of 50 nearly circular seminiferous tubules were measured in individual testes and compared to the mean
diameter of 50 seminiferous tubules in control testes.
Nuclear diameters of steps 5 and 7 were measured, and
the length between the apical and caudal ends of step
11 spermatid heads was determined in the carbendazimtreated and control testes collected at days 4.5, 7.5, and
10.5. Differences in the mean values between treated
and control testes were compared in individual parameters using the t-test. Stages of seminiferous tubules
were determined in the treated and control testis
collected at days 7.5 and 20.0, and the frequency of
stage VII tubules was compared using the x2 test. A
probability of less than 1% was considered significant
in both tests.
Eight Hours
Sloughing of immature elongate spermatids was
observed in stages I, III, IV, VI, VII, and X–XIV tubules,
but sloughing of steps 16 and 17 spermatids in stages
III and IV was rare. Another change seen at this
post-treatment interval was necrosis of meiotic spermatocytes in stage XIV tubules (Fig. 1). The necrosis
occurred most frequently in metaphase primary and
secondary spermatocytes, but newly formed secondary
spermatocytes occasionally showed necrotic features.
Back-calculation using STAGES software indicated that
the cells were meiotic spermatocytes in stage XIV at the
time of treatment.
Day 1.5
Immature elongate spermatids were often missing in
stages I–III, V–VII, and XIII–XIV, and were apparently
sloughed during the first 36 hours post-exposure. Further changes characteristic of this post-treatment interval were mainly observed in stage I. These changes
included simultaneous disappearance of step 1 and step
15 spermatids in the same stage I tubule, and empty
spaces of missing step 1 spermatids alone (Fig. 2a).
Round bodies that were PAS-positive and about the
same size as step 1 spermatids were often seen in stage
I tubules that also contained empty spaces of missing
step 1 spermatids (Fig. 2a). Round spermatids that
were larger in diameter than normal step 1 spermatids
(Fig. 2b) and binucleate step 1 spermatids were occasionally seen in stage I tubules (Fig. 2a). Backtracking
indicated that adversely affected step 1 spermatids had
most probably been meiotic spermatocytes at the time
of treatment.
Fig. 1. Stage XIV. a: At 8 hours post-treatment, necrosis of meiotic spermatocytes is noted by the
granular appearance of the cytoplasm (arrowheads). PAS-hematoxylin. 3390. b: A control stage XIV
tubule. PAS-hematoxylin. 3390.
Day 4.5
Areas of missing elongate spermatids were seen in
stage I–VII and XI–XIV tubules. At this post-treatment
interval, additional abnormalities were observed mainly
in stage V, where two spermatid steps (5 and 17) were
found missing in the same tubule, and empty spaces of
missing step 5 spermatids were observed (Fig. 3a).
Another characteristic feature common to stage V
tubules in the treated testis was the presence of large
round spermatids (megaspermatids, Fig. 3b). The megaspermatids were usually observed in tubules that also
showed missing either round or elongate spermatids.
These megaspermatids had normal morphology, but
were significantly larger in cellular and nuclear diameters (approximately 33%) than normal step 5 spermatids (Table 1). In addition, binucleate cells were often
seen in tubules containing megastep 5 spermatids. In
these cells, two neighboring round nuclei shared one
acrosome (Fig. 3b inset). Multinucleate giant cells
containing more than three nuclei were rarely observed
in stage V tubules. Backtracking indicated that step 5
spermatids had most probably been meiotic spermatocytes
at the time of treatment. In stage X tubules of the treated
testes, nuclei of elongate spermatids (step 19 in appearance) were seen in the basal level of the seminiferous
epithelium, indicating failure of sperm release (not shown).
Day 7.5
Areas of missing elongate spermatids were seen in all
stages except for stages VIII and IX. At this posttreatment interval, additional abnormalities were observed, mainly in mid- to late-stage VII, where both
step 7 and step 19 spermatids were missing or empty
spaces of missing step 7 spermatids were observed (Fig.
4a). The megastep 7 spermatids were often contained in
stage VII, exhibiting missing spermatids (Fig. 4b). As
observed at day 4.5, the megastep 7 spermatids showed
an appearance similar to that of normal step 7 spermatids, except for an increase in the cellular and nuclear
diameters (Table 1). Binucleate cells were often ob-
served in stage VII tubules containing the abovementioned abnormalities (Fig. 4b, inset). The neighboring nuclei often shared the acrosome. Multinucleate
giant cells with more than three nuclei were observed
in stage VII (Fig. 4a, inset). Backtracking indicated
that step 7 spermatids at day 7.5 had most probably
been meiotic spermatocytes at the time of treatment.
Failure of sperm release was also seen in stage X and
XIII tubules (not shown).
At day 7.5, frequency of stage VII tubules in the
treated testis was 23.8%, which was not significantly
different from that in the control (P , 0.01), suggesting
a normal progression of spermiogenesis after treatment
(Table 2).
Day 10.5
Areas of missing elongate spermatids were observed
in stage I–III, VII, and X–XIV tubules. Additional
abnormalities at this post-treatment interval were
observed mainly in stages X–XII. Megasteps 10–12
spermatids were observed, but megastep 11 spermatids
were the most numerous (Fig. 6a). However the number
of megaspermatids was less than previously observed
at steps 5 and 7. The heads of megastep 11 spermatids
were significantly longer in length than those of the
control (Table 1). Another striking observation at this
interval was the coexistence of abnormal steps of
spermatids in stages X–XI (approximately steps 7–10
or 11; Fig. 6b), indicating retardation of spermiogenesis. In these tubules, spermatids that showed the most
advanced development were used for stage identification. Abnormally shaped spermatid nuclei were seen in
stages X–XI. These abnormalities included various
distortions of nuclei (Fig. 6c), nuclei without chromatin
condensation (Fig. 6c), nuclear invaginations (Fig. 6e,
inset), and binucleate elongate spermatids that shared
an acrosome (Fig. 6d). Multinucleate giant cells with
round nuclei were rarely observed, but those of elongate
spermatids were not. The nuclear invaginations usually occurred at the caudal surface of the nucleus, and
Figs. 2–5
TABLE 1. Size differences between normal spermatids in
control and mega spermatids in carbendazim-treated
Spermatid steps
Normal spermatidsc
Mega spermatidsc
8.2 6 0.07
8.1 6 0.59
15.0 6 0.17
10.7 6 0.11d
10.8 6 0.14d
19.0 6 0.42d
aNuclear diameter.
bDistance between apical and caudal ends of the spermatid heads.
cMean (µm) 6 SEM. N 5 23 for mega step 11 spermatids. N 5 50 for the
dSignificantly different from normal (p , 0.01).
TABLE 2. Frequency of stage VII tubules in the control
and carbendazim-treated testes
intervals (days)
21.7 6 0.45
22.6 6 0.79
23.8 6 0.69b
24.2 6 0.40b
percent 6 SEM. N 5 2,301 for control at day 7.5, 2,432 for
carbendazim-treated at day 7.5, 2,265 for control at day 20.0, and 3,130 for
carbendazim-treated at day 20.0.
bNot significantly different from the controls at each interval (p , 0.01).
the invagination was directed toward the dorsal aspect
or apex of the nucleus. The invaginations contained
aggregates of microtubules (Fig. 6e). The ends of these
microtubules were embedded in electron-dense flocculent material that was similar in appearance to that
associated with normally positioned manchette microtubules (Fig. 6e). Spermatids with invaginated nuclei
often showed manchettes originating from the normally positioned nuclear ring. However, the nuclear
ring was displaced somewhat caudally in a few spermatids (Fig. 6e). Backtracking indicated that steps 10–12
spermatids had been meiotic spermatocytes at the time of
treatment. Failure of sperm release was seen in stage X.
Day 20.0
The major change in the germ cell population at this
post-treatment interval was missing elongate spermatids in stage I, II, VI, and VII tubules. In addition,
diakinetic spermatocytes were occasionally missing in
Fig. 2. Stage I tubules at day 1.5. a: Step 1 spermatids are missing,
leaving spaces in the epithelium. PAS-positive round bodies (arrowheads) and a binucleate cell (arrow) are seen. PAS-hematoxylin. 3350.
b: Large step 1 spermatids (arrowheads). Toluidine blue. 3870.
Fig. 3. Stage V tubules at day 4.5. a: Many step 5 spermatids are
missing. PAS-hematoxylin. 3350. b: Megastep 5 spermatids (arrowheads). Toluidine blue. 3870. Inset: Binucleate cell sharing an
acrosome. Toluidine blue. 3870.
Fig. 4. Stage VII tubules at day 7.5. a: Step 7 spermatids are
missing, leaving spaces in the epithelium. PAS-hematoxylin. 3350.
Inset: Multinucleate giant spermatid sitting near the lumen. PAShematoxylin. 3350. b: Megastep 7 spermatids (arrowheads) are
approximately 33% larger than normal. Toluidine blue. 3870. Inset:
Binucleate cell sharing an acrosome. Toluidine blue. 3870.
Fig. 5. Control seminiferous tubules. a: Stage I. PAS-hematoxylin.
3350. b: Stage V. PAS-hematoxylin. 3350. c: Stage VII. PAShematoxylin. 3350.
stage XIII, leaving behind large empty spaces in the
epithelium (Fig. 7a). Megastep 19 spermatids were not
identifiable in stage VII at this interval. Backtracking
indicated that step 19 spermatids had most probably
been meiotic spermatocytes, and that diakinetic spermatocytes had been either late type B spermatogonia or
preleptotene spermatocytes in stage VI. Again, failure
of sperm release was seen in stages X–XI (Fig. 7b).
Frequency of stage VII tubules at day 20.0 was not
significantly different from controls (Table 2).
Testes of the control animals showed normal histological structures (Figs. 1B; 5a–c; 7c,d), except for a rare
incidence of necrotic spermatocytes, failure of sperm
release, megaround spermatids, and binucleate spermatids. However, cells with these abnormalities occurred
independently in the control testes, not in clusters as in
the treated testes. Spermatids with nuclear invaginations, and multinucleate giant cells with more than
three nuclei were not observed in the controls.
Benzimidazole compounds, benomyl and carbendazim, are known to cause premature sloughing of germ
cells, along with cleaved cytoplasmic processes of Sertoli cells, (Hess et al., 1991; Nakai and Hess, 1994),
necrosis of meiotic spermatocytes (Parvinen and Kormano, 1974), occlusion of the efferent ductules (Hess et
al., 1991; Nakai et al., 1992; 1993), and seminiferous
tubular atrophy (Carter et al., 1987; Hess et al., 1991;
Nakai et al., 1992). It is the occlusion of efferent
ductules that is a crucial factor for the atrophy of
seminiferous tubules. In other words, continuation of
spermatogenesis depends on the degree of injury to the
efferent ductules (Hess et al., 1991; Nakai et al., 1992).
In the present study, at a dosage that does not induce
occlusions, we predicted that spermiogenesis would
continue in a normal manner, except for a temporal
absence of spermatids that were sloughed and meiotic
spermatocytes that showed necrosis. However, our observations reveal that carbendazim induces not only
necrosis of meiotic spermatocytes in stage XIV, but also
a number of various morphological abnormalities in
developing spermatids, and a partial failure of spermiogenesis. The incidence of some morphological abnormalities was greatly increased compared to the controls,
and others were newly induced by carbendazim. These
effects occurred in testes having intact efferent ductules. Therefore, carbendazim had direct effects on the
seminiferous epithelium independent of efferent ductule dysfunction.
Large, round spermatids have been observed in the
mouse testis after treatment with various chemotherapeutic agents, including microtubule-disrupting agents
(Lu and Meistrich, 1979; Meistrich et al., 1982). Large,
round spermatids are about the same size as secondary
spermatocytes, and are assumed to be diploid cells or
cells with two times the DNA content of haploid. In
Fig. 6. Spermatids in stage X–XI tubules at day 10.5. a: Megastep 11
spermatid (arrow). PAS-hematoxylin. 3370. b: Retardation of spermiogenesis. Different steps of spermatids (arabic numerals) coexist in this
tubule. PAS-hematoxylin. 3370. c: Abnormally shaped nuclei without
chromatin condensation (arrowheads). Toluidine blue. 3920. d: Elongating binucleate spermatid sharing an acrosome (arrow), and round
spermatid (step 7) showing retardation of development (arrowhead).
Toluidine blue. 3920. e: An electron micrograph of elongating
spermatid with a nuclear invagination containing microtubules (asterisk) and an abnormally positioned manchette. The ends of microtubules within the nuclear invagination are embedded in a flocculent
material (small arrow). Nuclear ring of the ectopic manchette is
displaced caudally (open arrow). Large arrow 5 flagellum. 311,300.
Inset: A light micrograph of a nucleus with a nuclear invagination
(arrow). Toluidine blue. 3920.
addition, it is reported that near-diploid spermatozoa,
especially those with giant heads, contain near-diploid
amounts of DNA, and that they are originally due to an
abnormal meiotic division (Stolla and Gropp, 1974).
This is supported by recent studies indicating that
colchicine and vinblastine induce aneuploidy in spermatocytes (Miller and Adler, 1992; Leopardi et al.,
1993). Although the number of chromosomes and the
amount of DNA were not determined, megaspermatids
observed in the present study are probably the same
cell type as large round spermatids; and, therefore, it is
possible that carbendazim induces spermatids with
aneuploidy. In the mouse, carbendazim caused an increase in the number of diploid cells of the testis at 7
days post-treatment (Evenson et al., 1987). Presumably
these cells could include a subpopulation of megaspermatids. The induction of aneuploidy by carbendazim or
benomyl is also known in other cell types (Hummler
and Hansmann, 1988; Zelesco et al., 1990; Zuelke and
Perreault, 1995).
Megaspermatids seemed to develop normally up to
step 12. It is reported that aneuploid spermatogenic
precursor cells (secondary spermatocytes) develop to be
mature spermatozoa despite the genetic defect (Stolla
and Gropp, 1974). The origin and fate of megaspermatids must be clarified to evaluate the potential risks of
chromosomal aberrations in spermatozoa induced by
Fig. 7. Seminiferous tubules at day 20.0. a: Diakinetic spermatocytes are missing in stage XIII tubule (asterisks). PAS-hematoxylin.
3390. b: Step 19 spermatids in stage XI tubule, indicating failure of
sperm release. PAS-hematoxylin. 3390. c: Control stage XIII tubule.
PAS-hematoxylin. 3390. d: Control stage XI tubule. PAS-hematoxylin. 3390.
Failure of Spermiogenesis
ment to retard their development, and that the retardation likely becomes obvious in spermatids later than
step 7 spermatids. Retardation may be attributable to
abnormality of spermatids, dysfunction of Sertoli cells,
or both. However, if retardation is due to dysfunction of
Sertoli cells, it should be observed in other stages and
post-treatment intervals besides stage X–XI at day
10.5. Therefore, it is likely that the cause of retardation
mainly resides in the spermatids. The fate of retarded
spermatids is unknown, but they are probably removed
from the seminiferous epithelium in the subsequent
stages by phagocytosis or sloughing, because retarded
spermatids did not occur in stage VII at day 20.0.
When specific types of germ cells are destroyed, there
appear windows or spaces where cells are missing in
later stages of spermatogenesis (Russell et al., 1990). In
the present study, necrosis of meiotic spermatocytes
resulted in windows of missing round spermatids in the
expected stages at individual post-treatment intervals.
Although these spaces were seen neither in stages
X–XII at day 10.5 nor in stage VII at day 20.0, the
elongate spermatid steps 10–12 and 19 were missing in
these tubules, respectively. In addition, the frequency of
stage VII tubules in the testicular sections at days 7.5
and 20.0 did not differ from that of control. These
suggest that spermatids that were not in meiotic division at the time of treatment continued with normal
development, which is consistent with the claim that
arrest and retardation of spermatogenesis does not
generally occur (Russell et al., 1990).
However, three or four different steps of spermatids
were sometimes observed to coexist in stage X–XI
tubules at day 10.5, but not in stage VII at day 7.5, nor
earlier. This suggests that carbendazim causes some
spermatocytes that are in meiosis at the time of treat-
Binucleate Cells
Multinucleate giant cells of round spermatids are a
common abnormality in the testis under a variety of
experimental and pathological conditions (Smith, 1962;
Russell et al., 1987; Singh and Abe, 1987; Russell et al.,
1991). They are assumed to be formed by opening of
intercellular bridges among spermatids (Russell et al.,
1990), and this phenomenon has been attributed to the
dysfunction of cytoskeletal elements, especially actin
filaments supporting the bridges (Russell et al., 1987;
Singh and Abe, 1987). Formation of multinucleate giant
cells observed in the present study could be explained
by this manner. However, it is uncertain whether
formation of multinucleate giant cells is a result of the
direct effect of carbendazim, because they are first
observed at day 4.5 (although rarely) and become more
common at days 7.5 and 10.5.
On the other hand, binucleate cells, one of the forms
of multinucleate cells, were often observed in the
present study. If these cells are formed by the same
mechanism as mentioned above, each nucleus should
have its own acrosome, which is not the case in the
present study. It is reasonable, therefore, to assume
that they are formed by failure of cytokinesis of secondary spermatocytes following nuclear division, as explained for other binucleate cells (Carter, 1967; Meistrich et al., 1982; Russell et al., 1987). This is supported
by the present observation that binucleate cells occur
as early as day 1.5.
Whereas multinucleate giant cells other than binucleate cells always contain round nuclei, nuclei of binucleate cells show nuclear elongation, at least up to the step
10 spermatid. Although the fate of binucleate spermatids is not known, it is possible that they develop into
binucleate spermatozoa.
Abnormally Positioned Manchette
Abnormally positioned manchette microtubules (‘‘ectopic manchette’’ by Meistrich et al., 1990), including
those in the nuclear invagination, have been observed
in testes treated with colchicine (Handel, 1979) and
taxol (Russell et al., 1991), and in the testes of mutant
mice (Bryan, 1977; Cole et al., 1988; Meistrich et al.,
1990). In the testes treated with these microtubule
poisons, the time lag between treatment and the incidence of abnormality is between 1 and 3 days. If
carbendazim acts directly on spermatids that are just
beginning their nuclear elongation and development of
manchette, then ectopic manchettes should occur at
earlier intervals. In the present study, however, step
10–11 spermatids with ectopic manchettes occurred in
stages X–XI at day 10.5. Thus, carbendazim does not
seem to act directly on the developing manchettes.
Rather, it seems that the germ cells originally affected
by carbendazim are the spermatocytes in meiosis at the
time of treatment, and that these effects are not expressed until the spermatid steps at the start of nuclear
elongation and manchette development, which is similar to the ectopic manchette in the mutant spermatozoa
(Meistrich et al., 1990). The underlying mechanisms of
carbendazim-induced manchette displacement are currently unknown, but they may be different from those of
other microtubule poisons, although the outcomes of
treatments are similar.
The present study did not focus on germ cells earlier
than meiotic spermatocytes. However, backtracking for
missing elongate spermatids (data not shown) and
diakinetic spermatocytes suggests that spermatocytes
and spermatogonia could be a target of carbendazim, as
reported in the mouse testis (Evenson et al., 1987).
In conclusion, the present study demonstrates that
despite the presence of intact efferent ductules, car-
bendazim induces various morphological abnormalities
in developing spermatids, which is more serious than
first expected. The observed abnormalities in spermatids are delayed effects of carbendazim that cause
severe disruption of spermatogenesis in the affected
tubules, either due to direct effects on meiotic spermatocytes, or indirect effects through the known effects on
Sertoli cells (Nakai and Hess, 1994; Nakai et al., 1995).
The authors thank Dr. K. Toshimori, Department of
Anatomy, Miyazaki Medical College, for his valuable
comments on the manuscript.
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