Ontogenic changes in the circulating concentrations of insulin-like growth factor (IGF)-I, IGF-II, and IGF-binding proteins in the chicken embryo.

The ontogeny of circulating concentrations of insulin-like growth factor (IGF-)-I, IGF-II, and IGF-binding proteins (IGF-BPs) was examined in the chick embryo. Distinct ontogenic changes in the circulating concentrations of IGF-I and IGF-II were observed. The present study confirms the overall profile for circulating concentrations of IGF-I. During middevelopment, plasma concentrations of IGF-I increased to a maximum which was attained on Day 14.5 of incubation. Thereafter, plasma concentrations of IGF-I declined with decreases (P < 0.05) between Days 14.5 and 15.5 and between Days 16.5 and 17.5 of incubation. In contrast to the monophasic profile for IGF-I, plasma concentrations of IGF-II were maximal on Day 10.5 of incubation and declined to a nadir on Day 17.5 of incubation. In late developmental stages (17.5 or 18.5 days of incubation), three IGF-BPs, having molecular weights of 22, 28, and 36 kDa, were detected in the plasma of chick embryos. No significant ontogenic changes in the circulating levels of the 28- and 36-kDa IGF-BPs were observed. However, it should be noted that prior to Day 17.5 of incubation, the 22-kDa IGF-BP was nondetectable in the circulation. The role of these changes in the functioning of IGF in embryonic development is discussed.

Ontogeny of 17B-estradiol (E2)- and estrogen receptor (ER)- immunostained cells in the hypothalamus and adenohyophyseal pars distalis of the chick embryo.

Brain sections of 6.5-18.5 day-old (stages 30-45: Hamburger and Hamilton, 1951) male and female chick embryos were examined immunocytochemically for the presence of 17B-estradiol (E2)- and estrogen receptor (ER)-containing cells in the hypothalamus and adenohypophyseal pars distalis. Single (E2 or ER)- and double (E2 and ER)-immunostained cells were observed in five nuclei of the anterior-, mid-, and posterior-hypothalamus and in the rostral and caudal pars distalis. The number of E2-immunostained cells were determined in each of these brain areas from Days 10.5 through 18.5 (stages 37 through 45); during this time period no differences were noted between the cell counts of males and females. In both sexes there was a significant increase (P<0.05) from Days 10.5 through 12.5-13.5 (stages 37 through 39-40) in the mean number of E2-immunoreactive cells. These findings agree with the observations of Wilson and Glick (1970) that prior to the 13th day of embryonic development E2 contributes, via its effect on the hypothalamic neural circuitry (organization) to posthatch mating behavior.

Ontogeny of immunocytochemically demonstrable androgen- and androgen receptor-containing cells in the hypothalamus and adenohypophysis of the chick embryo.

Brain sections of male chick embryos, 6.5-18.5 days of age, were examined immunocytochemically for the presence of androgen- and androgen receptor-containing cells in the hypothalamus and adenohypophyseal pars distalis. Using antibodies (Ab) against both androgens (T-Ab) and the androgen receptor (AR-Ab), single- and double-immunostained cells were located in a total of five nuclei of the anterior-, mid-, and posterior-hypothalamus, as well as in the rostral and caudal lobes of the adenohypophyseal pars distalis. From Days 9.5-12.5, the mean number of androgen-immunostained cells within the hypothalamus and pars distalis increased significantly (P < 0.01), while from Days 12.5-18.5 there was no further statistically significant increase. The results of the present investigation support previous findings which suggest that in the chick embryo the negative feedback loop of the hypothalamo-adenohypophyseal-testicular (HATest) axis is functional by the 13th day of development (Woods et al., 1989a,b). They also agree with the observations of Wilson and Glick (1970) that in the male chick embryo testosterone organizes masculine mating behavior prior to Day 13.0.

Effects of surgical decapitation and chicken growth hormone (cGH) replacement therapy on chick embryo growth.

The effects of decapitation and chicken growth hormone (cGH) replacement therapy on chick embryo growth has been investigated. Removal of the prosencephalon at 33-38 hrs (1.38 to 1.58 Days) of incubation decreased body (torso) and liver weights as well as skeletal growth as indicated by tibial length. A single pituitary gland transplanted onto the chorioallantoic membrane (CAM) partially restored torso growth and completely reversed the increase in body water content which characterizes decapitated embryos. Replacement therapy with cGH did not influence body weight but did, on Day 16.5 of incubation, increase tibial length and liver DNA content and concentration. These latter findings suggest that there may be limited hypothalamoadenohypophyseal (GH) axis function in the chick embryo. The effects of decapitation on torso growth are also discussed in conjunction with decapitation effects on albumen swallowing (absorption) and yolk absorption.

Immunocytochemical demonstration of T4 content and TSH-binding by cells of the thyroid of the developing chick embryo.

The numerical densities (Nv) of immunostained thyroxine (T4)-positive cells and thyrotropin-stimulating hormone (TSH)-positive cells were determined in chick embryos on Day 5.5 through Day 11.5 and Days 5.5, 7.5, 9.5, 10.5, 11.5, 12.5, 13.5, 14.5, 15.5, 16.5, and 17.5, respectively. The data demonstrate that selected cells of the "prefollicular" thyroid contain T4 and bind TSH at least as early as Day 5.5. The Nv of immunocytochemically demonstrable T4-positive cells increases gradually from Day 5.5 to Day 10.5, exhibiting a statistically significant increase (P less than 0.05) between Day 10.5 and Day 11.5. Similarly, the Nv of TSH-binding cells (Nv TSH) rises slowly from Day 5.5 until Day 11.5, followed by a statistically significant (P less than 0.05) increase between Day 11.5 and Day 12.5. The peak Nv TSH value on Day 12.5 is followed by a decline (P less than 0.05) on Day 13.5, and from Day 14.5 through Day 17.5, Nv TSH values remain relatively constant. Changes in the Nv of T4-positive and TSH-binding cells over developmental time are discussed with regard to pituitary regulation of the chick embryo thyroid and the possible contribution of the yolk to circulating T4 levels.

FSH- and TSH-binding cells in the ovary of the developing chick embryo.

The numerical density (Nv) and location of thyroid stimulating hormone (TSH)- and follicle stimulating hormone (FSH)-binding cells in the left ovary of the chick embryo were determined on Days 6.5 through 19.5 of incubation. From Days 6.5 to 11.5, TSH- and FSH-positive cells were located in the medullary cords and there was no statistically significant difference in the Nv of these two cellular populations. However, beginning on Day 12.5 and continuing through Day 19.5, TSH and FSH were bound principally to cells of the cortical cords and there was a significant difference (P less than 0.05) between the Nv of TSH-positive and FSH-positive cells. Results are discussed in terms of a prevalent hypothesis that throughout chick embryo development the left ovary lacks specific receptors for TSH and FSH.

Avian beta-endorphin: alterations in immunoreactive forms in plasma and pituitary of embryonic and adult chickens.

1. A heterologous radioimmunoassay for beta-endorphin (beta-endo) was established. Plasma and/or extracts of pituitaries from embryonic (days 14.5 and 17.5 of incubation), newly hatched, and adult chickens were chromatographed on a Sephadex G-75 column with 0.1 M acetic acid. 2. Embryonic plasma had only a single immunoreactive peak that eluted similar to a beta-lipotropin (beta-LPH) standard. In contrast, adult plasma had 2 peaks, co-eluting with beta-LPH (34%) and beta-endo (66%). 3. Chromatography of pituitary extracts demonstrated two immunoreactive peaks in both embryonic and adult birds. Although 70% of immunoreactivity eluted with beta-endo for embryonic birds, 80% eluted with beta-LPH from adults. 4. The smaller proportion of beta-endo in adult pituitaries may reflect a higher rate of secretion of this hormone into the blood.

Hypothalamic regulation of the adenohypophyseal-testicular axis in the male chick embryo.

An antibody against luteinizing hormone-releasing hormone (LHRH) as well as naloxone, an opioid antagonist, were added to the chorioallantoic membrane (CAM) of 11.5- and 14.5-day-old male chick embryos and plasma testosterone (T) concentrations were determined. This protocol was designed to demonstrate: (1) Whether LHRH is essential in the regulation of the adenohypophyseal-testicular axis in the male embryo and (2) if LHRH is operative in this unit's function, are opiatergic pathways involved in the secretion of LHRH by the hypothalamus. Both anti-LHRH and naloxone lowered plasma T levels in 14.5-day-old embryos, but not 11.5-day-old embryos. This indicates that the hypothalamus, via LHRH, begins to regulate the pituitary-testicular unit at some time between Days 11.5 and 14.5, i.e., the hypothalamo-adenohypophyseal-testicular axis is established. The results also strongly suggest that the normal secretory pattern of LHRH is dependent upon opiatergic innervation of the hypothalamus at the same embryonic time.

Plasma LH and gonadal LH-binding cells in normal and surgically decapitated chick embryos.

Plasma luteinizing hormone (LH) concentrations and the numerical density (Nv) of gonadal LH-binding (LH-positive) cells were determined in intact male and female chick embryos from Days 10.5 through 18.5 of incubation for plasma LH and from Days 6.5 through 19.5 for LH-binding cells, as well as in Day 15.5 decapitated ("hypophysectomized") embryos with pituitary transplants for both plasma LH and LH-binding cells. The data demonstrate that LH is already present in the plasma of male and female embryos as early as Day 10.5, the first day examined. Plasma LH levels in hypophysectomized embryos were statistically significantly lower than those of intact embryos, while pituitary transplants to the chorioallantoic membrane of hypophysectomized embryos elevated plasma LH concentrations to levels not statistically different from those of intacts. The "role(s)" of plasma LH levels and changes in form and numerical density (Nv) of gonadal LH-binding cells in the maturation of the pituitary-gonadal axes of the chick embryo are discussed and evaluated.

Ontogenesis of immunocytochemically demonstrable somatotrophs in the adenohypophyseal pars distalis of the developing chick embryo.

Growth hormone (GH)-containing cells in the hypophyseal pars distalis of the chick embryo were identified immunohistochemically using anti-chicken GH sera. GH cells are first demonstrable in Rathke's Pouch as early as Day 4.5 of incubation. By Day 9.5, when the two lobes (rostral and caudal) of the pars distalis are easily recognized, GH cells are confined exclusively to the caudal lobe. Although the number of GH cells increases gradually during embryonic development, there is a statistically significant difference between Days 10.5 and 12.5 in both the cellular density and the percentage of somatotrophs. GH cells, which contain coarse granules evenly distributed throughout the cytoplasm, varied in diameter from 9.3 micron on Day 4.5 to 11.8 micron on Day 20.5, while the nuclear diameter of these cells increased from 2.8 micron on Day 4.5 to 4.9 micron on Day 20.5. There was a statistically significant difference in the nuclear/cytoplasmic ratio between Days 4.5 and 13.5 and between Days 13.5 and 20.5 of incubation. Aspects of the regulation of growth hormone synthesis and secretion in the chick embryo are discussed.

Ontogenesis of thyroid function and regulation in the developing chick embryo.

B.H. Willier (Analysis of Development, W.B. Saunders, Philadelphia, 1955, pp. 574-619) hypothesized that the early differentiation and initial function of the glandular components of vertebrate endocrine axes are autonomous and that functional interdigitation of these glands occurs later in developmental time. The present review summarizes findings that indicate that the hypothalamus, pituitary, and thyroid glands of the developing chick embryo synthesize their appropriate hormones early in development, i.e., before day 5.5 of incubation. Observations also indicate that it is not until the days 10.5-11.5 embryonic period that the feed-forward and feed-backward components of the adenohypophyseal-thyroid or the hypothalamo-adenohypophyseal-thyroid axis mature. Evidence is also presented for a midgestational beginning of hypothalamic regulation of adenohypophyseal-thyroid unit function.

Plasma iodothyronines in the domestic fowl: newly hatched to early adult stages, with special reference to reverse triiodothyronine (rT3).

Circulating concentrations of thyroxine (T4), triiodothyronine (T3) and reverse triiodothyronine (rT3) were measured in chicks before, during, and after hatching, up to 9 weeks of age. T4 decreased prior to hatching, rose after emergence, and was variable in the immature domestic fowl. T3 increased prior to emergence, decreased until 5 days after hatching, and increased again by 1 week of age, after which the levels declined. Plasma rT3 declined prior to hatching, remained low until 5 days after emergence, and then increased, again, to 0.14-0.19 ng/ml between 1-9 weeks of age.

Hypothalamo-adenohypophyseal-thyroid interrelationships in the developing chick embryo. VII. Immunocytochemical demonstration of thyrotrophin-releasing hormone.

Thyrotrophin-releasing hormone (TRH) was demonstrated immunocytochemically in the infundibulum of the chick embryo as early as Day 4.5 of incubation. From Days 4.5 through 19.5 of embryonic development there is a gradual increase within the developing hypothalamus in the number of TRH-positive perikarya as well as the amount of immunoreactive-TRH (IR-TRH) per cell. There are no abrupt changes in either parameter during the critical time period (Days 10.5-13.5 of incubation) in the maturation of the pituitary-thyroid axis. Thus, although TRH is probably not directly responsible for the dramatic increase in the number of thyrotrophin-producing cells which occurs in the pars distalis of 10.5- to 11.5-day-old embryos (R. C. Thommes, J. B. Martens, W. E. Hopkins, J. Caliendo, M. J. Sorrentino, and J. E. Woods (1983). Gen. Comp. Endocrinol. 51, 434-443) the marked change in the activity of the pituitary-thyroid unit at this time may well reflect the response of these newly differentiated thyrothrophs to low levels of plasma TRH. This hypothesis is supported by the observations that between Days 10.5 and 11.5 the hypothalamic-adenohypophyseal-thyroid (HAT) axis is first responsive to cold (R. C. Thommes, J. B. Martens, J. B. Hopkins, D. A. Griesbach, D. J. Williams, M. J. Sorrentino, P. Wernke, and J. E. Woods. In "Proceedings, Ninth International Symposium on Comparative Endocrinology Hong Kong, 7-11 December 1981" (B. Lofts, ed.). Hong Kong Univ. Press, Hong Kong, in press) and also that the pituitary-thyroid unit exhibits a marked increase in its sensitivity to exogenous TRH (R. C. Thommes, D. J. Williams, and J. E. Woods (1984). Gen. Comp. Endocrinol. 55, 275-279).

Ontogeny of hypothalamo-adenohypophyseal-gonadal (HAG) interrelationships in the chick embryo.

In the male chick embryo the components of the hypothalamo-adenohypophyseal-testicular axis initially function independently of each other. It is not until days 12.5-13.5 that the adenohypophysis begins to regulate testosterone synthesis and secretion; on day 13.5 plasma testosterone reaches a maximum embryonic level. This feed forward regulation of the pituitary-testicular unit appears to involve a cause and effect relation between a statistically significant increase in the number of testicular interstitial cell LH receptors on days 12.5 and 13.0 and an increase in plasma LH levels on day 13.5 (up-regulation). Subsequently, events occur that are interpreted as indicative of the feedback phase of this endocrine axis. Plasma LH levels decrease after day 13.5. Also on day 13.5 and all subsequent embryonic days, there is a significant decline in the volume density of testicular LH receptor-positive interstitial cells (IC) associated with an internalization of the LH receptor complexes and a marked decline in plasma testosterone levels (down-regulation). It is strongly suggested that the decline in the number of LH receptor-positive ICs and the internalization of the LH receptor complexes is indicative of a "desensitization" of the ICs followed by a decrease in testosterone synthesis and secretion. Comparable events that occur in the female embryo with respect to the development of the hypothalamo-adenohypophyseal-ovarian axis are also discussed in this minireview.

Hypothalamo-adenohypophyseal-thyroid interrelationships in the chick embryo. VI. Midgestational adenohypophyseal sensitivity to thyrotrophin-releasing hormone.

The functional abilities of 9.5-, 10.5-, 11.5-, and 12.5-day-old chick embryos to respond to exogenous thyrotrophin-releasing hormone (TRH) were evaluated by means of changes in plasma total thyroxine (T4). T4 concentrations were determined 3.0, 6.0, 12.0, and 24.0 hr after TRH treatment. The data of the present investigation show that chick embryo adenohypophyseal sensitivity to exogenous TRH, as evidenced by changes in plasma T4 levels, increase during the 10.5- to 12.5-day incubation interval; however, the pattern and magnitude of plasma T4 response to administered TRH change markedly between Days 10.5 and 11.5 of development. This modification of the adenohypophyseal response pattern corresponds in embryonic time with the previously reported increases in immunocytochemically demonstrable TSH cells within the adenohypophysis(R. C. Thommes, J. B. Martens, W. E. Hopkins, D. A. Griesbach, D. J. Williams, M. J. Sorrentino, P. Wernke, and J. E. Woods, 1981, In 'Ninth International Symposium of Comparative Endocrinology"; R. C. Thommes, J. B. Martens, W. E. Hopkins, M. J. Sorrentino, J. C. Caliendo, and J. E. Woods, 1983, Gen. Comp. Endocrinol. 51, 434-443).

Hypothalamo-adenohypophyseal-thyroid and gonadal interrelationship in the chick embryo. I. Differential effect of ectopic pituitary grafts on plasma total T4 and testosterone levels.

Plasma total thyroxine (T4) and testosterone concentrations of Day 15.5 intact, surgically partially decapitated (hypothalamo-hypophyseoprivic) and partially decapitated chick embryos with adenohypophyseal transplants were determined by radioimmunoassay of the same plasma sample. Decapitation in both male and female chick embryos at 33-38 h of incubation results in plasma thyroxine and testosterone levels which are statistically significantly lower than those of intact embryos. The addition of a single adenohypophyseal transplant to the chorioallantoic membrane (CAM) of male and female decapitated embryos only partially restores plasma thyroxine levels to intact levels. However, the same procedure results in plasma testosterone levels which are higher than those of both decapitated and intact individuals. The data suggest that in the developing chick embryo there are differences in dependence of the thyroid and the gonads upon a functional hypothalamo-adenohypophyseal unit. The possible differential regulatory effects of the hypothalamus on the adenohypophyseal-thyroid and adenohypophyseal-gonadal components of the hypothalamic-adenohypophyseal-thyroid and hypothalamic-adenohypophyseal-gonadal axes are discussed.

Hypothalamo-adenohypophyseal-thyroid interrelationships in the chick embryo. IV. Immunocytochemical demonstration of TSH in the hypophyseal pars distalis.

The cells that synthesize thyroid-stimulating hormone (TSH) in the pars distalis of the chick embryo were identified immunocytochemically (immunoperoxidase and immunofluorescence) using anti-bovine TSH-beta and anti-human TSH-beta sera. TSH cells are first demonstrable on Day 6.5 of incubation. By Day 11.5, when the two lobes (rostral and caudal) of the pars distalis are easily recognized, TSH cells are confined exclusively to the rostral lobe. TSH cells identified by means of immunofluorescence were stained with the periodic acid-Schiff component of the performic acid-Alcian blue periodic acid-Schiff's Orange G stain. Immunoreactive TSH cells in the pares distales of Day 13.5 chick embryos, injected at 5.5 days of incubation with thiourea, were more intensively stained than their normal counterparts. The marked change in immunocytochemically demonstrable TSH on Day 11.5 corresponds with physiological and morphological events occurring within the hypothalamus, adenohypophysis, and the thyroid gland of the developing chick during this midincubational (midgestational) period. The data suggest that not only is hypophyseal TSH present in greater quantities after Day 10.5, but that adenohypophyseal synthesis and secretion of TSH may be stimulated by another factor (hypothalamic TRH) at this time, signaling functional maturation of the hypothalamo-adenohypophyseal-thyroid axis.

Functional development of the hypothalamic-adenohypophyseal-testicular (HAT) axis in the chick embryo.

Plasma testosterone concentrations in intact and surgically partially decapitated (hypothalamo-hypophyseoprivic) chick embryos were determined by radioimmunoassay for the incubation interval, Days 7.5-17.5. "Hypophysectomy" of male chick embryos at 33-38 hr of incubation resulted in plasma testosterone levels which were significantly lower than those of intact embryos on Days 13.5, 15.5, and 17.5. The addition of luteinizing hormone (LH) to the chorioallantoic membrane (CAM) of decapitated embryos increased plasma testosterone concentrations to normal levels by Day 13.5, while LH and pituitary transplants to the CAM elevated plasma testosterone to above normal values by Day 15.5. These observations, together with other findings, are interpreted as demonstrating that in the chick embryo the adenohypophyseal-testicular axis is functional by Day 13.5 of incubation. There are also indications that the hypothalamic-adenohypophyseal-testicular complex is functional at this same embryonic time.