Fig 1.
Identification of the Clytia MIHR.
(A) Diagram of a Clytia gonad showing the different tissues used for RNA-seq and GPCR expression comparisons. A total of 536 putative GPCRs identified in Clytia mixed-stages transcriptome were assigned to the three main GPCR classes (A, B, C) or “other” based on Pfam signatures. The 16 top candidate MIH GPCRs were selected based on oocyte enrichment, relatedness to known bilaterian class-A neuropeptide GPCRs, and Pfam information. (B) Luminescence response of CHO-K1 cells expressing the putative Clytia MIH GPCR treated with neuropeptide mixes lacking MIH activity (purple bars), MIH tetrapeptides identified from C. hemisphaerica (blue bars) or Cladonema radiatum (green bars), or related penta- and tripeptides previously shown to be ineffective in triggering oocyte maturation in vivo (red bars) [15]. Empty pcDNA3.1 vector was used as negative control and Platynereis FLamide and its receptor as a positive control (gray bars). Peptide concentrations all 1 μM. Absolute units of luminescence were normalized using the positive control; data are shown as mean ± standard error of the mean (n = 3). Full datasets are available in S1 Data. MIH tetrapeptides were selectively able to activate the Clytia GPCR, the responses closely matching the in vivo MIH activity of each peptide tested on Clytia oocytes as indicated (summarized results from [15]). (C) Dose–response curves of Clytia MIHR challenged with four variant Clytia MIH tetrapeptides. One of three independent experiments with equivalent results is shown. Luminescence values were normalized relative to the maximum of fitted dose–response curves and are shown as mean ± standard error of the mean (n = 3). Half maximal effective concentration (EC50) values were calculated as means of 3 independent experiments. Full datasets from three independent experiments are available in S2 Data. CHO-K1, Chinese Hamster ovary K1; GPCR, G protein–coupled receptor; MIH, maturation-inducing hormone; MIHR, MIH receptor; RNA-seq, RNA sequencing.
Fig 2.
Phenotypes of Clytia MIHR mutants.
Light and confocal microscope images of MIHR mutant F0 polyp colonies and jellyfish. (A) Morphology of a wild-type (WT) colony, Z11, and two MIHR mutant colonies (n5-8 and n5-13), as indicated. All MIHR mutant colonies contained gastrozooid and gonozooid polyps (pink and yellow arrows, respectively); however, the connecting stolons in some mutant colonies (see Table 1) were convoluted and frequently detached from the glass substrate, while stolons of WT colonies and the other mutant colonies were straight and adhered tightly. (B) Phenotypes of fully grown WT (top row) and mutant n5-8 or n5-24 (bottom row) jellyfish. Mutant and WT jellyfish show very similar morphology, but gonads developed poorly in these mutants, as shown by white arrows in the first column and at higher magnification in the second column. The third and fourth columns are confocal microscope images through the gonads of adult jellyfish from females and males, respectively; nuclei are stained with Hoechst 33342 (cyan) and F-actin with Phalloidin (red). In the n5-8 female gonad, small oocytes (arrows) can be detected between the endodermal (en) and ectodermal (ec) layers, but no large growing oocytes (oo) are present compared to the WT female gonad. In the n5-24 male gonad, the spermatogenic zone (asterisk) between endoderm and ectoderm is much thinner than in the WT male gonad. (C) Comparison of mutant n5-23 and n5-13 (bottom row) female medusae gonads (white arrows) swollen by an accumulation of large oocytes to WT (Z11) female medusae (top row). Right panels show gonads dissected from 3-week-old n5-23 medusae 10 hours after a light cue that induced spawning in the WT but not the mutant gonad. Black arrowheads indicate large growing oocytes in the WT gonad and black arrows indicate fully grown oocytes in the mutant gonad. (D) Adult mutant n5-10 and n5-6 male medusa (bottom row) compared to WT (Z13) male medusae (top row) have deformed gonads (white arrows). Right panels show isolated gonads, illustrating the thickened and irregular spermatogenic layer (arrowheads) in the mutant. Scale bars as indicated. MIHR, MIH receptor.
Table 1.
Characteristics of MIHR mutant colonies.
Fig 3.
Sites of MIHR expression in the Clytia medusa.
(A) In situ hybridization detection of MIHR mRNA in a young adult Clytia female medusa. Strong purple MIHR signal (orange arrows) was detected in oocytes within the gonads, as well as in scattered cells in tentacles. (B) Higher magnification images of MIHR mRNA detected in young adult medusae; with the gonad, each oocyte (e.g., at orange arrows) has an unstained nucleus. The intensity of labelling in individual oocytes decreases as they grow due to dilution of cytosol with yolk. In the tentacle, a row of individual MIHR-positive putative neural cells can be clearly distinguished leading from the center of the bulb on its oral face. (C) Comparison of the distribution of the MIH receptor and ligand expressing cells in different medusa structures as labelled, detected by in situ hybridization using probes to MIHR (top row) and to the MIH peptide precursors PP1 and/or PP4 as indicated (bottom row). Orange arrows point to oocytes, developing spermatozoa and tentacle MIHR cells, and white arrows indicate MIH cells. The focal plane in the male gonad image is through the center to illustrate the position of the MIH cells in the ectodermal layer. Weak staining at the base of the manubrium (black arrow) in A and B is frequently observed with probes for many genes and is probably due to a specific trapping of the color reagent. Scale bars: 100 μm. (D) Confocal images of the three main sites of MIH-expressing cells (white arrows) in medusae, visualized using anti-PRPamide antibody (MIH: white), anti-tyrosinated tubulin (magenta), and Hoechst staining of nuclei (blue). Summed z-stacks are shown in all cases except for the gonad tubulin and DNA staining, where a single plane was selected through the center of the gonad. All scale bars: 100 μm. MIH, maturation-inducing hormone; MIHR, MIH receptor.
Fig 4.
Oocyte maturation failure in MIHR mutant medusae.
Oocyte maturation assays performed using isolated gonads (A, B) or isolated oocytes (C) from MIHR mutant jellyfish compared to WT. In A and C, bar heights represent mean percentages of 3 independent experiments and error bars show standard deviations. Total gonad or oocyte numbers for each treatment are indicated in gray. (A) Spawning response of isolated gonads from WT and n5-23 female medusae. Three treatments were compared as indicated above each panel: Light: light stimulation after incubation in the dark; MIH: treatment of light-maintained gonads with 100 nM WPRPamide; cAMP: treatment of light-maintained gonads with 4 mM Br-cAMP. No oocyte maturation or spawning was observed in MIHR KO gonads upon light stimulation or MIH treatment, while Br-cAMP treatment provoked oocyte maturation and spawning. The Fisher exact test showed significant differences (F = 0) between wild-type and mutant responses to light and MIH, but not for the cAMP treatment (F = 1). Full datasets from three independent experiments are available in S3 Data. (B) Light microscope images illustrating gonads from an equivalent experiment performed with n5-13 female MIHR mutant medusae 120 minutes after the indicated treatments. Scale bar: 500 μm. (C) Response of fully grown oocytes isolated from WT and n5-23 MIHR mutant gonads to MIH and Br-cAMP treatments as in A. Both treatments triggered maturation of WT oocytes, visible after 20 to 30 minutes as germinal vesicle breakdown (GVBD), but only Br-cAMP induced maturation of MIHR KO oocytes. Control experiments using the Br-cAMP solvent (distilled water) showed a low level of spontaneous maturation in both cases. The Fisher exact test did not show significant differences between WT and mutant oocytes in the control (F = 0.101) or cAMP-treated (F = 0.216) groups, but did so in the MIH assays (F = 0). Full datasets from 3 independent experiments are available in S3 Data. Br-cAMP, bromoadenosine 3′,5′-cyclic monophosphate; cAMP, cyclic adenosine monophosphate; KO, knockout; MIH, maturation-inducing hormone; MIHR, MIH receptor; WT, wild-type.
Fig 5.
Involvement of Gαs in Clytia oocyte maturation.
(A) Results of an antibody inhibition experiment. Maturation response (scored as percent GVBD over time) of isolated oocytes injected with antibodies or buffer, then challenged first with a low dose of MIH (10 nM WPRPamide), then with a higher dose (100 nM WPRPamide), and finally with Br-cAMP to verify maturation competence, as indicated by the colored arrows and background shading. Oocytes injected with PBS (blue circles) or a control anti-GST antibody (orange squares) responded efficiently to MIH, whereas very few oocytes injected with anti-Gαs (black triangles) underwent GVBD after treatment with the low dose of MIH and only 29% following the high dose. The number of oocytes per group in this experiment was 30, 28, and 30, respectively. All of them had undergone GVBD by the end of the experiment. Times from the start of the first incubation are shown on the x-axis. Full datasets from five independent experiments are available in S4 Data. (B) Scheme illustrating the proposed cascade initiating Clytia oocyte maturation initiation. Following light stimulation after a dark period, Opsin9 mediates release of MIH neuropeptides from specialized cells (purple) of the gonad ectoderm (cyan). Activation of the MIHR (green) at the oocyte surface releases GαS to promote an increase in cytoplasmic cAMP, activating PKA. Unknown PKA substrates likely trigger in parallel Cdk1 activation and thus GVBD, and Mos1 synthesis to initiate the MAPK cascade.; Br-cAMP, bromoadenosine 3′,5′-cyclic monophosphate; GST, Glutathione-S-Transferase; GVBD, germinal vesicle breakdown; MIH, maturation-inducing hormone; MIHR, MIH receptor; PKA, protein kinase A.
Fig 6.
Relationship of Clytia MIHR to bilaterian neuropeptide hormone GPCRs.
(A) Sequence-similarity-based clustering using Clans2 of all identified class-A GPCRs from Clytia, human (olfactory receptors excluded), and Platynereis deorphanized GPCRs [28] BLASTP p-value < 1e-40. (B) Cluster map of the largest cluster (circled in red in A), keeping only sequences that show at least 2 connections with the central cluster. BLASTP p-value < 1e-40. (C) More stringent cluster map (p-value < 1e-50) of the same sequences as in (A) plus all Nematostella GPCR-A sequences. Only clusters containing at least 5 sequences from at least 2 species were kept. All connections with p-value <1e-40 are shown. (D) Maximum likelihood analysis of the sequences contained inside the dashed area shown in (C) using RaxML (PROTGAMMAGTR) with 500 Bootstrap replicates (BR). Rhodopsin beta GPCRs are rooted against rhodopsin gamma GPCRs [37]. Tree file provided in S2 Fig. Color code: Homo sapiens: blue, Platynereis dumerilli: green, Nematostella vectensis: black, Clytia: orange. Red star: Clytia MIHR. FSH, follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; GPCR, G protein–coupled receptor; GPR3, G protein–coupled receptor 3; LH, luteinizing hormone; MIHR, MIH receptor; NPY, neuropeptide Y; P2Y, purinoceptor; QRFP, pyroglutamylated RFamide peptide.
Fig 7.
Schematic comparison of GPCR regulation of Clytia and vertebrate oocyte maturation.
Simplified view of the tissues, hormones, and receptors involved in regulating oocyte maturation in Clytia and in fish/amphibians and mammals. For simplicity, we have not included protostome or echinoderm models. The principle peptide hormones of the reproductive hypothalamus-pituitary-gonadal axis (GnRH and LH/FSH) are in pink, and those for which the receptors group phylogenetically with Clytia MIHR in “Group A” (Fig 6) are in purple. Peptide hormones: Clytia MIH, Neuropeptide Y (NPY), GnIH, GnRH, LH, QRFP, NkB, and C-type natriuretic peptide (CNP). All their receptors, except the guanylyl cyclase natriuretic peptide receptor 2 (NPR2) activated by CNP, are GPCRs (green). Constitutively active (CA) GPCRs in vertebrate oocytes maintain cytoplasmic cAMP levels high prior to maturation. In mouse oocytes, a cAMP decrease upon hormone stimulation triggers maturation; however, in fish and frog oocytes the degree and role of this decrease is debated. Several types of oocytes receptor (orange) may respond to steroid hormones (Pg) in different species of amphibians and fish, but the relative importance of multiple downstream signalling pathways remains to be clarified [1,43,44,45,46]. See text for discussion. CNP, C-type natriuretic peptide; FSH, follicle-stimulating hormone; GnIH, gonadotropin inhibitory hormone; GnRH, gonadotropin-releasing hormone; GPCR, G protein–coupled receptor; LH, luteinizing hormone; LHR, lutenizing hormone receptor; MIH, maturation-inducing hormone; MIHR, MIH receptor; NkB, neurokinin B; QRFP, pyroglutamylated RFamide peptide.