Observations on ovarian cholesterol depletion (OCD) as a test for luteinizing hormone activity.

In the young rat rendered pseudopregnant by PMS and HCG, the doseresponse relationship between LH and OCD is multiphasic rather than linear over a broad test dose range. The character of this doseresponse curve is independent of the strain of animal used as well as the day of pseudopregnancy on which the assay is performed. The multiphasic character of the dose-response curve makes this test impractical for use as a routine laboratory tool in its present stage of development. (Endocrinology 78: 218, 1966) B et al. (1) have recently reported that the ovarian cholesterol concentration of immature pseudopregnant Wistar rats falls acutely after intraperitoneal injection of small doses (0.06-0.5 XlO~ g) of LH. This response has been proposed as a sensitive and specific method for bio-assaying LH activity. Our experience demonstrates that the dose-response curve over a wider range of LH concentrations has reproducible nonlinear characteristics which may limit its usefulness as a bio-assay tool. Materials and Methods Pseudopregnancy was induced in intact immature female Wistar and Holtzman rats (35-50 g) by subcutaneous injection of 35-50 IU Pregnant Mare Serum Gonadotropin (PMSG) followed 72 hr later by 25-50 IU Human Chorionic Gonadotropin (HCG). The dosages of PMSG and HCG used were varied to produce pairs of ovaries which appeared well luteinized and weighed between 90 and 300 mg. Ovaries containing rich yellow corpora lutea appearing to be lipid-filled were considered well luteinized. Red-gray, fleshyappearing ovaries consistently had low cholesterol concentrations (less than 800 ^ig/100 mg ovary) regardless of the test dose of NIH-LH given. The dosage required to produce acceptReceived July 9, 1965. 1 Present address: Department of Medicine, The University of Chicago, Chicago, Illinois. 2 Present address: Peter Bent Brigham Hospital, Boston, Massachusetts. 3 NIH-LH-S3. 4 Gonadin, Haver Lockhart. 6 A.P.L., Ayerst. "NIH-LH-S1 and NIH-LH-S5 were generously supplied by the Endocrinology Study Section, National Institutes of Health. able ovaries in a number of animals adequate for the experiment varied for unknown reasons. All experiments were performed 11 days following HCG injection, except for a few done 7, 8, 9, 10 or 12 days following the administration of HCG. A stock mixture of NIH-LH and lactose (1 ng LH/mg lactose), containing a small amount of carmine red to ensure thorough mixing, was prepared. The mixture was separated into 5-10 mg portions (containing 5-10 ng LH) and stored in a desiccator at — 20 C. On the day of each experiment, one of the portions was dissolved in 0.9% NaCl to make a solution containing 1 /ig LH/ml. This solution was serially diluted, using siliconized glassware, so that each test dose was contained in 1 ml. Control animals were given 1 ml saline. Five hr after intraperitoneal injection of the test or control dose, the animals were sacrificed by dislocation of the cervical spine. Each pair of ovaries was removed, rapidly cleaned, blotted, weighed on a torsion balance to the nearest 1 mg, and homogenized in 10 ml acetone: ethanol (1/1, v/v). The homogenate was filtered and an aliquot of the filtrate taken for cholesterol determination (2). The results were expressed as ^g cholesterol/100 mg ovarian weight. The mean cholesterol concentration was calculated for each group of 5 animals except where ovaries were discarded. Ovaries were discarded at the time of removal if their combined weight was less than 90 mg or they appeared poorly luteinized.