Zn-C3 – SSR signal

RFRP-3, the mammalian ortholog of GnIH, induces cell cycle arrest at G2/M in porcine ovarian granulosa cells

Abstract

RFamide-related peptide-3 (RFRP-3), the mammalian ortholog of gonadotropin-inhibitory hormone (GnIH), has been proposed as a key inhibitory regulator of mammal reproduction. Our previous studies have demonstrated that RFRP-3 inhibited the expression of proliferation-related proteins in porcine granulose cells (GCs), but the inhibitory mechanism causing this has not been discovered. Here, we aim to elucidate the underlying mechanism and determine the cell cycle regulatory sites of action of RFRP-3 on porcine GC proliferation. To this end, the viability of porcine GCs was initially estimated by cell counting kit-8 (CCK-8). We conﬁrmed that diﬀerent doses of RFRP-3 decreased the cellular viability, suggesting that RFRP-3 could inhibit the proliferation of GCs. Subsequently, we evaluated the direct eﬀects of RFRP-3 on the expression of cell cycle regulators. Compared to the control treated cells, 10−6 and 10−8 M of RFRP-3 eﬀectively reduced the transcription of Cyclin B1 and CDK1 mRNAs. However, treatment with RFRP-3 did not alter Cyclin A2, Cyclin D1, CDK2, or CDK4 mRNA levels. These results suggest that RFRP-3 might be inducing G2/M-phase arrest in porcine GCs. Finally, to further determine the molecular mechanism underlying RFRP-3-mediated G2/M cell cycle arrest, we observed the levels of G2/M cell cycle regulatory factors in RFRP-3-treated porcine GCs. The results showed that RFRP-3 treatment signiﬁcantly increased the expression of Myt1, p-Wee1 and p-Cdc2, whereas the level of Cyclin B1 signiﬁcantly decreased in porcine GCs treated with 10−6 M of RFRP-3. Taken together, our data suggest that RFRP-3 regulates the phosphorylation or expression of G2/M cell cycle regulatory factors to induce G2/M-phase arrest via in- hibition Cyclin B-CDK1 complex activation in porcine GCs, which might provide an unfavorable condition for porcine GC proliferation.

1. Introduction

Reproductive physiology is very complex and is regulated by mul- tiple factors, including a number of hypothalamic neuropeptides. In the last few decades, various neuropeptides have been discovered to be involved in stimulation or inhibition of reproduction. Among verte- brates, recent ﬁndings have indicated that gonadotropin-releasing hormone (GnRH) was not the primary factor responsible for the hy- pothalamic control of gonadotropin secretion. In 2000, Tsutsui dis- covered Gonadotropin-inhibitory hormone (GnIH) in quail brain, in- hibiting gonadotrophin secretion from cultured pituitary cells, and its discovery opened a new window in reproductive neuroendocrinology [1]. GnIH orthologs, such as RFRP-3, are similar to GnIH in structure and function and were later discovered in other vertebrates from ﬁsh [2,3] to mammals [4–6] including pigs [7].

EXtensive study of GnIH/RFRP-3 has conﬁrmed that GnIH/RFRP-3 not only inhibits gonadotropin release in the hypothalamo-hypophysial system, mediated by its receptor GPR147 but also acts directly on the gonads of vertebrates [8–10]. There are accumulating data to indicate that GnIH/RFRP-3 and GPR147 are expressed in gonadal tissue. The binding sites for GnIH were localized in the interstitial layer and seminiferous tubules of the testis in males [11,12], as well as in ovarian theca and granulosa cells in females [13,14]. In vivo and in vitro studies have shown that GnIH/RFRP-3 is able to aﬀect gonadal steroid secre- tion, development, and maintenance. In birds, the administration of GnIH induced testicular apoptosis in mature birds and suppressed normal testicular growth in immature birds [15]. A similar study in mice showed that GnIH treatment inhibited ovarian activity and folli- cular development [16]. Furthermore, our previous studies have de- monstrated that RFRP-3 and its receptor are distributed in the granu- losa cells of antral follicles in the pig ovary [7]. We also demonstrated that treatment with RFRP-3 in cultured porcine granulosa cells de- creased the accumulation of the proliferation-related proteins ERK1/2, PCNA, and Cyclin B1 [17].

Collectively, the studies described above suggest that RFRP-3 plays an important role in cell proliferation. However, the information pro- vided above is mostly based on in vivo studies, and studies that in- vestigated the underlying mechanism of how RFRP-3 regulates cell proliferation are limited. Thus, in this study, we investigated the eﬀects of RFRP-3 on the cell cycle in porcine GCs and the roles of RFRP-3 in the regulation of cell cycle progression. The primary aim of this study was to elucidate the underlying mechanism and determine the cell cycle regulatory sites of action of RFRP-3 on the proliferation of porcine GCs.

2. Materials and methods

2.1. Drugs

Human RFRP-3 (RFRP-3 (Human), catalog No. 048-46, PhoeniX Pharmaceuticals, USA) was used in the present study, as well as its corresponding amino acid sequences (Val – Pro – Asn – Leu – Pro – Gln – Arg – Phe – NH2) coincidence with pig RFRP-3 sequences. Human RFRP- 3 and porcine RFRP-3 are completely homologous.

2.2. Porcine granulose cells isolation, in vitro culture, and treatment

Porcine GCs were isolated and cultured as described previously by Hatey et al. [18]. Brieﬂy, after a series of washes with D-Hanks’ ba- lanced salt solution and ethanol (70%), the granulosa cells were iso- lated from medium sized (3–5 mm) follicles from the ovaries of forty pubertal pigs. ApproXimately 1.0 × 106 live granulosa cells/well were cultured in 6-well plates in DMEM/F12 and 1% penicillin/streptomycin and incubated for 72 h at 37 °C in culture medium. Then, some cells were ﬁXed for immunohistochemistry of FSHR to rate the purity of the cell culture, while the medium was replaced with DMEM/F12 con- taining diﬀerent doses of RFRP-3 (10−6, 10−8, 10−10, or 10−12 M). After incubation for 24 h, the medium was collected and assayed for cell viability by CCK-8. The cells were harvested and kept frozen at −70 °C for Western blot and semi-quantitative RT-PCR.

2.3. CCK-8 assay

Porcine GCs were isolated as described in Section 2.2. Then, 2 × 104 cells/well were cultured in 96-well plates. After culturing for 3 days, the medium was replaced with DMEM/F12 containing diﬀerent doses of RFRP-3 (10−6, 10−8, 10−10, or 10−12 M), followed by incubation for an additional 24 h. The supernatant was removed, and each well was washed with PBS, followed by the addition 10 μL of Cell Counting Kit-8
(CCK-8, Beyotime Institute of Biotechnology, China) solution. After the cells were incubated for 4 h, the absorbance was measured at 450 nm using a spectrophotometer (Thermo Varioskan, Finland). Four samples were tested in each group for each incubation time.

2.4. Semi-quantitative RT-PCR

Total RNA extraction and reverse transcription were performed as described by our previous study [7]. Ampliﬁcation reactions were conducted in triplicate using gene-speciﬁc primers designed from the clone sequences shown in Table 1. The reaction protocols were per- formed using the following thermal cycling conditions: 5 min at 94 °C for enzyme activation, followed by diﬀerent cycles of 5 s at 95 °C, 30 s at the appropriate annealing temperature, 15 s at 72 °C (Table 1), and a ﬁnal extension step of 72 °C for 10 min. Products were electrophoresed on 1.2% agarose gel and analyzed.

2.5. Western blotting

After porcine GC treatment as described in Section 2.2, cells were washed with cold PBS and lysed in a cell lysis buﬀer (Beyotime) con- taining 1 mM of phenylmethylsulfonyl ﬂuoride. An equal amount of protein (15–50 μg) was fractionated by SDS-PAGE and then blotted onto nitrocellulose membranes (Pall). Blotted membranes were blocked with 5% bovine serum albumin and then incubated with primary antibody at the appropriate dilution (Myt1 and Phospho-Wee1 (dilution 1:300, Cell Signaling Technology); Cyclin B1 (dilution 1:500, Cell Sig- naling Technology); Phospho-CDK1 (dilution 1:200, Cell Signaling Technology); and GAPDH (dilution 1:2000, Cell Signaling Tech- nology)). After incubation with horseradish peroXidase-labeled goat anti-rabbit or anti-mouse IgG secondary antibody (dilution 1:2000, Cell Signaling Technology), the immunoreactive proteins were detected using ECL chemiluminescent chromogenic kit (Biouniquer) and ex- posure to X-ray ﬁlm (Fuji Photo Film Co., Ltd., Tokyo, Japan). Densi- tometric quantiﬁcation was performed using ImageJ (National In- stitutes of Health, Bethesda, MD) with corresponding GAPDH as the internal control for normalization. These experiments were performed in triplicate (n = 3), and a representative blot of three independent experiments has been depicted.

2.6. Statistical analysis

All data are shown as the means ± S.E.M. for at least three separate experiments. The diﬀerences were considered to be signiﬁcant when P < 0.05. The statistical analysis was performed using one-way ANOVA and Tukey method with SPSS Statistics 17.0. 3. Results 3.1. RFRP-3 inhibits the proliferation of porcine granulosa cells To investigate the inﬂuence of RFRP-3 on the proliferation of por- cine GCs, porcine GCs treated with diﬀerent doses of RFRP-3 were harvested at diﬀerent times and were measured by CCK-8 assay. As shown in Fig. 1A, RFRP-3 inhibited the proliferation of porcine GCs in a dose-dependent manner after 12, 24 and 48 h treatment. Because the highest absorbance was measured after 24 h treatment, statistical ana- lysis was performed on this group data. As shown in Fig. 1B, 10−6, 10−8 and 10−10 M RFRP-3 treatment signiﬁcantly (P < 0.001) re- duced the porcine GC proliferation ability compared with the control, and 10−12 M RFRP-3 also showed a sharp inhibition (P < 0.01). These results indicate that RFRP-3 is closely related to porcine GC proliferation. 3.2. RFRP-3 aﬀects the expression of cyclins and CDKs in porcine granulosa cells Cyclin-dependent kinases (CDKs) and Cyclins play important roles in cell cycle progression. To further determine if RFRP-3 treatment induces cell cycle arrest, and if so, in which phase, we examined the relative levels of the CDKs and Cyclins mRNAs after treatment with diﬀerent doses of RFRP-3. As shown in Fig. 2, 10−6 and 10−8 M RFRP-3 treatment showed a sharp (P < 0.01) decrease in Cyclin B1 mRNA expression, but inhibition of lower doses (10−10 and 10−12 M) of RFRP- 3 showed no signiﬁcant diﬀerence when compared to the control. A similar tendency was detected in CDK1 mRNA expression (Fig. 3). Additionally, 10−6 and 10−8 M RFRP-3 treatment showed a marked inhibition (P < 0.05). However, the expression levels of Cyclin A2, Cyclin D1, CDK2 and CDK4 did not show signiﬁcant diﬀerences be- tween the control and RFRP-3 treatment groups. The results showed that CDK1 and Cyclin B1 decrease signiﬁcantly after RFRP-3 treatment, and Cyclin B1-Cdk1 complexes acting as a main coordinator advance the cell cycle into the M phase, suggesting that the cell cycle might be arrested at the G2/M phase in RFRP-3-treated porcine GCs. 3.3. RFRP-3 induces cell cycle arrest at the G2/M phase via inhibition of cyclin B-CDK1 complex activation To further determine the molecular mechanism underlying the RFRP-3-induced cell cycle arrest at the G2/M phase, we detected the levels of G2/M cell cycle regulatory factors in RFRP-3-treated porcine GCs. As shown in Fig. 4, RFRP-3 inhibited Cyclin B1 protein expression in a dose-dependent manner as 10−6 (P < 0.01) and 10−8 (P < 0.05) M RFRP-3 treatment showed a marked inhibition. However, 10−6 M RFRP-3 signiﬁcantly (P < 0.01) increased the expression of p- CDK1 compared with the control. Furthermore, CDK1 phosphorylation caused inactivation of Cyclin B-Cdk1 complexes, which is catalyzed by the protein kinases Wee1 (Phospho-Wee1 more active) and Myt1[19]. We also detected the levels of p-Wee1 and Myt1 in RFRP-3-treated porcine GCs. The results showed that all doses of RFRP-3 treatment induced a signiﬁcant (P < 0.01) addition of p-Wee1 expression, while the expression of Myt1 was only increased signiﬁcantly at 10−6 M and 10−10 M RFRP-3 treatment. Taken together, these results suggest that RFRP-3 treatment induces cell cycle arrest at the G2/M phase through increasing p-Wee1 and Myt1 expression, which inhibits Cyclin B-CDK1 complex activation. 4. Discussion The discovery of GnIH in the avian brain allowed us to pursue new avenues in studies of reproductive neuroendocrinology across species. Based on extensive research on birds and mammals, GnIH and its or- thologs are considered to be important neurohormones that down- regulate reproduction by the suppression of gonadal steroid production and germ cell diﬀerentiation and maturation [8,10,20–22]. In pig, we ﬁrst cloned the genes GnIH and GPR147, conﬁrming that the GnIH/ GPR147 system exists in the female pig reproductive axis and may play a signiﬁcant role in the regulation of reproduction at the level of the pig hypothalamus-pituitary-ovary axis, similar to its function in other identiﬁed vertebrates [7]. Furthermore, our recent study has de- termined that RFRP-3 has a direct eﬀect on the porcine GCs in vitro, and we surprisingly found that RFRP-3 decreased the accumulation of the proliferation-related proteins ERK1/2, PCNA and Cyclin B1 [17]. Thus, we presume that RFRP-3 could directly control the proliferation and diﬀerentiation of porcine GCs. To validate and further determine the molecular mechanism of this hypothesis, in this study, we systemically characterize the cell cycle regulatory sites of action of RFRP-3 on por- cine GC proliferation in vitro. Our CCK-8 results showed that RFRP-3 signiﬁcantly reduced the porcine GC proliferation ability at one of 12, 24 or 48 h, suggesting that RFRP-3 may be involved in regulating cell proliferation. Although little information is available regarding the direct eﬀect on germ cell pro- liferation, some studies have shown that the administration of GnIH/ RFRP-3 suppressed gonadal activity. Ubuka et al. demonstrated that peripheral administration of GnIH to mature birds induced testicular apoptosis, primarily observed in Sertoli cells, spermatogonia, and spermatocytes, and decreased spermatogenic activity in the testis. However, daily peripheral administration of GnIH for 2 weeks sup- pressed normal testicular growth and the rise in plasma testosterone concentrations in immature birds [15]. In agreement with the ﬁndings in birds, Anjum et al. showed that the RFRP-3 treatment in vivo caused dose-dependent histological changes in the spermatogenesis of adult mice, such as a decline in germ cell proliferation and survival markers and an increase in apoptotic markers in testis [23,24]. In a similar study using female mice, GnIH also caused dose-dependent histologic changes in follicular development and luteinization [16]. Together, these data suggest that the inhibitory eﬀect of GnIH/RFRP-3 in the gonadal ac- tivity may be mediated through inﬂuence of germ cell proliferation. The cell cycle progression is essential for porcine GC functions, which further impact porcine GC diﬀerentiation and proliferation. Throughout the cell cycle, progression is regulated by a series of Cyclins and CDKs [25]. The Cyclin A-CDK2 complex is the main Cyclin-CDK complex in the S phase, and the activity of the complex is required for S- phase transition [26]. The G2/M transition in the cell cycle is positively controlled by the Cyclin B-CDK1 complex, and the Cyclin D-CDK4 complex plays a crucial role in the G1 transition [27]. To determine if RFRP-3 treatment induces the arrest of the cell cycle and in which phase of porcine GCs, we examined the relative levels of the cell cycle regulators under the treatment of diﬀerent doses of RFRP-3. Our PCR results showed that Cyclin B1 and CDK1 were down-regulated after RFRP-3 treatment, without any changes on the other CDKs/cyclins, suggesting that RFRP-3 is involved in G2/M phase transition, which regulates the cell cycle to arrest at the G2/M phase in porcine GCs. Fig. 1. RFRP-3 inhibits the proliferation of porcine GCs. Cell proliferation was determined by CCK-8 assay. (A) Diﬀerent doses of RFRP-3 (10−6, 10−8, 10−10 and 10−12 M) inhibited the proliferation of porcine GCs after 12, 24 and 48 h treatment. (B) RFRP-3 treatment signiﬁcantly reduced porcine GCs proliferation ability in a dose-dependent manner after 24 h treatment. The data are expressed as the mean ± S.E.M., n = 4, with signiﬁcant diﬀerences from the control designated **P < 0.01 and ***P < 0.001. Fig. 2. RFRP-3 aﬀects the expression of Cyclin mRNAs in porcine GCs. (A-D) Agarose gel electrophoresis images of GAPDH (A), Cyclin A2 (B), Cyclin B1 (C) and Cyclin D1 (D). (E) Relative levels of the GAPDH and Cyclins mRNAs under the treatment of diﬀerent doses of RFRP-3. M, DL2000 DNA marker. The data are expressed as the mean ± S.E.M., n = 3, with signiﬁcant diﬀerences from the control designated *P < 0.05 and **P < 0.01. Although we suggested the importance of RFRP-3 in cell cycle arrest in the G2/M phase, it remains unclear just how it regulates. To further investigate how RFRP-3 contributes to the G2/M transition, we ana- lyzed the expression of the mitosis-regulating factors under the treat- ment of diﬀerent doses of RFRP-3. We found that RFRP-3 treatment resulted in the down-regulation of the mitotic marker proteins Cyclin B1, whereas it increased p-CDK1 expression in porcine GCs. Progression into mitosis is an accurately regulated process ultimately driven by Cyclin B1-CDK1. In normal cells, the Cyclin B1-CDK1 complex remains inactive due to inhibitory phosphorylation of CDK1 until the end of the G2 phase [28]. Our results indicated that RFRP-3 is involved in in- activating the Cyclin B1-CDK1 complex by phosphorylating CDK1 and inhibiting Cyclin B1, which are consistent with our previous study. In addition, two kinases, Wee1 and Myt1, are responsible for the in- hibitory phosphorylation of Cdk1 prior to mitotic entry. Myt1 and Wee1 kinases phosphorylate Cdk1 on T14 and Y15, thereby inhibiting Cyclin B/Cdk1 activity [19]. We showed that RFRP-3 increased levels of the phosphorylated Wee1 and Myt1, which are important regulators of the G2/M checkpoint. This suggests that RFRP-3 is associated with the phosphorylation of Cdk1 by Wee1 and Myt1. Interestingly, the 10−10 and 10−12 M doses of RFRP-3 have an eﬀect on the granulosa cell proliferation regulatory factors phosphorylated, Wee1 and Myt1, but no detectable eﬀect on Cyclin B1 and CDK1 mRNA expression. One pos- sible explanation for the discrepancy in the results is that RFRP-3-in- creased phosphorylated Wee1 and Myt1 may impact the phosphoryla- tion of Cdk1 in the protein level but not in the mRNA level. In addition, the diﬀerent methods of detecting protein and mRNA levels may cause the diﬀerent sensitivity of RFRP-3 eﬀect on the granulosa cell pro- liferation. Taken together, the mechanism of RFRP-3 controls the pro- liferation of porcine GCs possibly through increasing p-Wee1 and Myt1 expression that inhibit Cyclin B-CDK1 complex activation. Fig. 3. RFRP-3 aﬀects the expression of CDKs mRNAs in porcine GCs. (A-D) Agarose gel electrophoresis pictures of GAPDH (A), CDK1 (B), CDK2 (C) and CDK4 (D). (E) Relative levels of the GAPDH and CDK mRNAs under the treatment of diﬀerent doses of RFRP-3. M, DL2000 DNA marker. The data are expressed as the mean ± S.E.M., n = 3, with signiﬁcant diﬀerences from the control designated *P < 0.05 and **P < 0.01. Fig. 4. RFRP-3 aﬀects the levels of G2/M cell cycle regulatory factors in RFRP-3-treated porcine GCs. (A) Western blot assay was performed to evaluate the eﬀect of diﬀerent doses of RFRP-3 treatment on Cyclin B1, p-CDK1, p-Wee1 and Myt1 proteins expression in porcine GCs, using GAPDH as an internal control for protein loading. (B) Relative protein level was quantitated using ImageJ. The data are expressed as the mean ± S.E.M., n = 3, with signiﬁcant diﬀerences from the control designated *P < 0.05 and **P < 0.01. In summary, our data presented here demonstrate that RFRP-3 treatment inhibited the viability of porcine GCs and elicited the down- regulation of the major mitotic promoting factor, the cyclin B1/Cdk1 complex. However, it up-regulated of the cell cycle arresting proteins, p-CDK1, p-Wee1 and Myt1. These ﬁndings identify RFRP-3 as a med- iator of reproductive neuroendocrinology, which suppresses gonadal function ability by suppressing porcine GC proliferation and inducing cell cycle arrest in vitro. This study provided the ﬁrst molecular evi- dence to elucidate the underlying mechanism and determine the cell cycle regulatory sites of action of RFRP-3 on the porcine GC prolifera- tion. Our ﬁndings may lead to a better understanding of the physiolo- gical functions of the RFRP-3 in porcine GC proliferation and Zn-C3 diﬀerentiation.