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Prenatal development of the skeleton in long-evans rats.

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PRENATAL DEVELOPMENT O F T H E SKELETON
I N LONG-EVANS RATS1
HOWARD V. WRIGHT, C. WILLET ASLING, HARRY L. DOUGHERTY,
MARJORIE M. NELSON AND HERBERT M. EVANS
I n s t i t u t e of Experimental Biology and Department of A n a t o m y ,
University of California, Berkeley
SEVEN FIGURES
The fetal skeleton is a useful indicator of embryonic development and frequently reflects changes in the maternalfetal environment. Standards of normal skeletal development
are required to judge alterations in progress toward the adult
“definitive” form. Strong (,as) studied the rate and sequence
of fetal ossification in the albino (Wistar) rat, using cleared
but unstained fetuses. The present study was undertaken to
provide similar data on fetal ossification f o r the Long-Evans
strain, using alizarin red and toluidine blue staining to determine the earliest stages of both chondrification and ossification.
MATERIAL AND METHODS
Female rats of the Long-Evans strain were bred and the
fetuses were removed at gestational ages of 14 to 21 days at
half-day intervals. The fetuses were fixed in 95% ethyl alcohol
for 5 to 10 days, and then cleared and stained with toluidine
blue for cartilage or alizarin red for bone.
‘Aided by grants from the U. S. Public Health Services (A-841 and A-664).
‘The stock diet used f o r pregnancy is a modification of McCollum’s Diet I
and is composed of 67.5% ground whole wheat, 15.0% technical grade casein,
7.5% skim milk powder, 6.75% hydrogenated vegetable oil (Crisco or Primex),
1.5% calcium carbonate, 0.75% iodized sodium chloride, and 1.0% fish oil (vitamin
A-D concentrate). Lettuce is given ad libitum twice weekly.
The day on which sperm is found in the vagina is considered to be day zero.
659
660
WRIGHT AND OTHERS
The methods used for clearing and staining were modified
from those reported by Miller ( '21) and Dawson ('26) and are
as follows :
T'oluidiNe blue: After fixation the fetuses were skinned,
eviscerated, and then placed in toluidine blue solution (1: 400
in acidified 70% ethyl alcohol) for 10 days. The fetuses were
decolorized for 7 t o 10 days by washing first with acid 70%
alcohol, then with 80% and 95% until the alcohol showed only
a slight bluish tinge. The fetuses were transferred to 2%
KOH for clearing until the cartilages were visible and then
into Mall's solution f o r two or three days; the fatty deposits
were removed at this time. Finally, the specimens were placed
in glycerine for additional clearing and for storage.
Alizarin red: After fixation in 95% ethyl alcohol, the fetuses were skinned and eviscerated. The fetuses were then
placed in 1%KOH for clearing until the bone centers were
visible as whitish areas; the time necessary for this varied
with the age and size of specimen. After clearing, the fetuses
were transferred to Mall's solution (20% glycerine, 1%
KOH, 79% distilled water) and the solution of alizarin red
(1:10,000 in distilled water) added drop by drop to a dark
red color, i.e., until specimen was barely visible. Specimens
were left overnight or longer for staining. Fetuses were then
placccl in fresh Mall's solution for decolorization which usually required 3 to 10 days ; Mall's solution was changed every
two o r three days until alizarin red was absent from both the
soft tissues and the solution. F a t deposits were removed at
this time. After decolorization, the fetuses were placed in
glycerine for additional clearing and for storage.
These staining methods allowed the study of both intrarnembraneous and endochondral ossification in the formation
of the skeleton. The number of fetuses studied for each halfday interval varied from 6 to 16, with representatives from at
least three litters for each interval. Both intralitter and interlitter variability in chondrification and ossification were encountered. Hence, the average condition for each age interval
has been used in the following description and figures.
661
RAT PRENATAL SKELETAL DEVELOPMENT
RESULTS
The data have been summarized in a series of bar graphs,
with the development of the cranial skeleton shown in figure 1
and of the facial skeleton in figure 2. Figure 3 illustrates the
development of the post-cranial axial skeleton with the girdle
bones and figure 4 that of the free appendages.
I n the rat, as in other mammals, bone formation occurs in
either membrane (intramembraneous osteogenesis) o r in cartilage (endochondral osteogenesis) . Some membrane bones
develop in the neighborhood of cartilage, although not replacing it, e.g., the relationship of the body of the mandible
to Meckel’s cartilage. I n the charts only those cartilages
FETAL AGE
BONE
1-5
-I
BASIOCCIPITAL
8
EXOCCIPITAL
E
15%
- 16
IN DAYS
16% 17 17% 18
18% 19
20
21
0
SUPRAOCCIPITAL
PARIETAL
FRONTAL
PRESPHENOID
p BODY
0
LAT. PROCESS
n
$
INTER. PTERYGOID
m
ALAR PROCESS
SQUAMOSAL
TYMPANIC WLLA
ETHMOIO
KEY TO SYMBOLS
-
I
<.: .......
I
CARTILAGE
OSSIFICATION CENTER
EXPANDING BONE
OEFlNlTlVE BONE
Fig. 1 Development of the cranial bones with key to symbols.
662
WRIGHT AND OTHERS
which are actual precursors of bone are represented (stippled
areas in the figures). The establishment of the ossification
center, whether in membrane or on a cartilage model, is indicated by diagonal lines. It cannot be stated with certainty
whether the first material stainable with alizarin red was
bone or the calcified cartilage which immediately precedes it.
However, good evidence of the accuracy of the bone ages recorded in the charts was obtained from study of Mallorystained histologic sections. The expansion of the first osseous
centers has been indicated by cross-hatching. The term “definitive bone,’’ represented by the solid bar in the figures,
FETAL AGE IN DAYS
BONE
1
15 15% 16
16k 17 17% 18 18% 19 19% 20
21
a LAT.PLATE
J
2
5
PALATINE PROC.
5
ZYGOMATIC PROC.
a LAT. PLATE
-I
Z MED.PLATE
4
a
HORIZ. PLATE
PERPEN. PLATE
PALATINE
ZYGOMATIC
LACRIMAL
VOMER
NASAL
BODY
m
p
CORONOID PROC.
4
CONDYLOID PROC.
ANQULAR PROC.
HYOlO
Fig. 2
Development of the facial bones. See figure 1 f o r key to symbols.
663
RAT PRENATAL SKELETAL DEVELOPMENT
indicates that the bone has essentially attained the anatomical
contours found in the adult skeleton.
The earliest appearance of skeletal cartilage, as shown by
stainability with toluidine blue, was in the 3rd to the 9th ribs
(fig. 3 ) , at 15 days. The earliest appearance of bone in the
entire skeleton was that in the body of the mandible, likewise
at 15 days (fig. 2). During days 15% to 16 the majority of
bones made their first appearance, whether initially in membrane, as in the facial bones and vault of the skull, o r as a
cartilage model, as in the base of the skull and the postcranial skeleton. Several of the remaining bones appeared
BONE
FETAL AGE IN DAYS
I
15 15% 16 16% 17 17% 18 18% 19 19% 20
21
CLAVICLE
SCAPULA
CERVICAL
2f THORACIC
0
a LUMBAR
a
o SACRAL
z
4
8
CAUDAL
5 CERVICAL
A THORACIC
a
LUMBAR
w
SACRAL
>
CAUDAL
RIBS
ST E RNEBRAE
XlPHOlD
ILIUM
ISCHIUM
PUBIS
Fig. 3
symbols.
Development of the trunk and girdle bones. See figure 1 for key to
664
WRIGHT A N D OTHERS
on day 1736. The last bone to develop during the fetal period
was the presphenoid for which the cartilage model appeared
on day 181/2,followed by ossification on day 19. Some osseous
centers, notably the ethmoid, the patella and other sesamoids,
and the epiphyseal centers of the long bones, have no dis-
BONE
I
1
FETAL AGE IN DAYS
15 15% 16 16% 17 17% 18 18% 19
1914 20
21
HUMERUS
ULNA
RADIUS
CARPAL
METACARPAL
$
PROXIMAL
z
3
a
2
MIDDLE
DISTAL
FEMUR
TIBIA
FIBULA
TARSAL
METATARSAL
PROXIMAL
(3
f
2
MIDDLE
DISTAL
Fig. 4 Development of the bones of the extremities. See figure 1 f o r key to
symbols.
cernible fetal representation and do not appear until after
birth. On the other hand, many bones attained the definitive
form, i.e., approximation of the adult bony contour, before
birth. I n the skull this condition was reached slightly earlier,
days 18 and 19, than in the post-cranial skeleton, days 191/2
to 21.
RAT PRENATAL SKELETAL DEVELOPMENT
665
For convenience, some bones have been grouped together
in the charts, e.g., the vertebral arches and bodies, the ribs and
sternebrae, and bones of the paws. The sequence of development of the bones within these groups was as follows:
Vertebral arches: I n general the wave of ossification
started at the first cervical arch and proceeded caudad. I n
the cervical and thoracic regions ossification was so rapid
that all arches showed some ossification by the 17th day, although the more rostra1 ones were better developed. Ossification next appeared in the lumbar region and, with some
delay, in the sacral and finally in the caudal region. In the
latter the first and second arches were ossified by day 20 and
the third by day 21; ossification of the more distal caudal
arches occurred after birth.
Vertebral bodies: In contrast to the sequence observed in
the arches, ossification of the bodies started in the midthoracic
region, thoracic 4-13 on day 18, and progressed caudad more
rapidly than rostrad. I n fact, ossification in cervical vertebral bodies 3-7 appeared one day later (day 21) than ossification in the caudal vertebral bodies 1-3 (day 20). The corresponding ossification in the first and second cervical vertebrae was delayed until after birth. The sequence of caudal
bodies was incomplete a t birth, the last prenatal centers to
appear being those of 4-5 on day 21.
Ribs: Cartilage models of all the ribs were observed on
day 15. Ossification of the ribs began with ribs 3-9 on day
15% and during the following two days spread in both directions, slightly more rapidly caudad than rostrad. The sequence was as follows : day 15% -ribs 3-9 ; days 16 -ribs
2-10 ; day 16% -ribs 2-12 ; day 17 -ribs 2-13 ; and day 171/2
-ribs 1-13.
Sterwuna : Ossification of the first two sternebrae appeared
on day 19, followed by the 3rd and 4th on day 191/2 and the 5th
on day 20. The xiphoid process appeared on day 191/~before
the sternebral sequence was complete.
Bones of the paws: The carpal and tarsal bones and middle
phalanges could be seen only as cartilage models in the fetus.
666
WRIGHT AND OTHERS
Ossification of metacarpals, metatarsals, and remaining phalanges showed a complex pattern. With an important exception, the sequence of ossification in the paws was proximal
to distal. This exception was found in the distal phalanges
which showed ossification after the metacarpals and metatarsals but before the proximal and middle phalanges. Wood
Jones ('42) has discussed this unexpected sequence as a general mammalian trait. Among the various digits, the 2nd and
3rd were the earliest to appear and usually ossified simultaneously, followed by the 4th, the 5th and finally the first digit.
I n general, ossification in the forepaws preceded that in the
hindpaws.
Figures 5, 6 and 7 are diagrams showing the progressive
development in both chondrogenesis and osteogenesis of representative bones. Attention should be directed to the morphogenesis of representative bones since acceleration and retardation may be demonstrated not just by the earlier or
later appearance of the ossification centers but also by changes
in form. I n experimental animals the bones abnormal in form
on day 2 1 may actually have the form normal for an earlier
age. The bones illustrated showed their definitive form by
day 191/, being identical in shape but smaller in size than was
observed at 21 days.
I n the ribs (fig. 5) development of the tubercle with its
associated articular facet and the medial flattening at the
angle indicated the definitive form. The vertebral arch (fig. 5)
showed its definitive form with the development of the articular facet. The density of bony modeling of the supra- and
infra-spinous fossae marked the definitive scapular shape
(fig.5). I n the humerus (fig. 6) the disappearance of cartilage
on the deltoid tuberosity and the flattened triangular area at
the distal end of the diaphysis indicated the definitive form.
The distinctive curvatures of the radius and ulna (fig. 6) demonstrated their definitive shapes. I n the femur (fig. 7) the
elevation corresponding to the lesser trochanter marked the
definitive form. The tibia (fig. 7 ) showed definitive form by
the bony modeling for the accommodation of muscles on the
lateral aspect and by tlic c1iai.acteristic curvature, whicli also
indicated the definitive fibula (fig. 7).
UISCITSSION
The cai*c4alstudies of Strong (’25) were the first in wliicli
fetal osteogencsis was studied serially in r a t s of known coil~
the sequence of fetal ossification reccptioii age. I Kgeneral,
ported in the pi’esent study f o r tlic Long-Evans r a t is in agreclment with that recorded by Strong for the Wistar rat. Ideiitical times w e r ~given f o r the lumbar vci*tel)ralliotlies, stci*nclwae, rnetacarpals and metatarsals, but in most instances
Strong reported the ossification ccnters appearing one-half
to oiie day later than were found in the present study. The
delay was seldom as rnnch as two days. Such differences niay
have arisen from several factors : e.g., (1) determination of
conception age ; ( 2 ) strain of rat, TT’istar o r Long-Evans ;
( 3 ) adequacy of sample to include both intralitter and interlitter variability; (4) method of visualizatioii, uiistaiiictl or
stained material.
Recently TTalker and TVii~tenschafter ( ’57) have incliitltbrl
studies of fetal ostcogeiiesis in their survey of skeletal dcvelopnient in the Long-Evans rat. I n their study the strain of nit,
nicthocl of determining conception age,4and maternal diet were
the sanic as tliose used in the present study and the alizariii
red staining procedurc was similar. Nevertheless, they asually observed the first appearance of ossification centers on(>,
two or even three days later than was found in the present
study. I t may he noted that the earliest fetal age examined iil
their studies was 17 days, a time ~ v l i e nossification was already
well established in a number of hones in our fetuses. Succcccliiig fctuscs were examined at daily rather tliaii half-day intervals. Adequacy of sanipliiig in their study cannot be dctermined, as the nnmher of fetuses examined for each fetal
111 n pcrwiial coiniiiuiiicntioii to the autlion, D. G. Walker describes details of
his iiietlioil of determiiiiiig coiiception agc, which differ in soiiie resptxets f r o m that
cmplo~etlin tlic prescnt study. It is possible, therchy, to account f o r differences
of 1 2 to 94 hours in tlic staged agcs.
668
WRIGHT A N D OTl-IEHS
age was iiot giveii. lye are uiiable to explaiii the many discrepaiicics between their study aiicl tlic present report although it is possible that slight differences in the techiiique
of staining o r in the evaluatioii of sigiiificaiice of staiiicxl material may be partially responsible.
The charts provided by tlie present study offer standards
uscful for tlctcrrniniiig skeletal age in rat fetuses of tlie
Long-Evaiis strain, a i i d particularly for detecting any retardation o r accelcratioii of bone developmciit resultiiig from
experimental procc.tlures. Tliese standards have heeii used
for studying the skeletal dwelopmeiit of fetuses from rats
subjected to a tlcficiciicy of ptei*oylglutamic acid ( P G A ) during pi.egnaiicy (Asliiig c t al., '%). For this purpose, tlic most
useful criteria WCL'C the times of first appearance of tlic ossificatioii centers. In additioii, the sliapes of bones w e i ~also
fouiicl to i*c.flect tlie degree of maturity siiicc cliffel-ential
growth on the diflei-eiit axes of tlie boiies is iiccessai*y foi*
iiormal growth aiicl differentiation. The changes in scapular
sliapcl serve as a11 example. In the early stages tlic greatest
leiigtli of the scapula is parallel to the long axis of tlic botly
hut later niore rapid growth elongates tlie transverse axis.
Thus, R scapula judged as malformed in a fetus a t term following experiniciital proccdures iiiay actually be oiily delayeti
iii its diffeiwitial growth. True nialfoimiations iiiay be superiniposed upon such delayed bony development, a s in the convexity of thc scapula oliserred in PGA-deficient fetuses (Asling et al., '54).
SUMMARY
Tlie tlevelopmoiit of tlic skeleton has been studied in rat
fetuses of thp Long-Evans stmin, raiigiiig from 14 to 21 days
fetal agv by half-day intervals. Both toluidine lilue and alizarin red staining have been used to visualize the earliest
stages of chondrogeiicsis and of osteogciiesis. The charts and
figurcs summarizing tlic data can be uscd to determine fetal
skeletal agc. aiitl to provide standards for studying the accelei.atioii, i*ctartlatioii, or malformation of the fetal skeleton
resnltiiig f r om experimental procedures.
R A T P K E S A T A L S K E L E T A L DEVELOPMEST
669
LITERATURE CITED
ASLING, C. W., M. 1 4 . NELSON, 11. V. WRIGHT A N D €1. hl. E v a r x 1955 Coiigenital skeletal :iiioiiialies i i i fetal r a t s resulting f r o m niateriial pteroylg1ut:rmic :Icid drfiriciicy duriiig gest:rtioii. Aimt. Rec., 1.21 : 773-800.
J)AWSON, A. B. 1926 A iiotc on the staining of clearcd speciiiicns with :rlizariii
rcd S. Stain Teelinol., I : 123-124.
MILLER,C. H. 1 9 2 1 Den1oiistr:ition of the cartilaginous skcletoii in iii:iiniii:iliaii
fetuses. Aiiat. Rec., ,%I: 415-419.
STRONG,
R. Ill. 1925 The or(lor, tinir, :iiiil rate of ossific:rtioli of the albino r a t
( M M S ~ i o ~ ~ c ~ , q ui lcbui ns u s ) skeletoii. Am. J. Aii:it., 36: 313WALKER,I). G., ASD %. T. \VIRTSCII.ZPTER
1997 The Gelirsis of t h e R a t Skrlctoii.
A L;iborntory Atlas. Charles C Tlionias, p ~ i l ~ l i s l i e ~ s .
WOODJOXES,
F. 1942 The l’riiiciples of Aii:rtoniy as Sccii ill the Hand. Willinnis
and Wilkiiis Co.. I3altiinorc.
15V2
16
16V2
17
lev2
19
21
.
16
17
16k
I8 I / Z
19
17%!
18
21
PL.\TE 2
16
16V2
17
I 71/2
18
I81/2
19
21
16’/2
17
17’12
18
18 ‘h
19
21
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