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Lack of effect of relaxin or steroids on the histology of the mouse parathyroid gland.

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Lack of Effect of Relaxin or Steroids on the
Histology of the Mouse Parathyroid Gland
J. P. MANNING, B. G. STEINETZ, SARA F, PRIESTER
MARGARET C. BUTLER
Department of Physiology, Warner-Lambest Research Institute,
Morris Plains, New Jersey
AND
ABSTRACT
Normal parathyroid glands exhibited two distinct cellular types: one
type of cell had a large round nucleus containing fine chromatin granules and a
nucleolus; the other type had a round or spindle-shaped nucleus with densely-packed
chromatin. The following hormonal preparations were tested and failed to alter parathyroid histology: relaxin extract (R), estradiol cyclopentylpropionate (ECP), combined ECP and R, or various combinations of these hormones with progesterone or
cortisone acetate. The pubic joints of the mice underwent the anticipated hormonallyinduced changes. Our results are at variance with those of Chase and Shanmugasundaram ('64) who ( a ) described only one cell type in the mouse parathyroid and ( b )
reported that relaxin alone or a combination of estrogen and relaxin induced cellular
changes suggestive of increased glandular function. The two studies are apparently
irreconcilable; however, we did note in about 30% of our mice variable lymphatic
aggregates in the thyro-parathyroid complex. Although not actually related to hormonal treatments, it is conceivable that in a small series of animals and without
serial sectioning, such lymphatic tissue might be mistaken for altered parathyroid
gland.
Presumptive targets for the action of relaxin include the pubic symphysis, cervix
uteri, uterus (myometrium and endometrium), subcutaneous connective tissue
and mammary glands (see Hall, '60, for review). Recent work implies effects of relaxin upon the thyroid gland (Plunkett,
Squires and Heagy, '63; Braverman and
Ingbar, '63) although the specificity of
this action remains to be demonstrated
(Braverman and Ingbar, '63). Chase and
Shanmugasundaram ('64) have now described an alteration in parathyroid gland
histology in mice treated with relaxin extract. The potential importance of this
latter finding is far-reaching, in view of
the bone erosion which occurs in the
pubic symphysis of mice treated with
estrogens and relaxin (Hall, '56; Crelin,
'63) and also in consideration of the implicit change in electrolytes of connective
tissue ground substance following relaxin
administration (Gersh and Catchpole, '60).
However, Chase and Shanmugasundaram
('64) used a relatively crude relaxin extract (porcine origin) in their studies, and
the possibility of a non-specific polypeptide
effect upon the parathyroids was by no
means excluded. These authors likewise
ANAT. REC.,153: 225-232.
failed to demonstrate whether or not an
estrogen alone might produce the same
alterations in parathyroid histology.
Therefore, it was important to establish the specificity of the effects of relaxin
extract upon the parathyroid glands and
to determine if administration of an estrogen by itself would induce similar changes.
Furthermore, it was of interest to explore
possible inhibition of the parathyroid effect by compounds known specifically to
prevent the action of relaxin upon the
pubic symphysis (Steinetz, Beach and
Kroc, '59).
MATERIAL AND METHODS
Thirty-eight female virgin mice (20-24
gm), purchased from the Charles River
Breeding Laboratories, were housed in a
quiet air-conditioned room ( 74-78 OF),
bedded on shavings and fed Rockland
mouse diet and tap water ad-lib.
The following hormonal preparations
were made up for subcutaneous injection:
1. Estradiol cyclopentylpropionate (Depo-Estradiol, Upjohn; 5 ug/O.l ml corn oil.
2. Relaxin (W1164, 48E-l105a, 140
units/mg, Warner-Lambert), 50 units
225
226
J. P . MANNING, B. G. STEINETZ, S . F. PRIESTER AND M . C . BUTLER
(0.36 mg)/0.2 ml 5% beeswax in sesame
oil.i
3. Cortisone acetate (Cortone, 25 mg/
ml, aqueous suspension, Merck S+D), 2.5
mg/O.l ml.
4. Progesterone, U.S.P., 5 mg/0.2 ml
sesame oil.
5. Low potency relaxin control extract
(W1164-565/1C, 0.5 unit/mg, WarnerLambert), 0.14 unit (0.36 mg)/0.2 ml
5% beeswax in sesame oil.
The mice were divided into groups of
four or five each and treated as follows:
Group I . Intact controls, no injections.
The animals were killed at the end of the
experiment.
Group 2. Relaxin treated. Each mouse
received daily a single subcutaneous injection of 50 units (0.36 mg) relaxin in
0.2 ml 5% beeswax in sesame oil for four
days. Mice were killed on day four.
Group 3 . Relaxin and cortisone acetate
treated. Received relaxin as in Group 2
and in addition, a daily subcutaneous injection of 2.5 mg cortisone acetate in 0.2
ml aqueous suspension for four days.
Mice were killed on day four.
Group 4. Relaxin and progesterone
treated. Received relaxin as in Group 2
and in addition, a daily subcutaneous injection of 5 mg progesterone in 0.2 ml
sesame oil for four days. Mice were killed
on day four.
Group 5. Low potency relaxin treated
control. Each mouse received daily a single subcutaneous injection of 0.14 (0.36
mg) unit relaxin in 0.2 ml 5% beeswax
in sesame oil for four days. Mice were
killed on day four. Note that this group
received the same amount of ovarian polypeptides as did the other relaxin-treated
groups.
Group 6 . Estrogen treated. Each mouse
received a single subcutaneous priming
injection of 5 ug estradiol cyclopentylpropionate (ECP) in 0.1 ml corn oil seven days
preceding the onset of relaxin administration to the other groups. Mice were
killed on day 11.
Group 7. Estrogen and relaxin treated.
Received estrogen as in Group 6 and in
addition, on day seven, a daily subcutaneous injection of 50 units (0.36 mg) relaxin in 0.2 ml 5% beeswax in sesame oil
for four days. Mice were killed on day
11.
Group 8 . Estrogen, relaxin and cortisone acetate treated. Received estrogen
and relaxin as in Group 7 and in addition, on day seven, a daily subcutaneous
injection of 2.5 mg cortisone acetate in
0.2 ml aqueous suspension for four days.
Mice were killed on day 11.
Group 9. Estrogen, relaxin and progesterone treated. Received estrogen and
relaxin as in Group 7 and in addition, on
day seven, a daily subcutaneous injection
of 5 mg progesterone in 0.2 ml sesame oil
for four days. Mice were killed on day 11.
At necropsy, the trachea, with the intact thyroid and parathyroid glands, was
dissected free of muscle and fascia and
fixed in Zenker's fluid. Paraffin embedded
serial sections cut at 7 CI in the transverse
plane, were stained with hematoxylin and
triosin.
Interpubic ligament length was determined according to the method of Steinetz,
Beach and Kroc ('62) utilizing a binocular
dissecting scope with a calibrated micrometer eyepiece and a lucite rod transilluminator.
RESULTS
Group 1. The parathyroids were usually located on the caudal-lateral aspect of
the thyroid gland. Each parathyroid was
surrounded by a connective tissue capsule
which separated it from the thyroid (figs.
1 , 2 and 3). The parathyroid was traversed
by variable numbers of connective tissue
septa which, more or less, isolated groups
or cords of densely packed polygonal cells.
The septa contained blood vessels which
appeared to arise from a central core of
highly vascularized tissue. In most of the
parathyroid cells, the nuclei were large
and round to oval with fine chromatin
granules and usually contained a large
nucleolus. In other cells, the nuclei were
round or spindle-shaped with denselypacked chromatin. Cytoplasmic detail was
obscured by the nucleus which occupied
nearly the entire area of the cell (fig. 6).
Whether these two cell types represent intermediate stages of the chief cell or are
1The dose employed was 50 times greater than
needed to induce inter-pubic ligament formation.
This dose was selected because it had been used by
Chase and Shnnmugasundaram ('64).
RELAXIN AND PARATHYROID HISTOLOGY
two separate and distinct cells we were
unable to determine.
In the parathyroids of two control animals, non-encapsulated or partially encapsulated lymphatic aggregations could be
demonstrated (fig. 4). They consisted
usually of a framework of reticular cells
and fibers infiltrated with lymphocytes.
The reticular cells were typical with
large pale nuclei containing nucleoli and
vague cytoplasmic outlines. The lymphocytes consisted almost entirely of a nucleus
which was rounded or ovoid in shape and
contained coarse clumps of chromatin
(fig. 4).
Groups 2-9. There were no detectable
alterations in the histological appearance
of the parathyroids (figs. 7 and 8), except
for a variable hyperemia which occasionally occurred in some of the animals.
This change, however, was not a consistent
finding in any of the mice in the various
treatment groups. Approximately 30% of
the treated mice demonstrated lymphatic
tissue in the parathyroid or in the immediate area (figs. 2, 3 and 5).
The pubic symphyses of the mice treated
with relaxin or ECP and relaxin showed
the expected development of long interpubic ligaments (Groups 2 and 7; table 1).
This process was inhibited by concurrent
treatment with cortisone acetate (Groups
3 and 8) or progesterone (Groups 4 and
9). The low potent control extract did not
significantly increase the interpubic distance.
TABLE 1
Effect o f the various hormonal treatments on
interpubic ligament formation
Group
Treatment
1
Ligament
length
mm
Control
R
RC
RP
LR
E
ER
ERC
ERP
+. S.E.
0.1650.01
3.90 -C 0.39
1.115 0.24
2.04k0.17
0.21 f0.02
0.26 -C 0.01
4.91 f0.25
0.73 f0.15
0.7750.16
1 R, relaxin; C, cortisone acetate; P, progesterone.
E, estradiol cyclopentylpropionate; LR, low potenc;
relaxin.
227
DISCUSSION
The histology of the normal mouse parathyroid was very adequately described by
Fekete ('41) and Larionow ('40) and
agrees, in general, with that reported in
this communication.
The response of the pubic ligament to
the various hormone treatments is summarized in table 1. As reported by Hall
('56), Steinetz and Beach ('63) and
Steinetz, Manning, Butler and Beach ('65),
maximal ligament formation was obtained
in the intact female mouse when the relaxin administration was preceded by a
dose of estrogen as in Group seven. The
depression of ligament lengthening by
progesterone and cortisone acetate is well
documented (Hall, '56; Steinetz, Beach
and Kroc, '59; Steinetz, Manning, Butler
and Beach, '65). The low potency extract was not effective in promoting the
pubic symphyseal transformation. In spite
of the normal response of the intact female mouse pubic symphysis to the hormone treatments, we were unable to demonstrate any alteration in parathyroid histology. Nuclear diameter, cell number and
staining quality were unchanged. As noted
in our results, there were a few animals
from some treatment groups which exhibited an increased hyperemia in localized
area. This change, however, was also recorded in one of the controls.
It was difficult for these investigators to
interpret the histological evidence of Chase
and Shanmugasundaram ('64), since a
control print was not included in the transcript. Judging exclusively the photographs
(figs. 1, 2 ) in their article, we feel a similar histological picture can be demonstrated in parathyroid glands infiltrated
with lymphocytes which was fairly common (30% ) in controls, as well as hormone-treated mice.
Chase and Shanmugasundaram ('64)
indicated zero millimeters separation of the
pubic symphysis in the control and untreated-ovariectomized mice. The present
study as well as those of Crelin ('63) and
Steinetz, Beach and Kroc ('59), indicate
an average interpubic distance of 0.165 to
0.2 mm (range: 0.1-0.3 mm).
An interaction of parathormone with relaxin was difficult to rationalize since the
reports in the literature indicate :
228
J. P. MANNING, B. G. STEINETZ, S. F. PRIESTER AND M. C . BUTLER
1. Relaxin administration to ovariectomized mice results in no interpubic ligament formation unless they are first
“primed’ with estrogen.
2. Estrogen was solely responsible for
the bony and cartilaginous alterations in
the pubic symphysis.
3. The action of relaxin was only for
pubic ligament formation. If relaxin stimulated secretion of parathyroid hormone,
one might expect to find changes in bones
of other parts of the body. Crelin (’54),
however, was unable to demonstrate such
a phenomenon.
LITERATURE CITED
Braverman, L., and S. Ingbar 1963 Effects of
preparations containing relaxin on thyroid
function in the female rat. Endocrinology, 72:
337-340.
Chase, E. B., and D. Shanmugasundaram 1964
The effect of relaxin on parathyroid histology
i n the mouse. Anat. Rec., 149: 309-312.
Crelin, E. S. 1954 The effects of estrogen and
relaxin on the public symphysis and transplanted ribs i n mice. Anat. Rec., 120: 23-32
1963 The deveIopment and hormonal
response of the autotransplanted interpubic
joint in mice. Anat. Rec., 146: 149-163.
Fekete, E. 1941 In: Biology of the Laboratory
Mouse. (G. D. Snell, ed.), pp. 100-101. Dover
Publications, Inc., New York.
Gersh, I., and H. R. Catchpole 1960 The nature of ground substance of connective tissue.
Perspectives i n Biol. and Med., III: 282-319.
Haines, A. L. 1957 The effect of estrogen on
cartilage and bone i n castrate CJH mice. Yale
J. of Biol. and Med., 30: 121-136.
Hall, K. 1956 An evaluation of the roles of
oestrogens, progesterone and relaxin in producing relaxation of the symphysis pubis of
the ovariectomized mouse, using the technique
of metachromatic staining with toluidine blue.
J. Endocrin., 1 3 : 384-393.
1960 Relaxin. J. Reproduction and
Fertility, I : 368-384.
Larionow, L. Th. 1940 The endocrine glands
in experimental cancer induced by benzpyrene. A study of the role of the endocrine
glands in the pathology of tumors. Am. J.
Cancer, 38: 492-505.
Plunkett, E. R., B. P. Squires and F. C. Heagy
1963 Effect of relaxin on thyroid function
in the rat. J. Endocrin., 26: 331-338.
Steinetz, B. G., and V. L. Beach 1963 Hormonal requirements for interpubic ligament
formation in hypophysectomized mice. Endocrinology, 72: 771-776.
Steinetz, B. G., V. L. Beach and R. L. Kroc
1959 In: Recent Progress in the Endocrinology of Reproduction. (C. H. Lloyd, ed),
pp. 3 8 9 4 2 7 . Academic Press, New York.
1962 In: Methods in Hormone Research. (R. I. Dorfman, ed), Vol. 2, pp,
559-590.
Academic Press, New York and
London.
Steinetz, B. G., J. P. Manning, Margaret Butler
and Vivian Beach
1965 Relationships of
STH, steroids and relaxin i n the transformation of pubic joint cartilage to ligament in
hypophysectomized mice. Endocrinology, 76:
876-882.
PLATE 1
EXPLANATION O F FIGURES
1
Parathyroid gland from a normal untreated intact female mouse.
225 X.
2
The parathyroid from an estrogen-relaxin treated animal with a n
adjacent lymphatic aggregation separated by a poorly defined capsule. 225 X.
3
A nonencapsulated lymphatic infiltration of the mouse parathyroid
gland. This particular animal received only relaxin. Similar histological appearances can be demonstrated in approximately 30% of
animals throughout all experimental groups. 225 X.
RELAXIN AND PARATHYROID HISTOLOGY
J. P. Manning, B. G. Steinetz, Sara F. Priester and Margaret C. Butler
PLATE 1
229
PLATE 2
E X P L A N A T I O N O F FIGURES
230
4
Lymphatic tissue from a partially encapsulated aggregation in a
control animal (lymphocytes, L ) . There is a striking similarity between this microphotograph and figure 2 in report of Chase and
Shanmugasundaram ('64). Note the large nuclei of the primitive
reticular cells ( P ) . 560 X.
5
Nonencapsulated lymphatic tissue from a relaxin-treated mouse
located i n a n area immediately cranial to the parathyroid gland
(lymphocytes, L ) . Note the large nuclei of the primitive reticular
cells ( P ) . 560 X.
6
The parathyroid from a control mouse (cell A, round nucleus and
a prominent nucleolus; cell B, spindle-shaped nucleus with dense
chromatin).
7
Estrogen and relaxin administration resulted in no detectable histological change in the parathyroid gland. 560 X.
8
A section of parathyroid gland from a n intact female mouse treated
with relaxin as i n Group 2. 560 X .
RELAXIN A N D PARATHYROID HISTOLOGY
J. P . Manning, B. G. Steinetz, Sara F. Priester and Margaret C. Butler
PLATE 2
231
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