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HISTOCHEMICAL STUDY OF MITOCHONDRIA IN THE FEMUR
DEVELOPMENT OF PIGEON EMBRYO (Columba livia )
Khansa M.Jawad , Fawzi S. Mohsen , Abduljabbar R. Huait
Department of Anatomy and Histology, College of Vetreinary Medicine ,University of
Basrah,Basrah,Iraq
(Received 24 January 2016,Accepted 21 April 2016)
Keywords: Fertilized eggs, Embryo, Mitochondria.
ABSTRACT
Twenty fertilized eggs of pigeon (Columba livia) embryo were collected from local
markets and pigeon breeders in Basrah governorate.They incubated in eggs incubator .The study
comprised histochemical description of mitochondria decreasing and increasing in it density in
femur bone development of pigeon embryo (Columba livia ) which included appearance of the
mitochondria during the embryological stages. The study showed the mitochondria is spindle in
shape and few in resting chondrocyte stage , while it was more in hypertrophy chondrocyte and
more density in ossification in osteoblast stage .The aim of this study is know the density of
mitochondria during the development of the femur bone of pigeon embryo.
INTRODUCTION
The bones of the axial and appendicular skeleton are formed by one of two processes,
intramembranous or endochondral bone formation. In intramembranous bone formation is
formed in the absence of a cartilage model while in endochondral bone formation, a cartilage
model is first formed and then replaced by bone tissue(1,2).The flat bones of the skull and face
are formed by intramembranous ossification(3).Osteoprogenitor cells,which gives rise to
osteoblasts and osteocytes are present within the mesenchyme (1, 4) . These cells aggregate at
the sites where new bone is to be formed and differentiate into osteoblasts that actively
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synthesize new bone matrix(5). Endochondral ossification involves the formation of cartilage
tissue from aggregated mesenchymal cells and the subsequent replacement of this cartilage tissue
by bone tissue (6) . All the skeletal components of the vertebral column, pelvis, and appendicular
skeleton (limbs)was developed via endochondoral ossification(7).The process of endochondral
ossification is divided into five stages(8,9). First, the mesenchymal cells are committed to
become cartilage cells.During the second phase of endochondral ossification, the committed
mesenchymal stem cells condense into compact nodules and differentiate into
chondrocytes.During the third phase of endochondral ossification, chondrocytes proliferate
rapidly to form the cartilage model that will eventually be replaced by bone tissue(10).As they
divide, chondrocytes secrete a cartilage- specific extracellular matrix. In the fourth phase, the
chondrocytes stop dividing and become hypertrophic. Hypertrophic chondrocytes have increased
production of collagen type X and fibronectin, thus altering the remaining cartilage matrix so
that it can be mineralized by calcium carbonate. Finally, in the fifth phase, blood vessels begin
the invasion of the cartilage model. The hypertrophic chondrocytes undergo apoptosis and the
spaces are invaded by ingrowing blood vessels(11). As the cartilage cells die, osteoprogenitor
cells differentiate into osteoblasts and begin to lay down bone matrix on the partially-degraded,
mineralized cartilage remnants (12,13). The mitochondria were most numerous in large
osteoblasts, in which they occupied every part of the cytoplasm except the juxta-nuclear vacuole,
and generally tended to be orientated in the long axis of the cell (14) .In the development of
cartilage , the mitochondria were most numerous in the perichondrial cells(15). In both the
osteoblastic and chondroblastic series of cells, the mitochondria were most numerous where the
rate of deposition of intercellular matrix was greatest.The mitochondrial content of the
mesenchymal cells, reticular cells and of the osteoblasts was increased as these cells became
osteoclasts(16,17,18).Chang stated that the osteoclasts contained more mitochondria than any
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other cell in the area of bone formation(14) .The aim of this study is detect the mitochondria
density, while the femur bone development of pigeon embryo have been occurred .
MATERIALS AND METHOD
Twenty fertilized eggs of pigeon embryo ( C. livia ) were collected after laying ,then
incubated in an automatic incubator at 37 ◦C with humidity (50%-70%) for different stages, in
order to obtain embryo at different ages. The incubated eggs with different ages (8,10,12,14,16,
18) days were tested by candling to know the presence of whole embryo to start histochemical
study, then all embryonated egg shell was broken at wide side by small scissors ,then washing
with normal saline to remove the yolk. The embryo were carefully taken out from the egg shell
by forceps and put in a petri dish containing formalin. The skin was removed and then the
viscera were taken out from the body by forceps carefully. Skeleton was cut to parts , the femur
was taken,because of bone hardness , it must be undergo decalcification by put it in diluted nitric
acid solution 5% for 3 day . This solution dissolve all the calcareous salts , but in the same time ,
the bone retains with shape and structure . It just becomes simple to cutting and
banding.`Histological sections of femur of embryo with different ages were prepared by steps
according to Luna(19) . Staining the sections with acid fuchsin , methyl blue and HCl according
to (20).
RESULT
Mitochondria are present as spindle elongated bodies red in color in the resting chondrocyte
stage , (figure 1,2), and increase in density in hypertrophy chondrocyte stage ,(figure 3,4), but
during the late stages of embryonic bone development , the mitochondria was more increasing in
density in osteoblast in ossification stage, (figure 5,6 )
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.
Figure (1):Longitudinal section in femur in pigeon embryo at 8 days
of incubation showed mitochondria (m) in resting chondrocyte stage
stained with acid fuchsin stain.1000x
Figure (2):Longitudinal section in femur in pigeon embryo at 10 days
of incubation showed mitochondria (m) in resting chondrocyte stage
stained with acid fuchsine stain.1000x
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Figure (3):Longitudinal section in femur in pigeon embryo at 12 days
of incubation showed mitochondria (m) in hypertrophy chondrocyte
stage stained with acid fuchsine stain.1000x
Figure (4):Longitudinal section in femur in pigeon embryo at 14
days of incubation showed mitochondria (m) in hypertrophy
chondrocyte stage stained with acid fuchsin stain.1000x
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Discussion
Figure (5):Longitudinal section in femur in pigeon embryo at 16
days of incubation showed mitochondria (m) in ossification stage
stained with acid fuchsin stain.1000x
Figure (6):Longitudinal section in femur in pigeon embryo at 18
days of incubation showed mitochondria (m) in ossification stage
stained with acid fuchsine stain.1000x
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DISCUSSION
Mitochondria has active role in the cytoplasmic transport of calcium ions which may
accumulate also inorganic deposits during ossification process, so mitochondria participates in
calcification of bone, also mitochondria provides the bone cells with energy that it produce in
form as adenosine triphosphate (ATP) during bone formation(21). The results of primary stages
of bone development are in agreement with the findings of Pritchard, (1952) (22).The formation
of the matrix, mitochondrial content increased rapidly in the osteoblasts. In the later stages of
development the density of mitochondria increase in the cells area of rapid bone formation. In
either areas, at the late stages of development, the density of mitochondria is parallel the rate of
deposition of the bone matrix. The cells with a large density of mitochondria is metabolically
very active cells(23). The osteoblasts are active cells in this observation. when considered in
view of the other histochemical findings reported in this investigation, strongly suggests that the
osteoblast is actively engaged in the synthesis and secretion of certain components of the bone
matrix. After the formation of the matrix on the 12 day of incubation, the density of
mitochondria in the osteocytes indicate that these cells continue to produce the bone matrix, not
as rapidly as osteoblasts.
CONCLUSIONS
The observations in this study confirmed that the mitochondria are increased in density
with development of bone. In the primary stages of development bone (8 days of incubation ) of
the femur bone , the chondrocyte contains slight density of mitochondria. During the 14 day of
incubation , the chondrocyte matured and contained increasing in the density of
mitochondria.The increasing of mitochondria density in ossification stage continue to 16 days of
incubation.
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(Columba livia) دراسة کیمونسیجیة لتطور المایتوکوندریا فی عظم الفخذ فی اجنة الحمام
خنساء محمد علی محمد جواد ، فوزی صدام محسن ، عبد الجبار رسمی حویط
فرع التشریح والانسجة ،کلیة الطب البیطری،جامعة البصره، البصرة، العراق.
الخلاصة
من الاسواق المحلیة ومربی الحمام فی محافظة (Columba livia) جمعت عشرون بیضة مخصبة لاجنة الحمام
البصرة. حضنت فی حاضنة للبیض. تضمنت الدراسة الوصف الکیمونسیجی للمایتوکوندریا من حیث زیادة او نقصان کثافتھا
،16 ،14 ،12 ،10 ، فی تطور عظم الفخذ لأجنة الحمام والتی تضمنت ظھور المایتوکوندریا خلال المراحل الجنینیة ( 8
18 )یوم . حیث بینت الدراسة ان المایتوکوندریا تکون مغزلیة الشکل قلیلة العدد فی المراحل المبکرة من التطور و ،من ثم
تزداد فی الکثافة فی مرحلة تضخم الغضروف. و تزداد کثافتھا فی مرحلة التعظم. ھدف الدراسة ھو معرفة کثافة
المایتوکوندریا من خلال تطور عظم الفخذ فی جنین الحمام.
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