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Selective effects of testosterone and isoproterenol upon regenerating submandibular gland isografts in BALBc mice.

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Selective Effects of Testosterone and Isoproterenol
upon Regenerating Submandibular Gland
Isografts in BALB/c Mice
KAZUMASA HOSHINO AND C. D. LIN
Department of Anatomy, Health Sciences Centre, University of
Western Ontario, London, Onturio, Canada
ABSTRACT
Single submandibular glands were isografted intraperitoneally to
normal BALB/c mice. From the day following transplantation, 20 m g testosterone
enanthate fortnightly, or daily intraperitoneal injections of 0.25 mg per gram body
weight of isoproterenol-HC1 were given. Control mice were untreated. The grafts were
removed from five mice in each group 1, 2, 4, or 8 weeks after grafting. Regardless
of treatments, only ductal cells were observed one week after transplantation. With
testosterone treatment for longer than two weeks, their remaining ductal cells tended
to accumulate secretory granules. Some appeared to be secretory tubules resembling
those in the glands of normal adult male mice. When the glands were transplanted
from females to male hosts, surviving grafts responded to endogenous androgen of
the hosts, and some secretory tubules reappeared without testosterone treatment.
Contrarily, with isoproterenol treatment for longer than two weeks, acinar cells reappeared in the grafts, but no secretory tubules were observed. The remaining ductal
cells often underwent hyperplastic changes with reappearing tubules or acinar cells
amongst them corresponding to post-transplantational treatment. Mechanisms of differentiation of these three different components of submandibular glands were fourid
to be different and each had specific affinity to testosterone or isoproterenol. Grafts
were removed from hosts treated with testosterone for two months, and when 4 or 8
of these grafts were retransplanted into each new host, they demonstrated a lethal
effect.
Intraperitoneal isografts of the submandibular glands in BALB/c mice lose their
characteristic histological features and
consist only of duct-like structures. Without any further treatment to the hosts
these duct-like structures often undergo
hyperplastic changes (Hoshino and Lin,
'68a). However, when the donors are
males, varying degrees of mortality of the
hosts are observed (Hoshino and Lin, '68b,
'69a); also the lethal factor is testosterone
dependent (Lin and Hoshino, '69).
Lacassagne ('40) first reported that the
mouse submandibular gland exhibits sexual dimorphism and also that testosterone
may stimulate the tubular portion of the
gland of the female mouse to make it
resemble that of a male mouse.
Selye et al. ('61) reported that chronic
treatment of the rat with isoproterenol-HC1
solution resulted in the enlargement of
the submandibular and parotid glands
mainly due to hypertrophy. An increased
mitotic proliferation of the acinar cells of
these glands was also reported.
ANAT. REC., 167: 489-496.
The specific responses of submandibular
isografts to androgen and isoproterenolHC1 in the BALB/c strain of mice were
studied in respect to histogenesis of regeneration of the surviving graft tissues.
The preliminary report was previously
presented (Hoshino and Lin, '69b).
MATERIALS AND METHODS
The animals used were adult BALB/c
mice of both sexes, and were raised and
maintained at our laboratory under a uniformly controlled environment and provided with Purina Laboratory Chow and
water ad libitum (Hoshino and Lin, '69).
Single submandibular glands were intraperitoneally isografted immediately after
their removal from the female donors,
while the hosts were under Nembutal
anesthesia. In control groups, no posttransplantational treatments were given
to the hosts. When the control hosts were
male, their grafts were removed 1, 2, or
4 weeks after transplantation. In another
Received Dec. 16, '69. Accepted March 2, '70.
489
490
K. HOSHINO A N D C. D. LIN
control group, where females were used
as the hosts, the grafts were removed 1,
2, 4, or 8 weeks following transplantation.
In the experimental groups, only female
hosts were used. In one group, 20 mg of
testosterone enanthate in 0.1 ml of sesame
oil was dorsal subcutaneously injected
into each mouse fortnightly, and the
grafts were removed from these hosts 1,
2, 4, or 8 weeks after transplantation. In
another group, daily intraperitoneal injections of 0.25 mg per gram body weight
of isoproterenol-HC1 in normal saline solution were administered. The grafts were
removed 1, 2, or 4 weeks after transplantation. In both groups, the first injection
was given on the day following transplantation.
The grafts were removed from five host
mice at each interval in all the groups
described above, fixed in Susa fixative,
embedded in paraffin, cut at 5 I.L and
stained with hematoxylin and eosin, or
with periodic acid-Schiff reagent (PAS)
accompanied with either light green or
Orange G and hematoxylin (Jacoby and
Leeson, '59) for histological observations.
The in situ submandibular glands were
removed from normal control adult mice
and studied in the same manner as the
grafts.
In the final group, either single or
quadruple submandibular glands were intraperitoneally isografted from female
donors to female hosts. Testosterone enanthate was administered to the hosts in a
manner identical to the one described
above. Two months after transplantation,
the grafts were removed from all of these
hosts, and either four or eight recovered
submandibular grafts were retransplanted
into each of 12 normal untreated female
mice. Each of the other six normal untreated female mice received eight submandibular grafts recovered from the
host mice without any post-transplantational treatments f o r two months. The
purpose was to investigate the possible
lethal effect which might be exhibited by
those grafts.
RESULTS
1. Transplantability of submandibular
glands in the hosts
When single submandibular glands were
transplanted intraperitoneally from female
donors to female hosts, all the grafts were
recovered at the time of autopsy one to
eight weeks after transplantation.
When four submandibular glands were
transplanted from female donors to each
of the ten female hosts, 90% of the 40
submandibular gland grafts were recovered
eight weeks after transplantation. Each
mouse had at least one graft remaining.
2. Histological observations
( a ) In situ submandibular glands in
normal control adult mice. The acinar
cells of the submandibular glands of normal control adult mice of both sexes consisted of usually pyramidal shaped cells
having basophilic cytoplasm (figs. 1, 2).
The acinar cells were PAS positive (figs. 3,
4). The secretory tubules consisted of
columnar cells with eosinophilic cytoplasm (figs. 1, 2). In female controls, the
small secretory tubules were sporadically
distributed (figs. 2, 4), whereas in male
controls the large secretory tubules filled
with eosinophilic or Orange G positive
granules were predominant (figs. 1, 3).
The intralobular ducts (fig. 3 ) and the
intercalated ducts were also stained with
Orange G .
( b ) In
experimental
mice. b-I:
Control grafts. The surviving and regenerating submandibular isografts in untreated hosts consisted only of duct-like
structures when examined one or two
weeks after transplantation. The amount
of duct-like structure in these grafts varied
from one to another, but one week grafts
tended to have surviving structures in their
peripheral regions (having the central portion degenerated). The central portion of
the grafts were gradually replaced by regenerating duct-like structures. After four
weeks or longer after transplantation,
some duct-like structures underwent hyperplastic changes (fig. 5 ) . When isografts were obtained from female donors
and placed into female hosts, acinar celltype structures were occasionally observed
among the predominant duct-like structures when removed four or eight weeks
after transplantation. However, when the
grafts were isografted from female donors
to male hosts, the secretory tubules were
also sometimes observed in addition to
the occasional acinar cells.
REGENERATING SUBMANDIBULAR ISOGRAFT
b-2: In hosts treated with testosterone
enanthate.
The grafts recovered from
the hosts treated with testosterone enanthate for one week following transplantation were not distinguishable from those
in control grafts. However, in the hosts
receiving the treatment for longer than
two weeks, the grafts began to have reappearing secretory tubules among the
duct-like structures. These secretory tubule-like structures first appeared in the
peripheral regions of the grafts and
gradually appeared in the central portion
as the period of androgen treatment to
the hosts was prolonged (figs. 6, 8, 9).
PAS-positive acinar cell-like structures
were only rarely observed.
b-3: In hosts treated with isoproterenoZ-HCZ. The surviving graft tissues consisted mainly of duct-like structures with
only an occasional presence of the acinarllke structures when examined one week
after transplantation with daily injections
of isoproterenoLHC1. When the treatment
was given to the hosts for longer than
two weeks, more acinar cell-like structures reappeared from the peripheral regions centralward (figs. 7, 10). However,
these structures usually did not reappear
in the central portion of the grafts, most
of which was occupied by the duct-like
structures.
3. The lethal effect exhibited by
retransplanted grafts
When the surviving grafts in the hosts
treated with testosterone-enanthate for
two months were retransplanted intraperitoneally into normal untreated female
mice, the mortality rates of these new
hosts were 25% (1/4) when four surviving grafts were retransplanted and
62.5% ( 5 1 8 ) when eight grafts were
used. In one control group, eight surviving grafts were retransplanted into each
of six normal untreated female mice from
those without any post-transplantational
treatments two months after the original
isografting. The mortality rate of the
second hosts was zero out of six.
DISCUSSION
The present experiments revealed that
testosterone and isoproterenol-HC1 could
exert specific stimulatory effects on the
49 1
different components of the submandibular
graft tissues, the former on the secretory
tubules and the latter on the acinar cells
as have been observed in the mouse submandibular glands in situ. Both endogenous and exogenous androgen were found
to have the same effect. Without any exogenous treatments, regeneration of both
secretory tubules and acinar cells were
remarkably impaired and only the ductlike structures survived. Hyperplastic alterations, as previously reported (Hoshino
and Lin, '68a), were also observed in the
duct-like structures surviving in the grafts
after four weeks or longer following transplantation regardless of the further treatment. These results seem to indicate that
the cellular origins of the secretory tubule,
the acinar cell, and the ductal cell may
be different, instead of differentiating
from the same stem cell. This, however,
cannot be answered from the present experiment. Jacoby and Leeson ('59) observed that in the rat submandibular gland
the acinar cells start to develop from one
week of age, whereas the secretory tubules
appear only after six weeks of age.
Previously we (Hoshino and Lin, '69a;
Lin and Hoshino, '69) have observed that
the lethal factor was not demonstrated
by submandibular isografts obtained from
immature male or adult females, but when
these donors had received testosterone pretreatment prior to transplantation a lethal
effect was exerted on the hosts. In the
present experiment, submandibular isografts obtained from non-pretreated female donors but treated post-transplantationally while in the hosts' environment
with testosterone for two months, again
demonstrated the lethal effect when retransplanted into the new hosts. This experiment demonstrated the capability of
the surviving submandibular gland tissue
not only to regenerate the secretory tubule-like structures from the duct-like
structures but also to restore their specific
biological activity such as a lethal effect.
Some other substances were also found
to be testosterone-dependent in the submandibular glands; protease (Junqueira,
et al., '49), nerve growth factor (LeviMontalcini et al., '64), renin-like princid e (Oliver, '67), and others (Lin et al.,
'69). The biological activities of these
492
K. HOSHINO AND C. D. LIN
LITERATURE CITED
substances in isografted submandibular
glands, however, have never been reported. Hoshino, K., and C. D. Lin 1968a Hyperplasiainducing factor in mouse salivary gland isoIn the text, the terms the secretory
grafts. Cancer Res., 28: 255f3-2558.
tubule-like structure, the acinar cell-like
- 1968b Transplantability of salivary
structure and the duct-like structure were glands of mice and its lethal effects o n the
used instead of the secretory tubule, the
hosts. Anat. Rec., 160: 474-475.
acinar cell and the ductal cell, respec1969a Lethal factor released from
tively. However, the data in the present
submandibular grafts in mice. Canad. J.
Physiol. and Pharm., 47: 329-334.
experiment seem to indicate that reap1969b Comparative effects of testospearing structures were not different from -terone and isoproterenol upon regenerating
those observed in the in situ submandisubmandibular gland isografts in female
lar glands. Therefore, the present experiBALB/c mice. Anat. Rec., 163: 200.
ment provided a simple means to speci- Jacoby, F., and C. R. Leeson 1959 The postnatal development of the rat submaxillary
fically produce a predominant population
gland. J. Anat., 93: 201-216.
of either the secretory tubules or the
L. C., A. Fajer, M. Rabinovitch and
acinar cells in order to study histogenesis Junqueira,
L. Frankenthal 1949 Biochemical and histoof regeneration, proliferation, biological
chemical observations on the sexual dimorand biochemical activities, and also carphism of mice submaxillary glands. J. Cell.
and Comp. Physiol., 34: 129-158.
cinogenesis of these different components
of the submandibular glands in the mouse. Lacassagne, A. 1940 Dimorphism sexuel de la
ACKNOWLEDGMENTS
This work was supported by grants from
the National Research Council of Canada,
Dental Research Committee and the Medical Research Council of Canada and also
partly by a grant from the National Cancer Institute of Canada. The authors are
also indebted to Mrs. Sharon Griffin for
her technical assistance. One of the authors, C. D. Lin, was a National Research
Council Graduate Dental Research Fellowship Awardee and is a recipient of the
Medical Research Council Research Fellowship.
glande sous-maxillaire chez la souris. Com.
rend. SOC.Biol., 133: 180-181.
Levi-Montalcini, R., and P. U. Angeletti 1964
Hormonal control of the NGF content i n the
submaxillary glands of mice. In: Salivary
Glands and Their Secretions. L. M. Sreebny
and J. Meyer, eds. Pergamon Press Book. The
Macmillan Co., N. Y.. pp. 129-139.
Lin, C. D., and K. Hoshino 1969 Testosterone
dependency of the lethal factor in mouse submandibular gland isografts. Canad. J. Physiol.
and Pharm., 47: 335-338.
Oliver, W. J., and F. Gross 1967 Effect of
testosterone and duct ligation on submaxillary
renin-like principle. Am. J. Physiol., 213: 341346.
Selye, H., R. Veilleux and M. Cantin 1961 Excessive stimulation of salivary gland growth
by isoproterenol. Science, 133: 44-45.
PLATE 1
EXPLANATION OF FIGURES
1
Photomicrograph of a n i n situ submandibular gland of a 124 day old
normal male BALB/c mouse. H & E. x 125.
2
Photomicrograph of a n i n situ submandibular gland of a 145 day old
normal female BALB/c mouse. H & E. x 125.
3 Photomicrograph of a n i n situ submandibular gland of a 223 day old
normal male BALB/c mouse. PAS-Orange G-hematoxylin. x 310.
4 Photomicrograph of a n in situ submandibular gland of a 164 day old
normal female BALB/c mouse. PAS-Orange G-hematoxylin. x 310.
Abbreviations
A, the acinar cell
T, the secretory tubule
D, the intralobular duct
REGENERATING SUBMANDIBULAR ISOGRAFT
K. Hoshino and C. D. Lin
PLATE 1
493
PLATE 2
EXPLANATION OF FIGURES
5 Photomicrograph of a submandibular gland isograft transplanted
intraperitoneally from a 140 day old normal female donor to a 140
day old normal female BALB/c host mouse and recovered four weeks
after transplantation. Note hyperplastic changes in remaining ductal
components. H & E. x 310.
6
Photomicrograph of a submandibular gland isograft recovered eight
weeks after a n intraperitoneal transplantation from a 269 day old
normal female BALB/c mouse to a 154 day old normal female which
subsequently received four separate injections of 20 mg of testosterone enanthate fortnightly. Arrows indicate reappearing secretory
tubules. H & E. x 125.
7
Photomicrograph of a submandibular gland isograft recovered two
weeks after a n intraperitoneal transplantation from a 358 day old
female mouse to a 340 day old normal female BALB/c mouse which
subsequently received isoproterenol treatment for two weeks. Arrows
indicate reappearing acinar cells. H & E. x 125.
8 Photomicrograph of a submandibular gland isograft transplanted intraperitoneally from a 271 day old normal female BALB/c mouse
to a 151 day old normal female host which subsequently received
two separate injections of 20 mg of testosterone enanthate given
fortnightly, and removed from the host four weeks after transplantation. A n arrow indicates reappearing secretory tubules amongst
hyperplastic ductal cells. H & E. x 310.
494
9
Photomicrograph of a submandibular gland isograft obtained from
the same group as in figure 8. Arrows indicate reappearing secretory
tubules. PAS-Orange G-hematoxylin. x 310.
10
Photomicrograph of the same graft as i n figure 7. Arrows indicate
reappearing acinar cells stained with PAS. PAS-light green. x 310.
REGENERATING SUBMANDIBULAR ISOGRAFT
K. Hoshino and C. D. Lin
PLATE 2
495
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upon, effect, submandibular, balbc, testosterone, isografts, selective, mice, gland, regenerative, isoproterenol
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