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Biological activities of several derivatives of ovine LH obtained by chemical modification of the amino groups were investigated using ovaries from pseudopregnant rats. Binding-inhibition activities and steroidogenic potencies of ethylated, isopropylated and guanidinated LH were in good agreement, whereas adenylate cyclase activities were relatively greater. When compared with previous results on binding-inhibition activities and steroidogenic potencies using isolated rat Leydig cells, the ovaries from pseudopregnant rats appeared to be more discriminating. Ethylated and isopropylated derivatives exhibited lower binding-inhibition activities and steroidogenic potencies in female gonads. This difference was particularly evident in the case of guanidinated LH which exhibited a very low binding-inhibition activity and consequently was unable to act as an inhibitor of the action of LH on the ovaries. Guanidinated porcine LH (in which all the lysine residues of the alpha-subunit were transformed into homoarginine, without modification of the beta-subunit which does not contain lysine) showed similar biological activities to guanidinated ovine LH in the isolated Leydig cells as well as in pseudopregnant ovaries. It can, consequently, act as an inhibitor of LH action on Leydig cells but not on the ovary of the pseudopregnant rat. Thus, the inhibitory properties of this derivative can be ascribed to the modification introduced in the alpha-subunit.

A comparative study was made of the stimulation of rat ovary adenyl cyclase by ovine luteinizing hormone (LH) and its methylated drrivative (obtained by reductive alkylation of the lysine residues). The apparent Km (half maximal activity) of native LH and methylated LH was similar but the maximal activity obtained with the methylated derivative was significantly higher (1.2-1.8 times). When EDTA was added to the incubation medium the maximal activity obtained with the methylated LH diminished and became even lower than that obtained with native LH.

The biological activities of nitroguanidinated derivatives prepared from ovine or porcine luteinizing hormone were investigated using rat Leydig cells and pseudopregnant rat ovaries. In these tissues nitroguanidyl ovine luteinizing hormone (NGoLH) or nitroguanidyl porcine luteinizing hormone (NGpLH) were unable to stimulate adenylate cyclase or steroidogenesis but were able to inhibit the binding of ovine or porcine native LH to their specific receptors. When added to incubations of isolated Leydig cells or pseudopregnant ovary slices, NGoLH as well as NGpLH inhibited the stimulating action of native LH on adenylate cyclase or steroidogenesis. However, these derivatives had no inhibiting action on the stimulation of adenylate cyclase and steroidogenesis induced in the Leydig cells by choleratoxin or on the stimulation of testosterone production induced by 8-bromo-cyclic AMP. Since NGpLH (which does not contain lysine residues or free alpha-amino groups in the beta-subunit) exhibits the same antagonist action as NGoLH, we conclude that the nitroguanidination of the alpha-subunit is sufficient to endow the derivative with antihormone properties.