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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.

The binding of luteinizing hormone (LH) to cow corpora lutea homogenates was studied using a tritium labelled hormone obtained by reductive methylation. The KD observed was 0.9 10(-10) M and the number of sites was the equivalent of 0.4 10(-15) moles per mg of wet tissue. The influence of the pH and temperature was examined. HCG and LH produced the same binding inhibition properties of the derivative used for labelling LH were similar to those of native LH. The inhibitory activity of the subunits was extremely low (alpha-LH: 4%, beta-LH: 1%). No significant inhibition was observed in the case of FSH or prolactin.

An inhibitory effect of PGF2alpha at a dose of 7 X 10(-7) M on LH stimulated synthesis of progesterone was observed in vitro after incubation of pseudopregnant rat ovaries for a period of 2 hours. A similar effect was seen with cyclic and gestant ewe corpora lutea at the same dose of PGF2alpha. This effect was observed both in the secretion of progesterone and on the amount of progesterone present in the tissue. This inhibitory effect of PGF2alpha on LH stimulated progesterone synthesis may explain the modification in the time course for gonadotropin action in luteal tissue at high and low doses.

Lysine residues appear to play an important role in the biological activity of luteinizing hormone or lutropin (LH). Some derivatives obtained by chemical modification such as N-methylated LH exhibit the same hormonal activity than LH in the different steps of the mechanism of steroidogenesis. Some others, on the contrary, preserve the hormonal activity only at some steps. In this work was investigated the action of ovine LH and some derivatives on isolated cells prepared from ovine corpora lutea. Guanidinated LH (which is able to bind to LH receptors in Leydig cells but whose steroidogenic potency is very low) exhibits no binding or steroidogenic activity in the female (sheep or rat). As a consequence guanidyl LH can act as an inhibitor of LH action in the male (Leydig cells).

The LH binding properties (determined using tritiated methylated LH) and the in-vitro steroidogenic activity of CL from ewes in the oestrous cycle or early pregnancy (Day 18) were compared. No significant alteration in the Kd values was observed. However, the number of sites was maximal at Day 10 of the cycle and in early pregnant animals which had not been pregnant for at least 3 months (dry ewes). Non-lactating or suckling ewes had half the numbers of binding sites. The increase of the number of receptor sites was accompanied by a steroidogenic response at lower LH concentration. During incubation or superfusion for 5 h, a refractoriness to LH stimulation appeared after 1 h with high LH concentrations and after 3 h with low concentrations. The opposite effect of the addition of indomethacin or PGF-2 alpha suggests the intervention of PGs in this phenomenon.

We studied the effect of guanyl nucleotides, divalent cations and luteinizing hormone (LH) on the regulation of adenylate cyclase (AC) in partially purified plasma membranes obtained from isolated interstitial cells of the rat testis. AC was activated to different degrees by guanosine triphosphate (GTP) and GMP-P(NH)P; the latter was about 10 times more active than the former. Enzyme activation by GTP was biphasic; the nucleotide was rapidly hydrolysed by membrane preparation. Activation by GMP-P(NH)P was hysteretic, requiring about 20-30 min to reach steady state; this lag-time was not dependent on nucleotide concentration. GDP beta S did not stimulate AC activity. Delayed addition of GDP beta S to a GMP-P(NH)P-stimulated enzyme at 17 min resulted in a drop of AC activity although the activity 40 min later was higher than that obtained by mixing both nucleotides at the time the reaction was initiated. This result was incompatible with the formation of a truly irreversible, active form of the enzyme in the presence of GMP-P(NH)P. The effect of LH on AC depended on guanine nucleotides and Mg2+. LH and GMP-P(NH)P acted synergically. Dose-response curves showed that apparent LH affinity was not modified by the presence of GMP-P(NH)P. LH accelerated the slow rate of activation of GMP-P(NH)P. The stimulation of AC by LH was closely dependent on Mg2+ concentrations; LH diminished the apparent Mg2+ requirement. Plasma membranes from rat testicular interstitial cells are an excellent model for the study of AC regulation by LH.

The adenylate cyclase activation by ovine native LH, natural analogs (porcine LH, hCG) and chemical derivatives of LH (methylated, ethylated, isopropylated, guanidinated) was studied in purified plasma membranes of ovine corpora lutea, including the regulatory effects of guanyl 5'-yl imidophosphate (Gpp(NH)p) and Mg2+. The Ka app. for native LH (about 15 nM) was independent of Gpp(NH)p and Mg2+. Similar maximal activation of the enzyme was obtained by using ovine LH or natural analogs, but differences were remarked concerning the Ka app. values of these hormones. Porcine LH was equipotent with ovine LH; on the contrary, hCG exhibited a lower Ka app. value (3 nM). All chemical derivatives (Me-LH, Et-LH, Iso-LH and Gu-LH) exhibited Ka app. higher than native (about 2- to 4-fold), but similar maximal activation. No modification was observed in the regulatory effects of Gpp(NH)p and Mg2+ on the adenylate cyclase activation as a consequence of structural modifications of the hormone. A comparison of the steroidogenic activity on intact luteal cells and the adenylate cyclase activation ability on purified plasma membranes of the derivatives mentioned above evidenced some interesting discrepancies. The drop in adenylate cyclase activation potency of Me-LH was not reflected in its steroidogenic activity (Me-LH was equipotent with native LH); on the contrary, the capacity of Gu-LH to stimulate adenylate cyclase was not so much decreased as was its steroidogenic potency which was almost abolished.

To understand more closely the structural requirements of the LH molecule necessary to stimulate adenylate cyclase, we studied the modulation of this enzyme in partially purified plasma membranes prepared from isolated interstitial cells of rat testis submitted to oLH and to some oLH derivatives and natural analogues. The role of Mg2+ was also investigated in relation to the structural modifications of oLH. Some new facts appeared in this study: 1. Methyl oLH, which exhibited the same ability as native oLH to stimulate cAMP accumulation and steroidogenesis in isolated cells, cannot induce the same level of maximal stimulation of adenylate cyclase as native oLH in plasma membranes. This phenomenon is related to the Mg2+ concentration, and the differences between maximal activation induced by methyl oLH and oLH were more apparent at a free Mg2+ concentration of 3.3 mmol/l than at lower concentrations. The maximal activity (in terms of native oLH) of other alkyl derivatives, such as ethyl or isopropyl oLH, on the contrary, was similar in isolated plasma membranes and in intact cells suggesting that the differential behaviour of the membranes specifically concerns the methyl derivative. 2. Guanidyl oLH and guanidyl porcine LH, which were able to induce cAMP accumulation in intact cells, did not exhibit any stimulating activity in plasma membranes. 3. Among the natural analogues, hCG and pLH are distinguished by a lower maximal activity (by comparison with oLH) particularly at high Mg2+ concentration. This work shows that changes in the LH structure have an impact not only on the parameters of the adenylate cyclase complex but also on the transduction of the hormone signal and its modulation by Mg2+.

Rat intestinal cells prepared from testes were incubated in the presence of different lutropin derivatives obtained by chemical modification of the amino groups. The cAMP accumulation and the testosterone biosynthesis were determined in the cell homogenates. Binding determinations were carried out by a radioligand receptor assay using tritiated methylated lutropin. The binding activities--relative to native LH--of three different derivatives obtained by reductive alkylation (methylated, ethylated and isopropylated LH) were in good agreement with the relative potencies assessed by their capacity to stimulate cAMP and testosterone production. Guanidinated LH (11-NH2 groups modified) exhibited a binding activity and a relative potency relatively high with regard to cAMP accumulation (as compared with that of native LH). Its steroidogenic potency, however, was very low. When Leydig cells were incubated in the presence of native and guanidinated LH, the testosterone production was similar to that induced by the derivative alone, indicating that the derivative exerted a competitive inhibitory action preventing the stimulation of steroidogenesis by native LH. These results suggest that a guanidinated derivative is able to bind to the LH receptor and the complex so formed is able to be coupled with an adenylate cyclase pool (or cAMP compartment) which is not connected with the steroidogenic pathway.

The present communication documents LH- and forskolin-induced activation of adenylate cyclase (AC) system and progesterone synthesis in corpora lutea from pregnant ewes. The activation of AC in plasma membranes by LH or forskolin was amplified by Gpp(NH)p. These results suggest that regulatory nucleotide component (Ns) of the AC complex is required for forskolin. Simultaneous addition of maximal concentrations of forskolin (10(-4) M), Gpp(NH)p (10(-4) M) and LH (10(-7) M) led to greater than additive (i.e. synergistic) responses: the experimental value was 4.71 +/- 0.19 nmoles cAMP/mg of membrane protein, whereas the theoretical additive effect was 3.17 +/- 0.10 nmoles/mg of membrane protein (p less than 0.001). These data reveal that more Ns or C component is being activated in these cells when combined treatments with these agents are applied. In intact cells maximum stimulatory concentrations of forskolin or LH caused similar increase in progesterone production with similar time courses. In striking contrast, the exposure of the luteal cells to LH and forskolin simultaneously led to a decrease in progesterone synthesis as early as 1h30 (40%, p less than 0.001). Thus, the synergism observed between LH and forskolin on the stimulation of plasma membranes AC activity did not occur in steroidogenesis. The AC responses in crude plasma membranes form these cells to different stimulants were enhanced (i.e. 15%, p less than 0.2 for Gpp(NH)p, 33%, p less than 0.01 for LH plus Gpp(NH)p and 52%, p less than 0.01 for forskolin). These findings suggest that an early desensitization of the AC system cannot explain the impaired steroidogenic response observed.

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.