Crop Sac Research Articles

EFFECT OF HYPOTHALAMIC LESIONS ON PITUITARY PROLACTIN CONTENT IN THE RABBIT.

Hypothalamic lesions have been shown to induce lactation, but their mechanisms of action have not been understood. In the present study the effects of lactation-inducing hypothalamic lesions on pituitary prolactin were assessed in mature New Zealand female rabbits. The rabbits were ovariectomized and primed 10 days with estradiol benzoate. On day 12 an electrolytic lesion was placed in the basal hypothalamus, and autopsy was performed on day 15. Mammary gland activation was classified both grossly and by histologic examination. Pituitary prolactin was assayed by an intradermal crop sac method employing White King squabs. In rabbits which failed to show mammary gland activation, the lesions lay outside the posterior tuberal area, and pituitary prolactin content was high. Conversely, the mammary glands were markedly activated in rabbits with lesions involving the posterior tuber, and pituitary prolactin was low. Results indicate that the activation of mammary glands by hypothalamic lesions is an effect exer...

AN IMPROVED BIO-ASSAY FOR PROLACTIN USING ADULT PIGEONS.

The effect of age and duration of injection of prolactin on the slope of the dose-response curve in the systemic crop sac assay for prolactin in pigeons was studied. In 6-week-old pigeons the slope at 4 days was 2.5 and at 7 days it was 5.4 g of crop sac weight increase per 10-fold dosage change. In 5- to 8-yr-old pigeons the slope at 4 days was 6.5 and at 7 days it was 17.4. The index of precision increased with the level of response of crop gland weight. In old pigeons at 7 days, and at crop gland weights below 5 g, λ was 0.05–0.1 or about 25% of that in young pigeons at 4 days.

Failure of Oxytocin to stimulate the Pigeon Crop Gland

IT is well known that the crop gland of the pigeon proliferates and increases in weight in response to prolactin. This effect of prolactin on the crop gland has been used as a qualitative and quantitative assay for this hormone1. Recently, Chaudhury and Chaudhury2 threw doubt on the specificity of action of prolactin on the pigeon crop gland by claiming that intradermal injections over the crop sac or systemic administration of oxytocin produced increases in crop-weight. This contradicts the earlier work of Riddle and Bates1, who reported that posterior pituitary hormones had no effect on the pigeon crop gland. Chaudhury and Chaudhury2 also suggested that oxytocin may induce a release of prolactin in pigeons, which is similar to the hypothesis of Benson and Folley3 in rats. They did not indicate whether typical crop sac proliferation was seen in the pigeons.

LOCALIZATION OF PROLACTIN WITHIN THE PITUITARY BY SPECIFIC FLUORESCENT ANTIPROLACTIN GLOBULIN

It has long been recognized that the pleomorphic changes which occur in the pituitary gland during estrogenic cycles in the experimental animal are probably associated with hormonal activity. While various histochemical staining techniques demonstrate the particular cells concerned in these changes, the presence of a particular hormone cannot be directly demonstrated by these methods. Using ovine prolactin as an antigen with Freund's adjuvant, it has been possible to produce precipitating antisera capable of inhibiting the action of prolactin on the mucosa of the pigeon crop sac. By means of fluorescent anti-prolactin globulin, prolactin has been demonstrated in the cytoplasm of the acidophilic (α) cells of the pars distalis of the cat and rat pituitary.

Effects of intrahypothalamic and intrahypophysial estrogen implants on pituitary prolactin and lactation in the rabbit.

A minute amount of solid estradiol benzoate was implanted into the hypothalamus or pituitary gland in female rabbits. The prolactin content of each pituitary gland was assayed on White King squabs by the intradermal crop sac method. The pituitary prolactin content was elevated following estrogen implantation in the posterior median eminence area, but the mammary glands were not activated. In this group there was marked atrophy of the ovary and uterus, but hypertrophy of the adrenal cortex. Estrogen implantation into the anterior hypophysis was followed by activation of mammary glands and initiation of milk secretion, while the pituitary prolactin content was, if anything, depressed. No significant changes occurred in the ovary, uterus or adrenal. The results indicate that estrogen action on the posterior tuberal hypothalamus promotes synthesis and storage of prolactin in the pituitary gland, whereas the direct action of the steroid on hypophysial cells stimulates release of the lactogenic hormone.

Immunological Investigations of Sheep Prolactin

ZONE electrophoresis on starch revealed that purified sheep prolactin contained, according to Cole and Li1, three, and, according to Ferguson and Wallace2, four, components. All these components were found to have biological activity when tested by the crop sac test in pigeons2. However, Reisfeld et al.3, testing the biological activity of purified components, found one of these components to be a highly active prolactin, whereas the two further components were biologically loss active; these authors considered the latter fractions altered the form of the active hormone.

Presence in the human placenta and term serum of a highly lactogenic substance immunologically related to pituitary growth hormone.

A previously undescribed substance has been discovered in the peripheral and retroplacental sera of human pregnancy at term. This substance is immunochemically closely related to human pituitary growth hormone (HGH) and has been isolated in relatively large amounts and purified form from term human placentas. It is highly lactogenic both in pigeon crop sac assays (local intradermal injections) and in milk-promoting activity in the pseudopregnant rabbit, but is devoid of growth-promoting activity in the hypophysectomized rat tibial plate growth assay at the dose levels tested. The possible biologic role of this lactogenic material in pregnant primates is discussed. The findings encountered give further support to the theory that a small portion of a protein hormone molecule may determine major differences in its physiologic function.

Further observations on the question of specificity of antihuman growth hormone rabbit serum.

It recently has been demonstrated by several investigators (10–14) that highly purified human growth hormone (HGH) possesses significant lactogenic (prolactin) activity. In the present investigation, it was shown by the local crop sac test in immature pigeons that a 6-μg daily dose of HGH (Li) elicited a good lactogenic (crop sac stimulating) reaction, and that this response could be eliminated by prior treatment of the hormone with anti-HGH (Raben) rabbit serum, whereas treatment of the hormone with an equivalent amount of normal rabbit serum did not have any effect on this activity of the HGH. Results of agar diffusion tests (Ouchterlony) with crude human pituitary extracts demonstrated that anti-HGH (Raben) serum contained antibodies to at least 2 closely related antigenic components other than serum proteins and also a small amount of antibodies to human serum albumin. However, agar diffusion tests, including immuno-electrophoresis, showed the presence of only one antigenic component in highly purifie...

A comparison of the biological activity of prolactin by three methods of assay.

SUMMARY Three preparations of prolactin have been assayed against a reference preparation by different techniques. These are the prolongation of dioestrus in the mouse, the in vitro method depending on the increase of β-glucuronidase in the rat testis, and the pigeon crop sac method. The results have been compared and discussed.

Hormonal specificity of the in vitro action of growth hormone on amino acid transport into rat muscle.

Various pituitary hormones were tested for their ability to stimulate in vitro the transport of AIB into the cells of isolated hypophysectomized rat diaphragms. Crude and purified TSH, purified FSH, purified LH, purified MSH, posterior pituitary extracts and synthetic oxytocin andvasopressin were all ineffective at the concentrations tested. Three ovine prolactin preparations stimulated AIB transport into muscle when present in the incubation medium at high concentrations. This activity was abolished, however, when the prolactin solutions were adjusted to pH 8 and heated for 20 minutes at 100 C. The heating procedure also eliminates the action of growth hormone on amino acid transport but does not alter the ability of prolactin to stimulate the pigeon crop sac. Thus, it is conceivable that the amino acid transport stimulating activity present in ovine prolactin is due to growth hormone contamination. These experiments indicate that growth hormone is the only pituitary hormone which interacts to any extent...

Preliminary investigations on the action of pepsin on human pituitary growth hormone.

A slight modification of the original procedure for the isolation of human pituitary growth hormone has been described. The purity of the preparation was further examined by chromatography on carboxymethylcellulose, zone electrophoresis on starch, and electrodialysis. The hormone was found to be stable in a pH 7.0 solution at 100 degrees C and in 10 M urea at 25 degrees C. Digestion with pepsin to an extent of 40 per cent did not diminish the biological activities of the hormone. Bioassays of an active core derived from such digests exhibited essentially the same specific growth-stimulating activity in the hypophysectomized rat and crop sac stimulation in the pigeon. It was concluded that the integrity of the protein is not required for the activity of human growth hormone.

Initiation of Lactation in Rats with Hypothalamie or Cerebral Tissue.

SummaryRat tissue containing the hypothalamus, cerebral tissue, kidney or liver were injected subcutaneously in saline suspension into rats for 5 days, after they had previously been injected with estradiol for 10 days to develop their mammary glands. Saline alone was injected into control rats. The tissue containing the hypothalamus initiated lactation in 12 out of 15 rats; cerebrum in 4 out of 15 rats; and liver, kidney or saline in none out of 5 rats each. Tissue containing the hypothalamus was injected intradermally over crop sacs of 5 pigeons for 5 days and failed to induce a response, indicating that the active principle is not prolactin. It is suggested that rat hypothalamus and possibly cerebral tissue produce release of prolactin and probably ACTH from the anterior pituitary in amounts sufficient to initiate lactation.

Treatment of Menstrual Disorders with Prolactin1

PROLACTIN is one of the least frequently used fractions of the anterior lobe, in spite of the fact that it was the first active pituitary principle which, after isolation in absolute purity, has been brought to the point of crystallization. Its physiological properties are well explored; in addition to its effect upon the crop sac of pigeons and the mammary glands of mammals, prolactin is also important for body growth in general and to bring about the development of normal-sized viscera (15,19). Prolactin, furthermore, is antigonadotropic (15, 19), not only in birds but also in rats. Subsequent experiments with the purified hormone showed that, in addition to the inhibiting effect upon the secretion, release or effect of the pituitary follicle-stimulating hormone, prolactin also stimulates the luteal cells of the ovary, possibly increasing the secretion of the lutein body or at least, contributing to its maintenance (3).

The Direct Mammotrophic Action of Lactogenic Hormone

SummaryThirty oophorectornized, virgin rabbits, injected subcutaneously 5 days weekly for 4 weeks with 200 I.U. of estrone and 1 I.U. of progesterone showed prolactational mammary growth approximating that of a 3-weeks′ pregnant rabbit. Three days after the last of these injections, 5 different levels of lactogenic hormone were tested on groups of 6 rabbits. The hormone was given intraductally in such a way that an individual sector in each animal received a constant volume of fluid (1.0 cc). Of the different doses of hormone tested, 6 and 3 I.U. caused localized sector lactation in all 6 animals; 1.5 I.U. in 5 of 6; and 0.75 I.U. in 3 of 6 animals. None of the 6 animals injected with 0.37 I.U. lactated; nor did the uninjected or control-injected sectors in any animal lactate. This experiment demonstrates for the first time that the hypophysial lactogenic hormone has a direct mammotrophic effect just as it was previously shown to have a direct crop sac stimulating action. With all other factors essentiall...

Effect of Lactogenic Hormone on Embryonic Tissues Cultivated in vitro.

Since lactogenic hormone is known to produce a specific effect on epithelial cells of the pigeon cropin vivo, experiments were designed to determine whether it would stimulate the growth of embryonic pigeon crop epithelium cultivated in vitro. If stimulation could be observed in this manner such a test might be more delicate and superior to the present methods employed for determining hormonal potency. Preliminary to testing the hormone on the specific crop epithelium, which reacts to it in vivo, experiments were carried out on the following non-specific tissues cultivated in vitro: (1) Embryonic chick connective tissue, (2) embryonic chick epidermis, (3) embryonic pigeon oesophageal epithelium. Finally the hormone was tested on cultures of embryonic epithelium taken from the crop sac areas known to be reactive in vivo in the hatched pigeons. In all cases the dilutions were performed in triplicate. Also, every experiment was repeated 3 times before the results were reported. Each flask contained about 12 fragments of tissue. (1) Embryonic Chick Connective Tissue. Chick heart tissue from 10- to 14-day-old embryos was used as the source of fibroblasts. The tissues were finely cut into fragments of about 1 mm. in diameter and immersed in Tyrode solution until ready for use. The embryonic fluid was prepared by mincing 14-day-old chick embryos in a tissue grinder and suspending the pulp in 4 parts of Tyrode solution. The suspension was centrifugated at high speed to throw down all cellular material after which the clear supernatant fluid was carefully removed with a pipette. This clear liquid was used for coagulating the plasma as well as a nutrient for the tissue cells. Guinea pig plasma was used in all experiments. This was collected by first drawing 1.0 cc. of a 1-1,000 solution of heparin into a 10.0 cc. syringe and then heart blood to the 10.0 cc. mark. The mixture was quickly centrifugated, the clear plasma removed with a pipette and kept in ice water until ready for use.

The Physiology of the Formation of "Pigeon's Milk"

The Physiology of the Formation of "Pigeon's Milk"

Decreased Mammotropin in Pituitaries of Thyroidectomized (Maternalized) Male Rats

SummaryThe anterior pituitaries of normal adult male rats, when implanted directly over the center of the crop sacs of squabs, evoke a greater response in the crop sacs, hence store more mammotropin, than do the glandules from thyroparathyroidectomized (maternalized) males of the same age.Acknowledgment is hereby made to Dr. Herbert McLean Evans for his helpful criticism.