Effects of Norethynodrel on Progestins in Ovaries and Ovarian Venous Blood of Rats

EFFECTS OF NORETHYNODREL ON PROGESTINS IN OVARIES AND OVARIAN VENOUS BLOOD OF RATS* MASANAO HIRAI, YOSHIKO MORITA, and TAKESHI NAKAOj France and Pincus [i] reported that administration of I7a-ethynyl5 (io)-estrene-i7/3-ol-3-one (norethynodrel) resulted in the inhibition of gonadotropin-induced ovulation, as determined by the significant decrease in the mean number ofova recovered from the fallopian tubes of rats, and ovarian weight was decreased in hypophysectomized, gonadotropin -primed rats treated with norethynodrel suggesting a direct inhibitory action on the follicular growth induced by exogenous gonadotropins . Since Zander's observation [2-4], workers have demonstrated the presence of 4-pregnen-2oa-ol-3-one (2Oa-OH-P) in different species [5-7]. Recently, Hilliard, Penardi, and Sawyer [8] suggested that this 2Oa-OH-P acts as a positive feedback agent to prolong and heighten LH discharge in the mated rabbit. The secretion ofprogesterone by ovarian corpora lutea and the essential role of this steroid in implantation and maintenance of fertilized ova have been well established. In addition to this progestogen, the mammarian ovary synthesizes and releases two structurally similar compounds, 2Oa-OH-P and/or its 0-isomer (20/3-OH-P), to which no specific physiological functions have yet been assigned. The present experiments were carried out to define the effect ofnorethynodrel on ovulation and to determine whether the norethynodreltreated rat would have altered progestins in ovarian venous blood and the ovaries in vivo. * This paper is dedicated to Dr. Gregory Pincus because of his devotion to reproductive endocrinology and because of his steadfast faith in scientific inquiry and his unswerving encouragement ofhuman dignity. f Department of Pharmacology, Jikei University School of Medicine, Tokyo, Japan. We are grateful to Yoshiko Masubuchi for technical assistance. 44I Materials and Methods EXPERIMENTAL ANIMALS AND THEIR TREATMENTS The methods used in this study are basically similar to those used in the first ofthese two papers. Female Sprague-Dawley strain rats (Central Laboratory ofExperimental Animals, Tokyo, Japan) weighing about 200-265 gm. (aged eight to ten weeks) were given norethynodrel, 1 mg. daily for fifteen days by stomach tube. Those in the control group were given only the vehicle. Diet consisted of Laboratory Chow (CLEA) and water ad libitum; the rats were housed in a temperature-controlled room for three weeks before the start ofthe experiment. Ovarian venous blood was obtained by cannulation with a polyethylene tube of the left ovarian vein of rats from which adrenal glands had been removed just prior to the cannulation. They were anesthetized with pentobarbital (3 mg/100 gm body weight, ip). Heparin (30 unit/100 gm body weight) was injected through the cannula and allowed to enter the general circulation. The blood flowed into a receiver which was surrounded by ice. Blood was collected at one and one-half hours. Both ovaries were removed and weighed just after the blood collection. In each study, fifteen to eighteen rats were employed with an equal number of controls. Vaginal smears were made each morning before 9:00 a.m., classification of the cycle was according to Velardo, that is, diestrus, proestrus, estrus, and metestrus. Ovaries and adrenals were cleaned and weighed rapidly and the weights were expressed as actual wet weight oftissue in milligrams. EXTRACTION, PURIFICATION, AND IDENTIFICATION The steroids in the whole blood and in the ovaries were obtained by extracting the aqueous phases thrice with seven times their volume of dichloromethane (Table 1). The residues resulting from evaporation of the solvent were freed of non-polar lipids by refrigerated centrifugation with 70 per centmethanol. The steroid extractswere dissolved in dichloromethane and washed once with 0.4 N NaOH to separate the phenolic fraction. The neutral fraction was evaporated and thenpurified in 2-system paper chromatography and isolated using 1- or 2-system thin layer chromatography. The ovarian progestins were identified as follows: 442 Masanao Hirai et al. · Effects ofNorethynodrel Perspectives in Biology and Medicine · Spring 1968 2Oa-OH-P.—(?) On the basis of Rf values from above-mentioned chromatographies; (2) by means ofthe absorption maximum at 240 ??µ according to the ultraviolet energy distribution (UV); (3) by means of the absorption maximum at 450 ??µ according to the method of 2,4dinitrophenylhydrazine (DNPH); (4) acetylation...