A Chinese Medicine, Tokishakuyakusan, Increases Bovine Oviductal Tonus via G Protein-Coupled Estrogen Receptor 1

Abstract

Early embryo and sperm transport through the oviductal isthmus depends on the contraction and relaxation of the smooth muscle layers. Dysfunction of the oviduct transport is considered to be one of the causes of infertility. For human infertility, Chinese medicine is used in East Asia. Although there are many clinical reports regarding Tokishakuyakusan (TSS), there is little scientific evidence that it affects infertility. In this study, we investigated the effect of TSS on bovine oviductal contraction and relaxation via the G protein-coupled estrogen receptor 1 (GPER1). We collected bovine oviductal isthmic tissues at four stages of the estrous cycle, classified based on a macroscopic observation of the ovary. The Magnus method was used to monitor longitudinal contractility (frequency, contraction force, and tonus). The effects of TSS solution, GPER1 agonist (G-1), and antagonist (G-15) on oviductal contractility were examined. The protein expression level of GPER1 in the oviductal isthmic smooth muscle of each estrous stage was assessed by Western blotting. Although TSS did not affect frequency and contraction force, the tonus was significantly increased by TSS or G-1 at all stages (p < 0.05), and the effect was especially highest at days 1–4 after ovulation. The addition of G-15 significantly suppressed the TSS-induced increase of oviductal tonus at all stages (p < 0.05). There was no significant difference in GPER1 protein expression among the estrous stages. TSS affects oviductal contractility by increasing tonus via GPER1, and it may accelerate gamete and early embryo transport by contracting the oviducts longitudinally.

INTRODUCTION

According to WHO, human infertility is defined as the failure to achieve a pregnancy after 12 months or more of regular unprotected sexual intercourse.¹⁾ It is estimated that approximately 48 million couples and 186 million individuals live with infertility globally.^2–4) Therefore, the cause of infertility needs to be elucidated to establish treatment methods.

Since the transport of gametes and early embryos in the oviduct at an appropriate time is important for the establishment of pregnancy, dysfunction of the oviduct is considered one of the causes of infertility.⁵⁾ Early embryo and sperm transport through the oviductal isthmus depends on contraction and relaxation by the smooth muscle layers.^6,7) For example, inhibition of contraction in the oviductal isthmus of mice and rats has been reported to induce the failure of sperm and egg transport.^8,9) Approximately 98% of human ectopic pregnancies occur in the oviduct, and one of the causes is abnormal embryonic transport in the oviduct.¹⁰⁾ However, a method for improving oviductal contractility has not yet been established.

Oviductal contraction and relaxation are controlled by several factors. Prostaglandin (PG) F2α and endothelin increase the contraction of oviductal smooth muscle, and PGE2 and nitric oxide promote its relaxation.¹¹⁾ Estradiol (E2) and progesterone (P4) are known to increase the secretion of PGF2α and endothelin from oviductal epithelial cells, which induce oviductal contraction.^12,13)

Chinese medicine is used as an infertility treatment in East Asia.¹⁴⁾ Tokishakuyakusan (TSS), which is composed of Japanese angelica root, peony root, cnidium rhizome, Atractylodes lancea rhizome, Poria sclerotium, and alisma tuber, is often prescribed for various gynecological symptoms, such as irregular menstruation.^15,16) Recent studies indicate that TSS has E2 like effect,^17,18) suggesting that TSS is involved in oviduct contraction. E2 acts on nuclear estrogen receptor (ER) α and ERβ which elicit genomic effects, and the membranous E2 receptor G protein-coupled estrogen receptor 1 (GPER1) which mediates acute responses non-genomically.^19,20) Since it is revealed that TSS does not act via ERα and ERβ,^17,18) TSS possibly acts via GPER1.

In general, using human tissues for research has some ethical issues and therefore it is difficult to obtain samples. In addition, the oviducts are deeply inside the body and consequently it is difficult to perform experiments in vivo. Bovines are used as a research model animal for human²¹⁾ since bovines have many similarities to human in reproduction such as folliculogenesis.²²⁾ In this study, we investigated the effect of TSS on bovine oviductal contraction and relaxation to clarify the potential of TSS for infertility treatment that targets oviductal transport capacity.

MATERIALS AND METHODS

Collection of Bovine Oviducts

Oviducts of healthy cows were collected at a local abattoir and transported to our laboratory on ice. The stages of the estrous cycle were classified as Stage I (days 1–4 after ovulation), Stage II (days 5–10 after ovulation), Stage III (days 11–17 after ovulation), and Stage IV (days 18–20 after ovulation), based on macroscopic observation of the ovary and the uterus.²³⁾ The oviducts ipsilateral to the corpus luteum or the dominant follicle were used. After trimming the oviductal isthmus, the samples were utilized for the following experiments because oviductal isthmus observed developed smooth muscle layer and active contraction and relaxation.²⁴⁾

Preparation of TSS Solution

TSS extract powder (Lot No. 2180023010) was supplied by the Tsumura & Co (Tokyo, Japan). TSS is a mixture of six constituent crudedrugs: four parts of Peony Root (root of Paeonia lactiflora Pallas, Paeoniaceae), four parts Atractylodes Lancea Rhizome (rhizome of Atractylodes lanceae De Candolle, Compositae), four parts Alisma Tuber (tuber of Alisma orientale Juzepczuk, Alismataceae), four parts Poria Sclerotium (sclerotium of Poria cocos Wolf, Polyporaceae), three parts Cnidium Rhizome (rhizome of Cnidium officinale Makino, Umbelliferae), and three parts of Japanese Angelica Root (root of Angelica acutiloba Kitagawa, Umbelliferae). These six crude drugs were standardised in the Japanese Pharmacopoeia, 17th edition. HPLC data of TSS has been reported in previous paper.²⁵⁾

Ten or 100 mg TSS powder was dissolved in 1 mL of Krebs–Ringer solution (136.9 mM NaCl, 5.4 mM KCl, 1.5 mM CaCl₂, 1.0 mM MgCl₂, 23.8 mM NaHCO₃, 5.6 mM glucose), followed by vortex mixing for approximately 2 h at room temperature. The supernatants were collected after centrifugation (20 °C, 10000 × g, 10 min) as 10 and 100 mg/mL TSS solutions, respectively. These solutions were diluted and utilized for the following experiments.

Isometric Contraction Test

The isometric contraction test of the oviductal smooth muscle was performed as described previously^26,27) with some modifications. Briefly, isthmic tissues of bovine oviducts were cut open, and three or four pieces of 5 mm longitudinal strips adjacent to the uterine tubal junction (UTJ) were prepared. Each strip was fixed and equilibrated for 1 h before the experiment in a Magnus tube filled with 10 mL Krebs–Ringer solution. The Krebs–Ringer solution was kept at 38.5 °C and aerated with 95% O₂ and 5% CO₂ during the experiment. Oxytocin (4084-v; Peptide Institute, Inc., Osaka, Japan, 0.1 µM) and noradrenalin (143-04741; Wako, Osaka, Japan, 1 µM) were utilized as a constrictor and a relaxant, respectively, to confirm the normality of oviductal contraction before this test. The effects of TSS solution (100, 1000 µg/mL) and GPER1 agonist (G-1; 10008933, Cayman, Ann Arbor, MI, U.S.A., 1 or 10 µM) on oviductal contractility were examined. We also examined the effects of TSS solution (1000 µg/mL) and pre-treatment with G-1 (1 or 10 µM) and GPER1 antagonist (G-15; 14673, Cayman, 25 or 250 nM). The doses of TSS, G-1, and G-15 were based on the concentrations in previous reports.^28–30) The isometric tension of each strip was continuously recorded using a force–displacement transducer (Minebea Co., Ltd., Nagano, Japan) connected to a polygraph (Yokogawa Electric Corp., Tokyo, Japan). Since there was no difference in the results between Stage II and Stage III, they were merged into the same group in experiments 1–3.

Isometric Contraction Test Data Collection

In this study, the frequency, contraction force, and tonus were measured every 2 min before and after the addition of the TSS solution (Fig. 1).

Fig. 1. Frequency, Contraction Force, and Tonus

Frequency is the number of peaks of rhythmic oviductal smooth muscle movement in 2 min. Contraction force is the length of oviductal smooth muscle movement waveforms in 2 min. Tonus is the sustained muscle tension in 2 min.

(i) Frequency

We counted the number of peaks (at maximum contraction) of rhythmic oviductal smooth muscle movement in 2 min.

(ii) Contraction Force

To calculate the contraction force, the distance from the bottom to the top of the waveform on a polygraph paper was measured. The first, middle, and last waveforms in 2 min were selected for analysis. To convert the distance (mm) to the force (mN), the following formula was applied:

Contraction force (mN) = mean of the distances of three waveforms in 2 min (mm)/the distance corresponding to 1 g weight (mm/g) × 9.8 (mN/g)

(iii) Tonus

To calculate the tonus, the distance from the reference line on a polygraph paper to the median of the three selected waveforms was measured (Fig. 2). The first, middle, and last waveforms in 2 min were selected for analysis. To convert the distance (mm) to the tonus (mN), the following formula was applied:

Fig. 2. Tonus Measurement Method

The distance from the reference line on a polygraph paper to the median of the three selected waveforms was measured. The first, middle, and last waveforms in 2 min before and after treatment were selected for analysis.

Tonus (mN) = (mean of the distance between the reference line and the median of three waveforms in 2 min − mean of the distance between the reference line and the median of three waveforms before treatment (mm)) × the distance corresponding to 1 g weight (mm/g) × 9.8 (mN/g)

Protein Fractionation and Extraction

Isthmic tissues of bovine oviducts were cut open, and 5 mm strips adjacent to UTJ were prepared. The mucosal areas of these strips were scraped off with a scalpel. The tissues were homogenized and ultrasonicated in radio immunoprecipitation assay buffer (20 mM Tris–HCl, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 0.1% sodium dodecyl sulfate, 1% NP-40, and protease inhibitor cocktail (1697498; Roch, Mannheim, Germany). The supernatant was collected by centrifugation (4 °C, 20000 × g, 20 min), and the protein concentrations were determined by the BCA method (Pierce™ BCA Protein Assay Kit, 23225; Thermo Fisher Scientific, MA, U.S.A.).³¹⁾

Western Blotting

Oviduct isthmic tissue lysates were mixed with SDS gel-loading buffer (50 mM Tris–HCl, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, pH 6.8) and heated at 95 °C for 10 min. Each sample (25 µg protein/lane) was separated on SDS-polyacrylamide gel electrophoresis (8%), and then transferred to a polyvinylidene difluoride membrane (RPN303F; GE Healthcare., Little Chalfont, Buckinghamshire, U.K.). The membrane was washed in Tris-buffered saline (TBS) with 0.1% Tween 20 (TBS-T, pH 7.5), followed by incubation in blocking buffer (NYPBR01, TOYOBO, Osaka, Japan) for 1 h at room temperature. After washing, the membranes were incubated with anti-GPER-immunoglobulin G (IgG)-rabbit diluted at 1 : 2000 (SAB2700363; Sigma-Aldrich, St. Louis, MO, U.S.A.) by Can Get Signal Solution 1 (NKB-201, TOYOBO), or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-IgM-mouse diluted at 1 : 40000 (NB300-221SS; NOVUS BIOLOGICALS, Briarwood Avenue, Centennial, CO, U.S.A.) by phosphate buffered saline (PBS) overnight at 4 °C. After washing with TBS-T, the membranes were incubated with horseradish peroxidase (HRP)-linked donkey anti-rabbit IgG diluted at 1 : 5000 (NA934; Amersham Biosciences Corp., San Francisco, CA, U.S.A.) for GPER, or HRP-linked goat anti-mouse IgM diluted at 1 : 100000 (ab97230, Abcam, Cambridge, U.K.) for GAPDH by PBS for 1 h at room temperature. After washing again with TBS-T, the signal was detected by chemiluminescence using the Immobilon Western Chemiluminescent HRP Substrate (P36599; Millipore, Billerica, MA, U.S.A.). The protein expression of GAPDH was used as an internal control. The intensity of the immunological reaction in the oviductal isthmic tissues was evaluated by measuring the optical density in the defined area with computerized densitometry using Image Lab™ Software version 4.0 (Bio-Rad Laboratories, Inc., Berkeley, CA, U.S.A.).

Statistical Analysis

All experimental data are shown as the mean ± standard error of the mean (S.E.M.). The statistical analyses were performed using GraphPad Prism version 8 (GraphPad Software, La Jolla, CA, U.S.A.). In Figs. 3–8, the Friedman test was followed by Dunn’s multiple comparisons test for comparisons among the three groups within the same time point. The time-dependent changes within the same treatment group were analyzed using the generalized linear model, and Tukey’s multiple comparisons test was then performed to analyze the effect of reagents. The statistical significance of the differences in Fig. 9 was assessed via one-way ANOVA followed by Tukey’s multiple comparisons tests. The statistical level of significance was set at 5% (p < 0.05).

RESULTS

Experiment 1: The Effect of TSS on Contractility in the Isthmus of Bovine Oviducts

An isometric contraction test was performed to examine the effect of TSS on bovine oviductal contraction and relaxation. The results of tonus, frequency, and contraction force are shown in Figs. 3, 4, and 5, respectively.

Fig. 3. Effect of Tokishakuyakusan (TSS) Solution (100 or 1000 µg/mL) on the Tonus of Bovine Oviductal Isthmic Tissues in a Longitudinal Direction

Data were collected every 2 min after treatment (n = 10). Each graph shows tonus compared to before treatment in the oviducts of Stage I (i), Stage II-III (ii), and Stage IV (iii). Data are expressed as mean ± S.E.M. Mean differences among each treatment group within the same time point are indicated by different letters (p < 0.05). Asterisks mean significant differences (p < 0.05) between 1–3 min and each time point of the same treatment group.

Fig. 4. Effect of TSS Solution (100 or 1000 µg/mL) on the Frequency of Bovine Oviductal Isthmic Tissues in a Longitudinal Direction

Data were collected every 2 min after treatment (n = 10). Each graph shows the frequency of contraction in the oviducts of Stage I (i), Stage II-III (ii), and Stage IV (iii). Data are expressed as mean ± S.E.M.

Fig. 5. Effect of TSS Solution (100 or 1000 µg/mL) on the Contraction Force of Bovine Oviductal Isthmic Tissues in a Longitudinal Direction

Data were collected every 2 min after treatment (n = 10). Each graph shows contraction force in the oviducts of Stage I (i), Stages II and III (ii), and Stage IV (iii). Data are expressed as mean ± S.E.M.

TSS increased tonus of smooth muscle strips of bovine oviduct at all stages (Fig. 3, p < 0.05). Compared with the control group, a significant increase was observed after 7–9 min of treatment in both 100 and 1000 µg/mL TSS groups in Stage I (p < 0.05). Furthermore, both TSS treatments increased tonus after 7–9 min compared with 1–3 min after the addition of TSS (p < 0.05). At the end of the measurement, the tonus rose approximately 14-fold in 100 µg/mL TSS group, and approximately 18-fold in the 1000 µg/mL TSS group respectively compared with 1–3 min of treatment. In Stages II-III and IV, the tonus in 1000 µg/mL TSS was increased significantly after 31–33 min of treatment compared with the control group and 1–3 min after the addition (p < 0.05). At the end of measurement, the tonus increased approximately 25-fold in Stage II-III, and approximately 4-fold in Stage IV compared with 1–3 min of treatment. No significant change in tonus was observed in any of the 100 µg/mL TSS groups in Stages II-III and IV. Further, TSS had no effect on frequency and contraction force in all stages (Figs. 4, 5).

Experiment 2: The Effect of GPER1 Activation on TSS- Increase of Tonus

Since Experiment 1 revealed that TSS increased oviductal tonus rapidly within an hour, and TSS reportedly has an E2-like effect without through ERα and ERβ,^17,18) we hypothesized that TSS acts via GPER1 which bring an acute E2 effect unlike ERα and ERβ. In Experiment 2, we investigated the effect of a GPER1 agonist and TSS on oviductal contractility. We added either TSS or G-1, and the tonus was measured up to 1 h after treatment to confirm whether the effect of GPER1 activation on the tonus is similar to that of TSS. The tonus changes after the treatment with TSS or G-1 are shown in Fig. 6. The tonus of the oviductal isthmus tissues at Stages I and II-III was increased by TSS and both doses of G-1 treatment compared with the control (Fig. 6, p < 0.05). Moreover, G-1 at both concentrations elevated the tonus significantly after 13–15 min at Stage I, and after 17–19 min at Stage II-III compared with 1–3 min after the addition (p < 0.05). The tonus increased approximately 22-fold in the 1 µM G-1 group and approximately 23-fold in the 10 µM G-1 group at the end of measurement, compared with immediately after the addition in Stage I. At Stage II-III, 1 and 10 µM G-1 increased tonus approximately 10- and 7-fold, respectively. By contrast, a significant increase was observed after 17–19 min of treatment in the TSS group, and at the end of the treatment in the 10 µM G-1 group compared with the control group at Stage IV (p < 0.05). There was no difference in tonus compared with 1–3 min after the addition in either of the G-1 treatment groups in Stage IV. These data suggested that the activation of GPER1 had an effect similar to TSS.

Fig. 6. Effect of G Protein-Coupled Estrogen Receptor 1 Agonist (G-1) and TSS Solution on the Tonus of Bovine Oviductal Isthmic Tissues in a Longitudinal Direction

Data were collected every 2 min after treatment (n = 6). Each graph shows tonus compared to before treatment in the oviducts of Stage I (i), Stage II-III (ii), and Stage IV (iii). Data are expressed as mean ± S.E.M. Mean differences among each treatment group within the same time point are indicated by different letters (p < 0.05). Asterisks mean significant differences (p < 0.05) between 1–3 min and each time point of the same treatment group.

Next, TSS was added 20 min after G-1 treatment. At 3 min after TSS treatment, a significant increase of tonus in the TSS and G-1+ TSS groups at Stage I was observed compared with the control without TSS and G-1 treatment (Fig. 7, p < 0.05). There was also a significant increase of tonus in the TSS and G-1+ TSS groups after 15–17 min of the treatment at Stage II-III and after 17–19 min of the treatment at Stage IV compared to the control (p < 0.05). There was no significant difference between the TSS-treated group and the G-1+ TSS-treated group at all stages. No effect of TSS or G-1 on frequency and contraction force was observed at all stages in either experiment (data not shown).

Fig. 7. Effect of TSS Solution and Pre-treatment with G Protein-Coupled Estrogen Receptor 1 Agonist (G-1) on the Tonus of Bovine Oviductal Isthmic Tissues in Longitudinal Direction

At 20 min before the addition of TSS, the oviducts were exposed to 1 or 10 µM G-1. Data were collected every 2 min after treatment (n = 10). Each graph shows tonus compared to before treatment in the oviducts of Stage I (i), Stage II and III (ii), and Stage IV (iii). Data are expressed as mean ± S.E.M. Mean differences among each treatment group within the same time point are indicated by different letters (p < 0.05).

Experiment 3: The Effect of GPER1 Antagonist on TSS-Induced Increase of Tonus

We investigated the effect of GPER1 antagonist (G-15) on oviductal contractility to confirm that TSS acts via GPER1. In stage I, G-15 (250 nM) significantly reduced the TSS-induced increase of tonus at 7 min after the treatment (Fig. 8, p < 0.05). There was also a significant reduction of oviductal tonus after 17 min of TSS treatment in Stages II-III and IV, compared with the TSS group (p < 0.05). The addition of G-15 alone did not affect on oviductal tonus (Supplementary Fig. S1). No effect of TSS or G-15 on frequency and contraction force was observed at all stages (data not shown).

Fig. 8. Effect of Tokishakuyakusan (TSS) Solution and Pre-treatment with G Protein-Coupled Estrogen Receptor 1 Antagonist (G-15) on the Tonus of Bovine Oviductal Isthmic Tissues in Longitudinal Direction

At 20 min before the addition of TSS, the oviducts were exposed to 25 or 250 nM G-15. Data were collected every 2 min after treatment (n = 10). Each graph shows tonus compared to before treatment in the oviducts of Stage I (i), Stage II-III (ii), and Stage IV (iii). Data are expressed as mean ± S.E.M. Mean differences among each treatment group within the same time point are indicated by different letters (p < 0.05).

Experiment 4: GPER1 Protein Expression in Bovine Oviductal Isthmic Smooth Muscle Tissues

In Experiment 4, the protein expression of GPER1 in each estrous stage of bovine oviduct isthmic smooth muscle tissues was analyzed by Western blotting. The protein expression of GPER1 with GAPDH as the internal control was observed at Stages I–IV, and the expression level did not significantly change throughout the estrous cycle (Fig. 9).

Fig. 9. Expression of G Protein-Coupled Estrogen Receptor 1 (GPER1) Protein in Bovine Oviductal Ischemic Smooth Muscle Tissues

(a): The protein level of GPER1 was normalized by the protein level of GAPDH (n = 15). Data are expressed as mean ± S.E.M. (b): Representative image of the expression of GPER1 and GAPDH in bovine oviductal isthmic smooth muscle tissues during the estrous cycle.

DISCUSSION

This is the first study to investigate the effect of TSS on mammalian oviductal contraction movements and its mechanism to clarify its potential for improving oviductal transport capacity. Our study revealed that TSS increased bovine oviductal tonus throughout the estrous cycle. These results indicate that TSS may accelerate the transport of gametes and embryos by shortening the length of the oviduct in the longitudinal direction. Pregnancy in mammals requires the successful completion of many steps, starting with the transport of gametes in the reproductive tract.⁵⁾ The transport of gametes and embryos at the right time is important for the establishment of pregnancy.⁵⁾ Therefore, TSS may be particularly effective for infertile patients due to delayed oviductal transport. In addition, it is reported that approximately 98% of ectopic pregnancies occur in the oviduct,¹⁰⁾ and one of the causes of ectopic pregnancies is oviductal transport disorders. Therefore, TSS may have the potential as an ectopic pregnancy inhibitor by improving oviductal transport.

The present study also clarified that the effect of TSS differed among estrous stages. At both low and high concentrations, TSS increased oviductal tonus faster at Stage I than at the other stages, indicating that immediately after ovulation (Stage I), the oviduct showed high sensitivity to TSS. It is reasonable that TSS shows a higher effect of increasing the oviductal tonus in Stage I, in which the early embryo is transported in the isthmus. It is likely that TSS might be more effective in infertile patients due to delayed early embryo transport if this medicine is taken after ovulation.

TSS increased the oviductal tonus within 1 h in this study. TSS has been shown to demonstrate an E2-like effect that improves ovarian function without through ERα and ERβ.^17,18) Steroid hormones, such as E2, act by nuclear receptor-mediated genomic and transmembrane receptor-mediated non-genomic effects, and non-genomic effects generally cause a faster response.²⁰⁾ GPER1 is a transmembrane E2 receptor,¹⁹⁾ whose expression has been detected in bovine oviductal smooth muscle.³²⁾ Taken together, we hypothesized that TSS acted via GPER1. The addition of the GPER1 agonist G-1 induced an increase in oviductal tonus similar to that of TSS. There was no significant difference between the TSS group and the TSS with G-1 group. This might be because the pre-addition of G-1 had already activated the signal pathway via GPER1 involved in the increase of the oviductal tonus. Further, the GPER1 antagonist G-15 significantly suppressed the effect of TSS. These results suggest that TSS increases oviductal tonus via GPER1.

Since another E2 receptor (ERα) expression in the bovine oviduct shows a cyclic change, and it is higher during follicular phase and lower during the luteal phase,³³⁾ we hypothesized that the protein expression of GPER1 might show the cyclic change, resulting in the difference of TSS effect. From the above, we measured GPER1 protein expression to investigate why the effect of TSS differed among estrous stages. However, there was no significant difference in the expression level of GPER1. A possible reason for this observation may be the difference in the expression of downstream factors of GPER1 among estrous stages. The expressions of downstream factors of G protein-coupled receptors, such as protein kinase A and protein kinase C, change throughout the estrous cycle in the distal colonic crypt cells of rats.³⁴⁾ These factors might be highly expressed in the oviduct at Stage I, resulting in a higher effect of TSS than at other stages. Another possibility is the difference in the hormonal environment of the oviduct among the estrous stages. Previous reports have shown that oocytes are transported very slowly through the isthmus as long as progesterone (P4) is the dominant hormone, and that a surge of E2 is required to modify the action of P4 and accelerate the transport of oocytes to the uterus.³⁵⁾ Moreover, E2 reportedly stimulates myometrium contractility in nonpregnant humans.³⁶⁾ The highest E2 concentration is observed during the follicular phase, and P4 increases in bovine oviducts after ovulation.³⁷⁾ We considered that a further increase in oviductal contractility after TSS treatment might be less observed at Stage IV compared to the other stages since the oviducts of this stage had already been sensitized to E2 before slaughter. Further research is needed to clarify the differences in the effects of TSS throughout the estrous cycle.

In the present study, while TSS increased the oviductal tonus, it did not affect the frequency and contraction force of bovine oviducts within 1 h. These results suggest that TSS stimulates pathways that are not involved in contraction frequency or contraction force. Contraction mechanisms of vascular smooth muscle via G protein-coupled receptors have been reported to release Ca²⁺ by activating the phospholipase C β (PLCβ)/inositol 1,4,5-trisphosphate (IP3) pathway and activating the RhoA/Rho-kinase (ROCK) pathway.^38,39) On the one hand, release of Ca²⁺ causes rhythmic phasic contractions that primarily affect frequency and contraction force.⁴⁰⁾ On the other hand, activation of RhoA/ROCK signaling results in sustained tonically contraction.⁴⁰⁾ However, since these mechanisms have not been reported in bovine oviductal smooth muscles, further studies on oviductal contraction are needed.

The present study has not revealed which ingredients and metabolites of TSS affect bovine oviductal tonus. TSS is extracted from six medicinal herbs; Japanese angelica root, peony root, cnidium rhizome, atractylodes lancea rhizome, poria sclerotium, and alisma tuber. In past report, TSS has many compounds such as paeoniflorin, albiflorin, ferulic acid, and benzoic acid.²³⁾ Ferulic acid, one of the components of angelicae radix and cnidium rhizome, shows E2-like activities in ovariectomized rats.⁴¹⁾ For this reason, ferulic acid may be involved in the increase in oviductal tonus via the E2 receptor. Moreover, Chinese medicine, such as TSS, are metabolized in the body after ingestion.⁴²⁾ Therefore, future studies related to the impacts of TSS on oviductal contraction should use in vivo experiments and detailed analysis to investigate the ingredients and metabolites of TSS that drive its oviductal tonus effects.

In conclusion, we demonstrated that TSS affected bovine oviductal contractility by increasing tonus via GPER1, and this effect was particularly pronounced at days 1–4 after ovulation, although its detailed mechanism remains unclear. TSS may accelerate gamete and early embryo transport by shortening the oviduct, which would be helpful for the treatment of infertility in females caused by disorders of gamete and early embryo transport.

Acknowledgments

The authors appreciate the Okayama Meat Center (Okayama, Japan) and Tsuyama Meat Center (Tsuyama, Okayama, Japan) for providing the bovine oviducts. We also thank Toshimitsu Hatabu (Okayama University) for supporting the isometric contraction test.

Conflict of Interest

K. K. received Grant support from Tsumura & Co. Y.Y. and S.K. have declared no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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