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A factor from human ovarian follicular fluid (hFF) has been characterized which stimulates testosterone production of human, rat, mouse and hamster Leydig cells. hFF was obtained from women participating in an in vitro fertilization programme. Basal and hCG stimulated testosterone production of rat interstitial cells, and Percoll purified Leydig cells were significantly stimulated by hFF. The steroidogenic response of the cells was 3\p=n-\5 fold higher than that obtained after stimulation with maximal doses of hCG. A serum pool from the same patients was found to be about 30 times less potent than hFF in stimulating steroidogenesis. The stimulatory activity was retained after precipitation with ammonium sulphate and dialysis. Precipitation with ethanol, ether and acetone resulted in a partial loss of activity, whereas extraction with charcoal or heating at 100\s=deg\Cfor 10 min resulted in significant loss of activity. When hFF was fractionated by gel chromatography, the stimulatory activity was eluted in a molecular weight region between 30 and 50 kD. The stimulatory factor was further purified by chromatofocusing and was eluted as a homogeneous peak with an isoelectric point between 8.8 and 9.5. The SDS-PAGE analysis of these fractions, however, revealed that the active substance was not homogeneous. The purified factor was immunologically distinct from hCG and hLH. These studies demonstrate for the first time the presence of a factor in the hFF which may potentiate the action of LH in the ovary.

Chromatography of 125I-bovine LH (LER-1716-2 and USDA-I-1) by means of anion exchange high performance liquid chromatography (HPLC) revealed two main peaks of radioactivity regardless as to whether or not the tracer was initially purified on cellulose CF11. The content of radioactivity in the first peak tended to increase as the storage time of the bLH preparation, either before or after iodination, increased. The first peak of radioactivity after HPLC fractionation either with or without cellulose adsorption consisted of material with low binding ability to bLH antiserum (6.9% +/- 0.5 and 13.0% +/- 1.0, respectively) and high binding ability to ovine LH alpha antiserum (51.0% +/- 2.7 and 35.2% +/- 3.6, respectively). The average ratio of alpha-subunit immuno-reactivity to 125I-bLH immunoreactivity in this material was 7.4 +/- 0.1 and 2.7 +/- 0.2, respectively (P less than 0.001). Peaks in 125I-bLH radioactivity and 125I-bLH immunoreactivity had different elution times. Radioimmunoassays with tracers obtained from fractions derived from the first radioactive peak after HPLC chromatography (i.e. 125I-bLH-LER-1716-2) both with and without cellulose adsorption, yielded significantly lower mean plasma LH levels in GnRH-treated cows compared with the control tracer routinely purified only on cellulose CF11 (e.g. 5.7 vs. 8.2 micrograms/; 4.6 vs. 8.2 micrograms/l). Plasma LH levels in GnRH-treated cows were significantly (P less than 0.001) lower as measured by radioimmunoassay utilizing 125I-USDA-blH-I-1 tracers than by radioimmunoassays utilizing 125I-blH-LER-1716-2 tracers (i.e. either Y = 0.17 + 0.75X or Y = 1.18 + 0.60X).

A cation-exchan e high-performance l i q u i d chromatography procedure i s described !or the separation o f b i o a c t i v e l u t r o p i n from f o l l i t r o p i n i n a human u r i n a r y gonadotropin standard p r e p a r a t i o n ( 1 s t IRP 70/45). The r e s u l t s c l e a r l y demonstrate t h a t recovered p r o t e i n r e t a f n e d most o f i t s b i o l o ical, a c t i v i t y f o l l o w i n g t h e chromatography. Due t o a l a c k o f e a f i c i e n t and quick methods f o r p u r i f y i n g p i t u i t a r o r u r i n a r y gonadotro ins. t h i s method could prove t o be valuabfe f o r t h i s purpose. TRis

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