Changes in activity of chicken medullary bone cell populations in relation to the egg-laying cycle

0221s8747/84 $3.00 + .OO Copyright 0 1984 Pergamon Press Ltd.

Metab. Bone Dis. & Rel. Res. 5, 191-193 (1984) Printed in the USA. All rights reserved.

Changes in Activity of Chicken Medullary Bone Cell Populations in Relation to the Egg-laying Cycle J.t? VAN DE VELDE, J.F!W. VERMEIDEN,

J.J.A. TOUW,’ and J.f? VELDHUIJZEN

Department of Orthodontics, Dental School, Vrije Universiteit, 1081 HVAmsterdam, The Netherlands. ‘Present address: Department for Education, Science and Planning, Agricultural University, 6701 DH Wageningen, The Nether/ands. Address for correspondence and reprints: J.i? van de Velde, Department of Orthodontics, Dental School, Vrije Universiteit, 1081 HV Amsterdam, The Netherlands

Abstract

directly from dietary intake, and the remaining 40% is of osseous origin. The egg-laying cycle of chickens and quails is about 24 h (Miller, 1977). This cycle (Fig. 1) starts with ovulation. Five hours later the ovum reaches the uterus, where eggshell formation takes place between 7 and 20 h after ovulation. The egg is laid 24 h after ovulation, and then a new ovulation starts. Two metabolic periods can be identified: the period of eggshell formation (active period) and the period when no eggshell formation is taking place (inactive period) (Miller, 1977). The activity of the osteoclast population in the medullary bone is synchronized with the egg-laying cycle to meet the demand for calcium. Since medullary bone has no weightbearing function, mechanical influences are excluded. Thus, the purely intrinsic factors that influence bone remodeling and the possible interaction of the cells involved can be studied in medullary bone. Little is known about the activity of the osteoblast population during the egg-laying cycle, and there is no consensus on this topic (Bloom et al., 1958; Kusuhara, 1975; Miller, 1977; Taylor, 1981). This study describes the osteoblastic and osteoelastic activity at two time points during the egg-laying cycle of the chicken, one in the active and one in the inactive period.

Osteoblastic and osteoclastic activity was studied in avian medullary bone in vivo. During the active period of eggshell calcification, medullary bone active resorption surface increased ninefold. This correlated with a sevenfold increase in the percentage of active osteoclasts. Osteoblast activity is also increased during the active period, as demonstrated by a twofold increase in the active osteoblastic surface. These findings and our observation that the medullary bone volume remains the same (k 13%) whether the eggshell is being formed (active period) or not (inactive period) led to the conclusion that the activities of osteoblasts and osteoclasts rapidly return to balance. Key Words: Bone Remodeling-Medullary Bone-OsteoblastOsteoclast.

Introduction

Materials and Methods

During bone remodeling in mammals thetime interval between bone resorption and bone apposition can be several weeks in one BMU (Basic Multicellular Unit of bone). The whole remodeling process in the BMU takes approximately 4 months (Frost, 1973). This in addition to the fact that osteoclasts and osteoblasts are at different sites in different phases of the boneremodeling process make it difficult to study the interaction between these bone cell populations. In the medullary bone of female birds the activities of bone cells are related to the egg-laying cycle (Miller, 1977). Medullary bone is a special type of woven bone, normally found only in female birds, mostly in the marrow of long bones (especially the femur and tibia). Shortly before the bird is capable of egg production, concomitant with the maturation of ovarian follicles, medullary bone is formed by the synergistic action of estrogens and androgens (Simkiss, 1967). A portion of the calcium needed for the eggshell is stored in medullary bone. According to several authors (Driggers and Comar, 1949; Jowsey et al., 1956) 60% of the eggshell calcium is derived

Nine-month-old white leghorns with a high laying frequency were used. Five animals were sacrificed for bone histomorphometry immediately after oviposition (the inactive period), and five animals were sacrificed 15 h after oviposition (the active period) (Fig. 1). The day before sacrifice the animals were fed a low calcium diet (1% Ca, 0.7 P, 1200 IU vitamin D kg-‘) to decrease Ca influx from the intestine and to stress the mle of bone during formation of the eggshell. Parts of the marrow, containing pieces of medullary bone, were dissected, fixed in Bouin’s fluid, decalcified in 10% EDTA, and embedded in paraffin, Since medullary bone is woven bone and calcification is very diffuse (Van de Veldt?, unpublished observation), paraffin histology was preferred. Serial 5 pm sections were mounted on glass slides and stained with Mayer hematoxylin and toluidine blue. Sections were micmphotographed (x 510) at 90 pm intervals (Fig. 2). For each chicken 15 prints(representing a total of 2 mm’of tissue) were used for bone histomorphometry. The parameters measured are shown in Table 1. The bone surface covered with cuboid active osteoblasts in which the Golgi apparatus is often clearly visible is the active osteoblastic surface. An active osteoclast was histologically identified as a 191

192

J. P. van de Velde et al.: Medullary bone cells and egg laying.

Discussion hr

t

I ovulation

1

1

?? egg in utero

I’

ovulation

2

oviposition

Fig. 1. Scheme of the 24 h laying cycle of chickens and quails The period of eggshell formation is shaded.

multinuclear giant cell with a clear zone and ruffled border facing the bone. An inactive osteoclast has a rounded appearance, no clear zone, and no ruffled border and is detached from the bone. The medullary bone surface covered with active osteoclasts is called the “active resorption surface.” Howship’s lacunae without osteoclasts and other bone surfaces not covered with osteoblasts or osteoclasts are consid-

ered as inactive. Measurementswere performed with a computerized X-Ytablet. Data were analyzed using a two-sided t-testfor two independent samples, with a significance level of P < ,005.

Results The morphometric data are shown in Table I, Significant changes (P < 0.001) between the active and inactive periods were found in the percentage of active osteoclasts, the resorption surface per osteoclast, the active resorption surface, and the active osteoblastic surface. The percentage of active osteoclasts increased during the active period from 9.8 to 63.90/o, and the resorption surface per osteoclast increased from 23.6 to 33.4 pm. As a result the active resorption surface of the medullary bone increased during the active period, specifically from 1.7 to 15.0%. The active osteoblastic surface of the medullary bone also increased during the active period, from 1O.Oto 19.1%. Thetotal medullary bonevolumewas not significantly increased during the active period. Additionally, no significant changes were found in the number of osteocytes per mm2 of medullary bone, the osteocytic lacunar size, the number of osteoclasts per mm2 section, the number of nuclei per osteoclast, or the osteoclast size.

Since the number of osteocytes in medullary bone and their lacunar sizes in both periods are the same, no net osteocytic resorption of medullary bone occurred (Belanger et al., 1963; Taylor and Belanger, 1969). The high number of osteoclasts (e.g., compared to human biopsies; Evans et al., 1979) emphasizes the fact that chicken medullary bone represents a very active bone-remodeling system. In both periods the number of nuclei per osteoclast and the number and size of osteoclasts are the same. Thus, activation of osteoclasts does not increase their size. However, during the active period, the resorbing surface (i.e., the ruffled border) per active osteoclast is increased. Histologic examination (Fig. 2B) shows that active osteoclasts have a more flattened appearance. The total number of osteoclasts remains constant, but in theactive period the percentage of active osteoclasts is increased (Holtrop and King, 1977; Miller, 1981). As a result of this increase in the number of active osteoclasts the total active resorption surface is increased 7-fold to 11-fold in order to meet the demand for calcium. In cases where bone is in equilibrium (no net bone loss or gain), the active osteoblastic surface is several times larger than the active resorption surface (Bordier and Tun Chot, 1972). The same situation is found in medullary bone during the inactive period-l 0% active osteoblastic surface as compared to 1.7% active resorption surface. During the active period the active osteoblastic surface is increased by a factor of 1.6-2.3. This is less than the increase in active resorption surface but apparently sufficient to compensate for the bone loss. The increase in active osteoblastic surface reflects only partially the increase in osteoblastic activity. Medullary bone is woven bone, and no clear bone formation front exists as in lamellar bone. Measuring the rate of bone formation with standard histologic techniques (e.g., undecalcified embedding in plastic after labeling with tetracycline) is impossible in medullary bone, since in this bonecalcification is too diffuse a process (Van de Velde, unpublished observation). Osteoblastic and osteoclastic activities have been reported to alternate during the laying cycle (Bloom et al., 1958; Taylor and Belanger, 1969). However, in this study we have observed that when bone resorption is low, bone formation is also low, and when bone resorption is high, bone formation is also high, as was suggested by Taylor (1981). We presume that during the period of eggshell formation, fully calcified medullary bone

Fig. 2. A. Histologic section of the marrow cavity with medullary bone of a chicken femur in the inactive period (no eggshell formation). Inactive osteoclasts ( @ ) are detached from the medullary bone ( Q ) and have a rounded appearance. In the marrow cavity hematopoietic cells can be seen, x 500. B.‘Histologic section of medullary bone in the active period (eggshell formation),+: active columnar or cuboidal shaped osteoblasts depositing bone. @: bone resorption by osteoclasts with ruffled borders (foamy appearance). Fat remains unstained. X41 0.

193

J. P. van de Velde et al.: Medullary bone cells and egg laying.

Table

I. Histomorphometric

data of chicken

medullary

bone for the active and inactive periodsa Inactive period Mean r?~SD

Osteocyteslmm’ medullary bone Osteocytic lacunar size (pm*) Osteoclasts/mm* sectlon Nuclei per osteoclast Osteoclastic size (firn’) of active osteoclasts Resorption surface per osteoclast (pm) Active resorption surface

o/0

Active osteoblasfic surface Medullary bone volume

2975 28.5 165 4.8 317 9.8 23.6 1.7 10.0 12.6

t 623 + 4.6 f 38 ?Z 0.7 & 34 f 1.8% f 1.7 2 0.5% f 2.9% f

1.0%

Active period Mean f SD 2570 26.6 157 5.0 347 63.9 33.4 15.0 19.1

f f f f r + f * f

592 3.7 79 0.4 49 14.10/ob 4.5b 3.30/ob 2.10/ob

13.9

f

1.6%

aMean values with standard deviations based on 5 animals per group (N = 5), 15 sections per animal

bP< 0001 is replaced by an organic matrix low in calcium. In this way net calcium gain is achieved. After completion of the eggshell, the poorly calcified organic matrix will be calcified during the subsequent inactive period. This idea is supported by the observations of Kusuhara(l976), who reported that during the inactive period (his stage I) medullary bone contains more calcium than during the active period (his stage II) and shortly after the active period (his stage Ill). We hypothesize that the mineralization rate is high when the rate of osteoclastic resorption is low, and vice versa (alternation of mineralization and resorption), and that matrix formation and osteoclastic resorption are in phase. Since the osteoblastic and osteoclastic activities are so well balanced in medullary bone, a very efficient coupling mechanism must exist similar to that shown earlier (Howard et al., 1980). Since the inactive period in chickens lasts only 9 h, it is probably too short a period of time to form the amount of medullary bone required.

We thank Miss Janneke Hondema for her technical assistance, the members of the Netherlands “Spelderholt” Institute of Poultry Research at Beekbergen for their assistance, and Prof. Dr. Birte Prahl-Andersen and Dr. Wayne Hickoryfor reviewing the article.

Acknowledgement:

References E&anger L.F., Roblchon J., Migicovsky B.B.. Copp D.H. and Vincent J.: Resorption without osteoclasts (osteolysis). In: Mechanism of Hard Tissue Destrucbon. RF. Scgnnaes, ed. American Association for the Advancement of Science, Washington, D.C., 1963, pp. 531-556. Bloom MA, Domm L.V., Nalbandov A.V, and Bloom W.: Medullary bone of laying chlckens. Am. J. Anat. 102:41 l-453. 1958

Bordier P.J. and Tun Chot S.: Quantitative histology of metabolic bone disease. C/in. Endocnnol. Metab. I: 197-215, 1972. Driggers J.C. and Comar C.L : The secretion of radioactlve calcium (‘%a) in the hen’s egg. Poultry So. 28:420-424, 1949. EvansRA., DunstanC.R. and Bay1inkD.J.: Histochemical identificatlonof osteoclasts in undecalcified sections of human bone Mrneral Electrolyte Metab. 2.179-185, 1979. Frost H.M: Bone remodeling and Its relationship to metabolic bone diseases. Orthopaedfc Lecture Series. Thomas, Springfield, IL, 1973, Vol. 3. Holtrop M E. and King G.J.: The ultrastructure of the osteoclast and its functional implications. C/in. Orfhop. 123:177-196, 1977. Howard G.A., Bottemiller B.L. and Baylink D.J.: Evidence for the coupling of bone formation to bone resorption in vitro. Metab. Bone Dis. Rel. Res. 2:131-135, 1980. Jowsey J.R., Berlie M.R., Splnks J.W.T. and O’Nell J.B.. Uptake of calcium by the laying hen, and subsequent transfer from egg to chick. Poultry SC/ 35:1234-l 238, 1956. Kusuhara S Enzyme histochemrcal studies of bone formatlon and resorption In laying hens. Jpn. J. Zootechn. So. 46(5):277-282. 1975. Kusuhara S. Histochemical and microradiographlcal studies of medullary bones in laying hens. Jpn. J. Zootechn. So. 47(3): 141-I 46, 1976. Miller SC.: Osteoclast cell surface changes during the egg-laying cycle In Japanese quail. J. Cell Biol. 75:104-l 18, 1977. Miller S.C.: Osteoclast cell-surface specializations and nuclear kinetics during egg-laying in Japanese quail. Am. J. Anat. 162:35-43. 1981. Simkiss K.: Calcium in Reproductive Physiology. Reinhold Publishing Co., New York, 1967. Taylor T.G.. The regulation of calcium metabolism In birds. Recent Adv. Avlan Endocrinol. Adv. Phys/ol. So. 33:31 l-320, 1981. Taylor TG.and BBlanger L.F.: The mechanisms of bone resorption in laying hens Calcif. Tissue Res. 4: 162-I 73, 1969.

Received: May 17, 1983 Revised: August 20, 1983 Accepted: October 5, 1983

RCSUMC Les activit6s de6 populatlons cellulalres ost6obtaatique et ost6oclaatlque cnt BtB BtudlBes In vlvo sur de 1’0s mddullalre avlalre. Pendant la p&lode “active” da caki6catlor1 de la coquilb d’oeuf, bs surfaces slhges d’une horptlon actlve da I’os medulldre aont muttipll6ee par 9. Cela est II6 h une eugmenkstlon du poumentage des oathclasba actlfs, multipli~ par 7. CactMt6 oat6oblaatique eat aussl augment& pendent la p&lode “active”, comma en thoigne b doubbment dea surfaces oot6oblestiques actives. Ces dorhhe, alnal que la constatatlon que b volume de l’os m6dullelre reste b m&ns (* 13%) sl la coqullb d’oeut solt en cows de formation (p&lode actlve) ou ne solt pas en fomwtlon (p&rio& Inactive), condulsent h la concluslon que bs ectivit~s des ost6oblastes et des osthclastae s’6qulllbrent rapldement.