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Histochemical types and sizes of fibers in the rectus abdominis muscle of guinea pigAdaptive response to pregnancy.

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THE ANATOMICAL RECORD 217:23-29 (1987)
Histochemical Types and Sizes of Fibers in the
Rectus Abdominis Muscle of Guinea Pig:
Adaptive Response to Pregnancy
G. LALATTA COSTERBOSA, A.M. BARAZZONI, M.L. LUCCHI, AND R. BORTOLAMI
Institute
of
Veterinary Anatomy, University of Bologna, 40126 Bologna, Ztaly
ABSTRACT
Effects of pregnancy stimulation upon histochemically assessed myofibi-illar ATPase and muscle fiber diameters were analysed in the rectus abdominis
(RA) muscle of guinea pig. Samples of the muscle were taken a t 30, 40, 50, 60, and
70 days of pregnancy and compared with samples of the same muscle taken from
nonpregnant guinea pigs. Changes in muscle fiber proportions were noted through
the course of pregnancy. Starting from 50 days of gestation a n increase in type I
fibers and a decrease in type IIB fibers were noted. Increase in muscle fiber diameters was also observed in type I, IIA, and IIB fibers. In addition, the RA muscle of
the male guinea pig was compared with that of the female guinea pig and showed
more type IIA and less type IIB fibers and all the three fiber types were larger than
those of the female.
In recent years a number of studies of skeletal muscle
have demonstrated that fiber type transformation is possible not only with cross-innervation and specific electrical stimulation (for references see Jolesz and Sreter,
1981; Mabuchi et al., 1982) but also with prolonged and
intense endurance training. In animals (Kowalski et al.,
1969; Barnard et al., 1970; Faulkner et al., 1972; Maxwell et al., 1973; Muller, 1974; Green et al., 1984) and
man (for references see Howald, 1982) the effects of
endurance training consisted in a n increase in aerobic
metabolism and/or transformation of type IIB into type
IIA fibers and also of type I1 into type I fibers.
Pregnancy, with its increase in abdominal contents,
may represent a physiological chronic stimulus imposed
on the muscles of the abdominal wall which should
result, in our opinion, in a fiber type transformation.
Data on this subject are not available, with the exception of a study by Martin (1979), who has been unable to
detect a significant change in rectus abdominis (RA)
muscle fiber proportions through the course of pregnancy in the rat. However, in this animal pregnancy
lasts only 21 days.
In order to verify whether a longer gestation period
could result in a muscle fiber transformation, we investigated the effect of pregnancy on RA muscle of the
guinea pig, as in this laboratory animal the pregnancy
lasts approximately 70 days, i.e., a time period which
should be long enough to allow possible changes in muscle fibers. RA muscle was chosen since it is the most
involved muscle in any stretch of the abdominal wall. A
preliminary report has been given elsewhere (Bortolami
et al., 1980).
The fiber type patterns in the RA muscle of male and
female guinea pig was also compared to investigate possible sex differences.
0 1987 ALAN R. LISS, INC.
MATERIALS AND METHODS
Animals and Muscle Samples
Adult nulliparous female and male albino guinea pigs
of the same age and same weight were used. Females
were placed in a cage with male guinea pigs for a period
of 3 days and the middle of the 3 days was counted as
day 0 of pregnancy. The animals, killed with a n overdose of Nembutal, were 1) three females at 30, 40, 50,
60, and 70 days of pregnancy, 2) nine nonpregnant control females, and 3) nine males. The control females and
the males were killed at time points corresponding to
days 0,40, and 70 of gestation of the pregnant animals.
Both the RA muscles were dissected from each subject
with their full abdominal length from the cartilago xifoidea to the cranial level of the pubis and a mediolatera1 extent from the linea alba to the superficial
epigastric arteries. The muscles appeared clearly segmented by six or seven spaced tendinous intersections.
The muscle samples were removed from the central portion of each segment in order to avoid the areas near the
tendinous intersections and rapidly frozen by immersion
in isopentane cooled with liquid nitrogen. Serial cross
sections were cut at 10 K r n on a cryostat microtome at
- 20°C.
Enzyme Histochemistry and Fiber Typing
Serial sections were assayed for myofibrillar adenosine triphosphatase (ATPase) activity following alkaline
(pH 10.4) and acid (pH 4.6) preincubation (Brooke and
Kaiser, 1970). Sections were also incubated for succinic
acid dehydrogenase (SDH) (Nachlas et al., 1957) and
Received February 25, 1986; accepted August 4, 1986.
24
G. LALATTA COSTERBOSA. A.M. BARAZZONI, M.L. LUCCHI, AND R. BORTOLAMI
menadione-linked a-glycerophosphate dehydrogenase
(a-GPDH) (Hess and Pearse, 1961).According to the terminology of Brooke and Kaiser (1970), four muscle fiber
types were considered (types I, IIA, IIB, IIC). Figure 1
illustrates the results of the staining procedures used in
this study. The type I fibers were dark after acid preincubation (Fig. 1A)but light after alkaline preincubation
(Fig. 1B); type IIA fibers showed the reverse pattern;
type IIB fibers were dark at pH 10.4 but intermediate at
pH 4.6. Fibers classified as type IIC remained stable
throughout the entire pH range used. The type IIAB
fibers were also observed and grouped with the type IIB
fibers (see Staron et al., 1984). Oxidative activity was
high in type I, IIA, and IIC fibers (Fig. lC), while glycolytic activity was low only in type I fibers (Fig. 1D).
In order to determine the percentage of each fiber
type, different areas of each RA muscle segment had
been randomly selected. The proportion of each muscle
fiber type was calculated as a percentage of the total
number of fibers in each segment analysed. A total of
about 4,000 fibers were considered for each subject. The
same areas utilized for the fiber type counting were then
photographed and x 100 magnification pictures were
used to obtain the mean fiber diameters for 60 fibers of
each type in each subject. The diameter measured was
the maximum diameter across the lesser aspect of the
muscle fiber (Brooke, 1970). This diameter was chosen
in preference to fiber area because it is not subject to
enlargement by oblique sectioning.
The data obtained from individual segments of RA in
each animal were pooled; this was possible since no
significant differences occurred between the different
segments in each animal. After having statistically
tested the homogeneity of the data within each experimental group by the one-way analysis of variance, the
results from the different groups were analysed and
compared with the controls by using Student’s t-test
(significance was accepted at the P < .05, P < .01,P <
.001 levels).
RESULTS
Muscle Fiber Types
Fiber typing was based on the ATPase reaction after
acid preincubation (Fig. 2). As can be seen in Table 1,
RA muscle of both sexes was composed predominantly
of type 11fibers, type IIB being prevalent; type IIC fibers
were always very few. The males, compared with the
females, showed more type IIA and less type IIB fibers
while no significant difference in percentage of type I
fibers was observed. In control animals killed at different moments corresponding to either 0 or 40 or 70 days
of pregnancy, no differences were observed in the percentages of RA muscle fiber types.
Changes in muscle fiber proportions were noted
through the course of pregnancy (Table 1).In fact, type
I fibers underwent first (30 days) a decrease followed by
a slow but consistent progressive increase in number
(Fig. 3C), which was statistically significant starting
from 50 days of pregnancy. Type IIA fibers remained
essentially the same in number and showed a significant decrease only at 60 days of pregnancy. The proportion of type IIl3 fibers increased up to 40 days of
pregnancy and then underwent a progressive decrease
which became statistically significant at 70 days (Fig.
4).
Muscle Fiber Diameters
In the RA muscle of both sexes the type IIB fibers
showed the largest diameter, while type I fibers presented the smallest diameter (Table 2). Significant differences were observed between the male and female
(Table 2): all the three fiber types were larger in male
than in female (Fig. 3A,B). In control animals killed at
different moments corresponding to either 0 or 40 or 70
days of pregnancy, no differences were observed in muscle fiber diameters. Changes in muscle fiber diameters
were observed during pregnancy in each muscle fiber
type (Figs. 2, 3C, 5). The type I, IIA, and IIB fiber
diameters increased significantly starting from 50 days
of pregnancy. At the end of pregnancy the type I fibers
showed the greatest relative increase in diameter
(30.80%), followed by type IIA (8.99%) and type IIB
(6.65%) fibers (Table 2).
DISCUSSION
The guinea pig RA muscle of both sexes contains a
high proportion of type I1 fibers which are also generally
larger than type I fibers. The male shows significantly
more type IIA and less type IIB fibers and all the three
fiber types are significantly larger than those of the
female.
In this investigation, the purpose of using the pregnancy of the guinea pig was to determine the effect of a
long-term stimulation, such as the effect of a 70-day
pregnancy, on the RA muscle. Data were collected at 30,
40, 50, 60, and 70 days of pregnancy. At 30-40 days of
pregnancy a transient slight decrease of type I fibers
and conversely a n increase of type IIB fibers were observed. Starting from 50 days of pregnancy the pattern
changed and a progressive increase in the proportions of
type I and a progressive decrease of type IIB fibers
occurred. Concerning the fiber diameters, all three fiber
types increased significantly starting from 50 days of
pregnancy and type I exhibited the greatest relative
increase.
It has been shown that fast muscles subjected to
chronic electrical stimulation exhibit a remarkable increase in their capacity for aerobic metabolism (Peckham et al., 1973; Pette et al., 1973; Riley and Allin,
1973) and a n increase in number of slow fibers (Munsat
et al., 1976; Rubinstein et al., 1978). Changes in the
ultrastructure of myofibrils as a result of a changed
impulse pattern have also been shown (Salmons et al.,
1978; Heilman and Pette, 1979; Sjostrom et al., 1980).
Recently, Green et al. (1984) have suggested that 15
weeks of a treadmill training program can provoke a n
increase in type I with a concomitant decrease in type
IIB fibers, which has been not only histochemically but
also biochemically assessed. Taken collectively, the results of all these studies show that inyreased activity is
capable of inducing true fiber type transformation.
This remarkable plasticity of the skeletal muscle fiber
has been histochemically demonstrated also in the present study. The RA muscle, in fact, during pregnancy
shows first a n adaptation which involves mostly the
type IIB fibers and successively, when the continuous
stimulation produced by pregnancy exerts its influence
on the abdominal wall in its stronger form, the muscle
then acquires a histochemical pattern, with increased
type I fibers, which appears to suit it to its new functional role, in which a weight-bearing activity becomes
GUINEA PIG RECTUS ABDOMINIS MUSCLE IN PREGNANCY
Fig. 1. Serial cross sections from rectus abdominis (RA) muscle of guinea pig (control female)
showing the staining patterns of type I, type IIA, type IIB, type IIAB, and type IIC fibers. A)
ATPase, section preincubated at pH 4.6. B) ATPase, section preincubated at pH 10.4.C) Succinic
acid dehydrogenase (SDH). D) Alpha-glycerophosphatedehydrogenase (a-GPDH).X 190.
25
26
G. LALATTA COSTERBOSA, A.M. BARAZZONI, M.L. LUCCHI, AND R. BORTOLAMI
Fig. 2. RA muscle of guinea pig. A) Control female. B) Regnant (50 days) female. C) Regnant
(60 days) female. D) Pregnant (70 days) female. A-D) ATPase, section preincubated at pH 4.6.
In the pregnant females there is a n increase in diameter of all fiber types. A muscle spindle is
identifiable in A (bottom right of the photograph). x 100.
27
GUINEA PIG RECTUS ABDOMINIS MUSCLE IN PREGNANCY
TABLE 1. Percentage distribution of fibers in rectus abdominis (RA) muscle of
nonpregnant female, male, and pregnant female guinea pig'
Sex
F contr.
M
FP.
FP.
FP.
FP.
FP.
Days of
gestation
Type 1
0
31.69
32.13
29.46*
30.10
33.94"
36.35* * *
38.64** *
-
30
40
50
60
70
(%o)
Type IIA
Type IIB
(%I
(%I
Type IIC
(%I
21.10
23.71***
21.40
19.50
22.09
18.83*
20.17
46.77
43.73***
48.78*
50.17***
43.56
44.18
40.78***
0.44
0.43
0.36
0.23
0.41
0.64
0.41
'Abbreviations: F contr., nonpregnant control female; M, male; Fp., pregnant female.
*Significant difference from control females (P < .05).
***Significant difference from control females ( P < ,001).
TABLE 2. Mean fiber diameters of RA muscle of nonpregnant female, male,
and pregnant female guinea pig'
Sex
F contr.
M
FP.
FP.
FP.
FP.
FP.
Days of
gestation
0
30
40
50
60
70
Type 1
M+SE
(pm)
41.88 & 0.39
43.59 & 0.42*
42.63 0.65
40.86 i 0.65
53.89 + 0.61**'k
50.58 + 0.58**'k
54.78 i 0.61**'&
Type IIA
M+SE
Type IIB
M+SE
(clm)
Gm)
54.06 k 0.40
58.72 f 0.48*
54.04 k 0.79
54.28 f 0.65
61.19 k 0.80***
58.31 + 0.69***
58.92 + 0.71***
57.90 & 0.42
69.09 + 0.51***
55.54 + 0.88
58.81 f 0.72
65.56 + 0.78***
60.36 + 0.66*
61.75 f 0.73***
lAbbreviations: F contr., nonpregnant control female; M,male; Fp., pregnant female.
*Significant difference from control females ( P < . O W
***Significant difference from control females ( P < ,001).
fundamental. The earlier transient increase in type IIB
fibers observed at 30-40 days of pregnancy could be
interpreted as a change regulated at least in part by the
involvement of endocrine secretions, such as the sex
hormones and thyroid hormones, since their effect on
muscle fiber sizes and types has been clearly shown (see
Kelly, 1983). Starting from 50 days of pregnancy, the
stretch of RA muscle in combination with the functional
overload due to the sizes and weight of the fetuses could
prevail over any possible hormonal influence and result
in a n increase of type I fibers. The increased proportion
of type I fibers might result either from a transformation
of type I1 into type I fibers or from the formation of new
fibers. However, since ectopic nuclei, fissures, groups of
fibers having a much reduced diameter, and all the
aspects which have been generally reported to occur in
the process leading to fiber division (Hall-Craggs, 1972)
were never observed in our material, the latter hypothesis, in our opinion, is rather unlikely.
The other possibility is that a fiber transformation
could take place. According to this hypothesis, the increase in number of type I fibers could be gradually
generated via IIC fibers by transformation from type
IIA (Jansson et al. 1978; Pierobon-Bormioli et al., 1981)
and a conversion of type IIB via IIAB to IIA fibers
(Ingjer, 1979) could also occur. This hypothesis could be
also supported by the immunological and biochemical
studies, which demonstrated that both fast and slow
myosin isozymes are present within single fibers during
transformation provoked by long-term electrical stimulation (Pette and Schnez, 1977; Rubinstein et al., 1978),
thus indicating that a transformation can take place
within the preexisting set of fibers.
Moreover, the RA muscle of the guinea pig at 50-70
days of pregnancy has significantly larger fibers of all
three types than the same muscle of nonpregnant animals. Since it is well known that fiber diameter is related to the forces the fibers are required to develop
(Saltin and Gollnick, 1983) and that the only resource
available for increasing total muscular strength is to
induce hypertrophy of the existing muscle fibers, the
hypertrophy of RA muscle fibers noted in pregnant
guinea pig should demonstrate that relatively high
forces are continuously produced. These forces are necessary to maintenance of tone in the abdominal wall a s
the RA muscle is stretched during pregnancy.
Finally, the duration of the pregnancy stimulation is
fundamental; in fact, while the 21-day pregnancy of the
rat provoked no change on RA muscle fiber proportions
and hypertrophy of only type I fibers CMartin,l979), our
data suggest that the long-term pregnancy of guinea pig
plays a significant role in RA muscle fibers, which react
to changes in functional demand with both type transformation and hypertrophy. In fact, when pregnancyinduced stretch and work become remarkable the hypertrophy of all the fiber types takes place and the greater
relative increase in diameter of the type I fibers shows
a preferential recruitment of this fiber type. Concomitantly, a n increase in number of this fiber type also
appears to be necessary and shows that the muscle becomes adapted for constant use, can contract and relax
more slowly, and is more fatigue-resistant.
28
G. LALAYIA COSTERBOSA, A.M. BARAZZONI, M.L. LUCCHI, AND R. BORTOLAMI
-1
---DA
......DE
0
30
40
DAYS OF m w N o I
50
60
70
Fig. 4. Mean frequencies of RA muscle fiber types in the guinea pig
during pregnancy. *Level of significance (P < .05).***Level of significance (P < .001).
E
5
70
I
30
40
50
60
70
DAYS OF PREGNANCY
Fig. 5. Mean diameters of RA muscle fiber types in the guinea pig
during pregnancy. *Level of significance (P < .05). ***Level of significance (P < .001).
Fig. 3. RA muscle of guinea pig x 100.A) Male guinea pig. B) Control
female. C) Pregnant (70 days) female. A-C) ATPase, section preincubated at pH 10.4. Type I fibers stain light; note that they are larger
and more numerous in the pregnant female. The male shows larger
fibers compared with the control female. A muscle spindle is identifiable in C (top portion of the photograph).
GUINEA PIG RECTUS ABDOMINIS MUSCLE IN PREGNANCY
ACKNOWLEDGMENTS
The authors wish to thank Prof. P. Monari of the
Department of Statistical Sciences, University of Bologna, for statistical analysis of data. The authors also
express their gratitude to Ms. M.L. Polsoni for typing
the manuscript and to Mr. E. Ferrari for his technical
assistance. This research was supported by grants from
C.N.R. and M.P.I. of Italy.
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