Tokishakuyakusan directly attenuates PACAP’s luteolytic action on luteal function in the rat ovary

Tokishakuyakusan directly attenuates PACAP’s luteolytic action on luteal function in the rat ovary

Satoshi Usuki

Abstract: We investigated the potential direct effects of Tokishakuyakusan (TS) on progestin [progesterone and 20[alpha]-hydroxyprogesterone (20[alpha]-OH-P)] and cyclic adenosine-3′,5′-monophosphate (cAMP) production in cultured rat luteal cells. In addition, we examined whether TS regulates the inhibitory effects of pituitary adenylate cyclase-activating polypeptide (PACAP), a newly found peptide, on luteinizing hormone (LH)-stimulated progesterone production. TS significantly stimulated progesterone, but not 20[alpha]-OH-P, production and cAMP accumulation through 24 hours of culture. PACAP-38 significantly elevated progesterone, 20[alpha]-OH-P and cAMP levels at all concentrations studied. On the other hand, PACAP-38 inhibited the production of progesterone and the accumulation of cAMP enhanced by LH, while the ratio of progesterone to 20[alpha]-OH-P was significantly decreased by PACAP-38 + LH. Concomitant treatment with TS and PACAP-38 + LH increased the ratio of progesterone to 20[alpha]-OH-P more than with PACAP-38 + LH. The present data have demonstrated that TS stimulates progesterone production in rat luteal cells, reconfirming our previous evidence that TS stimulates luteal steroidogenesis. The data further suggest that TS tends to attenuate PACAP’s inhibition of LH-stimulated progesterone production, suggesting a luteotrophic effect within the corpus luteum.

Keywords: Tokishakuyakusan; PACAP; Corpus Luteum; Luteotrophic Action; Steroidogenesis; LH; Rat.


It has been clarified that Tokishakuyakusan (TS) stimulates the corpus luteum to secrete progesterone in vivo and in vitro (Usuki, 1987 and 199 la). However, the detailed luteotrophic or luteolytic effect of TS is not yet been fully investigated. Very recently, we have demonstrated that both pituitary adenylate cyclase-activating polypeptide (PACAP)-38 and its type IA receptor are expressed within the rat corpus luteum and stimulates progestin secretion in the corpus luteum (Kotani et al., 1997 and 1999). PACAP is a novel neuropeptide belonging to the secretin/glucagon/vasoactive intestinal polypeptide (VIP) family of peptides (Arimura, 1992). It was first isolated from ovine hypothalamic tissue on the basis of its ability to stimulate adenylate cyclase in cultured rat pituitary cells (Miyata et al., 1989). There are two forms of PACAP with comparable biological activity, the longer PACAP-38 and the shorter PACAP-27 (Miyata et al., 1990). Both forms are derived from a single amino acid precursor of 176 residues (Kimura et al., 1990). Since its isolation, many studies have described PACAP activity in a variety of tissues (Rawlings and Hezareh, 1996). In a recent report, PACAP was found to be more potent than VIP in its ability to stimulate steroidogenesis and cyclic adenosine-3′,5′-monophophate (cAMP) accumulation in cultured rat granulosa cells (Kotani et al., 1998) and luteal cells (Usuki and Kotani, 2001). At present, however, the mutual effect of TS and PACAP on luteal cell function is still far from clear.

In order to learn more about TS’s role in luteal cell function, the present study was carried out to investigate whether TS stimulates progesterone production in cultured luteal cells from the ovaries of immature rats treated with pregnant mare’s serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). We further examined whether TS regulates PACAP-38′ role in progestin [progesterone and 20[alpha]-hydroxyprogesterone (20[alpha]-OH-P)] production and the accumulation of cAMP in both the basal and the LH-stimulated state.

Materials and Methods

Reagents and Hormones

Ovine LH (oLH) was kindly provided by the National Institute of Health, MD, USA. This compound (NIH-LH) is also known as NIADDK-oLH-24 or AFP-0754. NIH-LH has an LH potency 2.3 times greater than NIH-LH-SI. By weight, the potency of NIH-LH is less than 0.5% for follicle stimulating hormone, 0.1% for growth hormone, 0.1% for prolactin, and less than 0.5% for thyroid stimulating hormone. TS (pulverized extracts; the detailed constituents are described somewhere) was kindly donated by Tsumura Co., Ltd. (Tokyo, Japan). PACAP-38 was purchased from the Peptide Institute (Osaka, Japan) while PMSG and hCG were purchased from TeikokuZoki Co. (Tokyo, Japan). Modified TC-medium 199 was prepared with Medium 199 (1.0% glucose, Gibco Life Technologies Inc., NY, USA) supplemented with 0.1% BSA (Sigma Chemical Co., MO, USA), 0.22% NaHC[O.sub.3], 100 mg/ml streptomycin and 100 IU/ml penicillin (both available from Meijiseika Co., Tokyo, Japan). Collagenase was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and DNase type I was purchased from Sigma Chemical Co.

Animals and Treatments

Immature female Wistar-Imamichi rats (Imamichi Institute for Animal Reproduction, Ibaraki, Japan) were maintained in air-conditioned quarters with a 12-hour light cycle. At 23 days of age, the animals were given a subcutaneous injection of 20 IU PMSG. Forty-eight hours later, the animals were given an intraperitoneal injection of 40 IU hCG to induce superovulation. After one week, the animals were anesthetized with ether and decapitated. The ovaries were removed.

Harvesting and Incubation of Luteal Cells

The corpora lutea were gently teased apart with small scissors and fine forceps. The tissue was then placed in synthetic medium 199 containing 2 mg/ml collagenase and 0.5 mg/ml DNase type I. To facilitate the disaggregation of cells, the solution containing the tissue was placed in a 37[degrees]C water bath with constant shaking for 90 minutes. The dispersed cell suspension was transferred to several 15 ml conical centrifuge tubes and centrifuged at 200 x g for five minutes at room temperature. The cells were washed three times with modified TC-medium 199, resuspended in the same solution, and counted with a hemacytometer. Viability was assessed by observing the exclusion of Trypan blue dye. Luteal cells were placed in 24-well tissue culture dishes (Beckton Dickenson, CA, USA) in modified TC-medium 199 at a final concentration of 1 x [10.sup.6] viable cells/mi. One group of cells was treated with 10, 100 and 1000 ng/ml TS with or without 10, 100 and 1000 ng/ml PACAP-38 and 100 ng/ml LH. Another group of cells was not treated with TS, PACAP-38 or LH. The cells were then placed in a 37[degrees]C incubator with a humidified gas mixture of 5% C[O.sub.2] and 95% room air for 24 hours.

Measurement of Progestin and cAMP Levels

At the end of the 24-hour incubation period, the progestin content and the level of cAMP were measured in all culture wells. For the measurement of progestins, samples were collected, frozen, and stored at-20[degrees]C until they could be assayed. Progesterone was measured in duplicate samples using an enzyme immunoassay (EIA) kit (Cayman Chemical Co., MI, USA). The minimum detectable amount of the steroid assay was equal to 10.2 pg/ml. The level of 20[alpha]-OH-P was measured using a competitive EIA in duplicate samples. Horseradish-peroxidase (HRP)-labeled 20[alpha]-OH-P, specific antiserum to 20[alpha]-OH-P, and either a known standard or an unknown sample were added to microtest plates coated with anti-rabbit IgG and incubated at room temperature for 4 hours. After a saline wash, the bound enzyme activity was measured using o-phenylenediamine as a chromogen. After a distinct yellow color was observed, 6 N sulfuric acid was added to each well to halt the reaction. The optical density at 492 nm was determined in a plate reader. The cross-reactivity of the antiserum used in the 20[alpha]-OH-P assay was 100% for 20[alpha]-OH-P, 5% for 20[beta]-OH-progesterone, 3.6% for 17[alpha], 20[alpha] [(OH).sup.2]-progesterone, 1.2% for progesterone, 0.18% for pregnanediol, 0.15% for pregnenolone, 0.07% for 17[alpha]-OH-progesterone, 0.05% for 17[alpha]-OH-pregnenolone, 0.4% for C18 steroids and 0.02% for 17[beta]-estradiol. The minimum detectable amount was equal to 0.1 ng/ml.

For the measurement of extracellular cAMP, the culture media were boiled for 10 minutes at the end of the incubation period in order to inactivate phosphodiesterases before the samples were frozen and stored at -20[degrees]C. The levels of cAMP were analyzed using an EIA kit in duplicate samples according to the guidelines provided by the manufacturer (Amersham, Bucks., UK). The minimum detectable amount was equal to 0.12 pmol/ml. Intraassay and interassay coefficients of variation were less than 10%.

Data Analysis

The data are presented as the means [+ or -] SEM for four samples from each measurement group: progesterone, 20[alpha]-OH-P and cAMP. Data were analyzed by Student’s t-test. When p < 0.05, the differences were considered significant.


In initial studies, luteal cells were cultured for 24 hours with and without TS (10, 100 and 1000 ng/ml); 100 and 1000 ng/ml TS significantly increased progesterone production [p < 0.05 and p < 0.05, respectively (Table 1)], while 20[alpha]-OH-P production was not significantly altered (data not shown). The cAMP accumulation was also significantly stimulated with 100 and 1000 ng/ml TS [p < 0.05 and p < 0.05, respectively (Table 2)].

In second studies, luteal cells were cultured for 24 hours with and without LH (100 ng/ ml) and/or PACAP-38 in concentrations of 10, 100 and 1000 ng/ml. As shown in Figs. 1 and 2, PACAP-38 significantly stimulated basal production of both progesterone and 20[alpha]-OH-P in a dose-dependent manner (p < 0.05, p < 0.01 and p < 0.01 for progesterone, and p < 0.05, p < 0.05 and p < 0.01 for 20[alpha]-OH-P, respectively). LH significantly enhanced basal progesterone and 20[alpha]-OHP production [p < 0.01 and p < 0.05, respectively (Figs. 3A and 3B)']. In concentrations of 100 and 1000 ng/ml, PACAP-38 significantly inhibited LH-stimulated progesterone production in a dose-dependent manner [p < 0.05 and p < 0.05, respectively (Fig. 3A)], while 1000 ng/ml PACAP-38 significantly increased LH-stimulated 20[alpha]-OH-P production (p < 0.05; Fig. 3B).


As shown in Fig. 4, 1000 ng/ml PACAP-38 significantly inhibited the ratio of progesterone to 20[alpha]-OH-P (p < 0.01), while the ratio was unaltered with 10, 100 and 1000 ng/ml TS and 10, 100 and 1000 ng/ml PACAP-38 + 100 ng/ml LH.


The effect of TS on cAMP accumulation in luteal cells cultured for 24 hours was also examined. TS significantly stimulated basal cAMP accumulation at concentrations of 100 and 1000 ng/ml TS [p < 0.05 and p < 0.05, respectively (Table 2)]. PACAP-38 significantly stimulated basal cAMP accumulation in a dose-dependent manner at concentrations of 10, 100 and 1000 ng/ml [p < 0.05, p < 0.01 and p < 0.01, respectively (Fig. 5)]. LH significantly enhanced basal cAMP accumulation [p < 0.001 (Fig. 3C)]. In contrast, PACAP-38 significantly inhibited LH-stimulated cAMP accumulation in a dose-dependent manner at all concentrations studied [10, 100 and 1000 ng/ml; p < 0.05, p < 0.05 and p < 0.01, respectively (Fig. 3C)]. cAMP accumulation with 1000 ng/ml PACAP-38 + 100 ng/ml LH and 1000 ng/ml TS + 1000 ng/ml PACAP-38 + 100 ng/ml LH were 1.75 [+ or -] 0.06 and 2.02 [+ or -] 0.05 pmol/ml, means [+ or -] SEM, n = 4, respectively. TS supressed PACAP-38's inhibition of LH-stimulated cAMP accumulation, significantly (p < 0.05).



The present data indicate that TS stimulates progesterone production by rat luteal cells, cultured in vitro, of immature rat ovaries primed with PMSG-hCG. Although the actions of TS on the reproductive function have been revealed, the mechanism by which TS acts in the ovary is not well understood. Recently, TS has been shown to exert a direct effect on the ovary to regulate ovarian function (Usuki, 1987 and 1991a).

Since the major secretory product of luteal cells is progesterone (a C21 steroid), we examined whether PACAP-38 regulated the production of progesterone in these cells. Our results indicate that treatment of cultured luteal cells with PACAP-38 produces dose-dependent increases in the production of progesterone. This indicates that, in luteal cells, PACAP-38 may directly enhance progesterone synthesis. Hsueh et al. (1984) demonstrated that LH increases progesterone secretion in luteal cells. In order to examine the effects of PACAP-38 on LH-stimulated progesterone production, luteal cells were cultured in media containing LH (100 ng/ml) with or without PACAP-38 ([PACAP] = 0, 10, 100 and 1000 ng/ml). LH alone significantly stimulated the production of progesterone following 24 hours in culture. PACAP-38 significantly inhibited the LH-stimulated production of progesterone. This suggests that PACAP-38 may inhibit the luteotropic action of LH in luteal cells. PACAP-38 is luteotropic in a manner that is similar to the action of LH. PACAP-38’s inhibition of the LH-stimulated production of progesterone may be related to the initiation of luteolysis. Since PACAP-38 treatment alone stimulated progesterone production, it is unlikely that PACAP-38 simply inhibits steroidogenesis. The effect of PACAP treatment may be attributed to a decrease in LH receptors, attenuation of LH receptor binding, and/or modulation of postreceptor signal transduction systems. In this study, TS increased PACAP-38 -decreased production of LH-stimulated cAMP. It is unclear at present which mechanisms work within the luteal cells.

TS also stimulated intraovarian cAMP by cultured luteal cells as well as by preovulatory follicles or corpora lutea (Usuki, 1991a and b). Peripheral and local hormones that regulate ovarian steroidogenesis employ a number of intracellular signaling mechanisms including the adenylate cyclase-cAMP pathway (Leung and Steele, 1992). We were interested to know how TS would effect the levels of extracellular cAMP in cultured luteal cells. TS alone stimulated basal cAMP accumulation. We found that PACAP-38 also stimulated basal cAMP accumulation in cultured luteal cells. The effect of PACAP-38 to stimulate cAMP production was dose dependent within almost the same concentration’s range that caused PACAP-38 to influence the production of progesterone. Marsh (1975) demonstrated that CAMP and dibutyryl cAMP, a membrane-permeable cAMP analogue, induce luteal cells to synthesize progesterone. Our data suggest that direct augmentation of progesterone production by TS and PACAP-38 may also be associated with an enhancement of cAMP production. Marsh (1975) reported that the action of LH on gonadal steroid production is mediated through the second messenger cAMP and that cAMP accumulation may reflect enhanced adenylate cyclase activity. Although PACAP-38 also stimulates adenylate cyclase activity (Miyata et al., 1989), in our study PACAP-38 inhibited LH-stimulated cAMP accumulation in luteal cells. The role of the phospholipase C (PLC) pathway in the regulation of luteal cell function is well documented (Leung and Steele, 1992). Very recently, we reported that PACAP-38 is closely involved in extracellular signal-regulated kinase 1 and 2 (ERK 1 and ERK 2) belonging to the mitogen-activated protein kinases (MAPKs)(Kotani et al., 1999). Our unpublished data suggest that PACAP may work within the ovary as shown in Fig. 6. Therefore, PACAP’s inhibition of LH-stimulated production of cAMP may be mediated through the PLC pathway. The finding in this study that TS suppresses PACAP’s inhibition of LH-stimulated cAMP accumulation may also be involved in similar complicated pathways. Further studies will be necessary to determine the mechanisms of intracellular signaling pathways by which TS works in luteal cells. Although it has been demonstrated that TS enhances LH action on ovarian progesterone secretion (Usuki, 1986), no definite information about the interrelationship as to ovarian steroidogenesis between TS and PACAP-38 + LH is available at present. Although the detailed mechanism underlying this enhanced action is still unclear, this evidence may provide an intriguing key to understanding the direct action of TS on luteal function. Further studies are necessary. However, it may be defined that TS acts on the luteal cells partially through the mediation of cAMP. This also suggests that the direct effect of TS may be mediated in part by changes in cAMP.


In summary, our results suggest that, in rat luteal cells, TS directly stimulates cell function and suppresses PACAP’s luteolytic action which inhibits the effects of LH. Furthermore, our results demonstrate that TS may be an important ovarian regulator with special significance for luteal cells as a luteotrophic agent. Our results indicate that TS may be involved in luteal cell function via the intracellular signal mechanisms.

Table 1. Effect of Treatment with Tokishakuyakusan

on Progesterone Levels by Luteal Cells

Tokishakuyakusan Progesterone

(ng/ml) (ng/ml)

0 0.34 [+ or -] 0.02

10 0.36 [+ or -] 0.06

100 1.87 [+ or -] 0.13 *

1000 1.91 [+ or -] 0.12 *

Luteal cells were cultured for 24 hours with TS, as described

in the text. At the end of the culture, medium progesterone

was measured by EIA. Results are means [+ or -] SEM, n = 4

independent experiments. A Student’s t-test indicated that

these results are significant compared with the TS-untreated

control group (* p < 0.05).

Table 2. Effect of Treatment with Tokishakuyakusan on

Extracellular cAMP Levels in Cultured Rat Luteal Cells

Tokishakuyakusan Extracellular cAMP

(ng/ml) (pmol/ml)

0 1.32 [+ or -] 0.11

10 1.36 [+ or -] 0.16

100 1.87 [+ or -] 0.13 *

1000 1.91 [+ or -] 0.12 *

Values are means [+ or -] SEM for four duplicate samples.

A Student’s t-test indicated that these results are

significant compared with the hormone-untreated control

group (* p < 0.05).


The authors wish to thank the Hormone Distribution Office, NIADDK, MD, USA for providing us with oLH (NIH-LH-24). This work was supported by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, Sports and Culture (C-05671351, C-07671900, C-8671865 and C-11671596).


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Satoshi Usuki

Department of Obstetrics and Gynecology, Institute of Clinical Medicine School of Medicing, Cluster of Medical Sciences, University of Tsukuba Tennodai 1-1-1, Tsukuba, Ibaraki 305-8575, Japan

Eri Kotani

Department of Obstetrics and Gynecology, Hasakisaisei Hospital Hasaki 8968, Kashima, Ibaraki 314-0412, Japan

Correspondence to: Prof. Satoshi Usuki, Department of Obstetrics and Gynecology, Institute of Clinical Medicine, School of Medicine, Cluster of Medical Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba Science City, Ibaraki 305-8575, Japan. Tel: (+81) 0298-51-5850 and (+81) 0298-53-3179, Fax: (+81) 0298-51-5850 or 3622, E-mail:

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