Perspectives in Biology and Medicine

ISOLATION OF 4-PREGNEN-20C1.-OL-3-ONE FROM RATS' OVARIAN VENOUS BLOOD AND OVARIES* MASANAO HIRAI, YOSHIKO MORITA, and TAKESHI NAKAOj Introduction Ovulation can be inhibited by both progesterone and testosterone, but certain newer synthetic compounds related to these are extremely effective and produce fewer undesirable side effects. Among these are: norethindrone , which is also classified as a 19-nortestosterone, and norethynodrel, which is more closely related to the estrogens. Fairly large scale studies in Puerto Rico [1] and Haiti [2] have revealed that these compounds when taken by mouth prevent ovulation, and therefore conception, in a very high proportion of sexually active women. Field trials ofthese and similar oral contraceptive substances are currently proceeding in a number ofheavily populated parts ofthe world, and they are being watched with interest by all those who are concerned with the problems raised by our worldwide population explosion. Biochemical control of contraception as most widely practiced is attained by means ofthe orally active ovulation inhibitors. The problem of the mechanisms ofaction ofthe synthetic progestins has been extensively discussed in numerous publications [3]. However, fundamental significance ofnorethynodrel as ovulation inhibitor remains unknown. In rabbits the steroidogenic response of the ovaries to LH, mating, or electrical stimulation of the hypothalamus or medial amygdala [4] is extremely rapid and appears to have a lower threshold than the ovulatory response [5]. The marked stimulus by FSH of follicle growth seen in * A preliminary report ofthis paper was presented at the 3d Asian and Oceanic Congress ofEndocrinology at Manila, January, 1967. t Department of Pharmacology, Jikei University School of Medicine, Tokyo, Japan. We are grateful to: Yoshiko Masubuchi for technical assistance; Dr. Gregory Pincus for his advice and for a gift of 4-pregnen-2oa-ol-3-one; Drs. Yoshihito Omori and Sumio Shima for infrared spectra analysis; and Dr. Tomojy Yanagita, Central Laboratory for Experimental Animals, Tokyo, for the Sprague-Dawley rats. 427 prepubertal rabbits is not accompanied by a capacity to convert ?5pregnenolone to progesterone and 4-pregnen-20a-ol-3-one (2Oa-OH-P); LH is needed for progestational steroidogenesis [6]. Several investigators have recently reported finding significant conversions ofprogesterone to the 20a-epimer with negligible formation of the 20/3-epimer [7-9]. The explanation for these differences is unknown. Thatlarge amounts ofprogesterone metabolites with 20a-hydroxy groups were present is not surprising. Many studies have shown that 20a-dehydrogenase is a widespread enzyme, present even in atrophic human ovaries and von Kuppfer cells [10, 11]. This hydroxylated progestin appears to have a special functional significance in the reflexly ovulating rabbit, and Hilliard, Penardi, and Sawyer [12] mentioned that this progestin acts as a positive feedback agent to prolong and heighten LH discharge in the mated rabbit. The mammalian ovary synthesizes and releases two structurally similar compounds, 2Oa-OH-P and or its ßisomer , to which no specific physiological functions have yet been assigned . Little is known about the nature ofthe 2Oa-OH-P formed by rat ovarian tissue. Therefore, the present papers are carried out to define isolation and identification of 2Oa-OH-P from rats' ovarian venous blood and their ovaries in Part I and to find that administration ofnorethynodrel produces a significant decrease in secretion of2Oa-OH-P by the rat in Part II. Materials and Methods ANIMALS AND OVARIAN CANNULATION Adult female rats ofthe Sprague-Dawley strain (Central Laboratory of Experimental Animals, Tokyo, Japan) aged eight to nine weeks were used. Diet consisted ofLaboratory Chow (CLEA) and water ad libitum and the rats were housed in a temperature-controlled room for three weeks before the start ofthe experiment. Ovarian venous blood was obtained by cannulation of the left ovarian vein of rats from which the adrenal glands had been removedjust prior to the cannulation. They were anesthetized with pentobarbital. Blood was collected for one hour. Bilateral ovaries were removedandweighedjustafter the blood collection. A polyethylene tube was inserted into the left ovarian vein or into the left renal vein as shown in Figure 1; all the venules that are connected with the ovarian vein were ligated with thread. Ten minutes were suffi428 Masanao Hirai et al. · 4-Pregnen-20 a-ol-3-onefrom Ovaries Perspectives in Biology and Medicine · Spring...