Progesterone induces meiosis through two obligate co-receptors with PLA2 activity
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eLife assessment
This important study provides solid evidence for a non-genomic action of progesterone in Xenopus oocyte activation. The findings demonstrate that two non-genomic progesterone receptors, ABHD2 and mPRb, function as a novel progesterone-stimulated phospholipase A2. However, the findings are reliant on high concentrations of inhibitor drugs, and mechanistic details about the molecular interaction and respective functions of ABHD2 and mPRb are incomplete. The findings will be of broad interest to reproductive endocrinologists and physiologists.
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Abstract
The steroid hormone progesterone (P4) regulates multiple aspects of reproductive and metabolic physiology. Classical P4 signaling operates through nuclear receptors that regulate transcription. In addition, P4 signals through membrane P4 receptors (mPRs) in a rapid nongenomic modality. Despite the established physiological importance of P4 nongenomic signaling, its detailed signal transduction remains elusive. Here, using Xenopus oocyte maturation as a well-established physiological readout of nongenomic P4 signaling, we identify the lipid hydrolase ABHD2 (α/β hydrolase domain-containing protein 2) as an essential mPRβ co-receptor to trigger meiosis. We show using functional assays coupled to unbiased and targeted cell-based lipidomics that ABHD2 possesses a phospholipase A2 (PLA2) activity that requires both P4 and mPRβ. This PLA2 activity bifurcates P4 signaling by inducing mPRβ clathrin-dependent endocytosis and producing lipid messengers that are G-protein coupled receptors agonists. Therefore, P4 drives meiosis by inducing the ABHD2 PLA2 activity that requires both mPRβ and ABHD2 as obligate co-receptors. Nongenomic progesterone signaling is important for many physiological functions yet the details of its signaling remain elusive. Here we define the early signaling steps downstream of membrane progesterone receptor β (mPRβ) during Xenopus oocyte meiosis. We show that progesterone requires two cell membrane receptors to work in unison to signal. The co-receptor complex possesses lipase activity that produces lipid messenger and induces receptor endocytosis to trigger meiosis progression. Our findings have broad physiological implications because nongenomic progesterone signaling operates in many tissues and regulates reproduction and metabolism.
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eLife assessment
This important study provides solid evidence for a non-genomic action of progesterone in Xenopus oocyte activation. The findings demonstrate that two non-genomic progesterone receptors, ABHD2 and mPRb, function as a novel progesterone-stimulated phospholipase A2. However, the findings are reliant on high concentrations of inhibitor drugs, and mechanistic details about the molecular interaction and respective functions of ABHD2 and mPRb are incomplete. The findings will be of broad interest to reproductive endocrinologists and physiologists.
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Reviewer #1 (Public Review):
Summary:
Numerous pathways have been proposed to elucidate the nongenomic actions of progesterone within both male and female reproductive tissues. The authors employed the Xenopus oocyte system to investigate the PLA2 activity of ABHD2 and the downstream lipid mediators in conjunction with mPRb and P4, on their significance in meiosis. The research has been conducted extensively and is presented clearly.
Strengths:
While the interaction between membranous PR and ABHD2 is not a novel concept, this present study exhibits several strengths:
1. mPRbeta, a member of the PAQR family, has been elusive in terms of detailed signal transduction. Through mutation studies involving the Zn binding domain, the authors discovered that the hydrolase activity of mPRbeta is not essential for meiosis and oocyte maturation. …
Reviewer #1 (Public Review):
Summary:
Numerous pathways have been proposed to elucidate the nongenomic actions of progesterone within both male and female reproductive tissues. The authors employed the Xenopus oocyte system to investigate the PLA2 activity of ABHD2 and the downstream lipid mediators in conjunction with mPRb and P4, on their significance in meiosis. The research has been conducted extensively and is presented clearly.
Strengths:
While the interaction between membranous PR and ABHD2 is not a novel concept, this present study exhibits several strengths:
1. mPRbeta, a member of the PAQR family, has been elusive in terms of detailed signal transduction. Through mutation studies involving the Zn binding domain, the authors discovered that the hydrolase activity of mPRbeta is not essential for meiosis and oocyte maturation. Instead, they suggest that ABHD2, acting as a coreceptor of mPRbeta, demonstrates phospholipase activity, indicating that downstream lipid mediators may play a dominant role when stimulated by progesterone.
2. Extensive exploration of downstream signaling pathways and the identification of several potential meiotic activity-related lipid mediators make this aspect of the study novel and potentially significant.
Weaknesses:
However, there are some weaknesses and areas that need further clarification:
1. The mechanism governing the molecular assembly of mPRbeta and ABHD2 remains unclear. Are they constitutively associated or is their association ligand-dependent? Does P4 bind not only to mPRbeta but also to ABHD2, as indicated in Figure 6J? In the latter case, the reviewer suggests that the authors conduct a binding experiment using labeled P4 with ABHD2 to confirm this interaction and assess any potential positive or negative cooperativity with a partner receptor.
2. The authors have diligently determined the metabolite profile using numerous egg cells. However, the interpretation of the results appears incomplete, and inconsistencies were noted between Figure 2B and Supplementary Figure 2C. Furthermore, PGE2 and D2 serve distinct roles and have different elution patterns by LC-MS/MS, thus requiring separate measurements. In addition, the extremely short half-life of PGI2 necessitates the measurement of its stable metabolite, 6-keto-PGF1a, instead. The authors also need to clarify why they measured PGF1a but not PGF2a.
3. Although they propose PGs, LPA, and S1P are important downstream mediators, the exact roles of the identified lipid mediators have not been clearly demonstrated, as receptor expression and activation were not demonstrated. While the authors showed S1PR3 expression and its importance by genetic manipulation, there was no observed change in S1P levels following P4 treatment (Supplementary Figure 2D). It is essential to identify which receptors (subtypes) are expressed and how downstream signaling pathways (PKA, Ca, MAPK, etc.) relate to oocyte phenotypes.
These clarifications and further experiments would enhance the overall impact and comprehensiveness of the study.
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Reviewer #2 (Public Review):
Summary:
This interesting paper examines the earliest steps in progesterone-induced frog oocyte maturation, an example of non-genomic steroid hormone signaling that has been studied for decades but is still very incompletely understood. In fish and frog oocytes it seems clear that mPR proteins are involved, but exactly how they relay signals is less clear. In human sperm, the lipid hydrolase ABHD2 has been identified as a receptor for progesterone, and so the authors here examine whether ABHD2 might contribute to progesterone-induced oocyte maturation as well. The main results are:
1. Knocking down ABHD2 makes oocytes less responsive to progesterone, and ectopically expressing ABHD2.S (but not the shorter ABHD2.L gene product) partially rescues responsiveness. The rescue depends upon the presence of critical …
Reviewer #2 (Public Review):
Summary:
This interesting paper examines the earliest steps in progesterone-induced frog oocyte maturation, an example of non-genomic steroid hormone signaling that has been studied for decades but is still very incompletely understood. In fish and frog oocytes it seems clear that mPR proteins are involved, but exactly how they relay signals is less clear. In human sperm, the lipid hydrolase ABHD2 has been identified as a receptor for progesterone, and so the authors here examine whether ABHD2 might contribute to progesterone-induced oocyte maturation as well. The main results are:
1. Knocking down ABHD2 makes oocytes less responsive to progesterone, and ectopically expressing ABHD2.S (but not the shorter ABHD2.L gene product) partially rescues responsiveness. The rescue depends upon the presence of critical residues in the protein's conserved lipid hydrolase domain, but not upon the presence of critical residues in its acyltransferase domain.
2. Treatment of oocytes with progesterone causes a decrease in sphingolipid and glycerophospholipid content within 5 min. This is accompanied by an increase in LPA content and arachidonic acid metabolites. These species may contribute to signaling through GPCRs. Perhaps surprisingly, there was no detectable increase in sphingosine-1-phosphate, which might have been expected given the apparent substantial hydrolysis of sphingolipids. The authors speculate that S1P is formed and contributes to signaling but diffuses away.
3. Pharmacological inhibitors of lipid-metabolizing enzymes support, for the most part, the inferences from the lipidomics studies, although there are some puzzling findings. The puzzling findings may be due to uncertainty about whether the inhibitors are working as advertised.
4. Pharmacological inhibitors of G-protein signaling support a role for G-proteins and GPCRs in this signaling, although again there are some puzzling findings.
5. Reticulocyte expression supports the idea that mPR and ABHD2 function together to generate a progesterone-regulated PLA2 activity.
6. Knocking down or inhibiting ABHD2 inhibited progesterone-induced mPRinternalization, and knocking down ABHD2 inhibited SNAP2520-induced maturation.
Strengths:
All in all, this could be a very interesting paper and a nice contribution. The data add a lot to our understanding of the process, and, given how ubiquitous mPR and AdipoQ receptor signaling appear to be, something like this may be happening in many other physiological contexts.
Weaknesses:
I have several suggestions for how to make the main points more convincing.
Main criticisms:
1. The ABHD2 knockdown and rescue, presented in Fig 1, is one of the most important findings. It can and should be presented in more detail to allow the reader to understand the experiments better. E.g.: the antisense oligos hybridize to both ABHD2.S and ABHD2.L, and they knock down both (ectopically expressed) proteins. Do they hybridize to either or both of the rescue constructs? If so, wouldn't you expect that both rescue constructs would rescue the phenotype since they both should sequester the AS oligo? Maybe I'm missing something here.
In addition, it is critical to know whether the partial rescue (Fig 1E, I, and K) is accomplished by expressing reasonable levels of the ABHD2 protein, or only by greatly overexpressing the protein. The author's antibodies do not appear to be sensitive enough to detect the endogenous levels of ABHD2.S or .L, but they do detect the overexpressed proteins (Fig 1D). The authors could thus start by microinjecting enough of the rescue mRNAs to get detectable protein levels, and then titer down, assessing how low one can go and still get rescue. And/or compare the mRNA levels achieved with the rescue construct to the endogenous mRNAs.
Finally, please make it clear what is meant by n = 7 or n = 3 for these experiments. Does n = 7 mean 7 independently lysed oocytes from the same frog? Or 7 groups of, say, 10 oocytes from the same frog? Or different frogs on different days? I could not tell from the figure legends, the methods, or the supplementary methods. Ideally one wants to be sure that the knockdown and rescue can be demonstrated in different batches of oocytes, and that the experimental variability is substantially smaller than the effect size.
2. The lipidomics results should be presented more clearly. First, please drop the heat map presentations (Fig 2A-C) and instead show individual time course results, like those shown in Fig 2E, which make it easy to see the magnitude of the change and the experiment-to-experiment variability. As it stands, the lipidomics data really cannot be critically assessed.
[Even as heat map data go, panels A-C are hard to understand. The labels are too small, especially on the heat map on the right side of panel B. The 25 rows in panel C are not defined (the legend makes me think the panel is data from 10 individual oocytes, so are the 25 rows 25 metabolites? If so, are the individual oocyte data being collapsed into an average? Doesn't that defeat the purpose of assessing individual oocytes?) And those readers with red-green colorblindness (8% of men) will not be able to tell an increase from a decrease. But please don't bother improving the heat maps; they should just be replaced with more informative bar graphs or scatter plots.]
3. The reticulocyte lysate co-expression data are quite important and are both intriguing and puzzling. My impression had been that to express functional membrane proteins, one needed to add some membrane source, like microsomes, to the standard kits. Yet it seems like co-expression of mPR and ABHD2 proteins in a standard kit is sufficient to yield progesterone-regulated PLA2 activity. I could be wrong here - I'm not a protein expression expert - but I was surprised by this result, and I think it is critical that the authors make absolutely certain that it is correct. Do you get much greater activities if microsomes are added? Are the specific activities of the putative mPR-ABHD2 complexes reasonable?
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Reviewer #3 (Public Review):
Summary:
The authors report two P4 receptors, ABHD2 and mPRβ that function as co-receptors to induce PLA2 activity and thus drive meiosis. In their experimental studies, the authors knock down ABHD2 and demonstrated inhibition of oocyte maturation and inactivation of Plk1, MAPK, and MPF, which indicated that ABHD2 is required for P4-induced oocyte maturation. Next, they showed three residues (S207, D345, H376) in the lipase domain that are crucial for ABHD2 P4-mediated oocyte maturation in functional assays. They performed global lipidomics analysis on mPRβ or ABHD2 knockdown oocytes, among which the downregulation of GPL and sphingolipid species were observed, and enrichment in LPA was also detected using their metabolomics method. Furthermore, they investigated pharmacological profiles of enzymes predicted …
Reviewer #3 (Public Review):
Summary:
The authors report two P4 receptors, ABHD2 and mPRβ that function as co-receptors to induce PLA2 activity and thus drive meiosis. In their experimental studies, the authors knock down ABHD2 and demonstrated inhibition of oocyte maturation and inactivation of Plk1, MAPK, and MPF, which indicated that ABHD2 is required for P4-induced oocyte maturation. Next, they showed three residues (S207, D345, H376) in the lipase domain that are crucial for ABHD2 P4-mediated oocyte maturation in functional assays. They performed global lipidomics analysis on mPRβ or ABHD2 knockdown oocytes, among which the downregulation of GPL and sphingolipid species were observed, and enrichment in LPA was also detected using their metabolomics method. Furthermore, they investigated pharmacological profiles of enzymes predicted to be important for maturation based on their metabolomic analyses and ascertained the central role of PLA2 in inducing oocyte maturation downstream of P4. They showed the modulation of S1P/S1PR3 pathway on oocyte maturation and the potential role for Gαs signaling and potentially Gβγ downstream of P4.
Strengths:
The authors make a very interesting finding that ABHD2 has PLA2 catalytic activity but only in the presence of mPRβ and P4. Finally, they provided supporting data for a relationship between ABHD2/PLA2 activity and mPRβ endocytosis and further downstream signaling. Collectively, this research report defines early steps in nongenomic P4 signaling, which has broad physiological implications.
Weaknesses:
There were concerns with the pharmacological studies presented. Many of these inhibitors are used at high (double-digit micromolar) concentrations that could result in non-specific pharmacological effects and the authors have provided very little data in support of target engagement and selectivity under the multiple experimental paradigms. In addition, the use of an available ABHD2 small molecule inhibitor was lacking in these studies.
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