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The development and hormonal response of the autotransplanted interpubic joint in mice.

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The Development and Hormonal Response of the
Autot ranspianted Interpubic Joint in Mice'
E. S. CRELIN
Department of Anatomy, Yale University School of Medicine,
New Haven, Connecticut
ABSTRACT
The ventral portion of the immature pelvis of 12-day-old female mice,
consisting of the interpubic cartilage and the angles of the pubic bones, was excised
and either autoimplanted into its original pelvic site or autotransplanted to the thoracic
region. Half the number of mice with pelvic implants and half with thoracic transplants received subcutaneous injections of estrogen and relaxin prior to necropsy. All
were sacrificed at 63 days of age.
All the pelvic implants and the majority of thoracic transplants were present
at necropsy. The grafts from the untreated mice were similar to the intact pubic
symphysis of a 63-day-old mature mouse both in size and morphology. The grafts
from the hormone-treated mice had undergone a pregnancy-type response of cartilage
transformation to ligamentous tissue and resorption of the medial ends of the pubic
bones. The possible significance of the cells of the interpubic joint in its response to
hormones is discussed.
Autotransplantation of the pubic symphysis to the thoracic region of adult virgin
female mice revealed that the symphyseal
tissues are capable of undergoing typical
pregnancy changes at this ectopic site in
response to either exogenous hormonal
treatment or the endogenous hormones of
pregnancy (Crelin, '58a, '59).
The present experiment was designed to
determine if an autotransplanted immature
interpubic joint could develop into a mature pubic symphysis both morphologically
and physiologically at an ectopic site. Preliminary reports of this study have appeared elsewhere (Crelin, '60a, '61; Felts,
'61).
MATERIALS AND METHODS
One hundred twenty-four Brown-belt
stock virgin female mice were arranged into eight groups.
Group 1. Six mice sacrificed at 63 days
of age.
Group 2. Six mice each received subcutaneously injections of 2 wg estradiol
benzoate in sesame oil and 100 Guinea
Pig units of relaxina in benzopurpurine at
46, 49, 53, 57 and 61 days of age. They
were sacrificed at 63 days of age.
Group 3. Six mice sacrificed at 12 days
of age.
Group 4. Six mice each received subcutaneously injections of 1 ug estradiol
benzoate in sesame oil and 100 G.P. units
of relaxin in benzopurpurine at one, four,
seven, and ten days of age post partum.
They were sacrificed at 12 days of age.
Group 5. Twenty-five mice at 12 days
of age had the ventral aspect of their bony
pelvis exposed by a transverse incision in
the overlying skin and transection of the
abdominal wall and hindlimb musculature
close to their ventral pelvic attachments.
The attachments of the urinary bladder,
urethra, and clitoris to the interpubic joint
were next separated. A longitudinal incision was made through the medial half
of each obturator foramen by transecting
the cranial ramus of the pubic bone and
the cartilagenous junction between the
caudal ramus of the pubic bone and the
ramus of the ischial bone (fig. 3 ) . The
free ventral portion of the pelvis was then
grasped with forceps, completely removed
and immediately replaced into its original
position in the pelvis. The wound was
closed by approximating the skin edges
and clamping them together with an 11
mm nickel-silver wound clip. All of the
mice were sacrificed at 63 days of age.
Group 6 . Twenty-five mice at 12 days
of age had the same operation that was
performed on the mice of group 5. Before
1 Sup orted by United States Public Health Service
grant G%-04433-08.
ZReleasin. Courtesy of Dr. R. L. ,Kroc. WarnerLambert Research Institute, Morns Plams, New Jersey.
149
150
E. S. CRELIN
the mice were sacrificed at 63 days of age
each received the same hormonal treatment that the mice of group 2 received.
Group 7. Twenty-five mice at 12 days
of age underwent the same operation that
was performed on the mice of groups 5
and 6 except that instead of returning the
excised ventral portion of the bony pelvis
to its original site, it was immediately implanted into the left pectoralis major muscle of the same mouse from which it was
removed. The pectoralis muscle was exposed previous to the excision of the ventral portion of the pelvis and by blunt dissection a pocket was made in the belly of
the muscle, separating it approximately
into dorsal and ventral halves. The pelvic
tissue was placed into the muscular pocket
and the overlying skin was closed with a
wound clip. All of the mice were sacrificed
at 63 days of age.
Group 8. Twenty-five mice at 12 days
of age underwent the same operation that
was performed on the mice of group 7.
Before the mice were sacrificed at 63 days
of age each received the same hormonal
treatment that the mice of groups 2 and 6
received.
At necropsy the entire bony pelvis of
each mouse from every group was exposed.
The pelvic implants of groups 5 and 6 and
the pectoral transplants of groups 7 and
8 were examined in situ with a stereoscopic
microscope. The narrowest point of separation between the pubes at the interpubic
joint (interpubic distance) of the intact
pelves and of the autografts was measured
directly with a vernier caliper and the degree of flexibility of the interpubic tissue
was determined by manual manipulation.
The entire bony pelvis was excised and
prepared as a whole mount from two mice
each of groups 1, 2, 3, and 4 and 5 mice
each of groups 5, 6, 7, and 8. The pelvic
implants and pectoral transplants of the
above mice from groups 5 through 8 were
included in the whole mounts which were
prepared in the following manner: After
dissecting away most of the attached musculature, the pelvic bones with the interpubic joint intact were macerated in 2%
KOH, stained with alizarin and then
cleared in glycerin.
The interpubic joints of all the other
mice of the experiment were sectioned
serially at 7 in the coronal plane and
stained with hematoxylin and triosin.
RESULTS
Group 1. The bony pelves of the 63day-old untreated mice were the typical
adult virgin female type both in size and
shape (fig. 1) (Crelin, '60b; Crelin and
Blood, '61 ). The interpubic distance averaged 0.2 mm (range: 0.1-0.3 mm). The
symphyseal cartilage was only slightly
flexible. It consisted of a cap of hypertrophic cartilage along the medial surface
of each pubic bone and a narrow strip of
compact fibrocartilage intervening between the caps in the center of the joint.
In this central fibrocartilage each lacuna
and its contained single chondrocyte were
flattened with their widest diameter paralleling the sagittal plane of the joint (similar to fig. 17). At the narrowest point of
separation of the pubes located in the cranial part of the dorsal half of the joint, the
transition of the compact fibrocartilage to
hypertrophic caps was abrupt; in the other
regions of the joint the transition was
gradual. The central fibrocartilage contained densely packed bundles of birefringent collagen fibers, many of which
were arranged in a criss-cross pattern
similar to that of the interpubic cartilage in
the rat (Crelin and Brightman, '57).
Three of the joints had a longitudinal cleft
in the center of the compact fibrocartilage.
One cleft probably was a tissue preparation artifact because a bridge of matrix
subdivided it into two parts. The clefts
were probably splits in the matrix because
they lacked an epithelium. The articular
capsule of the joint contained elongated
fibrocytes and/or fibroblasts interspersed
between densely packed bundles of collagen fibers. Only occasional osteoclasts
were found in a limited area along the
cranio-dorsal surface of the medial ends
of the pubes. This is the area where the
pubes normally undergo resorption at puberty in the female mouse, probably as a
result of an increased output of estrogen
by the ovaries. The resorption reduces the
cranio-caudal length of the pubic angles
at the symphysis to about half that of an
adult male or of an adult female ovariectomized before puberty (Crelin, '60b).
The constant presence of some osteoclasts
AUTOTRANSPLANTED INTERPUBIC JOINT
along the cranio-dorsal surface of the pubes
in the adult intact female may indicate
that this area continues to undergo some
limited resorption and replacement in response to fluctuations in the estrogen blood
level related to the estrous cycles.
Group 2. The pregnancy-type response
of the interpubic joint tissues to the estradiol benzoate and relaxin treatment in the
63-day-old adult virgin mice was similar
to that found in previous experiments
(Crelin, '54a, '54b, '56, '57; Hall, '56).
The interpubic cartilage was transformed
into ligamentous tissue. Much or nearly
all of the cartilage matrix had undergone
a change to a more fluid state. As a result the collagen fibers were easily visualized and were loosely-arranged in a direction parallel to the coronal plane of the
joint; the originally centrally located, flattened chondrocytes were enlarged, spherical, and increased in number; a number
of chondrocytes released from the confines
of their lacunae were elongated and resembled large fibroblasts; the joint capsule
was thin, flaccid, and semi-transparent;
bone resorption occurred along the medial
ends of the pubes (similar to figs. 1922). Numerous osteoclasts were found
along the medial ends of the pubes where
the adjacent hypertrophic cartilage was
transformed into ligamentous tissue (similar to figs. 11 and 12). A transverse, fluidfilled cleft with irregular borders occupied
the center of each interpubic ligament
throughout its length. These clefts were
originally a longitudinal split in the center
of the compact fibrocartilage that was
present either before hormonal treatment
or one that formed during the initial phase
of response to treatment. A few chondrocytes and fibroblasts were in mitotic division in each interpubic ligament in about
the same number and distribution as
those observed in the ligament of normal
pregnancy (Crelin, '57). Associated with
the above changes was an increased interpubic distance (average: 3.0 mm, range:
2.5-4.0 mm) (fig. 2).
Group 3. The overall size of the bony
pelves of the untreated 12-day-old mice
was approximately half that of the 63-dayold adults (fig. 3). The pubes were extensively ossified, however, about a third
of each acetabulum was cartilagenous and
151
a bar of cartilage separated the ossified
caudal ramus of each pubis from the ossified ramus of each ischium. The interpubic distance averaged, 0.4 mm (range:
0.3-0.5 mm). The medial surfaces of the
bony pubic angles at the interpubic joint
were capped with hypertrophic cartilage
which was calcified along the junction of
the cartilage and bone. The hypertrophic
cartilage caps were continuous with a
centrally-situated band of cartilage containing uniformly smaller cells (fig. 13).
The chondrocytes of this central cartilage
were spherical and each lacuna contained
a single cell (fig. 16). Using polarized
light it was determined that the central
cartilage contained numerous collagen fiber bundles, many of which were arranged
in a crisscross pattern similar to that
found in the young adult virgin. However,
the bundles were not as densely packed
together as those of the virgin adult. Manipulation of the interpubic joint revealed
that the joint cartilage was slightly more
flexible than that of the virgin untreated
adult.
Group 4 . The interpubic joints of the
hormone-treated 12-day-old mice were the
same as those of the untreated 12-day-old
controls of group 3, demonstrating that
the interpubic joint of the 12-day-old
mouse was not only immature morphologically but had not yet acquired the ability
to respond to the hormonal treatment.
Group 5. All of the interpubic joints
excised and implanted into the pelvic area
at 12 days of age were present in the 63day-old untreated mice. Although no special attempt was made to have the cut
ends of the transected cranial pubic rami
perfectly approximate each other when
the excised portion of the pelvis was implanted, a bone to bone union did occur
bilaterally in five mice (fig. 4 ) . Such a
union was quite unexpected because of
possible movement of the implant when
the animals ran and climbed around their
cage shortly after recovering from the ether
anesthesia. In eight mice a bony union of
the cranial pubic rami occurred unilaterally (figs. 5 and 6). In all except one
of these eight mice the union was on the
right side. In addition, in six of these
eight mice the cranial pubic ramus of the
implant on the side which failed to form
152
E. S. CRELIN
a bony union grew in length as a narrow
tapered extension of the pubic angle (fig.
5), whereas in the other two mice only an
ossified angle was present (fig. 6). No
bony union occurred on either side in the
remaining 12 mice of this group. In the
majority of these mice both the cranial
and caudal pubic rami of the implants
grew as narrow tapered extensions of the
angles, whereas, in those where the rami
failed to grow only the angle was present
on one or both sides. The free ends of the
pubic and ischial rami of the unexcised
portions of the pelvis had grown in width
and length in about half of the mice where
no bony union of the implants occurred,
whereas, in the remainder of these mice
there was only evidence of a growth in
width. These were general findings for
the mice of the other groups where the
pelvic implant failed to form a bony union
or where the excised portion of the pelvis
was transplanted to the pectoral region.
Where the rami of the pelvic implants of
groups 5 and 6 failed to form a bony union
with their respective rami of the unexcised portions of the pelvis, they were
connected by a dense sheet of fibrous tissue which was continuous with each obturator membrane.
The interpubic distance in the mice of
this group averaged 0.2 mm (range: 0.10.3 mm). The interpubic joint was only
slightly flexible and was the same size as
that of the adult virgin control (figs. 46). Microscopically the joint was essentially identical to the adult virgin symphysis (figs. 14 and 17). The most pronounced change that occurred in the implanted joint from 12 to 63 days of age
was the formation of the central strip of
compact fibrocartilage in which the originally spherical chondrocytes became flattened disks and the bundles of collagen
fibers became densely packed. In the
center of five joints there was a longitudinal cleft in the fibrocartilage. A few
osteoclasts were present along the craniodorsal surfaces of the medial ends of the
pubes, probably indicating that the normal post puberal pubic bone resorption
occurred. In the majority of the pelvic
implants, including those of group 6 and
the pectoral transplants of groups 7 and
8, the bone marrow consisted of essentially
normal myeloid tissue. Where i t did not,
the marrow cavities were filled with fibrous connective tissue.
Group 6. All of the pelvic implants
were present in the 63-day-old hormonetreated mice. In 13 mice no bony union of
the rami of the implant with the transected
ends of the rami of the unexcised portions
of the pelvis occurred (fig. 7). In two of
these mice bone was absent on one side
of the implant. The cranial pubic rami
of the implant formed a bony union bilaterally in four mice and unilaterally in six
mice. In two mice both the cranial and
caudal pubic rami on both sides formed
a bony union with the ends of their respective rami of the unexcised portions of
the pelvis. Although the bony union of
the rami in these pelves formed a complete coxal bone bilaterally, the ventral
portion of each was either abnormally
elongated or shortened. One was prepared
as a whole mount (fig. 8). These two
pelves were the only ones of groups 5 and
6 in which the caudal pubic ramus formed
a bony union with the ischial ramus. Cartilage was not present in any of the 63day-old pelves of groups 5 and 6 on the
ends of the caudal pubic rami of the implant or on the ends of the ischial rami of
the unexcised portions.
Microscopically the interpubic cartiIage
was variably transformed into ligamentous
tissue. In nine of the sectioned joints only
the central fibrocartilage matrix had undergone a change to a more fluid state;
the hypertrophic cartilage capping the
pubes was largely intact. In the other 11
sectioned joints the cartilage transformation was almost complete; only small portions of intact hypertrophic cartilage
matrix were present (fig. 19). These socalled interpubic ligaments resembled
those found in the normal post parturient
mouse. In the center of each of the joints
a fluid-filled cleft was present. Although
the majority of modified chondrocytes near
the center of the ligaments were enlarged
and separated from visible adjacent bundles of collagen fibers by clear, irregular
spaces resembling lacunae, there were
large elongated cells with a more basophilic cytoplasm dispersed among the collagen fibers not surrounded by a clear
space (fig. 21). The long axes of these
AUTOTRANSPJANTED INTERPUBIC JOINT
elongated cells were in the coronal place
of the joint. Since they were located near
the center of the ligament, at some distance from a now more edematous fibrous
articular joint capsule containing smaller
elongated fibroblasts and/or fibrocytes,
these large cells were probably chondrocytes released from their lacunae at an
early stage in the matrix alteration to a
more fluid state. There appeared to be an
increased number of cells in the ligament,
however, only a few mitoses were found.
In the joints where most of the hypertrophic cartilage was transformed into
ligamentous tissue the adjacent medial
ends of the pubes were lined with osteoclasts, indicating that bone resorption occurred in response to the estrogen treatment (fig. 11).
Grossly, the interpubic tissue of all of
the mice of this group was flaccid, thin,
and semi-transparent. It had little elasticity and could easily be ruptured. The interpubic distance averaged 2.5 mm (range:
2.0-4.0 mm). In general, the joints which
had the greatest interpubic distances also
showed the greatest amount of ligamentous tissue. In addition, the implants
which formed a bilateral bony union with
one or both of the rami of the unexcised
portions of the pelvis also had the greatest
interpubic distances.
Group 7. The ventral portion of the
pelvis transplanted to the left pectoralis
major muscle at 12 days of age was not
found in 6 of the 63-day-old untreated
mice when the muscle was exposed and
thoroughly dissected at autopsy. In 10 of
the 19 transplants which were recovered,
bone was present bilaterally. In six, bone
was present on one side and in three only
the joint cartilage persisted. Bone growth
of the transplants, including those of
group 8, was of two types. In one type
the angles and cranial pubic rami became
broad and thin with little or no growth of
the caudal rami having occurred (fig. 9);
in the other the angles were broad and
thin but both the cranial and caudal rami
were long, slender rod-like extensions of
the angles (fig. 10). The latter type occurred most frequently in both groups 7
and 8. Only a few osteoclasts were found
along the carnio-dorsal surfaces of the
angles. No cartilage was present on the
153
ends of the caudal pubic rami of the transplants or the ischial rami of the unexcised
coxal bones of this group and those of
group 8. The pubic and ischial rami of
the unexcised portions of each coxal bone
were widely separated but connected across
the midline of the body by a broad, dense
sheet of fibrous tissue that was continuous
with each obturator membrane (fig. 9).
This was also true for the unexcised portions of the coxal bones of group 8 (fig.
10).
The interpubic distances of the pectoral
transplants of this group were greater than
those of the pelvic implants in the untreated mice of group 5,for they averaged
0.4 mm (range: 0.3-0.7 mm) (fig. 9).
However, the joints were only slightly
flexible. The above figures include the
narrowest point in width of the three transplants consisting of only joint cartilage.
Microscopically, the joints were similar to
those of the normal untreated adult and
of the pelvic implants of group 5 except
that the central fibrocartilage was not as
compact and the transition between the
central fibrocartilage and the adjacent hypertrophic cartilage was gradual throughout the joint (figs. 15 and 18). This probably accounted for the wider interpubic
distances of this group compared to those
of group 5. A longitudinal cleft in the
center of the fibrocartilage was present in
only two joints.
Group 8. The ventral portion of the
pelvis transplanted to the left pectoralis
major muscle at 12 days of age was not
found at autopsy in five of the 63-day-old
hormone-treated mice. Of the 20 transplants which were recovered bone was
present bilaterally in 12 (fig. 10). In six,
bone was present on one side and in two
it was absent. The size and shape of the
persisting bone was similar to that described for the transplants of group 7. The
fact that all of the pelvic implants persisted and all possessed bone at least on one
side could have been due in part to a more
rapid and abundant initial vascularization
of the implants when compared to the
pectoral transplants. The degree of tissue
trauma inflicted at operation and the
amount of vascularization occurring during tissue repair were much greater at the
pelvic than at the pectoral grafting sites.
154
E. S . CRELIN
Microscopically the interpubic cartilage
of the transplants was variably transformed into ligamentous tissue. In 8 of
the 15 sectioned joints only small portions
of intact hypertrophic cartilage matrix
were present (fig. 20). The typical central
cleft was present in each joint. On a
cellular level the joint changes were similar to those that occurred in the hormonetreated, unoperated adults and in the
treated mice with pelvic implants (fig.
22). In the transplants where most of the
hypertrophic cartilage was transformed,
the adjacent medial ends of the pubes were
lined with osteoclasts (fig. 12). A few
chondrocytes were in mitotic division.
Grossly, the interpubic tissue of each
mouse in this group was similar to the
interpubic ligament of the hormone-treated
unoperated virgin adult female in that it
was flaccid and fragile. However, it differed by being thicker and less transparent. It was also slightly elastic because
the length of the ligament could be increased from five to eight tenths of a
millimeter by grasping its two ends with
forceps and stretching it. When stretched
the ligament became thinner and more
transparent. When the ligaments were
not under tension the interpubic distance
averaged 1.5 mm (range: 1.0-2.5 mm),
which was less than the average of the
unoperated, hormone-treated adults and
the hormone-treated mice with pelvic implants.
DISCUSSION
The interpubic ligaments of the pectoral
transplants tended to be shorter than
those of the pelvic implants and those of
the intact pelves probably because of the
lack of laterally-directed tension on the
interpubic tissue of the pectoral transplants
while it was undergoing changes in response to the hormonal treatment. Tension has been shown to be necessary for
the ligament to attain its maximum length
in the mouse and guinea pig (Crelin, ’5412;
Van Der Meer, ’50).
In all except two mice with pelvic implants the caudal pubic rami of the implants failed to form a bony union with
the ischial rami of the coxal bones. One
reason for the failure could have been the
presence of cartilage attached to the ends
of the bones for an unknown period of
time, since the original transection was
made through the cartilage bar connecting
the two ossifying rami at 12 days of age.
The potentiality of the immature interpubic joint of mice to grow and differentiate into a mature symphysis with the
ability to respond to estrogen and relaxin
treatment is undoubtedly attained much
earlier in development than 12 days post
partum. Fell (’56) showed that the
blastema of limb buds can develop joints
in vitro.
Whether or not the original tissue of
the interpubic joint transplanted to the
pectoral area was maintained throughout
the experimental period can only be inferred. The original cartilage was probably maintained in as much as both autografts and homografts of adult and growing cartilage apparently can survive for
long periods of time with no evidence of
replacement (Bacsich and Wyburn, ’55a,
’55b; Laskin et al., ’52). It is more difficult to estimate whether the original bone
was maintained and grew by giving rise
to new bone, or whether it induced the
surrounding connective tissue at the intramuscular graft site to give rise to new
bone which replaced the original, since
studies of small pieces of autotransplanted
bone indicate that both can happen (Goldhaber, ’62; Hutchinson, ’52; Wiliams, ’62).
Although cartilages of the pubic symphysis and pubic bone are similar morphologically to their respective tissue counterparts in other parts of the body in mice,
their unique hormonal response demonstrates that they are physiologically quite
distinct. Therefore, the original transplanted interpubic joint tissue was physiologically “tagged.” It is quite plausible
then to conclude that new joint tissue
formed during the experimental period was
derived entirely, or at least primarily, from
the graft. Otherwise, it must be concluded
that the transplanted tissue not only induced the connective tissue cells at the intramuscular graft site to form cartilage and
bone but in some manner transmitted to
the induced tissue the inherent characteristics of the transplanted tissue so that i t
could develop into a pubic symphysis similar to that of the normal adult in over-all
size and morphology, both grossly and mi-
AUTOTRANSPLANTED INTERPUBIC JOINT
croscopically, and with the unique ability
to respond to specific hormonal treatment.
In a previous experiment (Crelin, ’54a)
the eight-day-old interpubic joint of mice
was transplanted from one mouse to another of an originally inbred strain which
was not maintained by brother-sister matings. Therefore, each graft was probably
more of a homograft than an isograft.
After 98 days the grafts had increased in
size only slightly and both grossly and microscopically resembled more an eight-dayold joint than that of an adult. In addition, there was no evidence that the grafts
were affected in any manner by estrogen
and relaxin treatment the hosts received.
Even though the original cartilage may
have survived, its failure to grow and differentiate further precluded any hormonal
response because the interpubic cartilage
in the intact joint does not respond until
the joint reaches a fairly advanced stage
of differentiation into a symphysis (Crelin
and Levin, ’55). The pubic bones of these
grafts probably failed to respond to estrogen treatment because the original bone
may have been completely destroyed by an
immunological reaction and replaced by
bone derived from connective tissue cells
of the host. If so, bone was present that
morphologically resembled pubic bone but
physiologically it was not true pubic bone.
The results of Felts’ (’57) studies on isologous and homologous transplantations of
immature humeri in mice compare closely
in many ways to those of the studies on
the transplanted interpubic joint tissues.
Storey (’57) postulated that pubic bone
resorption is due to mechanical “fatigue”
because of the unusual stresses the bones
are subjected to during interpubic ligament formation. The fact that ectopically
transplanted pubic bones, lacking the normal stresses of the intact bones, still undergo resorption in response to estrogen
treatment, negates this hypothesis. Autotransplanted adult pubic bone of mice
undergoes resorption in response to estrogen treatment even when it is intimately
fused to the tibia, which responds simultaneously by undergoing increased ossification (Pinnell and Crelin, ’63).
According to the hypothesis of Gersh
and Catchpole (’60) connective tissue
ground substance is a heterogeneous col-
155
loidal system, consisting of a colloid-rich,
water-poor phase coexisting in equilibrium
with a colloid-poor, water-rich phase. The
quantities of the colloid-rich and waterrich phases, or the relative proportions of
presumed submicroscopic vacuoles in the
ground substance, would determine several important characteristics of a given
connective tissue such as its physical consistency, mass and total volume. By applying their hypothesis to the present
experiment it is supposed that the ground
substance of the symphyseal cartilage and
the articular capsule in a virgin adult
mouse is in a highly aggregated, dense
state with a preponderant shift to the
colloid-rich water-poor phase. This could
account for the compactness and firmness
of the cartilage and capsule. If the virgin
mouse is treated with estrogen and
relaxin, a shift to the less dense, disaggregated state would occur where the
colloid-poor, water-rich phase could be preponderant. This would account for the
softness and flexible consistency of the
resulting interpubic ligamentous tissue
containing loosely-arranged collagen fibers.
Gersh and Catchpole (’60) state that
because of the intimate proximity of both
epithelial and connective tissue in most
organs, it cannot be stated categorically
that the effect of hormones on ground
substance is a direct one or secondary to
an effect on the epithelium. However,
they feel that a secondary effect is implausible in the pubic symphysis. I agree
with this and would carry their hypothesis
further, even though it is completely unknown how estrogen and relaxin injected
into a mouse induces interpubic cartilage
transformation into ligamentous tissue.
Some substance or substances, which may
be different from the originally injected
hormones, could pass from the blood
stream and diffuse thraugh the cartilage
matrix to directly affect the chondrocytes.
The chondrocytes, which are responsible
for the original formation of their surrounding matrix and its continued maintenance, could then be induced to participate in a process by releasing one or more
catalytic substances which would result in
a matrix ground substance shift to a colloid-poor, water-rich phase. This explana-
156
E.
S. CRELIN
tion is in essence applicable to the fibrous
articular capsule.
An indication that estrogen and relaxin
may not have an exclusive, direct effect
on the cartilage matrix and extra-cellular
component of the articular capsule in mice
was the failure to obtain a response which
differed from the usual systemic effect
obtained following a subcutaneous injection when these hormones were injected
directly into the symphysis or when estrogen was directly applied to its dorsal surface (Crelin, '58b; Crelin and Haines, '55).
The results of in vitra studies of symphyseal cartilage from mice and guinea
pigs maintained in a medium containing
estrogen and relaxin have been negative
(Harkey and Crelin, '63a; Storey, '57; Van
Der Meer, '50). Experiments (Horn, '58,
'60; Horn and Barandes, '61) indicating
that thyroid hormone is necessary to obtain the maximum response of the symphyseal cartilage and articular capsule of
mice to estrogen and relaxin treatment
add further credence to the idea that the
ground substance alterations occurring
during treatment are the result of chondrocyte and fibroblast participation. The
cellular component of the pubic bone may
also directly participate in the matrix resorption occurring during estrogen treatment. If this hypothesis is substantiated,
it will be an additional indication that the
cellular component of the pectoral transplants recovered at necropsy consisted of
the original cells of the interpubic joint
tissue and/or their progeny. Also, the primary difference of interpubic cartilage
and pubic bone and their respective tissue
counterparts in other parts of the body
would then not be so much between their
matrices, which undoubtedly differ in
many respects, but between their cells
which are responsible for the composition
of their surrounding matrix. Haines' ('57)
histochemical study of interpubic joints
from mice treated with estrogen and
Bassett's ('59) studies on histochemical
changes occurring in fibroblasts of various
connective tissues responding to hormonal
treatment in different animals support the
suggestion that the cells participate in
bringing about the changes in the extracellular component of a given connective
tissue. Hall ('56) speculated that estro-
gens may cause the liberation locally of
an enzyme that induces alterations in the
symphyseal cartilage matrix of mice, but
she did not indicate the possible source of
such an enzyme.
A final point indicating the proposed
significance of the cells of the interpubic
joint in controlling its hormonal response
is that the response of the joint is conditioned by the gonadal hormones between
birth and sexual maturity (Crelin and
Grillo, '57; Harkey and Crelin, '63b). The
longer the time a female mouse had its
ovaries before puberty, the greater its
symphyseal response was to estrogen and
relaxin treatment received when an adult.
Conversely, the longer the time a male
mouse had its testes before sexual maturity, the more inhibited its symphyseal response was to estrogen and relaxin treatment received when an adult. Although
there are other possible interpretations,
these data could indicate that early in
development the interpubic joint cells acquire an inherent ability to respond in a
certain degree to specific hormonal treatment in the adult mouse and that this
ability is altered, or conditioned, during
later development by the endogenous gonadal hormones.
LITERATURE CITED
Bacsich, P., and G. M. Wyburn 1955a Subcutaneous homografts of the xiphoid cartilage in
the guinea pig. Transpl. Bull., 2: 4-5.
1955b The uptake of S35 in cartilage
homografts. Ibid., 2: 97.
Bassett, E. G. 1959 Fibroblast cells in pregnancy. Proc. U. Otago Med. Sch., 37: 15-16.
Crelin, E. S. 1954a The effects of androgen,
estrogen and relaxin on intact and transplanted pelves in mice. Am. J. Anat., 95:
47-74.
1954b The effects of estrogen and relaxin on the pubic symphyses and transplanted
ribs in mice. Anat. Rec., 120: 23-32.
1954c Prevention of innominate bone
separation during pregnancy in the mouse,
Proc. SOC.Exp. Biol. and Med., 86: 22-24.
1956 Fate of fibrocartilage cells in the
pubic symphysis of mice treated with estradiol
and relaxin. Anat. Rec., 124: 279.
1957 Mitosis in adult cartilage. Science,
125: 650.
1958a Interpubic ligament formation
in autologously transplanted pubic symphyses
of Brown-belt stock virgin mice. Anat. Rec.,
130: 290.
1958b Injections of estradiol benzoate
and relaxin into the pubic symphysis in mice.
Ibid., 130: 400401.
AUTOTRANSPLANTED INTERPUBIC JOINT
1959 The effezts of pregnancy on autotransplanted pubic symphyses of mice. Ibid.,
133: 369.
1960a The development of immature
interpubic tissue into a symphysis when transplanted in female mice. Ibid., 136: 179.
1960b The developmentof bony pelvic
sexual dimorphism in mice. Ann, N. y. Acad.
Sci., 84: 479-512.
lg61
Of pubic
symphysis in abnormal location. Highfights of
Res. Prog. in Gen. Med. Sci., '60. Public
' Health Ser. Pub. no. 815: 8-9.
Crelin, E. S., and D. 1;. Blood 1961 The influence of the testes on the shaping of the bony
pelvis i n mice. Anat. Rec., 140: 375-379.
Crelin, E. S., and M. B'.Brightman 1957 The
pelvis of the rat: its response to estrogen and
relaxin. Ibid., 128: 467-484.
Crelin, E. S., and M. A. Grillo 1957 The conditioning effect of puberty on the response of
the pubic symphysis in mice to estrogen and
relaxin. Ibid., 127: 407.
Crelin, E. S., and A. L . Haines 1955 The effects of locally applied estrogen on the pubic
symphysis and knee joint in castrated mice.
Endocrinology, 56: 461470.
Crelin, E. S., and J. Levin 1955 The prepubera1 pubic symphysis :and uterus in the mouse:
their response to estrogen and relaxin. Ibid.,
57: 730-747.
Fell, H. B. 1956 The Iliochemistry and Physiology of Bone. Ed. by G. H. Bourne. Academic
Press Inc,, New yorb. Chapter 14, 401440.
Felts, w. J. L. 1957 !, comparison of subcutaneous implants of isologous and homologous
immature whole mouse bones. Transpl. Bull.,
4: 131-135.
1961 International Review of Cytology.
Ed. by G. H. Bourne and J. F. Danielli. Academic Press Inc., New York, 12: 243-299.
Gersh, I., and H. R. Cat(2hpole 1960 The nature
of ground substance of connective tissue. Perspectives in Biol. and Med., 111: 282-319.
Goldhaber, P. 1962 Some current concepts of
bone physiology. New Eng. J. Med., 266:
924-931.
-
157
Haines, A. L. 1957 The effect of estrogen on
cartilage and bone i n castrate C3H mice. Yale
J. Biol. and Med., 30: 121-136.
Hall, K. 1956 An evaluation of the roles of
oestrogen, PIOgesterone and relaxin in producing relaxation of the symphysis pubis of the
ovariectomized mouse, using the technique of
metachromatic staining with toluidine blue.
J. Endocrin., 13: 384-393.
Harkey, M. E., and E. S. Crelin 1963a In vitro
study of pubic symphyseal cartilage from adult
virgin female mice. Anat. Rec., 145: 322-323.
1963b Hormonal response of the pubic
symphysis in adult mice gonadectomized at
different times before puberty. Ibid.. 145: 323.
Horn, E. H. 1958 Effects of -feeding thiouracil
and/or thyroid powder upon pubic symphyseal
separation in female mice. Endocrinology, 63:
481486.
1960 Reversibility of thiouracil-induced
inhibition of interpubic separation i n virgin
mice given thyroxine or large doses of relaxin.
Ibid., 66: 129-134.
Horn, E. H., and- M. Barandes 1961 Indifference of thiouracil-induced inhibition of pubic
symphyseal separation to changes in estrogenpriming dosages. Ibid., 69: 1102-1104.
Hutchinson, J. 1952 The fate of experimental
bone autoarafts and homomafts.
Brit. J. Sura..
- ,
158: 2-11;
Laskin, D. M., B. G. Sarnat and J. A. Bain 1952
Respiration and anaerobic glycolysis of transplanted cartilage. Proc. SOC. Exp. Biol. and
Med.9 79: 474476.
Pinnell, S. R., and E. S. Crelin 1963 Fate of
pubic bone autotransplanted to the tibia in
estrogen-treated
adult female mice. Anat. Rec.,
,.*.,r
143: 343.
Storey, E. 1957 Relaxation in the pubic symphysis of the mouse during pregnancy and
after relaxin administration, with special reference to the behaviour of collagen. J. Path.
and Bact., 74: 147-162.
Van Der Meer, C. 1950 Experiments on the
mechanism of action of relaxin. Acta Endocrinol., 4: 325-342.
Williams, R. G. 1962 Comparison of living
autogenous and homogenous grafts of cancellous bone heterotopically placed in rabbits.
Anat. Rec., 143: 93-106.
PLATE 1
E X P L A N A T I O N OF FIGURES
158
1
Coxal bones from a normal 63-day-old virgin female mouse. The interpubic distance at the symphysis ( S ) measured 0.2 mm. X 3.5.
2
Coxal bones from a 63-day-old virgin female mouse treated with estrogen and relaxin. The interpubic distance measured 3.0 mm at autopsy
A n interpubic ligament is attached to the medial ends (arrows) of
the pubic bones. x 3.5.
3
Top: Ilia1 ( I L ) , ischial ( I S ) and pubic ( P ) bones from a normal
12-day-old female mouse. Bottom: The angles of the pubes and interpubic cartilage (arrow) excised from a 12-day-old mouse to show
the amount of tissue that constituted a graft. x 5.
4
Coxal bones from a n adult mouse in which the interpubic joint was
excised and implanted to its original site a t 12 days of age. A bony
union occurred bilaterally between the cranial pubic rami of the implant and coxal bones. The ends of the caudal pubic rami of the
implant and ischial rami of the coxal bones (arrows) are connected
by dense fibrous tissue. The interpubic distance as the symphysis
( S ) measured 0.2 mm. x 3.5.
5
Coxal bones from a n adult mouse i n which the interpubic joint was
excised and implanted into its original site at 12 days of age. A bony
union occurred only between the right cranial pubic ramus of the
implant and that of the right coxal bone. The left cranial pubic
ramus of the implant is a narrow tapered extension of the angle. The
position of the left coxal bone obscures its pubic and ischial rami.
The interpubic distance a t the symphysis ( S ) measured 0.3 mm.
x 3.5.
6
Coxal bones from a n adult mouse i n which the interpubic joint was
excised and implanted into its original site at 12 days of age. A bony
union occurred between the right cranial pubic ramus of the implant
and that of the right coxal bone. Only a pubic angle is present on
the left side of the implant. The ends of the other coxal rami are
indicated by arrows. The interpubic distance at the symphysis (S)
measured 0.1 mm. X 3.5.
AUTOTRANSPLANTED INTERPUBIC JOINT
E. S. Crelin
PLATE 1
159
PLATE 2
EXPLANATION O F FIGURES
7
Coxal bones from a hormone-treated, adult mouse in which the interpubic joint was excised and implanted into its original site at 12 days
of age. The implant, consisting of bony pubic angles ( B ) , and a n
interpubic ligament ( L ) measuring 3.0 m m at autopsy, is suspended
between the coxal bones by retained strips of a n originally broad
sheet of connective tissue. The ends of the pubic and ischial coxal
rami are indicated by arrows. X 3.5.
8
Coxal bones from a hormone-treated adult mouse i n which the interpubic joint was excised and implanted into its original site at 12 days
of age. A bilateral bony union occurred between the cranial pubic
rami of the implant and those of the coxal bones and between the
caudal pubic rami of the implant and the coxal ischial rami. The
misshapen pubic angles are connected by a n interpubic ligament ( L )
which measured 4.0 mm at autopsy. X 3.5.
9
Coxal bones and interpubic joint from an adult mouse. The joint was
excised and transplanted to the thoracic region at 12 days of age.
The transplant consists of symphyseal cartilage ( S ) and bony pubic
angles with broad cranial rami. Caudal pubic rami are lacking. The
interpubic distance was 0.6 mm at autopsy. The coxal bones are connected by a retained strip of a n originally broad sheet of connective
tissue. The ends of the coxal rami are indicated by arrows. X 3.5.
10 Coxal bones and interpubic joint from a hormone-treated, adult
mouse. The joint was excised and transplanted to the pectoral region
at 12 days of age. The transplant consists of a n interpubic ligament
( L ) and pubic angles with long slender cranial and caudal bony
rami. The ligament measured 2.5 mm at autopsy. The coxal bones are
connected by a retained strip of a n originally broad sheet of connective tissue. The ends of the coxal rami are indicated by arrows.
X 3.5.
11 Section showing osteoclasts (arrows) located along the medial end
of a grafted left pubic bone ( B ) from a hormone-treated adult mouse
whose interpubic joint was excised and implanted into its original
site at 12 days of age. X 250.
12
160
Section showing a n osteoclast (arrow) adjacent to the medial end of
a left pubic bone ( B ) from a hormone-treated, adult mouse whose
interpubic joint was transplanted to the pectoral region at 12 days of
age. x 750.
AUTOTRANSPLANTED INrERPUBIC JOINT
E. S. Crelin
PLATE 2
161
PLATE 3
EXPLANATION O F FIGURES
162
13
Coronal section of the interpubic joint from a n untreated 12-day-old
mouse. Bracket demarcates a n area similar to that shown with higher
magnification in figure 16. X 50.
14
Coronal section of the cranial two-thirds of the interpubic joint from
a n adult mouse i n which the joint was excised and implanted into
its original site at 12 days of age. Bracket demarcates the central
compact fibrocartilage similar to that shown with higher magnification
in figure 17. x 50.
15
Coronal section of the cranial two-thirds of the interpubic joint from
a n adult mouse i n which the joint was transplanted to the pectoral
region at 12 days of age. Bracket demarcates the central compact
fibrocartilage similar to that shown with higher magnification i n figure 18. X 50.
16
Central area of a section of the interpubic joint from a 12-day-old
mouse similar to that shown in figure 13. Bracket demarcates a region containing spherical chondrocytes which normally appears in the
adult similar to the region indicated by a bracket i n figure 17.
X 150.
17
Central area of a section from a pelvic implant similar to that shown
in figure 14. Bracket demarcates the central strip of compact interpubic fibrocartilage containing flattened, disk-shaped chondrocytes.
The transition of compact fibrocartilage to hypertrophic cartilage on
each side is abrupt. x 150.
18
Central area of a section from an interpubic joint transplanted to the
pectoral region similar to that shown in figure 15. Bracket demarcates
the central strip of compact fibrocartilage which differs from that
shown i n figure 17 by being wider and having a more gradual transition to hypertrophic cartilage. X 150.
19
Coronal section of the interpubic joint from a hormone-treated, adult
mouse in which the joint was excised and implanted into its original
pelvic site at 12 days of age. Most of the joint cartilage was transformed into ligamentous tissue which resulted in a 3.0 mm distance
between the pubic bones ( B ) . X 20.
20
Coronal section of the interpubic joint from a hormone-treated adult
mouse in which the joint was excised and transplanted to the pectoral
region at 12 days of age. Nearly all of the joint cartilage was transformed into ligamentous tissue which resulted in a 2.5 m m distance
between the pubic bones (B). X 20.
21
Higher magnification of the center of the interpubic ligament shown
in figure 19. Enlarged chondrocytes are surrounded by a meshwork
of collagen fibers. Some cells are retained in irregular lacunae but
others (arrows) are not and resemble large elongated fibroblasts.
X 500.
22
High magnification of the center of the interpubic ligament shown
in figure 20. Explanation of figure 21 also applies to this figure.
X 500.
AUTOTRANSPLANTED INTERPUBIC JOINT
E. S. Crelin
PLATE 3
163
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