William R. Lyons

Preparation and Assay of Mammotropic Hormone

Conclusion A niethod has been described for extracting mammotropic hormone from sheep pituitaries involving its solubility in HC1-acetone and NH3-acetone, and its precipitation with isoelectric protein at pH 5.5. A fraction made by the same procedure hut precipitated at pH 6.5 has heen shown to contain relatively little mammotropin, and is identified in the following paper as the adrenocorticotropic hormone. The “local” intradermal squab test has several advantages over the “systemic” tests of Riddle, et nl., when only mammotropic hormone is to be tested for, but the latter test has the very great advantage of allowing one to assay for the gonadotropic, thyrotropic, adrenotropic and mammotropic hormones on the same bird.

Detection of Mammotropin∗ in the Urine of Lactating Women:

In the course of studies on the hormonal control of the mammary gland, it has been of interest to ascertain by urinalysis to what extent a lactating woman is under the influence of estrin and the hypophyseal mammotropic hormone. A crude estrin may be prepared from the urine and tested by smearing it in the vaginae of ovariectomized rats. The urines of 8 lactating women (4–13 days postpartum) have been tested and all found to contain mammotropin in amounts that make it appear that at least as much of this hormone is excreted daily as is extractable from a bovine anterior lobe. The urine may be treated as follows: (1) to 100 cc. add 200 cc. acetone and 3 cc. HCl (concentrated); centrifuge and discard insoluble material; (2) add acetone to 90%; discard supernatant; (3) extract precipitate with mixture of 10 cc. stronger ammonia water, 20 cc. water, and 60 cc. acetone; discard insoluble; (4) add one volume of acetone; discard supernatant; (5) wash precipitate with 85% acetone, absolute acetone and ethyl ether (2 × 25 cc. in each case); (6) dry precipitate in warm desiccator; dissolve in 5.0 cc. water; adjust to pH 7.6; discard any insoluble.† Inject in doses of 0.1 and 1.0 cc. intradermally over the right and left crop sacs of squabs one month from hatching‡ for presumptive testing. Sacrifice birds at 48 hours, and retest in accordance with results until the minimal effective dose is determined. Until the hormone is purified it may suffice to consider the minimal effective dose in a statistically adequate number of birds as the temporary unit of urinary mammotropin. Sufficient amounts of untreated urine or blood plasma may be injected into the crop areas to allow for detection of mammotropin, but this procedure sometimes prevents accurate reading of the reactions because of inflammatory processes.

Influence of lactogenic preparations on production of traumatic placentoma in the rat

Summary Normally occurring or artificially induced lutein tissue in the rat does not produce progestin, as shown by the placentoma test. The only pituitary preparation which has been shown to stimulate the production of progestin by such lutein tissue is lactogenic hormone. Therefore, besides its classical mammotrophic activity and crop-sac stimulating function, the “lactogenic hormone’ is important in activation of the corpus luteum, and for this reason should be considered a part of the gonadotrophic complex.

Maintenance of pregnancy in hypophysectomized rats with placental implants.

Summary Pregnancy has been maintained in rats hypophysectomized on day 6 of their first pregnancy and injected daily subcutaneously for 6 days with placentae from rats, 12 days pregnant. Five placentae or approximately 275 mg daily accomplished this in the majority of the test rats. One 12-day placenta plus 0.5 μg of estrone proved equally efficacious. Pregnancy was not maintained by similar treatment if the rats were also oöphorectomized.

Hormonal Requirements for Pregnancy and Mammary Development in Hypophysectomized Rats.

Summary The same daily doses of purified lactogenic hormone (60 I.U.) and estrone (10 I.U.) capable of inducing beginning lobulo-alveolar mammary growth in the hypophysec-tomized rat, also ensured successful implantation in rats hypophysectomized and injected from the day of sperm. Since only about one-half of the estimated number of implantations were found to have developed normally through mid-pregnancy it follows that these 2 pure substances in the doses used did not adequately substitute for the intact pituitary of a pregnant rat. When injected alone neither lactogenic hormone nor estrone permitted implantation to occur.

EMBEDDING STAINED MAMMARY GLANDS IN PLASTIC

A simplified method of embedding stained mammary spreads of small mammals in plastic (Selection) is presented. Two standard 50 × 75 × 1 mm. glass slides, separated by 2 narrow glass strips of similar thickness, are used to form the embedding chamber. The glands are stained in toto in alum-carmine, dehydrated, defatted, infiltrated with uncatalyzed plastic and embedded in catalyzed plastic. After baking and cooling, the glass chamber separates readily and provides a thin square slide of plastic suitable for low-power microscopic examination, projection, and filing.

Rapid Embedding with Hot Low-Viscosity Nitrocellulose

A rapid method for embedding with low-viscosity nitrocellulose is described. Advantage is taken of the greater penetrating power of low-viscosity nitrocellulose than that of common varieties of celloidin. Fixation, dehydration and infiltration are carried out in screw-topped jars in the incubator at 56° C. The whole procedure from fixing to sectioning can be finished in 15-30 hours, and sections as thin as 6 to 10 μ may be cut without difficulty.

Intravaginal Assay of Urinary Estrin

Since estrin is not commonly standardized on the basis of its ability to produce behavioristic estrus, but rather by virtue of its growth-promoting influence on the vaginal epithelium, there seems but little need to saturate the entire animal to gain this end. The vaginal epithelium reacts to extremely minute quantities of estrin applied locally,∗ although Pratt and Smeltzer 1 and Powers, et al., 2 found their methods of vaginal administration only one-third to one-half as efficacious as the subcutaneous. We have used the intravaginal method of assaying urinary estrin not only because minute amounts may be detected (even in male urine), but also to avoid the toxic effects produced by parenteral injection of crude extracts. Twenty-four-hour urines were obtained from 4 women† on days 7, 14, 21, and 28 of the menstrual cycle, extracted immediately, or stored at 0°C. Half of the 24-hour sample was evaporated in vacuo over a steam bath to a salty sludge, (a few drops of capryl alcohol being added to lessen foaming). The sludge was extracted with two 100 cc. portions of hot absolute C2H5OH and the insoluble material discarded. The alcohol was distilled off, and the residue neutralized with N/1 or stronger NaOH. A graded series of dilutions of this extract was prepared with distilled water so that the 0.02 cc. used in testing represented original urinary volumes of from 0.05 to 1.0 cc. 0.01 cc. of each dilution was introduced twice (24-hour interval) into the vaginae of 3 full-grown rats ovariectomized at least 10 days previously. The minimal effective urinary equivalent was considered the smallest amount causing cornification in the vaginae of at least 2 of the 3 rats, 24 hours after the second dose. After an interval of 5 days or more, the rats were used again.

Evidence for Progestogen Secretion by ACTH-Stimulated Adrenals.

Conclusions 1. Daily doses of 30 γ of an ACTH suspension in beeswax and peanut oil injected for a week stimulated the adrenals of immature hypophysectomized and oophorectomized rats to form progestogen(s) as shown by the deciduoma test. 2. MH in daily doses as low as 50 γ accomplished this effect in immature hypophysectomized-adrenalectomized rats, the ovaries of which had previously been luteinized with PMSG. 3. Progesterone and DOCA in 7 daily doses of one mg induced deciduoma formation in immature hypophysectomized rats; hydrocortisone in 7 daily doses of 5 mg did not. 4. Since both MH and ACTH induce progestogen secretion, an assayist should study the respective target tissue of each (corpus luteum or adrenal cortex) for histologic evidence of activation rather than use biologic or chemical evidence of progestogen formation. An alternative would be to test for MH progestogen in hypophysectomized-adrenalectomized animals, and for ACTH-progestogen after hypophysectomy and oophorectomy.

Effects of Androgen and Somatotrophin on the Os Penis of the Rat.

Summary In contrast to its excellent effect in stimulating cartilaginous and osseous growth in the rats penile ossicle, testosterone propionate, in a daily subcutaneous dose of 0.1 mg for 21 days following hypophysectomy and castration, caused no appreciable resumption of general skeletal growth. Somatotrophin (hypophyseal growth hormone) in a daily subcutaneous dose of 1.0 mg for the same period in similar animals caused excellent general skeletal growth, but did not correct appreciably the atrophic condition of the os penis. The two hormones synergized in causing better growth of the os penis than the androgen alone and a greater body weight increase than the growth hormone alone in doubly operated animals.