Mechanisms for type-II vitellogenesis-inhibiting hormone suppression of vitellogenin transcription in shrimp hepatopancreas: Crosstalk of GC/cGMP pathway with different MAPK-dependent cascades

Shrimp experiments were carried out according to the Care and Use of Agricultural Animals in Agricultural Research and Teaching and approved by the Science and Technology Bureau of China. All animal experiments were conducted in accordance with the guidelines and approval of the Ethics Committees of the South China Sea Institute of Oceanology, Chinese Academy of Sciences.

Pacific white shrimp (L. vannamei), with a body weight of 48.4±1.6 g and a body length of 18.3±0.3 cm, were collected from the Jinyang shrimp culture center, Maoming, China, and maintained in seawater at 28 °C under a 12D:12L photoperiod. Previtellogenic female shrimp, with the gonadosomatic index (GSI) of 0.62±0.10% were selected for in vivo injection and in vitro cell cultural experiments. Additionally, the Vg mRNA levels were detected in the hepatopancreas of female shrimp in different ovarian developmental stages, including stage O (immature, GSI of <0.3%), stage I (previtellogenesis, GSI of 0.4–0.8%), stage II (primary vitellogenesis, GSI of 0.9–2.4%), stage III (secondary vitellogenesis, GSI of 2.7–4.5%) and stage IV (maturation, GSI of >5.2%), were determined by using the paraffin sections stained with hematoxylin/eosin (H/E) staining as previously described before [48, 49]. For comparison, the Vg mRNA level was also examined in hepatopancreas of sexually immature male shrimp (GSI of <0.3%).

The L. vannamei VIH-2 recombinant proteins were expressed in Escherichia coli and purified by using immobilized metal ion affinity chromatography (IMAC) as previously described [15]. For pharmacological study, NOC-18, A-350619, 8-Br-cGMP, L-NAME, Zn(II)PPIX, Rp-8-Br-PET-cGMPS, SB02190, SP600125 and BI-87G3 were purchased from Sigma, while IBMX, forskolin, 8-Br-cAMP, PD169316↗, PD98059 and U0126 were acquired from Calbiochem. Test substances were prepared as 1 mM or 10 mM frozen stocks in distilled water or DMSO in small aliquots and diluted with a pre-warmed culture medium to appropriate concentrations 15 min prior to drug treatments.

VIH-2 recombinant protein was diluted in PBS (33‰ salinity). Only the previtellogenic female shrimp were used for VIH injection experiments. Each shrimp was injected with 100 μL of VIH-2 in PBS, and PBS injection alone was used as a negative control. Hepatopancreatic samples (n = 6) from each group were randomly sampled at 0−12 h and 0−24 h post injection to measure second messenger molecules, and mRNA and protein samples, respectively.

The L. vannamei hepatopancreatic cells were prepared as previously described [15]. Briefly, hepatopancreas from previtellogenic female shrimp (GSI = 0.62±0.10%, n = 10) were excised and washed three times in pre-chilled Ca²⁺/Mg²⁺-free Hanks balanced salt solution (HBSS, Sigma; containing 4 mM NaHCO₃, 9 mM HEPES, 154 mM NaCl, 100 U/mL penicillin and 100 μg/mL streptomycin, pH 7.63). Hepatopancreatic fragments were diced into 0.5-mm thickness, incubated in Ca²⁺/Mg²⁺-free HBSS with EDTA (1 mM) at room temperature for 5 min, and digested with collagenase type IV (1 mg/mL, Invitrogen) and DNase II (0.01 mg/mL, Sigma) in Ca²⁺/Mg²⁺-free HBSS at 28 °C for 30 min. Next, shrimp hepatopancreatic fragments were mechanically dispersed into single cells by gentle pipetting. Dispersed cells were then separated from the remaining fragments by filtration through a sterile mesh (30 μm pore size) and harvested by centrifugation at 100×g for 5 min at 4 °C. The cells obtained were resuspended in a Dulbecco-modified Eagle medium (DMEM)/F-12 (1:1, Gibco BRL; containing 14 mM NaHCO₃, 2 g/L bovine serum albumin, 154 mM NaCl, 100 U/mL penicillin and 100 μg/mL streptomycin, pH 7.63). Only the cell preparations with greater than 95% viability that assessed by using a trypan blue exclusion assay were used in subsequent experiments. Dispersed hepatopancreatic cells were diluted in DMEM/F-12 to a final density of 0.4×10⁶ cells/mL and cultured in polyethylenimine (PEI, 5 μg/mL, Sigma) pre-coated 24-well plates with 5% CO₂ and saturated humidity at 28 °C overnight for recovery.

On the second day after cell preparation, test substances prepared in DMEM/F12 medium were gently overlaid onto hepatopancreatic cells after the removal of the old culture medium. In pharmacological studies, the cells were incubated with test substances for another 12 h as previously validated [15].

For measurement of L. vannamei Vg (AY321153↗), p38 mitogen-activated protein kinases (P³⁸MAPK, JX990130↗), extracellular regulated protein kinases (ERK, JN035901↗) and c-Jun N-terminal kinase (JNK, JN035903↗) gene expressions in the hepatopancreas and in the hepatopancreatic primary cells, total RNA was isolated by using the TRIzol reagent (Invitrogen), digested with DNase I (Invitrogen), and reversely transcribed by using a PrimeScript RT kit (TaKaRa). Transcriptional expression of target genes were detected using SYBR Premix Ex Taq II (TaKaRa) in the RotorGene RG-3000 real-time PCR System (Qiagen) with primers and PCR conditions as shown in S1 Table. Serially diluted plasmid DNAs containing the ORF sequences for the target genes were used as the standards for the real-time PCRs. After reactions, the identities of the PCR products were routinely confirmed by analysis of the melting curve. In this study, real-time PCR of L. vannamei β-actin (JF288784↗) was used as an internal control because no significant changes were noted for β-actin mRNA in our studies. The raw data of the target gene transcripts were expressed in terms of fmol detected per tube, and the data were routinely normalized as the ratio of β-actin mRNA detected in the same sample.

VIH-2 effects on NO, cGMP and cAMP production were tested on hepatopancreatic tissues and primary cells from previtellogenic female L. vannamei. The hepatopancreatic tissues were sampled and frozen in liquid nitrogen immediately for further experiments. The hepatopancreatic cells were cultured at DMEM/F12 medium with a density of 1×10⁶ cells/mL in PEI-coated 6-well plates. To measure NO, after an overnight recovery, the old medium was replaced with a fresh medium containing the VIH-2 protein. NO production was estimated by spectrophotometrically measuring the levels of nitrite as a proxy of NO by using a Griess reagent system (Promega) as we previously described [50]. For measurement of GMP and cAMP, after an overnight recovery, the old medium was replaced with 0.9 mL fresh medium supplemented with IBMX (0.1 mM) and incubated at 28 °C for 15 min, and then 0.1 mL 10×stock solutions of VIH-2 was added. The cGMP and cAMP content were quantified using the Cyclic-GMP XP^® Assay Kit (Cell Signaling) and the Cyclic-AMP XP^® Assay Kit (Cell Signaling), respectively, as previously described [51].

Western blots were performed on the samples of previtellogenic female L. vannamei hepatopancreatic tissues and primary cells. For tissues samples, the shrimp hepatopancreas were collected and frozen in liquid nitrogen immediately, then dissolved in RIPA Lysis Buffer I (Sangon Biotech) with complete protease and phosphatase inhibitor cocktails (Roche) by extraction with a 23-gauge needle attached to a 1-mL syringe on ice. For cell samples, the shrimp hepatopancreatic cells were seeded in PEI-coated 6-well plates at a density of 1×10⁶ cells/mL/well. After incubating overnight, the culture medium was replaced with new medium containing the appropriate concentrations of test substances and cultured at 28 °C. The VIH incubation lasted from 0 to 24 h, and for other pharmacological studies, the treatment time was fixed at 1 h. Then, the cells were rinsed with PBS (pH 7.4) and cell lysate were prepared in RIPA Lysis Buffer IV (Sangon Biotech) with complete protease and phosphatase inhibitor cocktails by freezing and thawing for three times. The tissue and cell lysates were resolved on a 15% gel by using SDS-PAGE and transblotted onto a PVDF membrane (Roche) at 20 V for 1 h with a Trans-Blot SD Electrophoretic Cell (Bio-Rad). The membrane was then blocked using 3% BSA in TBST (Sangon Biotech) and incubated overnight at 4 °C with antibodies for Phospho-P³⁸MAPK (1:500, Cat#9211, Cell Signaling), total P³⁸MAPK (1:500, Cat#8690, Cell Signaling), Phospho-ERK (1:500, Cat#9102, Cell Signaling), total ERK (1:1000, Cat#9101, Cell Signaling), Phospho-JNK (1:1000, Cat#9251, Cell Signaling), total JNK (1:2000, Cat#9252, Cell Signaling) and ß-actin (1:2000, Cat#D190606, Sangon Biotech). On the following day, HRP-conjugated goat anti-rabbit (1:2000, Cat#DC03L, Calbiochem) or rabbit anti-mouse IgG (1:2000, Cat#AP160P, Calbiochem) was added and Immobilon Western (Pierce) was used as an HRP substrate for signal development.

The data expressed as the mean±S.E. and were analyzed by using Student’s t-test or one-way ANOVA followed by Fisher’s least significant difference (LSD) test with SPSS (IBM Software).

Vg transcript expressions were detected in the hepatopancreas from female shrimp with different ovarian maturation stages (stages I-IV), and the expressions in the hepatopancreas from sexually immature female (stage O) and male shrimp were used as the controls. The results showed that the hepatopancreatic Vg mRNA in sexually immature female shrimp were higher than in sexual immature male shrimp (Fig 1A). The hepatopancreatic Vg mRNA expression of female shrimp increased acutely and continuously during ovarian maturation (stages I-III) after eyestalk ablation (Fig 1B). The peak of Vg mRNA expression occurred at the stage III, followed by a rapid decrease at stage IV (Fig 1B).

The effects of VIH-2 on nitric oxide (NO), cGMP and cAMP production were measured in both the in vivo and in vitro experiments. By using the in vivo injection approaches, VIH-2 showed the ability to elevate the concentrations of cGMP (Fig 2B) but not NO (Fig 2A) or cAMP (Fig 2C) in the shrimp hepatopancreas. In the in vitro experiments that performed on the hepatopancreatic primary cells, VIH-2 administration was capable of inducing cGMP production within 1.5–6 h, with a peak of 3 h (Fig 2E). Meanwhile, this peptide was not able to change the production of NO (Fig 2D) or cAMP (Fig 2F). In a parallel experiment incubated with an increased concentration of substrates, VIH-2 showed a dose-dependent effect on cGMP production (Fig 2H), but no effects on NO (Fig 2G) or cAMP (Fig 2I). As positive controls, an NO donor (NOC-18), a soluble guanylyl cyclase (GC) activator (A-350619) and an adenylate cyclase (AC) activator (forskolin) effectively stimulated NO (Fig 2G), cGMP (Fig 2H) and cAMP (Fig 2I) production, respectively, in the hepatopancreatic primary cells.

The roles of the cGMP signaling pathway in VIH-2-regulated Vg gene expression in shrimp hepatopancreas were further investigated using pharmacological approaches. In this case, incubation of the NO donor (NOC-18), the GC activator (A-350619) or the membrane-permeable cGMP analog (cpt-cGMP) consistently reduced Vg mRNA expression in dose-dependent ways (Fig 3A–3C). On the other hand, when co-incubated with the inhibitors of GC [Protoporphyrin IX zinc(II)] or protein kinase G (PKG, Rp-8-Br-PET-cGMPS) but not with the non-specific inhibitor of NOS (L-NAME), the inhibitory effect of VIH-2 on Vg mRNA expression was abolished (Fig 3D–3F). Combined with the results above, VIH-2 may down-regulate Vg gene expression in shrimp hepatopancreas via the cGMP/PKG signal pathway without NO participation.

The functions of cAMP signaling in hepatopancreatic Vg gene expression were also developed. Interestingly, incubation of both the AC activator (forskolin) and the membrane-permeable cAMP analog (8-Br-cAMP) increased the mRNA levels of Vg in dose-dependent ways (Fig 4A & 4B). Additionally, co-incubation of the AC activator (forskolin) or the cAMP analog (8-Br-cAMP) reversed the inhibitory effects of VIH-2 on Vg gene expression (Fig 4C). In sum, the AC/cAMP cascade is a positive signaling for Vg gene expression in the shrimp hepatopancreas. However, this signal pathway is independent of the VIH-2 regulation on Vg expression.

The involvements of MAPK signal pathways including P³⁸MAPK, ERK and JNK in Vg regulation by VIH-2 in shrimp hepatopancreas were evaluated in this study. In both in vivo injection and in vitro primary cell incubation experiments, VIH-2 was effective at triggering JNK phosphorylation in a time-dependent manner without affecting the total JNK content (Fig 5A & 5B). In contrast, the enhanced P³⁸MAPK phosphorylation by VIH-2 was based on the increase of P³⁸MAPK content (Fig 5A & 5B). Furthermore, VIH-2 treatment changed neither ERK’s phosphorylation nor content in both in vivo and in vitro experiments (Fig 5A & 5B).

In parallel experiments, the mRNAs of shrimp P³⁸MAPK, ERK and JNK were measured after VIH-2 treatment. In this case, VIH-2 increased the expression only of P³⁸MAPK mRNA in both in vivo (Fig 5C) and in vitro (Fig 5F) experiments; the expressions of ERK (Fig 5D & 5G) and JNK (Fig 5E & 5H) transcripts were not affected. Therefore, it is logically to speculate that VIH-2 may enhance P³⁸MAPK phosphorylation by increasing the mRNA expression followed by protein production. However, JNK phosphorylation triggered by VIH-2 is independent of the regulation at the transcript level.

Further pharmacological experiments were performed on the hepatopancreatic primary cells at short and long time points, namely, 12 h and 36 h, respectively, to test the effect of three MAPK cascades blockage on the VIH-2-inhibited Vg gene expression. In this case, co-incubation of the P³⁸MAPK inhibitors (PD169316↗ and SB02190) partially reversed the inhibitory action of VIH-2 on Vg gene expression, with a more potent effects at 36 h than at 12 h (Fig 6A); while the JNK inhibitors (SP600125 and BI-87G3) partially reversed the VIH-2 action but were more potent at 12 h than at 36 h (Fig 6C). In contrast, the ERK inhibitors (PD98059 and U0126) did not change the Vg mRNA levels when VIH-2 was added (Fig 6B). In addition, when the P³⁸MAPK inhibitor (PD169316↗) and the JNK inhibitor (SP600125) were added together, the inhibitory role of VIH-2 on Vg gene expression was completely blocked at both 12 h and 36 h time (Fig 6D), suggesting that the inhibitory effect of VIH-2 on Vg gene expression was mediated by a combination of the P³⁸MAPK and JNK cascades.

The relationship between the GC/cGMP/PKG and MAPK cascades in the signaling of VIH-2 regulation on Vg gene expression was determined further using pharmacological approaches. In the hepatopancreatic primary cells, incubation of the GC activator (A-350619) and the cGMP analog (cpt-cGMP) mimicked VIH-2’s effect on JNK phosphorylation (Fig 7A). On the other hand, when the GC and PKG activities were blocked by GC inhibitor [Protoporphyrin IX zinc(II)] and PKG inhibitor (Rp-8-Br-PET-cGMPS), respectively, the phosphorylation trigged by VIH-2 treatment might be eliminated (Fig 7B).

The mediated role of GC/cGMP/PKG on P³⁸MAPK mRNA expression was similar to that on JNK phosphorylation. Both the GC activator (A-350619) and the cGMP analog (cpt-cGMP) could dose-dependently stimulate P³⁸MAPK transcript expression (Fig 7C & 7D). In parallel experiment, the VIH-2-induced P³⁸MAPK gene expression could be blocked by co-treatment of the GC inhibitor [Protoporphyrin IX zinc(II), Fig 7E] and PKG inhibitor (Rp-8-Br-PET-cGMPS, Fig 7F).

In contrast, co-incubation of all inhibitors of MAPK cascades including PD169316↗ and SB02190 for P³⁸MAPK (Fig 7G), PD98059 and U0126 for ERK (Fig 7H), and SP600125 and BI-87G3 for JNK (Fig 7I) were not able to block the stimulation of VIH-2 on cGMP production in the hepatopancreatic primary cells. The sum of these results implies that in the VIH-2 signaling, the GC/cGMP/PKG cascades are upstream and the MAPK cascades are downstream.

All relevant data are within the paper and its Supporting Information files.