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Carbenoxolone reduces cyclic nucleotides level, destabilizes maturation promoting factor and induces meiotic exit from diplotene arrest in rat cumulus oocytes complexes cultured in vitro
Biomedicine & Pharmacotherapy 94 (2017) 219–230
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Original article
Carbenoxolone reduces cyclic nucleotides level, destabilizes maturation promoting factor and induces meiotic exit from diplotene arrest in rat cumulus oocytes complexes cultured in vitro Meenakshi Tiwari, Shail K. Chaube* Cell Physiology Laboratory, Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi-221005, UP, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 June 2017 Received in revised form 14 July 2017 Accepted 19 July 2017
Background: Disruption of gap junction and transfer of cyclic nucleotides to the oocyte lead to meiotic exit from diplotene arrest (EDA) in mammals. In the present study, we examined whether a gap junction blocker, carbenoxolone (CBX) could induce EDA by reducing cyclic nucleotides level and destabilizing maturation promoting factor (MPF) in rat oocytes cultured in vitro. Methods: Diplotene-arrested cumulus oocyte complexes (COCs) were collected from ovary of immature female rats after 20 IU pregnant mare’s serum gonadotropins (PMSG) for 48 h. These diplotene-arrested COCs were incubated with various concentration of CBX for 3 h in vitro. The morphological changes, meiotic status of oocyte, inducible nitric oxide synthase (iNOS), total nitric oxide (NO), adenosine 30 ,50 cyclic monophosphate (cAMP), guanosine 30 ,50 -cyclic monophosphate (cGMP), cell division cycle 25 B (Cdc25B), changes in specific phosphorylation status of cyclin-dependent kinase 1 (Cdk1) and cyclin B1 levels were analyzed. Results: CBX induced EDA in a concentration-dependent manner. The iNOS expression, total NO and cyclic nucleotides level were significantly decreased. The reduced cyclic nucleotides level resulted in the decrease of Cdc25B expression level. The decreased Cdc25B was associated with the increased Thr14/ Tyr15 phosphorylated Cdk1 level. However, Thr161 phosphorylated Cdk1 as well as cyclin B1 levels were significantly reduced leading to MPF destabilization. The destabilized MPF finally induced EDA in rat COCs cultured in vitro. Conclusions: Our results suggest that CBX blocked gap junctions interrupted the transfer of cyclic nucleotides to the oocyte. Reduction of cyclic nucleotides level destabilized MPF and induced EDA in vitro. Thus, CBX could be used to induce meiotic maturation under in vitro culture conditions during assisted reproductive technology (ART) programs. © 2017 Elsevier Masson SAS. All rights reserved.
Keywords: Diplotene arrest CBX cAMP MPF Meiotic exit Rat oocytes
1. Introduction The ovarian follicle is the principal functional unit that is responsible for generating meiotically competent oocytes in mammals. The meiotic competency starts with the resumption from diplotene arrest [1,2]. The diplotene arrest is morphologically identified by the presence of germinal vesicle (GV) and nucleolus in the oocyte cytoplasm [3,4]. The diplotene arrest may last for several months to several years in mammals [1,5–10]. The cell cycle arrest for such a long period could be due to transfer of several inhibitory factors from encircling granulosa cells to the oocyte [2,11]. Encircling granulosa cells play imperative role in the synthesis and secretion of various signal molecules necessary for
* Corresponding author. E-mail address: [email protected] (S.K. Chaube). http://dx.doi.org/10.1016/j.biopha.2017.07.097 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
the maintenance of meiotic arrest at diplotene stage within the follicular microenvironment [3–5,10,12]. A cross-talk among encircling granulosa cells and between granulosa cells and oocyte depends heavily on functional gap junctions [2,12,13]. These connexin-coupled cell junctions directly mediate cell–cell communication by allowing the passage of ions, metabolites, nutrition and small signaling molecule between two adjacent cells [14]. In the growing follicles, connexin 43 (Cx43) primarily contributes to the formation of gap junctions among granulosa cells, whereas gap junctions that connect the oocyte to the surrounding cumulus cells are mainly composed of connexin 37 (Cx37) [15]. Studies using knockout mice demonstrated that mice lacking Cx43 posses reduced germ cell number, retarded oocyte growth and fertilization rate [16]. In addition, the ablation of Cx37 leads to an abolition of intercellular coupling between oocyte and granulosa cells, inhibition of follicular development
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Fig. 1. Representative photograph showing CBX-induced EDA in COCs cultured in vitro. COCs collected from PMSG-treated rats show diplotene arrest as evidenced by the presence of GV and nucleolus in the denuded oocyte (green arrow, D) of corresponding COCs (green arrow, A). These oocytes are tightly encircled with several layers of granulosa cells (red arrow, A). After 3 h of in vitro culture, EDA was observed in control (E) as well as in CBX-treated denuded oocytes (F) of their corresponding COCs (B, C) as evidenced by GVBD and absence of nucleolus (blue arrow). The diploid set of chromosomes confirms the diplotene arrest (green arrow, G), while formation of metaphase plate (yellow arrows) indicate spontaneous EDA (G) and CBX-induced EDA (I). CBX induced EDA in concentration-dependent manner. Data are mean SEM of three independent experiments and analyzed by one-way ANOVA followed by Bonferroni post-hoc analysis; “*”p < 0.05. The lower panel (Bar = 20 mm) photographs showing denuded oocytes of corresponding upper panel COCs photographs, Bar = 40 mm.
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and generation of incompetent oocytes [17]. The cyclic expression of Cx43 in the granulosa cells is regulated by gonadotropins and steroid hormones in many species including human [13,18]. Gap junctions among granulosa cells as well as between cumulus granulosa cells and oocytes are important for the transfer of cyclic nucleotides from granulosa cells to the oocyte [13,14,19]. The relative high levels of adenosine 30 ,50 -cyclic monophosphate (cAMP) and guanosine 30 ,50 -cyclic monophosphate (cGMP) within the oocyte are essential for the maintenance of meiotic arrest, while a transient decrease of intraoocyte cyclic nucleotides level enables resumption of meiosis [20,21]. This is supported by observations that the physical removal of encircling granulosa cells results in decreased intraoocyte cyclic nucleotide levels and spontaneous meiotic exit from diplotene arrest (EDA) in several mammalian species [3–10]. Luteinizing hormone (LH) disrupts the gap junctions thereby transfer of inhibitory molecules from encircling granulosa cells to the oocyte [19,22]. As a result, nitric oxide (NO) and cyclic nucleotides level in oocyte decrease that trigger EDA [6,7,23–26]. Drugs that disrupt gap junctions could mimic the action of LH. Although carbenoxolone (CBX; 3b-hydroxy-11-oxoolean-12-en30-oic acid 3-hemisuccinate) has been widely used as a
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pharmacological agent to induce oocyte meiosis [24,27–29], the mechanism by which CBX induce EDA under in vitro culture remains ill understood. Therefore, present study was aimed to find out in vitro effects of CBX on morphological changes, inducible nitric oxide synthase (iNOS) expression, total NO, cAMP, cGMP, cell division cycle 25 B (Cdc25B), total as well as specific phosphorylation status of cyclin-dependent kinase 1 (Cdk1) and cyclin B1 levels during EDA in rat cumulus oocyte complexes (COCs). 2. Material and methods 2.1. Chemicals and culture medium All chemicals used in current study were purchased from Sigma Chemical Co. St. Louis, MO, USA, unless otherwise stated. Culture medium (Medium-199, AL094A; HiMedia Laboratories, Mumbai, India) was prepared following company manual protocol. The sodium bicarbonate (0.035% w/v) was added to the culture medium-199 (M-199). The pH of M-199 was adjusted (7.2 0.05) and osmolarity (290 5 m Osmol) was checked and then supplemented with L-glutamine, penicillin and streptomycin (GPS; 1 mL/ml: cat. no. A007, HiMedia) and then stored at 4 C until use (discarded if
Fig. 2. Representative photograph showing effect of CBX on iNOS expression in COCs and total NO level in denuded oocytes. A significant reduction of iNOS expression was observed in COCs (A2) that underwent spontaneous EDA as compare to diplotene-arrested COCs (A1). CBX (200 mM) treatment reduced iNOS expression (A3) and immunofluorescence intensity was similar to control COCs that underwent spontaneous EDA (A2). The CTCF analysis of iNOS immunofluorescence intensity of denuded oocytes (B1–3) of corresponding COCs (A1–3) further confirms above observations (C). The total NO level was significantly decreased in control as well as CBX-treated denuded oocytes that underwent EDA after 3 h in vitro culture as compare to diplotene-arrested denuded oocytes collected from PMSG-treated rats (D). Data are mean SEM of three independent experiments and analyzed by Student's t-test, “#”p < 0.05.
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Sexually immature female rats, Rattus norvegicus (22–24 days old; 45 5 gm body weight) of Charles-Foster strain were housed in light-controlled room providing food and water ad libitum. Rats were subjected to a single subcutaneous injection of 20 IU pregnant mare's serum gonadotropin (PMSG) for 48 h to stimulate ovary for follicular growth and development. Thereafter, rats were euthanized and ovary was collected in 35 mm Petri dish containing 2 mL of pre-warmed M-199. All procedures confirmed to the terms of Institutional Animal Ethical Committee (wide letter no. F.Sc./ IAEC/2014-15/0248) of the university.
inhibit spontaneous resumption of meiosis in rat oocytes during collection and handling of COCs in vitro [6,30]. The COCs were washed quickly with fresh plain culture medium to remove IBMX and then exposed to various concentrations (0, 25, 50, 100, 200 mM) of CBX for 3 h in CO2 incubator (Galaxy 170R, New Brunswick, Eppendorf AG, Hamburg, Germany, UK; 37 C temperature, 5% CO2 and 100% humidity). After 3 h culture, some of the COCs were transferred to culture medium containing 0.01% hyaluronidase at 37 C and denuded quickly by repeated pipetting through a narrow-bore pipette. The denuded oocytes were washed three times with culture medium and then analyzed for morphological changes using a phase-contrast microscope (Nikon, Eclipse; E600, Tokyo, Japan) at 400 magnification. Three independent experiments were conducted using 50 experimental animals to collect COCs and denuded oocytes sufficient for morphological, biochemical and immunofluorescence analysis.
2.3. Collection of COCs and denuded oocytes
2.4. Determination of meiotic status of oocyte
Ovary was punctured using a 26-gauge needle attached to 1 mL of tuberculin syringe under a stereomicroscope (Nikon type 104, Japan) for the collection of diplotene-arrested COCs. A group of diplotene-arrested COCs (approximately 12–14) were collected using microtubing attached with disposable glass micropipette (Clay Adams, NJ) in pre-warmed M-199 containing 0.1 mM of 3isobutyl-1-methylxanthine (IBMX) to inhibit spontaneous EDA. The 0.1 mM IBMX (a phosphodiesterase inhibitor) has been used to
The meiotic status of oocytes was confirmed by propidium iodide (PI) using fluorescence microscope (Model, Ni-U, Nikon Eclipse Tokyo, Japan). For this purpose, 12–14 denuded oocytes collected from each group were washed twice with phosphatebuffered saline (PBS) and then incubated with PBS containing PI (1 mg/ml) for 1 min at room temperature. Thereafter, oocytes were washed 10 times with PBS and then checked for their meiotic status under fluorescence microscope at 540 nm (400 magnification).
not used within 15 days). M-199 was used for washing purpose for all experiments, while 5% fetal bovine serum (FBS) was added to M-199 just prior to in vitro culture. 2.2. Experimental animal and stimulation of ovary
Fig. 3. Effect of CBX on cyclic nucleotides level in denuded oocytes and their corresponding granulosa cells. A significant decrease of cAMP as well as cGMP levels were observed during spontaneous EDA as well as CBX-induced EDA after 3 h in vitro culture as compare to diplotene-arrested denuded oocytes collected from 20 IU PMSG-treated rats (A). Both cyclic nucleotides level remained high in their corresponding encircling granulosa cells of both the groups (B). Data are mean SEM of three independent experiments and analyzed by Student's t-test, “#”p < 0.05.
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Three independent experiments were conducted to validate the meiotic stage and representative photographs are shown in the results section. 2.5. Quantitative estimation of total NO concentration The total NO level was estimated using NO assay kit (Cat. No. KGE001) purchased from R&D Systems (MN) following previous published protocol [6]. Briefly, 36–42 COCs from each group were denuded by manual pipetting. The denuded oocytes were lysed in hypotonic lysis buffer (5 mM Tris, 20 mM ethyl diamine tetraacetic acid (EDTA), 0.5% Triton X-100, pH 8) and centrifuged at 10,000g at 4 C for 30 min. All samples, working standards and reagents were brought to room temperature and prepared by following company manual protocol. The reaction diluent (50 mL) was added to blank wells and nitrate standards (50 mL) or samples to remaining wells. The nicotinamide adenine dinucleotide (NADH; 25 mL) and then diluted nitrate reductase (25 mL) were added to each well. The microplate was incubated at 37 C for 30 min and
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then Griess reagent I and II (50 mL each) were added. The plate was then incubated for 10 min at room temperature. After the end of incubation period, the optical density (OD) was determined using a microplate reader (Model: Micro Scan MS5608A, ECIL, Hyderabad, India) set at 540 nm with wavelength correction at 690 nm. Three independent samples were run in one assay to avoid inter-assay and intra-assay variation was found to be 1.65%. 2.6. Quantitative estimation of cAMP and cGMP The cAMP as well as cGMP concentrations were analyzed using ELISA kits (cAMP: Cat. No. KGE002; cGMP: Cat. No. KGE003) purchased from R&D Systems (MN, USA) as per our previous published protocol [7]. In brief, approximately 36–42 COCs were collected and denuded by manual pipetting. The encircling granulosa cells and denuded oocytes were lysed separately in hypotonic lysis buffer (5 mM Tris, 20 mM EDTA, 0.5% TritonX-100, pH 8) and centrifuged at 10,000g at 4 C for 30 min. Reagents, samples and standards were prepared as per the company manual
Fig. 4. Representative photograph showing the effect of CBX on Cdc25B expression in COCs. A significant reduction of Cdc25B expression was noticed in COCs during spontaneous EDA (A2) as compare to diplotene-arrested COCs (A1). CBX treatment reduced Cdc25B expression in COCs (A3) and immunofluorescence intensity was similar to control COCs that underwent spontaneous EDA (A2). The CTCF analysis of denuded oocytes (B1–3) of corresponding COCs (A1–3) further confirms above observations (C). Data are mean SEM of three independent experiments and analyzed by Student's t-test, “#”p < 0.01. Bar = 80 mm.
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protocol. The OD was taken using microplate reader set at 450 nm within 10 min. Samples from three independent experiments were run in one assay to avoid inter-assay variation and intra-assay variations were found to be 2.1% and 1.85%, respectively. The cAMP as well as cGMP levels are represented as pmol/mg protein in the results section. 2.7. Analyses of iNOS, Cdc25B, specific as well as total phosphorylation status of Cdk1 and cyclin B1 levels The iNOS, Cdc25B, Thr161, Thr14/Tyr15 as well as total phosphorylated Cdk1 and cyclin B1 levels were analyzed using their specific antibodies purchased from Santa Cruz Biotechnology, Inc. CA, USA, following our published protocol [31] with some minor modifications. In brief, A group of 12–14 COCs and denuded oocytes fixed in 4% buffered formaldehyde on the slide were exposed to 100 mL of their respective primary antibodies (NOS2 (N-20), rabbit polyclonal antibody (sc-651) raised against a peptide mapping near the N-terminus of NOS2; Cdc25B (H-85), rabbit polyclonal antibody (sc-5619) raised against amino acids 93–177 mapping near the N-terminus of Cdc25B; p-Cdc2 p34 (Thr161),
rabbit polyclonal antibody (sc-12341) raised against a short amino acid sequence containing pThr161 of Cdc2 p-34; p-Cdc2 p34 (Thr14/Tyr15), rabbit polyclonal antibody (sc-12340) raised against a short amino acid sequence containing Thr14 and Tyr15 phosphorylated cdc2 p34; cdc2 p34 (PSTAIRE), rabbit polyclonal antibody (sc-53) raised against a peptide epitope mapping within the conserved PSTAIRE domain of cdc2 p34; cyclin B1 (H-433), rabbit polyclonal antibody (sc-752) raised against amino acids 1– 433 representing full length of cyclin B1; Actin (C-2), mouse monoclonal antibody (sc-8432) specific for an epitope mapping between amino acids 350–375 at the C-terminous of actin; 1:500 dilutions in blocking buffer) at 37 C for 1 h in CO2 incubator. After 6–8 washes with PBS, slides were exposed to 100 mL of specific anti-rabbit fluorescein isothiocyanate (FITC)-labeled (sc-3839) secondary antibody for detection of iNOS, Cdc25B, Thr161, Thr14/Tyr15 as well as total phosphorylated Cdk1 and cyclin B1 levels and anti-mouse tetra methyl rhodamine isothiocyanate (TRITC)-labeled (sc-3796) secondary antibody for detection of b-actin at 37 C in humidified chamber (1:1000 dilutions in blocking buffer). After 1 h of incubation, slides were washed 5 times with pre-warmed PBS, mounted with fluorescence
Fig. 5. Representative photograph showing the effect of CBX on Thr161 phosphorylated Cdk1 level in COCs. Spontaneous EDA results in the decrease of Thr161 phosphorylated Cdk1 level (A2). CBX (200 mM) treatment decreased Thr161 phosphorylated Cdk1 level (A3) and immunofluorescence intensity was comparable to the control COCs that underwent spontaneous EDA (A2). The CTCF analysis of immunofluorescence intensity of denuded oocytes (B1–3) further confirms above observations (C). Data are mean SEM of three independent experiments and analyzed by Student's t-test, “#”p < 0.01. Bar = 80 mm.
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mounting medium by VECTASHIELD (Vector laboratories, US) and then observed under fluorescence microscope at 465 nm (FITC) and 540 (TRITC) at 400 magnification. Immunofluorescence intensity of b-actin was analyzed in parallel as a control. The experiment was repeated three times to confirm our results. A total of 18–20 denuded oocytes of corresponding COCs were used for corrected total cell fluorescence (CTCF) analysis following previous published method [32]. All parameters for each oocyte of COCs were kept constant and the whole area was selected for the analysis of immunofluorescence intensity using ImageJ software (version 1.44; National Institute of Health, Bethesda, USA). 2.8. Statistical analyses Data are expressed as mean standard error of mean (S.E.M) of three independent experiments. All percentage data were subjected to arcsine square-root transformation before statistical analysis and then analyzed either by Student’s t-test or One-way analysis of variance (ANOVA) followed by Bonferroni post-hoc analysis using SPSS software, version 17.0 (SPSS, Inc. Chicago, IL,
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USA). A probability of p < 0.05 was considered to be statistically significant. 3. Results 3.1. CBX induced meiotic resumption from diplotene arrest in COCs cultured in vitro As shown in Fig. 1, COCs collected from PMSG (20 IU for 48 h)treated rats show diplotene stage as evidenced by the presence of GV and nucleolus in the denuded oocyte (Fig. 1D; green arrow) of corresponding COCs (Fig. 1A; green arrow). These oocytes are tightly encircled with several layers of granulosa cells (red arrow; Fig. 1A). Spontaneous EDA and progression to metaphase-I (M-I) stage was observed in control (Fig. 1E) as well as in CBX-treated denuded oocytes (Fig. 1F) of their corresponding COCs (Fig. 1B and C) as evidenced by germinal vesicle breakdown (GVBD) and absence of nucleolus (blue arrow) after 3 h of in vitro culture. The meiotic stages such as diplotene arrest (Fig. 1G), spontaneous EDA (Fig. 1H), CBX-induced EDA (Fig. 1I) were confirmed using PI
Fig. 6. Representative photograph showing the effect of CBX on Thr14/Tyr15 phosphorylated Cdk1 level in COCs. A significant increase of Thr14/Tyr15 phosphorylated Cdk1 level was observed in COCs that underwent spontaneous EDA (A2) as compare to diplotene-arrested COCs (A1). 200 mM of CBX increased Thr14/Tyr15 phosphorylated Cdk1 level in COCs (A3) and immunofluorescence intensity was similar to control COCs that underwent spontaneous EDA (A2). The CTCF analysis of denuded oocytes (B1–3) of corresponding COCs (A1–3) further confirms above observations (C). Data are mean SEM of three independent experiments and analyzed by Student's t-test, “*”p < 0.01. Bar = 80 mm.
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staining in the present study. CBX induced resumption of meiosis from diplotene arrest in concentration-dependent manner (Oneway ANOVA, F = 148.23, p < 0.001; Fig. 1J). Maximum resumption of meiosis from diplotene arrest (73.60 1.56%) was noticed if the COCs were exposed to 200 mM of CBX as compare to control COCs and only few oocytes (24.60 1.22%) showed spontaneous EDA after 3 h of in vitro culture (Fig.1J). 3.2. CBX reduced iNOS expression and NO level in COCs cultured in vitro As shown in Fig. 2A, a significant (p < 0.01) reduction of iNOS expression was observed in COCs that underwent spontaneous EDA (Fig. 2A2) as compare to diplotene-arrested COCs (Fig. 2A1). CBX (200 mM) treatment for 3 h in vitro reduced iNOS expression (Fig. 2A3) and immunofluorescence intensity was similar to control COCs that underwent spontaneous EDA (Fig. 2A2). The CTCF analysis of iNOS immunofluorescence intensity of denuded oocytes (Fig. 2B1–B3) of corresponding COCs (Fig. 2A1–A3) further confirms above observations (Fig. 2C). The total NO level was significantly (p < 0.05) decreased in control (50.47 2.27 mM/mg protein) as well as CBX-treated denuded oocytes (47.50 2.44 mM/ mg protein) that underwent EDA after 3 h in vitro culture as
compare to diplotene-arrested denuded oocytes collected from 20 IU PMSG-treated rats (Fig. 2D). 3.3. CBX decreased cyclic nucleotides level As shown in Fig. 3A, a significant decrease (p < 0.05) of cAMP as well as cGMP levels were observed in control as well as CBXtreated denuded oocytes that underwent EDA after 3 h in vitro culture as compare to diplotene-arrested denuded oocytes collected from 20 IU PMSG-treated rats (Fig. 3A). On the other hand, both cyclic nucleotides level remained high in corresponding encircling granulosa cells of both the groups (Fig. 3B). 3.4. CBX reduced Cdc25B expression As shown in Fig. 4, a significant reduction (p < 0.01) of Cdc25B expression was noticed in COCs during spontaneous EDA (Fig. 4A2) as compare to diplotene-arrested COCs (Fig. 4A1). CBX treatment reduced Cdc25B expression in COCs (Fig. 4A3) and immunofluorescence intensity was similar to control COCs that underwent spontaneous EDA (Fig. 4A2). The CTCF analysis of denuded oocytes (Fig. 4B1–B3) of corresponding COCs (Fig. 4A1–A3) further confirms above observations (Fig. 4C).
Fig. 7. Representative photograph showing the effect of CBX on total phosphorylated Cdk1 level. CBX treatment for 3 h in vitro did not change total Cdk1 level (A3) and the fluorescence intensity was similar to both control COCs that underwent spontaneous EDA (A2) as well as diplotene-arrested COCs (A1) collected after PMSG (20 IU) treatment. The CTCF analysis of denuded oocytes (B1–3) of corresponding COCs (A1–3) further confirms above observations (C). Bar = 80 mm.
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3.5. CBX decreased Thr161 phosphorylated Cdk1 level Fig. 5 shows effect of CBX on the immunofluorescence intensity of Thr161 phosphorylated Cdk1 in COCs cultured in vitro. A significant reduction (p < 0.01) of Thr161 phosphorylated Cdk1 level was noticed in COCs that underwent spontaneous EDA (Fig. 5A2) as compare to diplotene-arrested COCs (Fig. 5A1). CBX treatment reduced Thr161 phosphorylated Cdk1 level in COCs (Fig. 5A3) and immunofluorescence intensity was similar to control COCs that underwent spontaneous EDA (Fig. 5A2). The CTCF analysis of Thr161 phosphorylated Cdk1 immunofluorescence intensity of denuded oocytes (Fig. 5B1–B3) of corresponding COCs (Fig. 5A1–A3) further confirms above observations (Fig. 5C). 3.6. CBX increased Thr14/Tyr15 phosphorylated Cdk1 level Fig. 6 shows changes in Thr14/Tyr15 phosphorylated Cdk1 level in COCs cultured in vitro. A significant increase (p < 0.01) of Thr14/ Tyr15 phosphorylated Cdk1 level was noticed in COCs that underwent spontaneous EDA (Fig. 6A2) as compare to diplotene-arrested COCs (Fig. 6A1). Treatment of CBX increased
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Thr14/Tyr15 phosphorylated Cdk1 level in COCs (Fig. 6A3) and immunofluorescence intensity was similar to control COCs that underwent spontaneous EDA (Fig. 6A2). The CTCF analysis of denuded oocytes (Fig. 6B1–B3) of corresponding COCs (Fig. 6A1– A3) further confirms above observations (Fig. 6C). 3.7. CBX did not alter total Cdk1 level As shown in Fig. 7, CBX treatment for 3 h in vitro did not change total Cdk1 level in the present study (Fig. 7A3) and the fluorescence intensity was similar to both control COCs that underwent spontaneous EDA (Fig. 7A2) as well as diplotene-arrested COCs (Fig. 7A1). This is further supported by the immunofluorescence intensity of denuded oocytes (Fig. 7B1–B3) of corresponding COCs (Fig. 7A1–A3). The CTCF analysis further confirms above observations (Fig. 7C). 3.8. CBX decreased cyclin B1 level Fig. 8 shows a significant decrease (p < 0.01) of cyclin B1 level was observed in COCs (Fig. 8A2) that underwent spontaneous EDA
Fig. 8. Representative photograph showing the effect of CBX on cyclin B1 level in COCs. A significant decrease of cyclin B1 level was observed in COCs (A2) that underwent spontaneous EDA as compare to diplotene-arrested COCs (A1). CBX (200 mM) treatment reduced cyclin B1 level in COCs (A3) and immunofluorescence intensity was similar to control COCs that underwent spontaneous EDA (A2). The CTCF analysis of cyclin B1 immunofluorescence intensity of denuded oocytes (B1–3) of corresponding COCs (A1–3) further confirms above observations (C). Data are mean SEM of three independent experiments and analyzed by Student's t-test, “#”p < 0.01. Bar = 80 mm.
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as compare to diplotene-arrested COCs (Fig. 8A1). CBX (200 mM) reduced cyclin B1 level in COCs (Fig. 8A3) and immunofluorescence intensity was similar to control COCs that underwent spontaneous EDA (Fig. 8A2). The CTCF analysis of cyclin B1 immunofluorescence intensity of denuded oocytes (Fig. 8B1–B3) of corresponding COCs (Fig. 8A1–A3) further confirms above observations (Fig. 8C). 3.9. CBX did not alter b-actin level For all the immunofluorescence studies, b-actin was analyzed as a control in parallel. As shown in Fig. 9, CBX treatment did not alter b-actin expression neither in COCs (Fig. 9A1–A3) nor in corresponding denuded oocytes (Fig. 9B1–B3). The CTCF analysis of corresponding denuded oocytes further confirms our observations (Fig. 9C). 4. Discussion Granulosa cells play vital role in the synthesis and secretion of various signal molecules required for the maintenance of diplotene arrest within the follicular microenvironment [3–12]. Bi-directional communication between granulosa cells and oocyte is heavily dependent on functional gap junctions [2,12,13]. The physical removal of encircling granulosa cells results in decreased
NO and intraoocyte cyclic nucleotides level thereby spontaneous EDA in several mammalian species [3–11]. LH surge during ovarian stimulation protocol decreases the expression of Cx43 leading to the disruption of the gap junction communication between the cells [13] and induces EDA [19,22]. Drugs that disrupt gap junctions could mimic the action of LH and trigger EDA. This possibility was strengthened by our results suggest that CBX induced meiotic resumption in a concentrationdependent manner. The maximum induction of EDA (70%) was observed if COCs were exposed to 200 mM of CBX for 3 h in vitro. Gap junctions are important for the transfer of NO and cyclic nucleotides from encircling granulosa cells to the oocyte [13,14,25]. Disruption of gap junctions interrupts the transfer of NO and cyclic nucleotides to oocyte [19]. Our results suggest that CBX significantly reduced iNOS, total NO, cAMP as well as cGMP levels in the oocytes and result in EDA. Similarly CBX has been used to induce resumption of meiosis from diplotene arrest in mouse [27], rat [21,24], bovine [29] and porcine oocytes in vitro [28]. Our results together with previous studies support the notion that cyclic nucleotides required for the maintenance of diplotene arrest of oocyte are synthesized by granulosa cells and are transported to the oocyte via gap junctions. CBX mimics the action of LH and triggers resumption of meiosis from diplotene arrest [24,27,28], but its downstream impact during
Fig. 9. Representative photograph showing the effect of CBX on b-actin expression in COCs. The b-actin was analyzed as a control for all immunofluorescence studies. CBX treatment did not change b-actin expression neither in COCs (A1-3) nor in corresponding denuded oocytes (B1-3). The CTCF analysis of corresponding denuded oocytes further confirms our observations (C). Bar = 80 mm.
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EDA remains poorly understood. We propose that the CBXmediated reduction of both cyclic nucleotides level in oocyte could modulate the downstream pathway to destabilize maturation promoting factor (MPF, a complex of the enzymatic subunit, Cdk1 (p34cdc2) and the regulatory subunit, cyclin B1) by altering Cdc25B phosphatase level. This possibility is supported by observations that the reduction in intraoocyte cyclic nucleotides level may result in the inactivation of Cdc25B that finally destabilize MPF leading to EDA [33,34]. This possibility is further strengthened by our findings that CBX reduced Cdc25B expression level during EDA. These data suggest that CBX reduced NO and cyclic nucleotides level that resulted in the inactivation of Cdc25B level during EDA in rat COCs cultured in vitro. High level of stabilized MPF is required for the maintenance of meiotic arrest at diplotene stage [6–10,35], while MPF destabilization results in EDA [6,7,36]. However, it remains unclear whether CBX-mediated decrease of Cdc25B could destabilize MPF during EDA. Previous studies suggest that Cdc25B dephosphorylates Thr14/Tyr15 residue of Cdk1 and Cdk1 activating kinase phosphorylates Thr161 residue of Cdk1, which stabilizes MPF [34,37,38]. The inhibition of Cdc25B induces Thr14/Tyr15 phosphorylation of Cdk1 and leads to MPF destabilization [36]. Based on these observations, we propose that the reduced level of Cdc25B might have induced accumulation of Thr14/Tyr15 but not Thr161 phosphorylated Cdk1 and thereby EDA. Data of the present study suggest that CBX increased Thr14/Tyr15 phosphorylated Cdk1 expression level and decreased Thr161 phosphorylated Cdk1 level during EDA under in vitro culture conditions. On the other hand, total Cdk1 level did not change during the entire course of in vitro studies. These results suggest CBX-mediated decrease of Cdc25B might have modulated specific phosphorylation status of Cdk1 and reduced cyclin B1 level during EDA. Similarly, changes in specific phosphorylation status of Cdk1 and reduction in cyclin B1 levels have been reported during EDA in rat [6,26], mouse [34,39] and bovine oocytes [40]. In conclusion, CBX-induced meiotic resumption was accompanied by a substantial decrease in iNOS expression, total NO and intraoocyte cyclic nucleotides level. The reduced cyclic nucleotides level decreased Cdc25B expression level. The decreased Cdc25B was associated with the accumulation of Thr14/Tyr15 phosphorylated Cdk1 level. However, Thr161 phosphorylated Cdk1 as well as cyclin B1 levels were reduced significantly leading to MPF destabilization. The destabilized MPF finally induced EDA in rat COCs cultured in vitro. Based on our results, we propose that the gap junctions are important for the maintenance of diplotene arrest and CBX could be used to induce meiotic maturation under in vitro culture conditions during assisted reproductive technology (ART) programs in several mammalian species including human. Competing interest None. Acknowledgments This study was financially supported by Department of Science and Technology, Ministry of Science and Technology, Government of India (Grant No. EMR/2014/000702). References [1] L.M. Mehlmann, Stops and starts in mammalian oocytes: recent advances in understanding the regulation of meiotic arrest and oocyte maturation, Reproduction 130 (2005) 791–799. [2] A.M. Luciano, F. Franciosi, S.C. Modina, V. Lodde, Gap junction-mediated communications regulate chromatin remodeling during bovine oocyte growth and differentiation through cAM-dependent mechanism(s), Biol. Reprod. 85 (2011) e1252–e1259.
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Original article
Carbenoxolone reduces cyclic nucleotides level, destabilizes maturation promoting factor and induces meiotic exit from diplotene arrest in rat cumulus oocytes complexes cultured in vitro Meenakshi Tiwari, Shail K. Chaube* Cell Physiology Laboratory, Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi-221005, UP, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 June 2017 Received in revised form 14 July 2017 Accepted 19 July 2017
Background: Disruption of gap junction and transfer of cyclic nucleotides to the oocyte lead to meiotic exit from diplotene arrest (EDA) in mammals. In the present study, we examined whether a gap junction blocker, carbenoxolone (CBX) could induce EDA by reducing cyclic nucleotides level and destabilizing maturation promoting factor (MPF) in rat oocytes cultured in vitro. Methods: Diplotene-arrested cumulus oocyte complexes (COCs) were collected from ovary of immature female rats after 20 IU pregnant mare’s serum gonadotropins (PMSG) for 48 h. These diplotene-arrested COCs were incubated with various concentration of CBX for 3 h in vitro. The morphological changes, meiotic status of oocyte, inducible nitric oxide synthase (iNOS), total nitric oxide (NO), adenosine 30 ,50 cyclic monophosphate (cAMP), guanosine 30 ,50 -cyclic monophosphate (cGMP), cell division cycle 25 B (Cdc25B), changes in specific phosphorylation status of cyclin-dependent kinase 1 (Cdk1) and cyclin B1 levels were analyzed. Results: CBX induced EDA in a concentration-dependent manner. The iNOS expression, total NO and cyclic nucleotides level were significantly decreased. The reduced cyclic nucleotides level resulted in the decrease of Cdc25B expression level. The decreased Cdc25B was associated with the increased Thr14/ Tyr15 phosphorylated Cdk1 level. However, Thr161 phosphorylated Cdk1 as well as cyclin B1 levels were significantly reduced leading to MPF destabilization. The destabilized MPF finally induced EDA in rat COCs cultured in vitro. Conclusions: Our results suggest that CBX blocked gap junctions interrupted the transfer of cyclic nucleotides to the oocyte. Reduction of cyclic nucleotides level destabilized MPF and induced EDA in vitro. Thus, CBX could be used to induce meiotic maturation under in vitro culture conditions during assisted reproductive technology (ART) programs. © 2017 Elsevier Masson SAS. All rights reserved.
Keywords: Diplotene arrest CBX cAMP MPF Meiotic exit Rat oocytes
1. Introduction The ovarian follicle is the principal functional unit that is responsible for generating meiotically competent oocytes in mammals. The meiotic competency starts with the resumption from diplotene arrest [1,2]. The diplotene arrest is morphologically identified by the presence of germinal vesicle (GV) and nucleolus in the oocyte cytoplasm [3,4]. The diplotene arrest may last for several months to several years in mammals [1,5–10]. The cell cycle arrest for such a long period could be due to transfer of several inhibitory factors from encircling granulosa cells to the oocyte [2,11]. Encircling granulosa cells play imperative role in the synthesis and secretion of various signal molecules necessary for
* Corresponding author. E-mail address: [email protected] (S.K. Chaube). http://dx.doi.org/10.1016/j.biopha.2017.07.097 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
the maintenance of meiotic arrest at diplotene stage within the follicular microenvironment [3–5,10,12]. A cross-talk among encircling granulosa cells and between granulosa cells and oocyte depends heavily on functional gap junctions [2,12,13]. These connexin-coupled cell junctions directly mediate cell–cell communication by allowing the passage of ions, metabolites, nutrition and small signaling molecule between two adjacent cells [14]. In the growing follicles, connexin 43 (Cx43) primarily contributes to the formation of gap junctions among granulosa cells, whereas gap junctions that connect the oocyte to the surrounding cumulus cells are mainly composed of connexin 37 (Cx37) [15]. Studies using knockout mice demonstrated that mice lacking Cx43 posses reduced germ cell number, retarded oocyte growth and fertilization rate [16]. In addition, the ablation of Cx37 leads to an abolition of intercellular coupling between oocyte and granulosa cells, inhibition of follicular development
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Fig. 1. Representative photograph showing CBX-induced EDA in COCs cultured in vitro. COCs collected from PMSG-treated rats show diplotene arrest as evidenced by the presence of GV and nucleolus in the denuded oocyte (green arrow, D) of corresponding COCs (green arrow, A). These oocytes are tightly encircled with several layers of granulosa cells (red arrow, A). After 3 h of in vitro culture, EDA was observed in control (E) as well as in CBX-treated denuded oocytes (F) of their corresponding COCs (B, C) as evidenced by GVBD and absence of nucleolus (blue arrow). The diploid set of chromosomes confirms the diplotene arrest (green arrow, G), while formation of metaphase plate (yellow arrows) indicate spontaneous EDA (G) and CBX-induced EDA (I). CBX induced EDA in concentration-dependent manner. Data are mean SEM of three independent experiments and analyzed by one-way ANOVA followed by Bonferroni post-hoc analysis; “*”p < 0.05. The lower panel (Bar = 20 mm) photographs showing denuded oocytes of corresponding upper panel COCs photographs, Bar = 40 mm.
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and generation of incompetent oocytes [17]. The cyclic expression of Cx43 in the granulosa cells is regulated by gonadotropins and steroid hormones in many species including human [13,18]. Gap junctions among granulosa cells as well as between cumulus granulosa cells and oocytes are important for the transfer of cyclic nucleotides from granulosa cells to the oocyte [13,14,19]. The relative high levels of adenosine 30 ,50 -cyclic monophosphate (cAMP) and guanosine 30 ,50 -cyclic monophosphate (cGMP) within the oocyte are essential for the maintenance of meiotic arrest, while a transient decrease of intraoocyte cyclic nucleotides level enables resumption of meiosis [20,21]. This is supported by observations that the physical removal of encircling granulosa cells results in decreased intraoocyte cyclic nucleotide levels and spontaneous meiotic exit from diplotene arrest (EDA) in several mammalian species [3–10]. Luteinizing hormone (LH) disrupts the gap junctions thereby transfer of inhibitory molecules from encircling granulosa cells to the oocyte [19,22]. As a result, nitric oxide (NO) and cyclic nucleotides level in oocyte decrease that trigger EDA [6,7,23–26]. Drugs that disrupt gap junctions could mimic the action of LH. Although carbenoxolone (CBX; 3b-hydroxy-11-oxoolean-12-en30-oic acid 3-hemisuccinate) has been widely used as a
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pharmacological agent to induce oocyte meiosis [24,27–29], the mechanism by which CBX induce EDA under in vitro culture remains ill understood. Therefore, present study was aimed to find out in vitro effects of CBX on morphological changes, inducible nitric oxide synthase (iNOS) expression, total NO, cAMP, cGMP, cell division cycle 25 B (Cdc25B), total as well as specific phosphorylation status of cyclin-dependent kinase 1 (Cdk1) and cyclin B1 levels during EDA in rat cumulus oocyte complexes (COCs). 2. Material and methods 2.1. Chemicals and culture medium All chemicals used in current study were purchased from Sigma Chemical Co. St. Louis, MO, USA, unless otherwise stated. Culture medium (Medium-199, AL094A; HiMedia Laboratories, Mumbai, India) was prepared following company manual protocol. The sodium bicarbonate (0.035% w/v) was added to the culture medium-199 (M-199). The pH of M-199 was adjusted (7.2 0.05) and osmolarity (290 5 m Osmol) was checked and then supplemented with L-glutamine, penicillin and streptomycin (GPS; 1 mL/ml: cat. no. A007, HiMedia) and then stored at 4 C until use (discarded if
Fig. 2. Representative photograph showing effect of CBX on iNOS expression in COCs and total NO level in denuded oocytes. A significant reduction of iNOS expression was observed in COCs (A2) that underwent spontaneous EDA as compare to diplotene-arrested COCs (A1). CBX (200 mM) treatment reduced iNOS expression (A3) and immunofluorescence intensity was similar to control COCs that underwent spontaneous EDA (A2). The CTCF analysis of iNOS immunofluorescence intensity of denuded oocytes (B1–3) of corresponding COCs (A1–3) further confirms above observations (C). The total NO level was significantly decreased in control as well as CBX-treated denuded oocytes that underwent EDA after 3 h in vitro culture as compare to diplotene-arrested denuded oocytes collected from PMSG-treated rats (D). Data are mean SEM of three independent experiments and analyzed by Student's t-test, “#”p < 0.05.
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Sexually immature female rats, Rattus norvegicus (22–24 days old; 45 5 gm body weight) of Charles-Foster strain were housed in light-controlled room providing food and water ad libitum. Rats were subjected to a single subcutaneous injection of 20 IU pregnant mare's serum gonadotropin (PMSG) for 48 h to stimulate ovary for follicular growth and development. Thereafter, rats were euthanized and ovary was collected in 35 mm Petri dish containing 2 mL of pre-warmed M-199. All procedures confirmed to the terms of Institutional Animal Ethical Committee (wide letter no. F.Sc./ IAEC/2014-15/0248) of the university.
inhibit spontaneous resumption of meiosis in rat oocytes during collection and handling of COCs in vitro [6,30]. The COCs were washed quickly with fresh plain culture medium to remove IBMX and then exposed to various concentrations (0, 25, 50, 100, 200 mM) of CBX for 3 h in CO2 incubator (Galaxy 170R, New Brunswick, Eppendorf AG, Hamburg, Germany, UK; 37 C temperature, 5% CO2 and 100% humidity). After 3 h culture, some of the COCs were transferred to culture medium containing 0.01% hyaluronidase at 37 C and denuded quickly by repeated pipetting through a narrow-bore pipette. The denuded oocytes were washed three times with culture medium and then analyzed for morphological changes using a phase-contrast microscope (Nikon, Eclipse; E600, Tokyo, Japan) at 400 magnification. Three independent experiments were conducted using 50 experimental animals to collect COCs and denuded oocytes sufficient for morphological, biochemical and immunofluorescence analysis.
2.3. Collection of COCs and denuded oocytes
2.4. Determination of meiotic status of oocyte
Ovary was punctured using a 26-gauge needle attached to 1 mL of tuberculin syringe under a stereomicroscope (Nikon type 104, Japan) for the collection of diplotene-arrested COCs. A group of diplotene-arrested COCs (approximately 12–14) were collected using microtubing attached with disposable glass micropipette (Clay Adams, NJ) in pre-warmed M-199 containing 0.1 mM of 3isobutyl-1-methylxanthine (IBMX) to inhibit spontaneous EDA. The 0.1 mM IBMX (a phosphodiesterase inhibitor) has been used to
The meiotic status of oocytes was confirmed by propidium iodide (PI) using fluorescence microscope (Model, Ni-U, Nikon Eclipse Tokyo, Japan). For this purpose, 12–14 denuded oocytes collected from each group were washed twice with phosphatebuffered saline (PBS) and then incubated with PBS containing PI (1 mg/ml) for 1 min at room temperature. Thereafter, oocytes were washed 10 times with PBS and then checked for their meiotic status under fluorescence microscope at 540 nm (400 magnification).
not used within 15 days). M-199 was used for washing purpose for all experiments, while 5% fetal bovine serum (FBS) was added to M-199 just prior to in vitro culture. 2.2. Experimental animal and stimulation of ovary
Fig. 3. Effect of CBX on cyclic nucleotides level in denuded oocytes and their corresponding granulosa cells. A significant decrease of cAMP as well as cGMP levels were observed during spontaneous EDA as well as CBX-induced EDA after 3 h in vitro culture as compare to diplotene-arrested denuded oocytes collected from 20 IU PMSG-treated rats (A). Both cyclic nucleotides level remained high in their corresponding encircling granulosa cells of both the groups (B). Data are mean SEM of three independent experiments and analyzed by Student's t-test, “#”p < 0.05.
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Three independent experiments were conducted to validate the meiotic stage and representative photographs are shown in the results section. 2.5. Quantitative estimation of total NO concentration The total NO level was estimated using NO assay kit (Cat. No. KGE001) purchased from R&D Systems (MN) following previous published protocol [6]. Briefly, 36–42 COCs from each group were denuded by manual pipetting. The denuded oocytes were lysed in hypotonic lysis buffer (5 mM Tris, 20 mM ethyl diamine tetraacetic acid (EDTA), 0.5% Triton X-100, pH 8) and centrifuged at 10,000g at 4 C for 30 min. All samples, working standards and reagents were brought to room temperature and prepared by following company manual protocol. The reaction diluent (50 mL) was added to blank wells and nitrate standards (50 mL) or samples to remaining wells. The nicotinamide adenine dinucleotide (NADH; 25 mL) and then diluted nitrate reductase (25 mL) were added to each well. The microplate was incubated at 37 C for 30 min and
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then Griess reagent I and II (50 mL each) were added. The plate was then incubated for 10 min at room temperature. After the end of incubation period, the optical density (OD) was determined using a microplate reader (Model: Micro Scan MS5608A, ECIL, Hyderabad, India) set at 540 nm with wavelength correction at 690 nm. Three independent samples were run in one assay to avoid inter-assay and intra-assay variation was found to be 1.65%. 2.6. Quantitative estimation of cAMP and cGMP The cAMP as well as cGMP concentrations were analyzed using ELISA kits (cAMP: Cat. No. KGE002; cGMP: Cat. No. KGE003) purchased from R&D Systems (MN, USA) as per our previous published protocol [7]. In brief, approximately 36–42 COCs were collected and denuded by manual pipetting. The encircling granulosa cells and denuded oocytes were lysed separately in hypotonic lysis buffer (5 mM Tris, 20 mM EDTA, 0.5% TritonX-100, pH 8) and centrifuged at 10,000g at 4 C for 30 min. Reagents, samples and standards were prepared as per the company manual
Fig. 4. Representative photograph showing the effect of CBX on Cdc25B expression in COCs. A significant reduction of Cdc25B expression was noticed in COCs during spontaneous EDA (A2) as compare to diplotene-arrested COCs (A1). CBX treatment reduced Cdc25B expression in COCs (A3) and immunofluorescence intensity was similar to control COCs that underwent spontaneous EDA (A2). The CTCF analysis of denuded oocytes (B1–3) of corresponding COCs (A1–3) further confirms above observations (C). Data are mean SEM of three independent experiments and analyzed by Student's t-test, “#”p < 0.01. Bar = 80 mm.
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protocol. The OD was taken using microplate reader set at 450 nm within 10 min. Samples from three independent experiments were run in one assay to avoid inter-assay variation and intra-assay variations were found to be 2.1% and 1.85%, respectively. The cAMP as well as cGMP levels are represented as pmol/mg protein in the results section. 2.7. Analyses of iNOS, Cdc25B, specific as well as total phosphorylation status of Cdk1 and cyclin B1 levels The iNOS, Cdc25B, Thr161, Thr14/Tyr15 as well as total phosphorylated Cdk1 and cyclin B1 levels were analyzed using their specific antibodies purchased from Santa Cruz Biotechnology, Inc. CA, USA, following our published protocol [31] with some minor modifications. In brief, A group of 12–14 COCs and denuded oocytes fixed in 4% buffered formaldehyde on the slide were exposed to 100 mL of their respective primary antibodies (NOS2 (N-20), rabbit polyclonal antibody (sc-651) raised against a peptide mapping near the N-terminus of NOS2; Cdc25B (H-85), rabbit polyclonal antibody (sc-5619) raised against amino acids 93–177 mapping near the N-terminus of Cdc25B; p-Cdc2 p34 (Thr161),
rabbit polyclonal antibody (sc-12341) raised against a short amino acid sequence containing pThr161 of Cdc2 p-34; p-Cdc2 p34 (Thr14/Tyr15), rabbit polyclonal antibody (sc-12340) raised against a short amino acid sequence containing Thr14 and Tyr15 phosphorylated cdc2 p34; cdc2 p34 (PSTAIRE), rabbit polyclonal antibody (sc-53) raised against a peptide epitope mapping within the conserved PSTAIRE domain of cdc2 p34; cyclin B1 (H-433), rabbit polyclonal antibody (sc-752) raised against amino acids 1– 433 representing full length of cyclin B1; Actin (C-2), mouse monoclonal antibody (sc-8432) specific for an epitope mapping between amino acids 350–375 at the C-terminous of actin; 1:500 dilutions in blocking buffer) at 37 C for 1 h in CO2 incubator. After 6–8 washes with PBS, slides were exposed to 100 mL of specific anti-rabbit fluorescein isothiocyanate (FITC)-labeled (sc-3839) secondary antibody for detection of iNOS, Cdc25B, Thr161, Thr14/Tyr15 as well as total phosphorylated Cdk1 and cyclin B1 levels and anti-mouse tetra methyl rhodamine isothiocyanate (TRITC)-labeled (sc-3796) secondary antibody for detection of b-actin at 37 C in humidified chamber (1:1000 dilutions in blocking buffer). After 1 h of incubation, slides were washed 5 times with pre-warmed PBS, mounted with fluorescence
Fig. 5. Representative photograph showing the effect of CBX on Thr161 phosphorylated Cdk1 level in COCs. Spontaneous EDA results in the decrease of Thr161 phosphorylated Cdk1 level (A2). CBX (200 mM) treatment decreased Thr161 phosphorylated Cdk1 level (A3) and immunofluorescence intensity was comparable to the control COCs that underwent spontaneous EDA (A2). The CTCF analysis of immunofluorescence intensity of denuded oocytes (B1–3) further confirms above observations (C). Data are mean SEM of three independent experiments and analyzed by Student's t-test, “#”p < 0.01. Bar = 80 mm.
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mounting medium by VECTASHIELD (Vector laboratories, US) and then observed under fluorescence microscope at 465 nm (FITC) and 540 (TRITC) at 400 magnification. Immunofluorescence intensity of b-actin was analyzed in parallel as a control. The experiment was repeated three times to confirm our results. A total of 18–20 denuded oocytes of corresponding COCs were used for corrected total cell fluorescence (CTCF) analysis following previous published method [32]. All parameters for each oocyte of COCs were kept constant and the whole area was selected for the analysis of immunofluorescence intensity using ImageJ software (version 1.44; National Institute of Health, Bethesda, USA). 2.8. Statistical analyses Data are expressed as mean standard error of mean (S.E.M) of three independent experiments. All percentage data were subjected to arcsine square-root transformation before statistical analysis and then analyzed either by Student’s t-test or One-way analysis of variance (ANOVA) followed by Bonferroni post-hoc analysis using SPSS software, version 17.0 (SPSS, Inc. Chicago, IL,
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USA). A probability of p < 0.05 was considered to be statistically significant. 3. Results 3.1. CBX induced meiotic resumption from diplotene arrest in COCs cultured in vitro As shown in Fig. 1, COCs collected from PMSG (20 IU for 48 h)treated rats show diplotene stage as evidenced by the presence of GV and nucleolus in the denuded oocyte (Fig. 1D; green arrow) of corresponding COCs (Fig. 1A; green arrow). These oocytes are tightly encircled with several layers of granulosa cells (red arrow; Fig. 1A). Spontaneous EDA and progression to metaphase-I (M-I) stage was observed in control (Fig. 1E) as well as in CBX-treated denuded oocytes (Fig. 1F) of their corresponding COCs (Fig. 1B and C) as evidenced by germinal vesicle breakdown (GVBD) and absence of nucleolus (blue arrow) after 3 h of in vitro culture. The meiotic stages such as diplotene arrest (Fig. 1G), spontaneous EDA (Fig. 1H), CBX-induced EDA (Fig. 1I) were confirmed using PI
Fig. 6. Representative photograph showing the effect of CBX on Thr14/Tyr15 phosphorylated Cdk1 level in COCs. A significant increase of Thr14/Tyr15 phosphorylated Cdk1 level was observed in COCs that underwent spontaneous EDA (A2) as compare to diplotene-arrested COCs (A1). 200 mM of CBX increased Thr14/Tyr15 phosphorylated Cdk1 level in COCs (A3) and immunofluorescence intensity was similar to control COCs that underwent spontaneous EDA (A2). The CTCF analysis of denuded oocytes (B1–3) of corresponding COCs (A1–3) further confirms above observations (C). Data are mean SEM of three independent experiments and analyzed by Student's t-test, “*”p < 0.01. Bar = 80 mm.
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staining in the present study. CBX induced resumption of meiosis from diplotene arrest in concentration-dependent manner (Oneway ANOVA, F = 148.23, p < 0.001; Fig. 1J). Maximum resumption of meiosis from diplotene arrest (73.60 1.56%) was noticed if the COCs were exposed to 200 mM of CBX as compare to control COCs and only few oocytes (24.60 1.22%) showed spontaneous EDA after 3 h of in vitro culture (Fig.1J). 3.2. CBX reduced iNOS expression and NO level in COCs cultured in vitro As shown in Fig. 2A, a significant (p < 0.01) reduction of iNOS expression was observed in COCs that underwent spontaneous EDA (Fig. 2A2) as compare to diplotene-arrested COCs (Fig. 2A1). CBX (200 mM) treatment for 3 h in vitro reduced iNOS expression (Fig. 2A3) and immunofluorescence intensity was similar to control COCs that underwent spontaneous EDA (Fig. 2A2). The CTCF analysis of iNOS immunofluorescence intensity of denuded oocytes (Fig. 2B1–B3) of corresponding COCs (Fig. 2A1–A3) further confirms above observations (Fig. 2C). The total NO level was significantly (p < 0.05) decreased in control (50.47 2.27 mM/mg protein) as well as CBX-treated denuded oocytes (47.50 2.44 mM/ mg protein) that underwent EDA after 3 h in vitro culture as
compare to diplotene-arrested denuded oocytes collected from 20 IU PMSG-treated rats (Fig. 2D). 3.3. CBX decreased cyclic nucleotides level As shown in Fig. 3A, a significant decrease (p < 0.05) of cAMP as well as cGMP levels were observed in control as well as CBXtreated denuded oocytes that underwent EDA after 3 h in vitro culture as compare to diplotene-arrested denuded oocytes collected from 20 IU PMSG-treated rats (Fig. 3A). On the other hand, both cyclic nucleotides level remained high in corresponding encircling granulosa cells of both the groups (Fig. 3B). 3.4. CBX reduced Cdc25B expression As shown in Fig. 4, a significant reduction (p < 0.01) of Cdc25B expression was noticed in COCs during spontaneous EDA (Fig. 4A2) as compare to diplotene-arrested COCs (Fig. 4A1). CBX treatment reduced Cdc25B expression in COCs (Fig. 4A3) and immunofluorescence intensity was similar to control COCs that underwent spontaneous EDA (Fig. 4A2). The CTCF analysis of denuded oocytes (Fig. 4B1–B3) of corresponding COCs (Fig. 4A1–A3) further confirms above observations (Fig. 4C).
Fig. 7. Representative photograph showing the effect of CBX on total phosphorylated Cdk1 level. CBX treatment for 3 h in vitro did not change total Cdk1 level (A3) and the fluorescence intensity was similar to both control COCs that underwent spontaneous EDA (A2) as well as diplotene-arrested COCs (A1) collected after PMSG (20 IU) treatment. The CTCF analysis of denuded oocytes (B1–3) of corresponding COCs (A1–3) further confirms above observations (C). Bar = 80 mm.
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3.5. CBX decreased Thr161 phosphorylated Cdk1 level Fig. 5 shows effect of CBX on the immunofluorescence intensity of Thr161 phosphorylated Cdk1 in COCs cultured in vitro. A significant reduction (p < 0.01) of Thr161 phosphorylated Cdk1 level was noticed in COCs that underwent spontaneous EDA (Fig. 5A2) as compare to diplotene-arrested COCs (Fig. 5A1). CBX treatment reduced Thr161 phosphorylated Cdk1 level in COCs (Fig. 5A3) and immunofluorescence intensity was similar to control COCs that underwent spontaneous EDA (Fig. 5A2). The CTCF analysis of Thr161 phosphorylated Cdk1 immunofluorescence intensity of denuded oocytes (Fig. 5B1–B3) of corresponding COCs (Fig. 5A1–A3) further confirms above observations (Fig. 5C). 3.6. CBX increased Thr14/Tyr15 phosphorylated Cdk1 level Fig. 6 shows changes in Thr14/Tyr15 phosphorylated Cdk1 level in COCs cultured in vitro. A significant increase (p < 0.01) of Thr14/ Tyr15 phosphorylated Cdk1 level was noticed in COCs that underwent spontaneous EDA (Fig. 6A2) as compare to diplotene-arrested COCs (Fig. 6A1). Treatment of CBX increased
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Thr14/Tyr15 phosphorylated Cdk1 level in COCs (Fig. 6A3) and immunofluorescence intensity was similar to control COCs that underwent spontaneous EDA (Fig. 6A2). The CTCF analysis of denuded oocytes (Fig. 6B1–B3) of corresponding COCs (Fig. 6A1– A3) further confirms above observations (Fig. 6C). 3.7. CBX did not alter total Cdk1 level As shown in Fig. 7, CBX treatment for 3 h in vitro did not change total Cdk1 level in the present study (Fig. 7A3) and the fluorescence intensity was similar to both control COCs that underwent spontaneous EDA (Fig. 7A2) as well as diplotene-arrested COCs (Fig. 7A1). This is further supported by the immunofluorescence intensity of denuded oocytes (Fig. 7B1–B3) of corresponding COCs (Fig. 7A1–A3). The CTCF analysis further confirms above observations (Fig. 7C). 3.8. CBX decreased cyclin B1 level Fig. 8 shows a significant decrease (p < 0.01) of cyclin B1 level was observed in COCs (Fig. 8A2) that underwent spontaneous EDA
Fig. 8. Representative photograph showing the effect of CBX on cyclin B1 level in COCs. A significant decrease of cyclin B1 level was observed in COCs (A2) that underwent spontaneous EDA as compare to diplotene-arrested COCs (A1). CBX (200 mM) treatment reduced cyclin B1 level in COCs (A3) and immunofluorescence intensity was similar to control COCs that underwent spontaneous EDA (A2). The CTCF analysis of cyclin B1 immunofluorescence intensity of denuded oocytes (B1–3) of corresponding COCs (A1–3) further confirms above observations (C). Data are mean SEM of three independent experiments and analyzed by Student's t-test, “#”p < 0.01. Bar = 80 mm.
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as compare to diplotene-arrested COCs (Fig. 8A1). CBX (200 mM) reduced cyclin B1 level in COCs (Fig. 8A3) and immunofluorescence intensity was similar to control COCs that underwent spontaneous EDA (Fig. 8A2). The CTCF analysis of cyclin B1 immunofluorescence intensity of denuded oocytes (Fig. 8B1–B3) of corresponding COCs (Fig. 8A1–A3) further confirms above observations (Fig. 8C). 3.9. CBX did not alter b-actin level For all the immunofluorescence studies, b-actin was analyzed as a control in parallel. As shown in Fig. 9, CBX treatment did not alter b-actin expression neither in COCs (Fig. 9A1–A3) nor in corresponding denuded oocytes (Fig. 9B1–B3). The CTCF analysis of corresponding denuded oocytes further confirms our observations (Fig. 9C). 4. Discussion Granulosa cells play vital role in the synthesis and secretion of various signal molecules required for the maintenance of diplotene arrest within the follicular microenvironment [3–12]. Bi-directional communication between granulosa cells and oocyte is heavily dependent on functional gap junctions [2,12,13]. The physical removal of encircling granulosa cells results in decreased
NO and intraoocyte cyclic nucleotides level thereby spontaneous EDA in several mammalian species [3–11]. LH surge during ovarian stimulation protocol decreases the expression of Cx43 leading to the disruption of the gap junction communication between the cells [13] and induces EDA [19,22]. Drugs that disrupt gap junctions could mimic the action of LH and trigger EDA. This possibility was strengthened by our results suggest that CBX induced meiotic resumption in a concentrationdependent manner. The maximum induction of EDA (70%) was observed if COCs were exposed to 200 mM of CBX for 3 h in vitro. Gap junctions are important for the transfer of NO and cyclic nucleotides from encircling granulosa cells to the oocyte [13,14,25]. Disruption of gap junctions interrupts the transfer of NO and cyclic nucleotides to oocyte [19]. Our results suggest that CBX significantly reduced iNOS, total NO, cAMP as well as cGMP levels in the oocytes and result in EDA. Similarly CBX has been used to induce resumption of meiosis from diplotene arrest in mouse [27], rat [21,24], bovine [29] and porcine oocytes in vitro [28]. Our results together with previous studies support the notion that cyclic nucleotides required for the maintenance of diplotene arrest of oocyte are synthesized by granulosa cells and are transported to the oocyte via gap junctions. CBX mimics the action of LH and triggers resumption of meiosis from diplotene arrest [24,27,28], but its downstream impact during
Fig. 9. Representative photograph showing the effect of CBX on b-actin expression in COCs. The b-actin was analyzed as a control for all immunofluorescence studies. CBX treatment did not change b-actin expression neither in COCs (A1-3) nor in corresponding denuded oocytes (B1-3). The CTCF analysis of corresponding denuded oocytes further confirms our observations (C). Bar = 80 mm.
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EDA remains poorly understood. We propose that the CBXmediated reduction of both cyclic nucleotides level in oocyte could modulate the downstream pathway to destabilize maturation promoting factor (MPF, a complex of the enzymatic subunit, Cdk1 (p34cdc2) and the regulatory subunit, cyclin B1) by altering Cdc25B phosphatase level. This possibility is supported by observations that the reduction in intraoocyte cyclic nucleotides level may result in the inactivation of Cdc25B that finally destabilize MPF leading to EDA [33,34]. This possibility is further strengthened by our findings that CBX reduced Cdc25B expression level during EDA. These data suggest that CBX reduced NO and cyclic nucleotides level that resulted in the inactivation of Cdc25B level during EDA in rat COCs cultured in vitro. High level of stabilized MPF is required for the maintenance of meiotic arrest at diplotene stage [6–10,35], while MPF destabilization results in EDA [6,7,36]. However, it remains unclear whether CBX-mediated decrease of Cdc25B could destabilize MPF during EDA. Previous studies suggest that Cdc25B dephosphorylates Thr14/Tyr15 residue of Cdk1 and Cdk1 activating kinase phosphorylates Thr161 residue of Cdk1, which stabilizes MPF [34,37,38]. The inhibition of Cdc25B induces Thr14/Tyr15 phosphorylation of Cdk1 and leads to MPF destabilization [36]. Based on these observations, we propose that the reduced level of Cdc25B might have induced accumulation of Thr14/Tyr15 but not Thr161 phosphorylated Cdk1 and thereby EDA. Data of the present study suggest that CBX increased Thr14/Tyr15 phosphorylated Cdk1 expression level and decreased Thr161 phosphorylated Cdk1 level during EDA under in vitro culture conditions. On the other hand, total Cdk1 level did not change during the entire course of in vitro studies. These results suggest CBX-mediated decrease of Cdc25B might have modulated specific phosphorylation status of Cdk1 and reduced cyclin B1 level during EDA. Similarly, changes in specific phosphorylation status of Cdk1 and reduction in cyclin B1 levels have been reported during EDA in rat [6,26], mouse [34,39] and bovine oocytes [40]. In conclusion, CBX-induced meiotic resumption was accompanied by a substantial decrease in iNOS expression, total NO and intraoocyte cyclic nucleotides level. The reduced cyclic nucleotides level decreased Cdc25B expression level. The decreased Cdc25B was associated with the accumulation of Thr14/Tyr15 phosphorylated Cdk1 level. However, Thr161 phosphorylated Cdk1 as well as cyclin B1 levels were reduced significantly leading to MPF destabilization. The destabilized MPF finally induced EDA in rat COCs cultured in vitro. Based on our results, we propose that the gap junctions are important for the maintenance of diplotene arrest and CBX could be used to induce meiotic maturation under in vitro culture conditions during assisted reproductive technology (ART) programs in several mammalian species including human. Competing interest None. Acknowledgments This study was financially supported by Department of Science and Technology, Ministry of Science and Technology, Government of India (Grant No. EMR/2014/000702). References [1] L.M. Mehlmann, Stops and starts in mammalian oocytes: recent advances in understanding the regulation of meiotic arrest and oocyte maturation, Reproduction 130 (2005) 791–799. [2] A.M. Luciano, F. Franciosi, S.C. Modina, V. Lodde, Gap junction-mediated communications regulate chromatin remodeling during bovine oocyte growth and differentiation through cAM-dependent mechanism(s), Biol. Reprod. 85 (2011) e1252–e1259.
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