Phosphatases modified by LH signaling in ovarian follicles: testing their role in regulating the NPR2 guanylyl cyclase†.

In response to luteinizing hormone (LH), multiple proteins in rat and mouse granulosa cells are rapidly dephosphorylated, but the responsible phosphatases remain to be identified. Because the phosphorylation state of phosphatases can regulate their interaction with substrates, we searched for phosphatases that might function in LH signaling by using quantitative mass spectrometry. We identified all proteins in rat ovarian follicles whose phosphorylation state changed detectably in response to a 30-min exposure to LH, and within this list, identified protein phosphatases or phosphatase regulatory subunits that showed changes in phosphorylation. Phosphatases in the phosphoprotein phosphatase (PPP) family were of particular interest because of their requirement for dephosphorylating the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase in the granulosa cells, which triggers oocyte meiotic resumption. Among the PPP family regulatory subunits, PPP1R12A and PPP2R5D showed the largest increases in phosphorylation, with 4-10 fold increases in signal intensity on several sites. Although follicles from mice in which these phosphorylations were prevented by serine-to-alanine mutations in either Ppp1r12a or Ppp2r5d showed normal LH-induced NPR2 dephosphorylation, these regulatory subunits and others could act redundantly to dephosphorylate NPR2. Our identification of phosphatases and other proteins whose phosphorylation state is rapidly modified by LH provides clues about multiple signaling pathways in ovarian follicles.

Phosphatases modified by LH signaling in ovarian follicles: testing their role in regulating the NPR2 guanylyl cyclase.

In response to luteinizing hormone, multiple proteins in rat and mouse granulosa cells are rapidly dephosphorylated, but the responsible phosphatases remain to be identified. Because the phosphorylation state of phosphatases can regulate their interaction with substrates, we searched for phosphatases that might function in LH signaling by using quantitative mass spectrometry. We identified all proteins in rat ovarian follicles whose phosphorylation state changed detectably in response to a 30-minute exposure to LH, and within this list, identified protein phosphatases or phosphatase regulatory subunits that showed changes in phosphorylation. Phosphatases in the PPP family were of particular interest because of their requirement for dephosphorylating the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase in the granulosa cells, which triggers oocyte meiotic resumption. Among the PPP family regulatory subunits, PPP1R12A and PPP2R5D showed the largest increases in phosphorylation, with 4-10 fold increases in signal intensity on several sites. Although follicles from mice in which these phosphorylations were prevented by serine-to-alanine mutations in either Ppp1r12a or Ppp2r5d showed normal LH-induced NPR2 dephosphorylation, these regulatory subunits and others could act redundantly to dephosphorylate NPR2. Our identification of phosphatases and other proteins whose phosphorylation state is rapidly modified by LH provides clues about multiple signaling pathways in ovarian follicles. Summary sentence: Quantitative mass spectrometric analysis of phosphatases whose phosphorylation state is rapidly modified by luteinizing hormone provides clues about how LH signaling dephosphorylates NPR2 as well as a resource for future studies.

Epitope-tagged and phosphomimetic mouse models for investigating natriuretic peptide-stimulated receptor guanylyl cyclases.

The natriuretic peptide receptors NPR1 and NPR2, also known as guanylyl cyclase A and guanylyl cyclase B, have critical functions in many signaling pathways, but much remains unknown about their localization and function in vivo. To facilitate studies of these proteins, we developed genetically modified mouse lines in which endogenous NPR1 and NPR2 were tagged with the HA epitope. To investigate the role of phosphorylation in regulating NPR1 and NPR2 guanylyl cyclase activity, we developed mouse lines in which regulatory serines and threonines were substituted with glutamates, to mimic the negative charge of the phosphorylated forms (NPR1-8E and NPR2-7E). Here we describe the generation and applications of these mice. We show that the HA-NPR1 and HA-NPR2 mice can be used to characterize the relative expression levels of these proteins in different tissues. We describe studies using the NPR2-7E mice that indicate that dephosphorylation of NPR2 transduces signaling pathways in ovary and bone, and studies using the NPR1-8E mice that indicate that the phosphorylation state of NPR1 is a regulator of heart, testis, and adrenal function.

A CRISPR toolbox for generating intersectional genetic mouse models for functional, molecular, and anatomical circuit mapping.

BACKGROUND: The functional understanding of genetic interaction networks and cellular mechanisms governing health and disease requires the dissection, and multifaceted study, of discrete cell subtypes in developing and adult animal models. Recombinase-driven expression of transgenic effector alleles represents a significant and powerful approach to delineate cell populations for functional, molecular, and anatomical studies. In addition to single recombinase systems, the expression of two recombinases in distinct, but partially overlapping, populations allows for more defined target expression. Although the application of this method is becoming increasingly popular, its experimental implementation has been broadly restricted to manipulations of a limited set of common alleles that are often commercially produced at great expense, with costs and technical challenges associated with production of intersectional mouse lines hindering customized approaches to many researchers. Here, we present a simplified CRISPR toolkit for rapid, inexpensive, and facile intersectional allele production. RESULTS: Briefly, we produced 7 intersectional mouse lines using a dual recombinase system, one mouse line with a single recombinase system, and three embryonic stem (ES) cell lines that are designed to study the way functional, molecular, and anatomical features relate to each other in building circuits that underlie physiology and behavior. As a proof-of-principle, we applied three of these lines to different neuronal populations for anatomical mapping and functional in vivo investigation of respiratory control. We also generated a mouse line with a single recombinase-responsive allele that controls the expression of the calcium sensor Twitch-2B. This mouse line was applied globally to study the effects of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on calcium release in the ovarian follicle. CONCLUSIONS: The lines presented here are representative examples of outcomes possible with the successful application of our genetic toolkit for the facile development of diverse, modifiable animal models. This toolkit will allow labs to create single or dual recombinase effector lines easily for any cell population or subpopulation of interest when paired with the appropriate Cre and FLP recombinase mouse lines or viral vectors. We have made our tools and derivative intersectional mouse and ES cell lines openly available for non-commercial use through publicly curated repositories for plasmid DNA, ES cells, and transgenic mouse lines.

Phosphatase inhibition by LB-100 enhances BMN-111 stimulation of bone growth.

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations in the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase both result in decreased production of cyclic GMP in chondrocytes and severe short stature, causing achondroplasia (ACH) and acromesomelic dysplasia, type Maroteaux, respectively. Previously, we showed that an NPR2 agonist BMN-111 (vosoritide) increases bone growth in mice mimicking ACH (Fgfr3Y367C/+). Here, because FGFR3 signaling decreases NPR2 activity by dephosphorylating the NPR2 protein, we tested whether a phosphatase inhibitor (LB-100) could enhance BMN-111-stimulated bone growth in ACH. Measurements of cGMP production in chondrocytes of living tibias, and of NPR2 phosphorylation in primary chondrocytes, showed that LB-100 counteracted FGF-induced dephosphorylation and inactivation of NPR2. In ex vivo experiments with Fgfr3Y367C/+ mice, the combination of BMN-111 and LB-100 increased bone length and cartilage area, restored chondrocyte terminal differentiation, and increased the proliferative growth plate area, more than BMN-111 alone. The combination treatment also reduced the abnormal elevation of MAP kinase activity in the growth plate of Fgfr3Y367C/+ mice and improved the skull base anomalies. Our results provide a proof of concept that a phosphatase inhibitor could be used together with an NPR2 agonist to enhance cGMP production as a therapy for ACH.

Cellular Heterogeneity of the Luteinizing Hormone Receptor and Its Significance for Cyclic GMP Signaling in Mouse Preovulatory Follicles.

Meiotic arrest and resumption in mammalian oocytes are regulated by 2 opposing signaling proteins in the cells of the surrounding follicle: the guanylyl cyclase natriuretic peptide receptor 2 (NPR2), and the luteinizing hormone receptor (LHR). NPR2 maintains a meiosis-inhibitory level of cyclic guanosine 5'-monophosphate (cGMP) until LHR signaling causes dephosphorylation of NPR2, reducing NPR2 activity, lowering cGMP to a level that releases meiotic arrest. However, the signaling pathway between LHR activation and NPR2 dephosphorylation remains incompletely understood, due in part to imprecise information about the cellular localization of these 2 proteins. To investigate their localization, we generated mouse lines in which hemagglutinin epitope tags were added to the endogenous LHR and NPR2 proteins, and used immunofluorescence and immunogold microscopy to localize these proteins with high resolution. The results showed that the LHR protein is absent from the cumulus cells and inner mural granulosa cells, and is present in only 13% to 48% of the outer mural granulosa cells. In contrast, NPR2 is present throughout the follicle, and is more concentrated in the cumulus cells. Less than 20% of the NPR2 is in the same cells that express the LHR. These results suggest that to account for the LH-induced inactivation of NPR2, LHR-expressing cells send a signal that inactivates NPR2 in neighboring cells that do not express the LHR. An inhibitor of gap junction permeability attenuates the LH-induced cGMP decrease in the outer mural granulosa cells, consistent with this mechanism contributing to how NPR2 is inactivated in cells that do not express the LHR.

Follicle-stimulating hormone and luteinizing hormone increase Ca2+ in the granulosa cells of mouse ovarian follicles†.

In mammalian ovarian follicles, follicle stimulating hormone (FSH) and luteinizing hormone (LH) signal primarily through the G-protein Gs to elevate cAMP, but both of these hormones can also elevate Ca2+ under some conditions. Here, we investigate FSH- and LH-induced Ca2+ signaling in intact follicles of mice expressing genetically encoded Ca2+ sensors, Twitch-2B and GCaMP6s. At a physiological concentration (1 nM), FSH elevates Ca2+ within the granulosa cells of preantral and antral follicles. The Ca2+ rise begins several minutes after FSH application, peaks at ∼10 min, remains above baseline for another ∼10 min, and depends on extracellular Ca2+. However, suppression of the FSH-induced Ca2+ increase by reducing extracellular Ca2+ does not inhibit FSH-induced phosphorylation of MAP kinase, estradiol production, or the acquisition of LH responsiveness. Like FSH, LH also increases Ca2+, when applied to preovulatory follicles. At a physiological concentration (10 nM), LH elicits Ca2+ oscillations in a subset of cells in the outer mural granulosa layer. These oscillations continue for at least 6 h and depend on the activity of Gq family G-proteins. Suppression of the oscillations by Gq inhibition does not inhibit meiotic resumption, but does delay the time to 50% ovulation by about 3 h. In summary, both FSH and LH increase Ca2+ in the granulosa cells of intact follicles, but the functions of these Ca2+ rises are only starting to be identified.

Multiple cAMP Phosphodiesterases Act Together to Prevent Premature Oocyte Meiosis and Ovulation.

Luteinizing hormone (LH) acts on the granulosa cells that surround the oocyte in mammalian preovulatory follicles to cause meiotic resumption and ovulation. Both of these responses are mediated primarily by an increase in cyclic adenosine monophosphate (cAMP) in the granulosa cells, and the activity of cAMP phosphodiesterases (PDEs), including PDE4, contributes to preventing premature responses. However, two other cAMP-specific PDEs, PDE7 and PDE8, are also expressed at high levels in the granulosa cells, raising the question of whether these PDEs also contribute to preventing uncontrolled activation of meiotic resumption and ovulation. With the use of selective inhibitors, we show that inhibition of PDE7 or PDE8 alone has no effect on the cAMP content of follicles, and inhibition of PDE4 alone has only a small and variable effect. In contrast, a mixture of the three inhibitors elevates cAMP to a level comparable with that seen with LH. Correspondingly, inhibition of PDE7 or PDE8 alone has no effect on meiotic resumption or ovulation, and inhibition of PDE4 alone has only a partial and slow effect. However, the fraction of oocytes resuming meiosis and undergoing ovulation is increased when PDE4, PDE7, and PDE8 are simultaneously inhibited. PDE4, PDE7, and PDE8 also function together to suppress the premature synthesis of progesterone and progesterone receptors, which are required for ovulation. Our results indicate that three cAMP PDEs act in concert to suppress premature responses in preovulatory follicles.

Luteinizing hormone signaling phosphorylates and activates the cyclic GMP phosphodiesterase PDE5 in mouse ovarian follicles, contributing an additional component to the hormonally induced decrease in cyclic GMP that reinitiates meiosis.

Prior to birth, oocytes within mammalian ovarian follicles initiate meiosis, but then arrest in prophase until puberty, when with each reproductive cycle, one or more follicles are stimulated by luteinizing hormone (LH) to resume meiosis in preparation for fertilization. Within preovulatory follicles, granulosa cells produce high levels of cGMP, which diffuses into the oocyte to maintain meiotic arrest. LH signaling restarts meiosis by rapidly lowering the levels of cGMP in the follicle and oocyte. Part of this decrease is mediated by the dephosphorylation and inactivation the NPR2 guanylyl cyclase in response to LH, but the mechanism for the remainder of the cGMP decrease is unknown. At least one cGMP phosphodiesterase, PDE5, is activated by LH signaling, which would contribute to lowering cGMP. PDE5 exhibits increased cGMP-hydrolytic activity when phosphorylated on serine 92, and we recently demonstrated that LH signaling phosphorylates PDE5 on this serine and increases its activity in rat follicles. To test the extent to which this mechanism contributes to the cGMP decrease that restarts meiosis, we generated a mouse line in which serine 92 was mutated to alanine (Pde5-S92A), such that it cannot be phosphorylated. Here we show that PDE5 phosphorylation is required for the LH-induced increase in cGMP-hydrolytic activity, but that this increase has only a modest effect on the LH-induced cGMP decrease in mouse follicles, and does not affect the timing of meiotic resumption. Though we show that the activation of PDE5 is among the mechanisms contributing to the cGMP decrease, these results suggest that another cGMP phosphodiesterase is also activated by LH signaling.

Dephosphorylation of the NPR2 guanylyl cyclase contributes to inhibition of bone growth by fibroblast growth factor.

Activating mutations in fibroblast growth factor (FGF) receptor 3 and inactivating mutations in the NPR2 guanylyl cyclase both cause severe short stature, but how these two signaling systems interact to regulate bone growth is poorly understood. Here, we show that bone elongation is increased when NPR2 cannot be dephosphorylated and thus produces more cyclic GMP. By developing an in vivo imaging system to measure cyclic GMP production in intact tibia, we show that FGF-induced dephosphorylation of NPR2 decreases its guanylyl cyclase activity in growth plate chondrocytes in living bone. The dephosphorylation requires a PPP-family phosphatase. Thus FGF signaling lowers cyclic GMP production in the growth plate, which counteracts bone elongation. These results define a new component of the signaling network by which activating mutations in the FGF receptor inhibit bone growth.

Dephosphorylation is the mechanism of fibroblast growth factor inhibition of guanylyl cyclase-B.

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations of guanylyl cyclase-B (GC-B, also called NPRB or NPR2) cause dwarfism. FGF exposure inhibits GC-B activity in a chondrocyte cell line, but the mechanism of the inactivation is not known. Here, we report that FGF exposure causes dephosphorylation of GC-B in rat chondrosarcoma cells, which correlates with a rapid, potent and reversible inhibition of C-type natriuretic peptide-dependent activation of GC-B. Cells expressing a phosphomimetic mutant of GC-B that cannot be inactivated by dephosphorylation because it contains glutamate substitutions for all known phosphorylation sites showed no decrease in GC-B activity in response to FGF. We conclude that FGF rapidly inactivates GC-B by a reversible dephosphorylation mechanism, which may contribute to the signaling network by which activated FGFR3 causes dwarfism.

Regulation of Mammalian Oocyte Meiosis by Intercellular Communication Within the Ovarian Follicle.

Meiotic progression in mammalian preovulatory follicles is controlled by the granulosa cells around the oocyte. Cyclic GMP (cGMP) generated in the granulosa cells diffuses through gap junctions into the oocyte, maintaining meiotic prophase arrest. Luteinizing hormone then acts on receptors in outer granulosa cells to rapidly decrease cGMP. This occurs by two complementary pathways: cGMP production is decreased by dephosphorylation and inactivation of the NPR2 guanylyl cyclase, and cGMP hydrolysis is increased by activation of the PDE5 phosphodiesterase. The cGMP decrease in the granulosa cells results in rapid cGMP diffusion out of the oocyte, initiating meiotic resumption. Additional, more slowly developing mechanisms involving paracrine signaling by extracellular peptides (C-type natriuretic peptide and EGF receptor ligands) maintain the low level of cGMP in the oocyte. These coordinated signaling pathways ensure a fail-safe system to prepare the oocyte for fertilization and reproductive success.

Luteinizing Hormone Causes Phosphorylation and Activation of the cGMP Phosphodiesterase PDE5 in Rat Ovarian Follicles, Contributing, Together with PDE1 Activity, to the Resumption of Meiosis.

The meiotic cell cycle of mammalian oocytes in preovulatory follicles is held in prophase arrest by diffusion of cGMP from the surrounding granulosa cells into the oocyte. Luteinizing hormone (LH) then releases meiotic arrest by lowering cGMP in the granulosa cells. The LH-induced reduction of cGMP is caused in part by a decrease in guanylyl cyclase activity, but the observation that the cGMP phosphodiesterase PDE5 is phosphorylated during LH signaling suggests that an increase in PDE5 activity could also contribute. To investigate this idea, we measured cGMP-hydrolytic activity in rat ovarian follicles. Basal activity was due primarily to PDE1A and PDE5, and LH increased PDE5 activity. The increase in PDE5 activity was accompanied by phosphorylation of PDE5 at serine 92, a protein kinase A/G consensus site. Both the phosphorylation and the increase in activity were promoted by elevating cAMP and opposed by inhibiting protein kinase A, supporting the hypothesis that LH activates PDE5 by stimulating its phosphorylation by protein kinase A. Inhibition of PDE5 activity partially suppressed LH-induced meiotic resumption as indicated by nuclear envelope breakdown, but inhibition of both PDE5 and PDE1 activities was needed to completely inhibit this response. These results show that activities of both PDE5 and PDE1 contribute to the LH-induced resumption of meiosis in rat oocytes, and that phosphorylation and activation of PDE5 is a regulatory mechanism.

Dephosphorylation of juxtamembrane serines and threonines of the NPR2 guanylyl cyclase is required for rapid resumption of oocyte meiosis in response to luteinizing hormone.

The meiotic cell cycle of mammalian oocytes starts during embryogenesis and then pauses until luteinizing hormone (LH) acts on the granulosa cells of the follicle surrounding the oocyte to restart the cell cycle. An essential event in this process is a decrease in cyclic GMP in the granulosa cells, and part of the cGMP decrease results from dephosphorylation and inactivation of the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase, also known as guanylyl cyclase B. However, it is unknown whether NPR2 dephosphorylation is essential for LH-induced meiotic resumption. Here, we prevented NPR2 dephosphorylation by generating a mouse line in which the seven regulatory serines and threonines of NPR2 were changed to the phosphomimetic amino acid glutamate (Npr2-7E). Npr2-7E/7E follicles failed to show a decrease in enzyme activity in response to LH, and the cGMP decrease was attenuated; correspondingly, LH-induced meiotic resumption was delayed. Meiotic resumption in response to EGF receptor activation was likewise delayed, indicating that NPR2 dephosphorylation is a component of the pathway by which EGF receptor activation mediates LH signaling. We also found that most of the NPR2 protein in the follicle was present in the mural granulosa cells. These findings indicate that NPR2 dephosphorylation in the mural granulosa cells is essential for the normal progression of meiosis in response to LH and EGF receptor activation. In addition, these studies provide the first demonstration that a change in phosphorylation of a transmembrane guanylyl cyclase regulates a physiological process, a mechanism that may also control other developmental events.

Intercellular signaling via cyclic GMP diffusion through gap junctions restarts meiosis in mouse ovarian follicles.

Meiosis in mammalian oocytes is paused until luteinizing hormone (LH) activates receptors in the mural granulosa cells of the ovarian follicle. Prior work has established the central role of cyclic GMP (cGMP) from the granulosa cells in maintaining meiotic arrest, but it is not clear how binding of LH to receptors that are located up to 10 cell layers away from the oocyte lowers oocyte cGMP and restarts meiosis. Here, by visualizing intercellular trafficking of cGMP in real-time in live follicles from mice expressing a FRET sensor, we show that diffusion of cGMP through gap junctions is responsible not only for maintaining meiotic arrest, but also for rapid transmission of the signal that reinitiates meiosis from the follicle surface to the oocyte. Before LH exposure, the cGMP concentration throughout the follicle is at a uniformly high level of ∼2-4 µM. Then, within 1 min of LH application, cGMP begins to decrease in the peripheral granulosa cells. As a consequence, cGMP from the oocyte diffuses into the sink provided by the large granulosa cell volume, such that by 20 min the cGMP concentration in the follicle is uniformly low, ∼100 nM. The decrease in cGMP in the oocyte relieves the inhibition of the meiotic cell cycle. This direct demonstration that a physiological signal initiated by a stimulus in one region of an intact tissue can travel across many layers of cells via cyclic nucleotide diffusion through gap junctions could provide a general mechanism for diverse cellular processes.

Dephosphorylation and inactivation of NPR2 guanylyl cyclase in granulosa cells contributes to the LH-induced decrease in cGMP that causes resumption of meiosis in rat oocytes.

In mammals, the meiotic cell cycle of oocytes starts during embryogenesis and then pauses. Much later, in preparation for fertilization, oocytes within preovulatory follicles resume meiosis in response to luteinizing hormone (LH). Before LH stimulation, the arrest is maintained by diffusion of cyclic (c)GMP into the oocyte from the surrounding granulosa cells, where it is produced by the guanylyl cyclase natriuretic peptide receptor 2 (NPR2). LH rapidly reduces the production of cGMP, but how this occurs is unknown. Here, using rat follicles, we show that within 10 min, LH signaling causes dephosphorylation and inactivation of NPR2 through a process that requires the activity of phosphoprotein phosphatase (PPP)-family members. The rapid dephosphorylation of NPR2 is accompanied by a rapid phosphorylation of the cGMP phosphodiesterase PDE5, an enzyme whose activity is increased upon phosphorylation. Later, levels of the NPR2 agonist C-type natriuretic peptide decrease in the follicle, and these sequential events contribute to the decrease in cGMP that causes meiosis to resume in the oocyte.

Between-female variation in house sparrow yolk testosterone concentration is negatively associated with CYP19A1 (aromatase) mRNA expression in ovarian follicles.

Maternally-derived yolk androgens influence the development and long-term phenotype of offspring in oviparous species. Between-female variation in the amounts of these yolk androgens has been associated with a number of social and environmental factors, suggesting that the variation is adaptive, but the mechanisms behind it are unknown. Using two different approaches, we tested the hypothesis that variation in yolk androgen levels across individuals is associated with variation in their capacity to synthesize androgens. First, we injected female house sparrows with exogenous gonadotropin-releasing hormone (GnRH) to maximally stimulate ovarian steroidogenesis. Second, we collected pre-ovulatory follicle tissue and quantified the mRNA expression of four key enzymes of the steroid synthesis pathway: steroidogenic acute regulatory protein (StAR), cytochrome P450-side chain cleavage enzyme (CYP11A1), 17ß-hydroxysteroid dehydrogenase (HSD17B1), and aromatase (CYP19A1). Thirty minutes after GnRH injection, androgen concentrations in both the plasma and in the yolks of pre-ovulatory follicles were significantly elevated compared to controls. However, this measure of steroidogenic capacity did not explain variation in yolk testosterone levels, although physiological differences between house sparrows and more widely studied poultry models were revealed by this approach. Steroidogenic enzyme mRNA levels were detectable in all samples and were significantly lower in the most mature pre-ovulatory follicles. Of the four measured genes, CYP19A1 expression exhibited a significant negative relationship with yolk testosterone concentrations in laid eggs, revealing a key mechanism for between-female variation in yolk testosterone. Furthermore, this suggests that any factors which alter the expression of CYP19A1 within an individual female could have dramatic effects on offspring phenotype.