Endogenous rhythms of luteal and adrenal cholesterol ester hydrolase and serum PRL, LH and progesterone in mature pseudopregnant rats.

Luteal and adrenal cholesterol ester hydrolase (CEH) activity and serum PRL, LH and proges terone were measured at 3 h intervals throughout Day 6 of pseudopregnancy in mature rats. The nocturnal PRL surge which peaked at 0600 h was correlated (r = 0.95; P<0.01) with a significant increase in total luteal CEH activity which began between 0600 and 0900 h and attained its highest activity at 1200 h. Serum progesterone concentrations and total adrenal CEH activity did not change significantly at time intervals measured. A transient, but significant, increase in serum LH was observed at 1200 h. The data suggest that the nocturnal PRL surge initiates the subsequent increase in luteal CEH activity. Although the LH spurt could not initiate the increased CEH activ ity, it could have a modulatory role. PRL AND LUTEAL CHOLESTEROL ESTER HYDROLASE 1023 regulatory mechanisms which can exist, they do not necessarily demonstrate mechanisms which actually do exist in normal ovarian functioning. Hence, it appeared that the study of effects of PRL on CEH in mature, female rats with functioning luteal tissue would give the best indication of relationships which actually exist among PRL, CEH and serum progesterone. The experiment presented herein demon strates endogenous rhythms in serum PRL, LH, and progesterone concentrations, and in luteal and adrenal CEH activities throughout Day 6 of pseudopregnancy, as we!! as measures correla tions among these serum hormonal concen trations and tissue enzymatic activities. The data indicate the possibility that the morning PRL surge initiates a subsequent increase in lutea! CEH activity. Animals weighed on a torsion balance, placed into a half-dram (2 ml) shell vial and rapidly frozen in a dry ice-acetone bath. Adrenal glands were freed of surrounding fat and their connective tissue capsule, blotted, weighed and rapidly frozen. Tissue samples were stored at —? 60°C until used. Cholesterol Ester Hydrolase Assay After thawing on ice, luteal and adrenal tissues were processed as previously described (Klemcke and Brinkley, 1980) to prepare the 149,000 X g super natant which served as the source of CEH. For luteal tissue, 20, 40 and 60 Ml aliquots, and for adrenal tissue, 5, 10 and 20 Ml aliquots of this supernatant were delivered by pipet into tubes containing suffi cient 0.05 M Tris HCI buffer, pH 7.4 at 37°C,contain ing 4.2 mg/ml bovine serum albumin (BSA), to give a final volume of 270 Ml. Details of the CEH assay and subsequent extraction and purification techniques have been previously reported (Klemcke and Brinkley, 1980). Enzymatic activity is computed by multiplying the specific activity (dpm/min/mg protein) by the total amount of luteal or adrenal protein present, and is reported as dpm/min/total luteal or adrenal tissue/rat. Total CEH activity is presented since serum proges terone concentrations are significantly and positively correlated with total amounts of rat luteal tissue (DeGreef and Zeilmaker, 1974; Elbaum et al., 1975). Therefore, the most meaningful correlations would be those between total luteal CEH and serum progester one concentrations. For both luteal and adrenal tissue, saturating substrate concentrations were added. Time period of initial velocity was 0—5min for luteal tissue and 0—6 mm for adrenal tissue. Rate of product formation was linear for 65—260 Mg of luteal and 18—108 @gof adrenal, 149,000 X g supernatant protein. Protein was quantitated by the method of Lowry et al. (1951) using BSA as the standard.