The influence of ‘hypophysectomy’ by means of surgical decapitation on skeletal growth in the developing chick embryo

In the developing chick embryo, ‘hypophysectomy’ by surgical decapitation has been shown to retard growth (Fugo, 1940; Case, 1952; Vogel, 1957, 1965; Thommes & McCarter, 1966; Betz, 1967, 1968). This retardation may be as great as 53-55 % during the latter half of development (Vogel, 1957, 1965; Betz, 1967; Thommes & McCarter, 1966). Partial restoration of growth in ‘hypophysectomized’ embryos has been achieved by transplanting pituitaries from donor chick embryos onto the chorioallantoic membrane of pituoprivic hosts of the same age (Thommes & McCarter, 1966; Betz, 1967, 1968). Moreover, Enemar (1967) has demonstrated a growth-promoting influence of chick embryo pituitaries transplanted into amphibian tadpoles.

Although little specific data on pituitary regulation of skeletal development exists, it might be anticipated that the generalized effects on growth noted above include changes in bone growth. Fugo (1940), for example, noted that limb bones of ‘hypophysectomized’ embryos appeared shorter than normal although they maintained their normal proportions. Betz (1968), also, observed that the 3rd toe length of ‘hypophysectomized’ animals was 23–26% below normal by day 20 of incubation, while those of operated embryos bearing pituitary transplants were only 10–13% below that of intact controls. More recently, Mehall (1970) reported that ‘hypophysectomy’ retarded the normal increase in long bone length and width, and in size of articular-epiphyseal caps, the zone of cartilage hypertrophy and the epiphyseal plate, and caused periosteal disorganization. Hypophyseal transplants reduced these skeletal defects in pituoprivic animals.

The present investigation was undertaken to evaluate the effects of ‘hypophysectomy’ by surgical decapitation on several other gross parameters of skeletal growth. These studies include an analysis of matrix, total mineral, and calcium content in the long bones of normal and ‘hypophysectomized’ chick embryos.

Embryos were obtained from White Leghorn eggs incubated at 38 ± 0·5 °C in a ‘Jamesway’ incubator. The age of the embryos at the time of ‘hypophysectomy’ and sacrifice represents the actual time the eggs remained in the incubator.

Experimental embryos were ‘hypophysectomized’ at 33–38 h of incubation by the partial decapitation method of Fugo (1940). A transverse cut was made through the mesencephalic portion of the brain and the severed portion removed. Eyes, upper beak and all other derivatives of the prosencephalon, including both rudiments of the hypophysis (Rathke’s pouch and infundibulum), were eliminated. Controls consisted of intact embryos.

Embryos were killed at 24 h intervals from 12·5 to 19·5 days of incubation. Incompletely ‘hypophysectomized’ animals, characterized by the partial or complete presence of prosencephalic derivatives, were discarded. Tibiae were isolated from hind limbs and cleaned of adhering tissue. They were demarrowed by expressing marrow mechanically through a transverse cut, followed by flushing the marrow cavity with Ringer’s solution under pressure. All bones were stored in isotonic Ringer’s solution at −20 °C until used, to prevent tissue deterioration and mineral loss.

After determination of wet weight, bones were dried at 55 °C in a forced-draft oven until constant weight was attained (24 h), and ashed at 500 °C for 4–6 h, or until the gray-ash stage was reached. The residue was moistened with dilute HNO₃, dried, and heated at 500 °C until the ash was white (1–2 h). The mineral residue was dissolved in 1·0 ml of 0·08 M-HCI, and diluted with distilled water. Total calcium was determined at 422·8 nm on a Beckman DU spectrophotometer with flame attachment.

Statistical methods employed were Analysis of Variance (with log transformations) and the Duncan New Multiple Range Test (Duncan, 1955).

Bone development in normal chick embryos is characterized by progressive mineralization and matrix elaboration. From days 12·5 to 19·5, wet, dry and ash weights and total calcium increase with an elevation noted from days 16·5–19·5 (Table 1, Fig. 1). That rates of mineralization and matrix synthesis remain similar during development is shown by the stability of the ratios, ash/dry weight and calcium to ash and dry weights (Table 2, Fig. 2).

‘Hypophysectomy’ markedly influenced bone development. Abnormally low tibial wet, dry and ash weights, as well as total calcium content, were a consistent consequence of this operation. By day 18·5, growth retardation was 56 % when the dry mass of tibiae from operated embryos was compared with that of controls. Ash weights were significantly depressed by day 16·5. While all weight values of bones from ‘hypophysectomized’ animals slightly increased on day 17·5, on days 18·5 and 19·5, the experimental values were again much lower (P < 0·01) than those of intact embryos (Table 1, Fig. 1). Total calcium content was also very low; the reduction was statistically significant by day 14·5, and was 39-2% below controls by 18·5 days. These data indicate that lack of pituitary influence during the latter half of development causes a general retardation in bone growth.

In addition to the abnormally slow bone growth in the absence of the hypophysis, an influence on specific skeletal compartments was noted. In 12·5-to 14·5-day tibiae, the amount of calcium relative to total bone mineral (ash) was elevated (P < 0·01) above that of intact embryos. However, by day 15·5 the calcium/ash ratio approached control levels, and paralleled the normal pattern through the remainder of the incubation periods studied (Table 2, Fig. 2). These data indicate that during early ossification in pituoprivic embryos, there may be an abnormal balance of mineral ions available for bone crystal formation.

The calcium/dry weight ratio in bones of ‘hypophysectomized’ animals was slightly but consistently greater than that of controls from days 13·5 to 16·5, and was significantly higher the last few days of development. In contrast, the ash/dry weight ratio was lower than normal (P < 0·05) in bones of experimental animals from days 12·5 to 16·5. By day 18·5, however, this relationship had reversed, and the ratio became significantly greater (P < 0·05) than that of normal embryos (Table 2, Fig. 2).

Long-bone growth in the embryonic chick is accompanied by progressive elevations in bone mass, as demonstrated by wet, dry, ash and calcium determinations. ‘Hypophysectomy’ results in abnormal bone development; during the last half of incubation the long bones of pituoprivic embryos were significantly lower in wet, dry, ash, and calcium values than controls.

Since dry weight includes both mineral and matrix phases of bone, the lower dry weights of tibiae from ‘hypophysectomized’ embryos might indicate that either mineralization and/or matrix synthesis have been impeded. Total mineral and calcium content are less than normal, indicating that bone crystal deposition is retarded in the operated embryos; matrix synthesis is also inhibited. While normal tibiae increased 35 % and 43 % in calcium and dry weight respectively (a ratio less than 1) from days 17·5–19·5, those of experimental animals only increased 30 % and 27% (a ratio greater than 1). ‘Hypophysectomy’ thus results in a failure of bone mass to increase normally as a consequence of reduced mineralization and matrix elaboration.

An analysis of ratios between bone compartments reveals that they are differentially affected by ‘hypophysectomy’. From days 12·5 to 14·5, bone calcium is greater than normal, even though total mineral is reduced. This change in the calcium/mineral ratio may be due to either an influx of calcium to the mineral compartment, or depletion of other mineral components in bone crystal. This unbalanced mineral content returns to normal the last week of development.

When the calcium/dry weight ratio is examined it must be recognized that dry weight includes both mineral and matrix. If, as noted above, calcium increases in operated embryos on days 12·5–14·5, an elevation in the calcium/dry weight ratio might be anticipated. This was observed; the lack of statistical significance may be due to masking of small changes in calcium by the larger amount of matrix present. Subsequently, as matrix synthesis becomes more limited, the calcium/dry weight ratio is elevated. Similar interpretations may be drawn from an analysis of the ash/dry weight ratio.

Whether the nature of the pituitary influence on bone development is via the gland itself, or through trophic hormones, is not established. However, events related to several pituitary-endocrine axes correspond in time to the bone changes observed in surgically decapitated embryos. ‘Hypophysectomy’ modifies the following adrenal parameters between days 10 and 14: (1) ultrastructural differentiation and △⁵-3β-hydroxysteroid dehydrogenase levels (Straznicky, Hajós & Bohus, 1966) ; (2) adrenal ascorbic acid content (Case, 1952) ; (3) cortical cord development and free cholesterol levels (Adjovi, 1970); (4) allantoic fluid levels of corticosteroids (Woods, DeVries & Thommes, 1971). Moreover, stimulation of ACTH release by use of corticosteroid synthesis inhibitors leads to an initial hypertrophy of the pituitary (Stoll, Faucounau & Maraud, 1964) and decreased conversion of cholesterol to adrenal steroids (Adjovi & Eidelman, 1969) on day 12. Whether or not establishment of the pituitary-adrenal axis during days 10–14 relates to bone changes following ‘hypophysectomy’ is problematical, since glucocorticoids may arrest skeletal growth and ossification (Karnofsky, Ridgway & Patterson, 1951; Buño & Goyena, 1955; Siegel, Smith & Gerstl, 1957; Sobel & Freund, 1958; Reynolds, 1966; Badran & Provenza, 1969); in their probable absence in this study, similar results occurred.

The day 10–14 period is also critical in thyroid development. At this time, thyroxin is present in relatively large amounts and iodide uptake sharply increases (Trunnell & Brayer, 1953; Trunnell & Wade, 1955); ‘hypophysectomy’ eliminates the latter (Mess & Straznicky, 1970). Moreover, the pituitarythyroid axis may be established at this time, i.e. pituitary grafts to the chorioallantoic membrane now stimulate the thyroid in ‘hypophysectomized’ embryos (Studitskii, 1946), and conversely, thyroid grafts now respond to pituitary TSH (Martindale, 1941). Additionally, the thyroids of ‘hypophysectomized’ embryos are arrested in growth at this time (Fugo, 1940), and retain the vascular pattern characteristic of younger embryos (Thommes, 1958). Electron-microscopic data also indicate that thyroidal fine-structure becomes pituitary-dependent about day 11 (Hajós, Straznicky & Mess, 1964).

Thyroid hormones are known to influence skeletal development. Fell & Mellanby (1955, 1956), Fell, Galton & Pitt-Rivers (1958) and Lawson (1961 a, b) have shown that lack of, or presence of, thyroid hormones depresses or stimulates, respectively, growth, differentiation, ossification and matrix formation in chick embryo bones. Moreover, Lengemann (1962) has demonstrated that incorporation of radiocalcium in embryonic chick tibiae is inhibited by thyroxin. Therefore growth, matrix synthesis, mineralization, and mineral balance may be regulated by thyroid hormones in vivo. Such an analysis allows interpretation of the present data, since the pituitary-thyroid axis appears to be established during that time when ‘hypophysectomy’ first influences bone development.

The possibility that pituitary somatotrophin (STH) may regulate bone growth in the embryonic chick cannot be excluded. As reported by Ito, Takamura & Endo (1959, 1960), the addition of mammalian STH to bone organ cultures increases bone length and sulfur uptake into matrix. STH also increases wet and dry weights and total nitrogen of embryonic bone in vitro (Hay, 1958), elevates the mitotic index and bone thickness (Blumenthal, Hsieh & Wang, 1954), and alleviates cortisone-induced skeletal teratologies in vivo (Sobel, 1958). In contrast, other studies indicate that growth hormone may have little influence on avian bone development. For example, Chen (cited by Fell, 1954, 1955 and Hay, 1958) could detect no change in bone length with STH treatment in vitro, and Vogel (1965) could not restore depressed growth rates of ‘hypophysectomized’ embryos to normal with an undefined bovine growth ‘factor’. Additionally, it is not certain that the avian pituitary possesses a growth hormone comparable to that found in mammals, although Enemar (1967) observed a ‘growth hormone effect’ in tadpoles receiving transplants of the caudal region of the embryonic chick pars distalis. The role of STH in avian embryonic growth and bone elaboration is thus unclear: (1) since there is uncertainty as to the presence or absence of STH in birds and (2) since a lack of response in the chick embryo to non-avian STH’s may be a result of the species-specific nature of growth hormones. It is also unclear whether the striking effects of ‘hypophysectomy’ in the chick embryo are a result of the cleidoic nature of the avian egg, for in the relatively ‘open’ maternal-fetal relationship of mammals, ‘hypophysectomy’ appears to have little effect on growth (see McWhinnie & Thommes, 1973).

The role of the hypothalamus in adenohypophyseal regulation of embryonic systems remains problematical. Some data on the chick embryo indicate that in the absence of the hypothalamus, adenohypophyseal function is maintained; for example, pituitaries transplanted to ‘hypophysectomized’ embryos (no hypothalamus present) produce sufficient TSH to cause an abrupt rise in thyroidal iodide-trapping (Mess & Straznicky, 1964); ACTH secretion may also be independent of the hypothalamus (Betz, 1967; Woods et al. 1971). However, these data are as yet inconclusive, and it is unknown whether secretion of different adenohypophyseal hormones may be independent of, or partially or totally dependent upon, hypothalamic release factors. Hence, the possibility that normal bone development in the chick embryo may require a functional hypothalamic-adenohypophyseal relationship cannot be excluded.

In conclusion, this investigation indicates that the pituitary gland (or hypothalamus-pituitary complex) is essential for skeletal growth and development in the chick embryo. In its absence, bone matrix synthesis is severely retarded and a lag in bone crystal deposition occurs. Whether the nature of this pituitary influence is direct or through its trophic hormones remains to be elucidated.

This investigation was supported in part by Training Grant 5-TOI-HD00293 from the National Institute of Child Health and Human Development, and the Brown-Hazen Fund of the Research Corporation of America.