Hills Road, Cambridge CB2 2QD (Received 9 January 1975)

J. Physiol. (1975), 250, pp. 669-679 669 With 2 text-ftgurew Printed in Great Britain

IONIC DEPENDENCE OF LUTEINIZING-HORMONE-INDUCED STEROIDOGENESIS IN THE RABBIT OVARY BY J. R. BEDWANI* AND P. Y. D. WONGt From the Department of Pharmacology, Medical School, Hills Road, Cambridge CB2 2QD (Received 9 January 1975)

SUMMARY 1. Investigations were made into the influence of the ionic environment on the steroidogenic response of the rabbit ovary to luteinizing hormone (LH). 2. Removal of Ca2+ from the medium was without effect on the response to LH. A similar result was obtained in Ca2+-free medium containing EGTA. 3. A tenfold increase in [Ca2+]. to 25-6 mm, or the addition of La3+ or Eu3+ (0-25 mM) to medium containing the normal concentration of Ca2+, caused a marked inhibition of the response to LH. 4. Removal of Na+ from the medium, and replacement by choline, had no effect on the response to LH. Replacement of Na+ by Li+ inhibited the response to the hormone strongly, but the addition of 4 mM-Li+ to normal medium was without effect. 5. Removal of K+ from the medium inhibited LH-induced steroidogenesis, whereas a twentyfold increase in [K+]., to 100 mm had no effect. The response to LH was also unaffected by the absence of C1-. 6. Ouabain (10-4 M) inhibited the response to LH, but nupercaine (10-4 M) was without effect. 7. The inhibitory effect of ouabain was reversed by the addition of 2 mm-NADP+ to the medium. In contrast, the inhibitory effect of Eu3+ persisted in the NADP+-rich medium. 8. It is suggested that the intracellular ratio of Na+ (or Li+) to K+ is important for the expression of the steroidogenic response of the ovary to LH. Altered concentrations of these ions might affect the formation or availability of NADP+. The inhibitory effects of high [Ca2+]0 and lanthanide ions, however, are probably due to inhibition of hormone- stimulated adenyl cyclase. * Present address: Department of Physiology, University College, Cardiff. t Present address: Department of Physiology, University of Hong Kong. 24 PHY 250 670 J. R. BEDWANI AND P. Y. D. WONG

INTRODUCTION Inorganic ions play an important role in may biological systems (see Williams, 1970). Apart from the well-known effects of Ca2+ and Na+ on membrane permeability and structure in eukaryote cells, and the importance of Ca in exocytosis (Douglas & Poisner, 1964; Hales & Milner, 1968; Samli & Geschwind, 1968), Na+ and K+ are thought to be important in regulating some metabolic processes (Wyatt, 1964; Bygrave, 1967). In this context, it has been found that some glycolytic enzymes are activated by K+ and inhibited by Na+ and Li+ (Mahler & Cordes, 1966). Matthews & Saffran (1973) have studied the importance of the ionic environment for the expression of the steroidogenic response to ACTH in the adrenal cortex. However, little attention had been paid to the ionic dependence of steroidogenesis in the ovary. Accordingly, we have explored the effects of mono- and polyvalent ions on the steroidogenic response of the rabbit ovary to LH. METHODS Ovaries were taken from up to four freshly killed rabbits, and cut into slices 200 jam thick. The slices were pooled, and pre-incubated for 40 min at 370 C in normal Krebs-bicarbonate or Krebs-Tris buffer (solutions B and J, Table 1). After this, the tissue was distributed among incubation flasks containing, in quadruplicate, 5 ml. normal buffer or 'test' buffer (of altered composition), with and without LH (NIH-LH-S18, 2 ,ug/ml.). The amount of tissue added to each flask varied in different experiments, but was within the range of 9-40 mg. The contents of the flasks were incubated for 1 hr at 370 C, in an atmosphere of 02 or 5 % C02 in 02 as appropriate (see Table 1). Following this, the tissue was separated from the incubation medium by centrifugation and weighed. Aliquots of the super- natant (0.4 ml.) were extracted with 10 vol. diethyl ether and assayed in duplicate by the radio-immunological method of Abraham, Swerdloff, Tulchinsky & Odell (1971). The antiserum was obtained from Dr Abraham, list no. 257, no. 2. This antiserum measures mainly progesterone, but also shows a 7 % cross-reaction with 20a-hydroxypregn-4-en-3-one (20a-HP), which is the other major secretary product of the rabbit ovary. The material measured by the reaction with this antiserum is called hereafter 'progestin', and its concentration in the incubation medium was taken as a measure of steroidogenesis. Two experiments were carried out in which larger amounts (150-350 mg) tissue were incubated, and the production of progesterone and 20a-HP were measured separately. Ovaries were sliced and pre-incubated as before, and distributed among four flasks. Each flask contained 10 ml. appropriate control or 'test' buffer, in the presence or absence of LH (2 #g/ml.). After a 1 hr incubation, the tissue was homo- genized in the medium, and progesterone and 20a-HP extracted and separated chromatographically using methods described by Dorrington & Kilpatrick (1966a). 20a-HP was assayed by light absorption at 240 nm; progesterone was assayed similarly or by the radio-immunological method. The authenticity of the steroids extracted from rabbit ovaries in this manner has been verified by combined gas chromatography-mass spectrometry (Bedwani & Horton, 1971). IONS AND OVARIAN STEROIDOGENESIS 671

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24-2 672 J. R. BEDWANI AND P. Y. D. WONG

RESULTS The recovery of 50 ng progesterone from 04 ml. Krebs-bicarbonate buffer by the methods used in the immunological assays was 88-3 + 7-8 % (mean+ S.D. of observation, n = 6). The results which follow are not corrected for recovery. Blanks consisting of 0 4 ml. buffer extracted with diethyl ether gave values corresponding to < 12 pg progesterone, which was the limit of sensitivity of our assay.

TABLS 2. Effects of Ca2+ and lanthanide ions on progestin secretion by rabbit ovaries in vitro, in the presence and absence of LH (2 fig/ml.). Each result is the mean ± s.E. of mean of four or eight replicates. The compositions of the media are shown in Table 1 No. Progestin secretion of (ng/mg tissue. hr) repli- Baa -l Incubation medium cates Basal LH-stimulated Krebs-bicarbonate (B) 4 1*20+0-17 j 2'02 + 0-23 Ca2+-free (G) 4 0.77 + 0-27 n.s. 2-05 ± 0-23 n.s. Krebs-Tris (J) 8 1-25+±0.13 2*10 + 0-21n Ca2+-free + EGTA (2 mM) 8 1-75 ± 0 16 P < 0*05 2-80+0361ns Krebs-Tris (J) 4 1-61 ± 0.08 2 72 + 0*26 P < 0.02 Ca2+, 25-6 mm (H) 4 1.59+0.29 n.s. 1*53 + 0.20~ Krebs-Tris (J) 4 0-18± 0.028 J+ La8+ (0.25 mu) 4 0.18 + 0.02 n*s. 0253+±01030-28±00o3}<00P < 0-02 Krebs-Tris (J) 4 0.77+0.091 160 + 027P < 005 J+ Eu3+ (0.25 mm) 4 089 ±0*1 0-91 + 0.09fP<05 Significance calculated by Student's t test; n.s., not significant (P > 0.05).

Effects of Ca2+ and lanthanide ions The results of experiments in which the concentration of Ca2+ was altered, or the lanthanide ions, LaO+ or Eu3+, were present, are shown in Table 2. Removal of Ca2+ from the medium (solution 0) had no effect on basal progestin secretion or the response to LH. When EGTA (2 mm) was added to the Ca2+-free medium to remove membrane-bound Ca, there was a small increase in basal progestin secretion, but the response to LH was not altered significantly (P > 0 05, Student's t test). A tenfold increase in the Ca2+ concentration of the medium to 25-6 mm (solution H) had no effect on basal steroidogenesis but inhibited the response to LH. A similar result was obtained when LaO+ or Eu3+ (each 0-25 mm) was added to Krebs-Tris buffer containing the normal concentration of Ca +. IONS AND OVARIAN STEROIDOGENESIS 673

Effects of monovalent ions The effects of altering the concentrations of K+, Na+ and Cl- are shown in Table 3. A twentyfold increase in K+ concentration to 100 mm (solution C) was without effect on basal progestin secretion or the response to LH. Removal of K+ from the medium (solution A), however, reduced the response to the hormone while having little effect on basal steroidogenesis. In a subsequent experiment, ovaries were pre-incubated in normal or K+-free medium for 2 hr, before measuring the response to LH during a 1 hr incubation as before. This was done to produce a greater depletion of intracellular potassium. Under these conditions, the effect of LH was abolished completely in the K+-free medium, and there was also a small decrease in basal progestin secretion.

TABLE 3. Effects of monovalent ions on progestin secretion by rabbit ovaries in vitro. Each result is the mean + s.E. of mean of four replicates. Other details as for Table 2 Progestin secretion (ng/mg tissue. hr) Incubation medium Basal LH-stimulated Krebs-bicarbonate (B) 0-34+ 003 2*40 + 0*20 K+, 100 mM (C) 0-29+ 0021 *s 2-90± 0-16 n s

Krebs-bicarbonate (B) 1*91 _ 0-23 5.31 ± 0*38 P<00 K+-free (A) 2-05+ 021 n.s. 3*90±030 < Krebs-bicarbonate (B)* 0-96 +0*05 ~<00 2 10± 0-33 P < 0 005 K+-free (A)* 0*62 ±0 12P0f05< 0-57+0*05 1<*0 Krebs-bicarbonate (B) 4*40 + 0*24 6-20 +0*28 Choline, 138 mM (E) 450 +0±29 n.s. 6-80+ 0*11 nsn s Krebs-bicarbonate (B) 1-59 + 0*10 2*50+0.20 Li+, 138 mm (F) 1-27+ 0-13 n.s. 1 56 + 0 09 0J01P < Krebs-bicarbonate (B) 3-12+ 026y 690 +0*47n B+Li+, 4mM 3-65+ 021 n.s. 6-17 +0-59J Krebs-bicarbonate (B) 0-49 + 0*06l 1-32± 019n Cl--free (D) 0.50+0*06 n.s. 135+ 0-18 n s. Significance calculated by Student's t test; n.s. not significant (P > 0.05). * Tissue pre-incubated in this medium for 2 hr. When Na+ was removed from the medium and replaced by choline, there was no effect on progestin secretion either in the presence or absence of LH. However, when the Na+ was replaced by Li+ (solution F) the response to LH was inhibited strongly. There was no effect on basal progestin secretion. When a small concentration (4 mM) Li+ was added to buffer containing the normal concentration of Na+, there was no effect on either basal secretion or the response to LH. 24-3 674 674 ~J.B. BED WANI AND P. Y. D. WONG Replacement of Cl- by isethionate and sulphate (solution D) was without effect on either basal or LH-stimulated steroidogenesis. Effects of ouabain and nupercaine The effects of these drugs are shown in Table 4. Guabain (I10-4 M) inhibited the steroidogenic response to LH strongly, but had no significant effect on basal progestin secretion (P > 0-05, Student's t test). The local anaesthetic nupercaine (10-4m) appeared to reduce the response to LII slightly, but this effect was not significant statistically (P > 0-05 < 0-1, Student's t test).

TABLE 4. Effects of ouabain and nupercaine on progestin secretion by rabbit ovaries in vitro. Each result is the mean +S.E. of mean of four replicates. Other details as for Table 2 Progestin secretion (ng/mg tissue. hr)

Incubation medium Basal LH-stimulated Krebs-bicarbonate (B) 0-22 + 0.02 0-93 +0-17k B+ouabain (I10-4M) 0-35±+0-091ns 0*43 0.10jP < 0-05 Krebs-bicarbonate (B) 1-75 ± 0-09 3-32 +0-65 B+nupercaine (10-4m) 1-71±0-22) ns 2-37+0-26 ns Significance calculated by Student's t test; n.s. not significant (P > 0.05). Influence of NADP+ on the effects of ouabain and EU3+ The results of experiments designed to test whether the inhibitory effects of ouabain and EU3+ could be reversed by the addition of NADP+ to the medium are shown in Fig. 1. As reported by others (Dorrington & Kilpatrick, 1966b) NADP+ (2 mm) caused a slight stimulation of steroidogenesis. This response was not affected by either ouabain (10-4 M) or Eu3+ (0-25 mm). A further increase in steroidogenesis occurred when LH was added to the NADP+-rich medium, and under these conditions ouabain had no effect on the response to the hormone. However, the inhibitory effect of EU3+ on the response to LH was still apparent in the medium containing exogenous NADP+. Effects of ouabain and EU3+ on the synthesis of progesterone and 20cc-HP Since our radio-immunological assay measured mainly progesterone, additional experiments were carried out with EU3+ (0-25 mm) and ouabain (10-4 m) respectively, in which progesterone and 20cc-HP were isolated and assayed individually. The extraction and isolation procedures we used gave recoveries of 78-85 % for progesterone and 80-86 % for 20cc-HP (three determinations). IONS AND OVARIAN STEROIDOGENESIS 675 The results are shown in Fig. 2. The values represent the total amount of each steroid detected in the tissue plus the incubation medium, and are not corrected for recovery. Both Eu3+ and ouabain caused a marked inhibition ofthe production of both progesterone and 20a-HP in the presence of LH.

NADP 2 mM LH

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Fig. 1. Effects ofouabain (Oua, 1O-4 M) and Eu3+ (Eu, 0-25 mM) on progestin secretion by rabbit ovaries in the presence of NADP+, 2 mm. Incubations were carried out in normal Krebs solutions (B and J respectively, Table 1) with additions as shown. LH was used in a concentration of 2 jug/ml. Results are the means ± S.E. of mean from four replicates.

DISCUSSION In the experiments reported here we have investigated the importance of inorganic ions for the expression of the steroidogenic response of the rabbit ovary to LH. This response involves the synthesis of two major steroids, progesterone and 20z-hydroxypregn-4-en-3-one (20a-HP). We have quantified the effect of LH by measuring the formation of material binding to an antiserum showing selectivity for progesterone. The antiserum showed only a 7 % cross-reaction with 20a-HP. As we have been measuring mainly progesterone, therefore, it is important to consider whether the effects seen in solutions of altered composition could have 676 J. R. BEDWANI AND P. Y. D. WONG

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Control LH Eu Eu+LH Control LH Oua Oua+LH Fig. 2. Effects of Eu3+ (Eu, 0-25 mM) and ouabain (Oua, 104 M) on the production of progesterone (open blocks) and 20a-hydroxypregn-4-en-3-one (hatched blocks) by rabbit ovaries. Incubations were carried out in normal Krebs solutions (J and B respectively, Table 1) with additions as shown. LH was used in a concentration of 2 /ig/ml. been due to changes in the interconversion of the two steroids, rather than to direct effects on the steroidogenic action of LH. We investigated this possibility by carrying out additional experiments with ouabain and Eu3+, which we consider to be two agents which inhibit the response to LH by different mechanisms (this will be discussed later). In these experiments, we used much larger amounts of tissue, enabling us to isolate progesterone and 20a-HP and to assay them separately. If the reduction in 'progestin' secretion seen with ouabain and Eu3+ was due to an increased conversion of progesterone to 20a-HP, they should have caused a decrease in progesterone production accompanied by an increase in 20ac-HP production. However, we were able to confirm that ouabain and Eu3+ inhibit the formation of both steroids in the presence of LH. It should also be noted that we measured the total production of progesterone and 20a-HP in these experiments, by assaying the tissue in combination with the incubation medium. The effects of ouabain and Eu3+ were similar to those seen in the experiments where we measured 'progestin' in the incubation medium alone. We consider, therefore, that the assay of this material in the medium provides a valid measure of the steroidogenic response of the tissue. In our first experiments, we found that the response of the rabbit ovary IONS AND OVARIAN STEROIDOGENESIS 677 to LH was not impaired by the absence of extracellular Ca2+. This is in contrast to the response of the adrenal cortex to ACTH, where chelation of the extracellular Ca2+ with EDTA blocks the steroidogenic action of the trophic hormone completely (Carchman, Jaanus & Rubin, 1971; Matthews & Saffran, 1973). The situation in the ovary is also different from that in many other secretary tissues, for example the endocrine and exocrine pancreas (Curry, Bennett & Grodsky, 1968; Hales & Milner, 1968; Hokin, 1966), the neurohypophysis (Douglas & Poisner, 1964) and the adrenal medulla (Douglas & iRubin, 1961). In these tissues the secretary cells have a particle storing mechanism, and hormone secretion is dependent upon exocytosis which is inhibited by deprivation of external Ca. In steroidogenic tissues, however, the secretary product does not appear to be stored in any particulate form, but is released from the cell soon after formation (Holzbauer, 1957; Rhodin, 1971). The Ca dependence of adrenal steroidogenesis referred to above seems to be due to a requirement for this cation for the biosynthetic response to ACTH, and not for the release of pre- formed hormones (see Matthews & Saffran, 1973). We found that a tenfold increase in [Ca2+]0 to 25*6 mm inhibited the response of the ovary to LH, without affecting basal steroidogenesis. In this case, the situation was similar to that found in the adrenal cortex, where a tenfold increase in [Ca2+]0 abolished the response to ACTH (Matthews & Saffran, 1973). This effect of high extracellular Ca2+ probably reflects an action on the membrane, rather than a direct effect on the pathway of steroid biosynthesis. There is strong evidence that cyclic 3',5'-AMP is the mediator of the steroidogenic action of LH in the ovary (Savard, Marsh & Rice, 1965; Marsh, Butcher, Savard & Sutherland, 1966). It is possible that high [Ca2+]0 inhibits membrane-bound adenyl cyclase and thus prevents hormone-induced cyclic 3',5'-AMP formation. The slight increase in basal progestin secretion which we observed in the presence of EGTA might be due to removal of adenyl cyclase inhibition by membrane- bound Ca. It has been found that the adenyl cyclase system in many other tissues is sensitive to external Ca (Ramwell & Shaw, 1970). The inhibition of steroidogenesis seen in the presence of La3+ or Eu3+ probably has the same basis. These lanthanide ions resemble Ca2+ closely, and have been shown to inhibit hormone-stimulated adenyl cyclase in another tissue, the toad bladder (Wong, Bedwani & Cuthbert, 1972). The steroidogenic response of the ovary to LH did not appear to require the presence of extracellular Na+, since replacement of Na+ with choline was without effect. In contrast, replacement of Na+ with Li+ reduced the response to LH markedly. This effect of a high concentration of Li+ might have been due to inhibition of adenyl cyclase, as Li+ has been reported to inhibit the stimulation of this enzyme by hormones in other tissues 678 J. R. BEDWANI AND P. Y. D. WONG (Birnbaumer, Pohl & Rodbell, 1969; Dousa & Hechter, 1970; Wolff, Berens & Jones, 1970; Forn & Valdecasas, 1971). However, the inhibition of steroidogenesis seen with Li+ might also have been due to other effects on cellular metabolism. Keynes & Swan (1959) have shown that cells can accumulate Li+ ions, which enter passively but cannot be removed by the Na-pump; [Li+]i therefore rises steadily. Some of the glycolytic enzymes involved in the rapid turnover of ATP and NADPH essential for steroid biosynthesis are sensitive to monovalent cations (Mahler & Cordes, 1966). These enzymes are activated by K+, but are inhibited by Li+ or Na+. Ouabain blocks the Na-pump, thereby causing [Na+]i to increase, and we found that this drug also inhibits the steroidogenic effect of LH. The K+ dependency of these glycolytic enzymes might explain our finding that the response to LH is inhibited under conditions which lead to a decrease in [K+]i. It was of interest that the inhibitory effect of ouabain was reversed completely by the addition of 2 mm NADP+ to the incubation medium. This would also suggest that ouabain inhibits steroidogenesis by decreas- ing the availability of essential pyridine nucleotides. In contrast, the inhibitory effect of Eu3+ could not be reversed by the addition of NADP+. This is consistent with our supposition that lanthanide ions antagonize LH by a different mechanism, namely inhibition of adenyl cyclase.

We are grateful to the National Institutes of Health, Bethesda, U.S.A., for a gift of LH. P.Y.D. Wong is a Research Fellow of Jesus College, Cambridge.

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