Stimulatory Effect Of Luteinizing Hormone Research Articles

Arbutin inhibits androgen biosynthesis by rat immature Leydig cells in vitro

Arbutin, a widely used skin lightening agent, has raised concerns regarding its potential side effects. In this study, we investigated the impact of arbutin on Leydig cell function using an in vitro model. We measured medium androgen levels, as well as the gene and protein expression related to Leydig cell steroidogenesis. Rat immature Leydig cells from age of 35 days were exposed to arbutin at concentrations ranging from 0.5 to 50 μM for a duration of 3 hrs. Following treatment, we observed a significant inhibition of androgen secretion by Leydig cells at both the 5 and 50 μM concentrations of arbutin. Furthermore, at a concentration of 50 μM, arbutin effectively blocked the stimulatory effects of luteinizing hormone (LH) and 8Br-cAMP on androgen secretion. Subsequent analysis revealed that arbutin downregulated the expression of crucial genes involved in androgen production, including Lhcgr, Hsd3b1, Cyp17a1, and Srd5a1. In silico computer program analysis predicted that arbutin exhibits good absorption, possesses a long elimination half-life, and may have other potential toxicity such as hepatoxicity. Taken together, our results demonstrate that arbutin negatively influences Leydig cell function and androgen production, potentially impacting male reproductive health.

Stimulatory effect of luteinizing hormone, insulin-like growth factor-1, and epidermal growth factor on vascular endothelial growth factor production in cultured bubaline luteal cells

The purpose of this study was to evaluate the temporal (24, 48, and 72 hours) and dose-dependent (0, 5, 10, and 100 ng/mL of LH, insulin-like growth factor 1 [IGF-1], and EGF) in vitro expression and secretion patterns of vascular endothelial growth factor (VEGF) in luteal cell culture during different stages of estrous cycle in water buffaloes. Corpus luteum samples from ovaries of early luteal phase (ELP; Days 1–4), midluteal phase (Days 5–10), and late luteal phase (Days 11–16) were collected from a local slaughterhouse. The samples were then processed and cultured in (serum containing) appropriate cell culture medium and incubated separately with three factors (LH, IGF-1, or EGF) at the previously mentioned three dose–duration combinations. At the end of the respective incubation periods, VEGF was assayed in the spent culture medium by ELISA, whereas the cultured cells were used for VEGF mRNA expression by quantitative real-time polymerase chain reaction. The results of the present study disclosed dose- and time-dependent stimulatory effects of LH, IGF-1, and EGF on VEGF production in bubaline luteal cells. The VEGF expression and secretion from the cultured luteal cells were highest during the ELP, intermediate in the midluteal phase, and lowest in the late luteal phase of the estrous cycle for all the three tested factors. Comparison of the results of the three treatments depicted EGF as the most potent stimulating factor followed by IGF-1 and LH. Immunocytochemistry findings in luteal cell culture of ELP agreed with the VEGF expression and secretion. In conclusion, mRNA expression, protein secretion, and immunolocalization of VEGF data clearly indicated for the first time that LH, IGF-1, and EGF play an important role in stimulating luteal angiogenesis in buffalo CL. The highest expression and secretion of VEGF in the ELP might be associated with the development of blood vessels in early growth of CL, which in turn gets augmented by the aforementioned factors emphasizing their regulatory role in luteal angiogenesis. Further studies are however necessary to divulge more information on other factors which regulate VEGF secretion in bubaline CL and the synergistic effects existing among such growth factors.

Stimulatory effect of luteinizing hormone, insulin-like growth factor-1, and epidermal growth factor on progesterone secretion and viability of cultured bubaline luteal cells

We evaluated the temporal (24, 48 and 72 hours) and dose-dependent (5, 10, and 100 ng/mL of LH, IGF-1, and EGF, respectively) production and secretion of progesterone (P4) in cultured luteal cells from different stages of estrous cycle as well as the expression of steroidogenic acute regulatory protein (STARD1), cytochrome P450 cholesterol side-chain cleavage (CYP11A1), and 3β-hydroxysteroid dehydrogenase (HSD3B), anti-apoptotic gene PCNA, and pro-apoptotic gene BAX in luteal cells of mid-luteal phase in buffalo. Samples from early luteal phase (ELP; Day 1 to 4; n = 4), mid-luteal phase (MLP; Day 5 to 10; n = 4), and late luteal phase (LLP; Day 11 to 16; n = 4) of estrous cycle were collected. Progesterone was assayed by RIA, whereas mRNA expression was determined by quantitative real-time polymerase chain reaction. Results depicted that highest dose (100 ng/mL) of LH, IGF-1, and EGF and longer duration of time brought about a (P < 0.05) rise in P4 level and expression of steroidogenic enzymes and PCNA compared with the lower level(s) and control while, all treatments (P < 0.05) inhibited BAX expression in a time dependent-manner. Analysis of interaction between stage and treatments revealed that LH treatment (P < 0.05) increased P4 production compared with IGF-1 and EGF in ELP and MLP. However in LLP, treatment with IGF-1 and EGF significantly (P < 0.05) increased P4 production compared with LH treatment. Summarizing, our study explores the steroidogenic potential of LH and growth factors across different luteal stages in buffalo, which on promoting steroidogenic enzyme expression and cell viability culminated in enhanced P4 production in luteal cells.

Tumor necrosis factor-α inhibits the stimulatory effect of luteinizing hormone and prostaglandin E 2 on progesterone secretion by the bovine corpus luteum

Tumor necrosis factor-α (TNF-α) is involved in the tissue remodeling that occurs in the corpus luteum (CL) during its development and regression. This cytokine is also implicated in the regulation of reproduction by its actions on ovarian steroidogenic cells. The aim of this study was to examine the influence of TNF-α on (1) progesterone (P 4) output by the bovine CL and on (2) the responsiveness of the CL to LH or prostaglandin E 2 (PGE 2) in vitro. In experiment 1, CL (days 8 to 10 of the estrous cycle) were perfused by using an in vitro microdialysis system with TNF-α (0.1, 0.5, or 1 μg/mL) alone or with TNF-α (1 μg/mL) followed by LH (1000 ng/mL) or PGE 2 (2 × 10 −5 M). Basal P 4 release ( P < 0.05) was increased by TNF-α (0.5 or 1 μg/mL). Moreover, TNF-α (1 μg/mL) inhibited the stimulatory effect of LH or PGE 2 on P 4 output ( P < 0.05). In experiment 2, 4 h after intrauterine infusion of TNF-α (0.01 μg/mL or 1 μg/mL), CL (days 8 to 10 of the estrous cycle) were collected by colpotomy, cultured, and stimulated with LH (10 ng/mL) or PGE 2 (10 −6 M). Intrauterine infusion of TNF-α at a concentration of 1 μg/mL increased basal P 4 output by CL ( P < 0.05). Moreover, the intrauterine infusion of TNF-α at a concentration of 0.01 μg/mL inhibited the stimulatory effect of LH or PGE 2 on P 4 output ( P < 0.05). These results indicate that TNF-α (1) does not have an effect on the autonomous, pulsatile release of P 4; (2) increases P 4 secretion by bovine CL with increasing doses, and (3) reduces in a dose-dependent manner the responsiveness of CL to luteotropic factors both directly (after infusion to CL) and indirectly (after intrauterine infusion).

VEGF modulates the effects of gonadotropins in granulosa cells

Follicle selection is associated with an increase in the expression of vascular endothelial growth factor (VEGF) and its receptors in granulosa cells, however, the roles of VEGF in regulating the function of these or other non-endothelial cells in the ovary have not been explored in detail. The current study used bovine cell cultures to investigate potential roles of VEGF in the regulation of granulosa cell function during follicle development. Granulosa cells were obtained from morphologically healthy follicles 4 to 8 mm or 9 to 14 mm in diameter (corresponding to diameters before and after the establishment of dominance, respectively, during a bovine follicular wave) and exposed to a range of VEGF concentrations (1 to 100 ng/mL) encompassing concentrations found naturally in bovine dominant follicles. A concentration of VEGF of 1 ng/mL induced significant proliferation of granulosa cells from 4- to 8-mm follicles ( P = 0.024) and increased the proliferative response of these cells to follicle-stimulating hormone (FSH; P = 0.045); whereas higher doses of VEGF had no effect on proliferation ( P = 0.9). Treatment with VEGF induced an overall increase in mean extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation ( P = 0.02). In contrast, VEGF, alone or in combination with FSH, had no effect on expression of the steroidogenic enzyme, CYP11A1, by cells from 4- to 8-mm follicles ( P = 0.9). Granulosa cells from 9- to 14-mm follicles responded to 1 ng/mL VEGF with an increase in expression of the ovulation-associated gene, PTGS2 ( P = 0.003) but higher VEGF doses had no effect ( P = 0.9). The PTGS2 response to 1 ng/mL VEGF was similar to that induced by treatment with luteinizing hormone (LH). Interestingly, the stimulatory effects of LH on ERK1/2 phosphorylation ( P = 0.003) and PTGS2 expression ( P < 0.01) in granulosa cells from 9- to 14-mm follicles were abolished ( P = 0.2) by specific chemical inhibition of VEGF receptor 2 (VEGFR2). These results suggest novel and important roles of VEGF and its receptor, VEGFR2, in mediating and/or enhancing the effects of gonadotropins in granulosa cells.

Stimulatory effects of LH on release of melatonin and activities of its synthesizing enzymes NAT and HIOMT in organ-cultured pineal glands of female rats.

The pineal hormone melatonin (N-acetyl-5-methoxytryptamine) exerts antigonadotropic effects in some mammalian species. To evaluate the effect of luteinizing hormone (LH) on melatonin release and its synthesizing enzyme activities in pineal glands, pineals of adult female rats undergoing diestrus were organ-cultured in a medium containing 10(-12), 10(-10) or 10(-8) M LH for 6 h. Melatonin release increased significantly in pineals cultured with 10(-12) and 10(-10) M LH, as compared to control values. Similarly, the activity of arylalkylamine N-acetyltransferase (NAT), the key regulatory enzyme in melatonin biosynthesis, was significantly higher in pineals cultured with 10(-12) and 10(-10) M LH for 6 h, while LH at 10(-8) M had no effect. Although LH at 10(-10) M increased pineal hydroxyindole-O-methyltransferase (HIOMT) activity, which catalyzes the final step of melatonin biosynthesis, LH at 10(-12) and 10(-8) M had no effect. These results demonstrate that at relatively low physiological levels, LH stimulates pineal melatonin synthesis and release, mainly by increasing NAT activity.

Stimulation of pregnenolone metabolism and aromatase activity by luteinizing hormone in mouse uterus.

In vitro metabolism of pregnenolone (P5) as well as production of 17beta-estradiol (E2) were studied in uteri of untreated and luteinizing hormone (LH)-treated mice that had been ovariectomized (OVX) at late-diestrus stage. In the uteri of untreated mice, [H]pregnenolone was shown to be metabolized to Delta-components such as 17alpha-hydroxypregnenolone (17alpha-P5) and dehydroepiandrosterone (DHEA), whereas LH treatment resulted in significant increases in the formation of progesterone (P4), 17alpha-hydroxyprogesterone (17alpha-P4), androstenedione (AD) and testosterone (T). This was assessed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The content and release of P4 was shown to be stimulated by LH. Trilostane, an inhibitor of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), inhibited LH-induced P4 synthesis and its release in a dose-dependent manner. A considerable increase in [H]estradiol formation from [H]testosterone was recorded in LH-stimulated uterine tissue as compared with the control, indicating the stimulatory effect of LH on aromatase activity. LH-stimulation in the synthesis of P4 and E2 in OVX mouse uteri was mimicked by dbcAMP (cell-permeable cAMP). Incubation with LH was shown to augment the conversion of P4 to various delta-3-oxosteroids. In vitro effects of LH on the synthesis and metabolism of P4, as well as on the stimulation of aromatase activity, were more pronounced in the uterine tissue of LH-primed OVX mice. Thus the results of the present study indicate that, under specific conditions, the uterus of the mouse behaves like steroidogenic tissue. Its prompt response to LH reveals the probable physiological relevance of the existence of LH receptors of high binding affinity in the uterine tissue of the mouse, as reported earlier.

Functional relevance of luteinizing hormone receptor in mouse uterus.

The presence of high-affinity luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptors has been reported in porcine, rabbit, rat and human uteri. We have demonstrated binding of [125I]LH to mouse uterus, which was saturable. Scatchard plot analysis indicated Kd to be 1.37 x 10(-10) mol/l and the maximum binding capacity to be 5.24 nmol/kg protein. Attempts have been made to observe the functional relevance of gonadotropin receptor in the mouse uterus. The size and weight of the uterus remarkably decreased as a result of ovariectomy; administration of LH to ovariectomized (OVX) mice significantly increased the uterine weight in comparison to the OVX control (p < 0.01), indicating a direct effect of LH on the uterus. There was a two-fold decrease of uterine ascorbic acid content in LH-treated OVX mice as compared to the intact control. The gain in uterine weight of OVX mice by LH was due to the increase in uterine protein synthesis. The stimulatory effect of LH on OVX mice uterus appears to be mediated via steroid hormones because it significantly augmented uterine mitochondrial steroidogenesis. Since 17 beta-estradiol (E2) is known to stimulate uterine protein synthesis, the circulatory level of E2 was determined in intact, OVX and OVX + LH-treated mice. A fall in the circulatory level of E2 occurred in OVX mice as compared to the control, while treatment of LH for 7 days (three injections) significantly elevated E2 levels in OVX mice (p < 0.001). This higher level of E2 in OVX mice remains unaltered on adrenalectomy, indicating that adrenals are not the source for increased E2 levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of oxygen tension and substrates on basal and luteinizing hormone stimulated progesterone production and energy metabolism by isolated corpora lutea of adult pseudopregnant rats.

The effect of substrate reduction on in vitro progesterone production by isolated corpora lutea of pseudopregnant rats was studied. Pseudopregnancy was induced by mating adult females with vasectomized males. The rats were killed at different luteal ages and the corpora lutea of pseudopregnancy were identified, dissected free and incubated for 3 h. Six-day-old corpora lutea were incubated in the presence of different oxygen tensions as well as different glucose and amino acid concentrations and basal as well as luteinizing hormone (LH)-stimulated progesterone production was studied. When the oxygen tension was reduced, both basal and LH-stimulated progesterone production was decreased. At 6 and 0 kPa, the stimulatory effect of LH on progesterone production was abolished. Lowering the oxygen tension decreased the levels of adenosine-5'-triphosphate (ATP) in the tissue and increased lactic acid accumulation in the medium. Omitting both glucose and amino acids while maintaining oxygen tension at 100 kPa abolished the stimulatory effect of LH on progesterone production and decreased the levels of ATP. The present study shows that the corpus luteum in vitro is dependent on a high oxygen tension for maintenance of its progesterone production and LH responsiveness. It is suggested that the amount of ATP available is critical to the degree of LH response. Hypoxia increased luteal glycolysis, but this could only marginally compensate for a low oxygen tension.

The influence of prostaglandin E2 and indomethacin on progesterone production and ovulation in the rabbit ovary perfused in vitro.

The effects of prostaglandin E2 (PGE2) on the ovulation process were studied in a recirculating perfusion model using ovaries from virgin rabbits. Ovulation frequency, time of ovulation, and progesterone release from the ovaries were examined after the addition of PGE2, either alone or with luteinizing hormone (LH) in the presence or absence of indomethacin. The stimulatory effect of LH on ovulation was totally blocked if the ovaries were exposed to indomethacin at the same time. Ovaries treated with PGE2 alone did not ovulate, and PGE2 was unable to restore indomethacin-blocked ovulation. Conversely, the frequency of ovulation was reduced in ovaries treated with PGE2 and LH compared with controls receiving only LH. There was no measurable difference in the pattern of progesterone release between ovaries simultaneously treated with LH and indomethacin and LH-treated controls. Ovaries exposed to PGE2 alone showed only a slight increase of progesterone release in the medium, while those treated with PGE2 in combination with LH in the perfusate showed a smaller progesterone release than those treated with LH alone. The results confirm the blocking effect on ovulation by indomethacin. PGE2 could not reverse this effect, but instead reduced the number of LH-induced follicular ruptures. Indomethacin had no effect on progesterone levels, while PGE2 (which alone showed a slight stimulating effect on the steroid concentration) together with LH counteracted the effect of LH on progesterone release.

Luteinizing hormone stimulates the formation of inositol trisphosphate and cyclic AMP in rat granulosa cells. Evidence for phospholipase C generated second messengers in the action of luteinizing hormone.

The following studies were conducted to determine whether luteinizing hormone (LH), a hormone which increases cellular levels of cyclic AMP, also provokes increases in 'second messengers' derived from inositol lipid metabolism (i.e. inositol phosphates and diacylglycerol). Rat granulosa cells isolated from mature Graafian follicles were prelabelled for 3 h with myo-[2-3H]inositol. LH provoked rapid (5 min) and sustained (up to 60 min) increases in the levels of inositol mono-, bis, and trisphosphates (IP, IP2 and IP3, respectively). Time course studies revealed that IP3 was formed more rapidly than IP2 and IP following LH treatment. The response to LH was concentration-dependent with maximal increases at LH concentrations of 1 microgram/ml. LiCl (2-40 mM) enhanced the LH-provoked accumulation of all [3H]inositol phosphates, presumably by inhibiting the action of inositol phosphate phosphatases. The effectiveness of LH, however, was dependent on the concentration of lithium employed; maximal increases in IP were observed at 10 mM-LiCl, whereas maximal increases in IP2 and IP3 were observed at 20 mM- and 40 mM-LiCl, respectively. The stimulatory effects of LH on inositol phosphate and progesterone accumulation were also compared with changes in cyclic nucleotide levels. LH rapidly increased levels of inositol phosphates, progesterone and cyclic AMP, but transiently reduced levels of cyclic GMP. These results demonstrate that LH increases both cyclic AMP and inositol trisphosphate (and presumably diacylglycerol) in rat granulosa cells. Our findings suggest that two messenger systems exist to mediate the action of LH in granulosa cells.

Adenosine potentiates the effects of LH and isoproterenol on luteal cells but not on isolated intact corpora lutea.

Adenosine potentiated the stimulatory effect of luteinizing hormone (LH) in a dose-dependent way on the production of cyclic AMP (cAMP) in isolated cells from heavily luteinized rat ovaries and from individual rat corpora lutea of various ages (2- and 5-day-old). Such an effect has earlier been reported only for cells from heavily luteinized ovaries (Behrman et al. 1983). A similar potentiating effect of adenosine was now seen on catecholamine-stimulated cAMP production in isolated cells from 2-day-old corpora lutea. Adenosine did not, however, potentiate LH- or catecholamine-stimulated cAMP production in isolated intact corpora lutea.

Effect of Lipoproteins, 25-Hydroxycholesterol and Luteinizing Hormone on In Vitro Follicular Steroidogenesis in the Hamster and Rat

In 4-h incubations with the medium changed every hour, proestrous Graafian follicles of the rat secreted greater amounts of progesterone (P4), androstenedione (delta) and estradiol (E2) than the hamster follicle (at H 1: 3.5, 3.6, 2.5 times, respectively). Follicles isolated from both species responded to 10-100 micrograms of human high-density lipoprotein (HDL) by enhanced P4 production, whereas these doses of HDL augmented delta and E2 secretion only in the hamster. One mg of human low-density lipoprotein (LDL) was as potent as 100 micrograms of HDL in stimulating P4 secretion in the hamster, but was unable to increase steroid synthesis in the rat. One to 50 micrograms of 25-hydroxy-cholesterol (25-OH) enhanced P4, delta and E2 secretion in a dose-dependent manner in the hamster follicle, but was without effect in the rat. In the hamster, 10-100 ng of luteinizing hormone (LH) increased steroid secretion in a dose-related fashion, while the rat follicle only responded to 100 ng of LH and the hamster follicle was much more responsive than the rat follicle. The responsiveness of the hamster follicle to 50 micrograms of 25-OH was less than to 100 ng LH (P4 production after LH stimulation was 12-fold greater than that after 25-OH stimulation). There was no additive effect of LH and HDL on follicular steroidogenesis in either species. A pharmacological dose of LDL (1000 micrograms) negated the stimulatory effect of LH on follicular steroidogenesis in both species, especially P4.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulatory effect of luteinizing hormone upon relaxin secretion by cultured porcine preovulatory granulosa cells.

Relaxin, a peptide hormone capable of causing connective tissue alterations, is produced by the corpus luteum and traditionally considered a hormone of pregnancy. We have cultured granulosa cells from preovulatory porcine follicles and have found that these non-pregnancy associated cells secrete relaxin, and that luteinizing hormone, which stimulates ovulation, enhances relaxin secretion by cells from large preovulatory follicles. These results suggest that relaxin secreted prior to ovulation may have a local ovarian effect, perhaps facilitating ovulation.

Stimulatory effect of luteinizing hormone and human chorionic gonadotropin on testicular prolactin receptor levels.

A possible effect of LII or hCG on testicular prolactin receptor levels was investigated in hypophysectomized adult rats. Hypophyseetomy leads to a progressive decrease of testicular prolactin receptor content to ‘\�25% of that of controls 12 days after surgery. In animals injected3 times a day for 10 days with highly purifiedpreparations of ovine LII or hCG, prolactin receptor levelsincreasedfrom 21 4 to 41 ± 4 and 71 7 fmol/testis, respectively (P<0.01 for both). The increase in the number of testicular prolactin receptor levels observed after treatment with hCG in hypophysectomized rats could not be duplicated by injection of estradiol-173 (0.5 pg), testosterone (200 pg) or 5u-dihydrotestosterone (200 pg) given twice a day for 5 days. Moreover, these steroids do not interfere with the stimulation of prolactin binding induced by hCG. Since treatment with prolactin is well known to stimulate testicular LII binding and steroidogenesis, the present data provide further evidence for a close interaction between prolactin and LII in the control of the testicular levels of receptors for these two hormones and their subsequent role in the control of testicular functions.

Reptilian ovarian steroidogenesis and the influence of mammalian gonadotrophins (follicle-stimulating hormone and luteinizing hormone) in vitro.

SUMMARY The synthesis of steroids from [7α-3H]cholesterol, [7α-3H]pregnenolone and [7α-3H]progesterone by lizard and turtle ovarian tissues in vitro was studied. Progesterone, 17α-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone, oestrone and oestradiol were identified as products. In the turtle (Pseudemys), conversion of pregnenolone to progesterone was efficient, but transformation of progesterone to other steroids was relatively slow as indicated by the accumulation of progesterone over the incubation period. In Dipsosaurus, accumulation of radioactivity was greatest in testosterone, the quantities of which continued to increase at each sampling period. The rate of utilization of pregnenolone as a substrate was similar for the two species studied and the quantities of oestrone and oestradiol formed were lower in Pseudemys. The use of progesterone as precursor by Dipsosaurus ovarian tissue revealed a similar pattern of Δ4-steroid metabolism to that obtained with pregnenolone as precursor. The effects of addition of purified follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on the metabolism of [14C]cholesterol in vitro was studied using Pseudemys follicular tissue. The pattern of cholesterol metabolism was similar to that for pregnenolone in this species. The synthesis of pregnenolone, progesterone, dehydroepiandrosterone and androstenedione in vitro was significantly enhanced in the presence of LH. Follicle-stimulating hormone had no effect on steroid synthesis except for a decrease of androstenedione formation. The stimulatory effect of LH on steroidogenesis in vitro is discussed in relation to the literature suggesting that mammalian FSH, but not LH, stimulates all phases of reptilian ovarian function when injected in vivo.

Changes in ovarian venous blood flow following cannulation; effects of luteinizing hormone (LH) and antihistamine.

SummaryThe present results indicate that cannulation of the ovarian vein and subsequent hemorrhage results in rapid decline of blood flow from the cannula. This decline is significantly retarded by the administration of LH. The stimulatory effect of LH is apparently histamine-mediated, since simultaneous administration of promethazine hydrochloride completely blocks the LH-induced increase in blood flow.

The Stimulatory Effect of Luteinizing Hormone on Adenyl Cyclase in the Bovine Corpus Luteum

Abstract Adenyl cyclase and cyclic 3',5'-nucleotide phosphodiesterase were shown in homogenates of bovine corpora lutea. In order to determine the site of action of luteinizing hormone, the adenyl cyclase activity was assessed in the absence of phosphodiesterase activity. This was accomplished by using a high concentration of theophylline and short incubation periods. Under these conditions, luteinizing hormone significantly increased the adenyl cyclase activity. NaF produced an even more marked stimulation of this enzyme. Epinephrine at a concentration of 0.2 mm produced a small but statistically significant stimulation of adenyl cyclase, but preparations of luteinizing hormone inactivated by hydrogen peroxide, bovine serum albumin, prolactin, adrenocorticotropin, and glucagon were inert in homogenates that responded to luteinizing hormone and NaF. The minimum effective dose of luteinizing hormone was 0.1 µg per ml. Luteinizing hormone had no effect on the phosphodiesterase activity when assayed under a variety of conditions, but NaF produced a slight but significant inhibition of this enzyme activity when the substrate cyclic AMP was added at a saturating level. These data indicate that the increase in endogenous cyclic AMP brought about by luteinizing hormone is due to a stimulation of the adenyl cyclase rather than to an inhibition of the phosphodiesterase.

The Stimulatory Effect of Luteinizing Hormone on Adenosine 3‘,5‘-Monophosphate Accumulation in Corpus Luteum Slices

The presence of adenosine 3‘,5‘-monophosphate was demonstrated in bovine luteal tissue. The addition of luteinizing hormone to incubating slices of a bovine corpus luteum caused a rapid accumulation of this cyclic nucleotide which preceded the increase in progesterone synthesis. This effect appeared to be specific for luteinizing hormone. Puromycin did not inhibit this action of luteinizing hormone on cyclic AMP accumulation. In view of the previous finding that exogenous cyclic AMP added to incubating slices can mimic the effect of luteinizing hormone on steroidogenesis, these results indicate that the cyclic nucleotide is a mediator of this action of luteinizing hormone. An increase in the concentration of cyclic AMP has also been demonstrated in a human corpus luteum stimulated in vitro by human chorionic gonadotropin.