Actin polymerization-enhancing drugs promote ovarian follicle growth mediated by the Hippo signaling effector YAP

Abstract

Hippo signaling pathway consists of conserved serine/threonine kinases to maintain optimal organ sizes. Studies have demonstrated that fragmentation of murine ovaries increases actin polymerization and disrupts Hippo signaling, leading to nuclear translocation of Hippo signaling effector Yes-associated protein (YAP) in ovarian follicles and follicle growth. For patients with polycystic ovarian syndrome showing follicle arrest, ovarian wedge resection and laser drilling promote follicle growth. Because these damaging procedures likely involve actin polymerization, we tested whether actin polymerization-promoting drugs could promote YAP translocation and stimulate follicle growth. Treatment of murine ovaries with μM Jasplakinolide (JASP), an actin polymerization-promoting cyclic peptide, or sphingosine-1-phosphate (S1P), a follicular fluid constituent known to promote actin polymerization, increased the conversion of globular actin to the filamentous form, followed by increased nuclear YAP and expression of downstream connective tissue growth factor (CCN2). After short-term treatments with JASP or S1P, in vitro cultured and in vivo grafted ovaries showed follicle growth. Furthermore, induction of constitutively active YAP in ovarian grafts of transgenic mice enhanced follicle development, whereas treatment of human ovarian cortices with JASP or S1P increased CCN2 expression. Thus, JASP and S1P stimulate follicle growth and are potential therapeutic agents for treating polycystic ovarian syndrome and other ovarian disorders.—Cheng, Y., Feng, Y., Jansson, L., Sato, Y., Deguchi, M., Kawamura, K., Hsueh, A. J. Actin polymerization-enhancing drugs promote ovarian follicle growth mediated by the Hippo signaling effector YAP.

The Hippo signaling pathway is essential for organ size control, and components of this pathway are conserved in metazoan animals (1–3). Hippo signaling consists of several negative growth regulators macrophage stimulating 1/2, Salvador [SAV] 1, and large tumor-suppressor homolog [LATS] 1/2) acting in a serine/threonine kinase cascade that phosphorylate and inactivate key transcriptional coactivators, Yes-associated protein (YAP), and transcriptional coactivator with PDZ binding motif (TAZ). When Hippo signaling is disrupted, nonphosphorylated YAP or TAZ accumulates in the nucleus and acts in concert with TEA domain–containing sequence-specific transcriptional factors to increase the expression of downstream CCN growth factors and BIRC (baculoviral inhibitors of apoptosis repeat containing) apoptosis inhibitors (1). These CCN proteins in turn stimulate cell growth, survival, and proliferation (4). In various tissues, intercellular junctions, cell polarity, and cytoskeletons regulate Hippo signaling to maintain optimal organ sizes (5). Recent genome-wide RNA interference screening demonstrated that induction of extra filamentous actin (F-actin) from globular actin (G-actin) disrupts Hippo signaling and induces overgrowth in Drosophila imaginal discs and proliferation of human HeLa cells (6, 7). Because polymerization of G-actin to F-actin is important for cell shape maintenance, adhesion, and locomotion, actin polymerization could link biophysical changes, such as alterations in intercellular tension of an organ after physical damage, to the suppression of Hippo signaling and resulting YAP nuclear localization. This in turn leads to changes in cell proliferation and organ sizes.

Damages to ovaries promote follicle growth in patients with polycystic ovarian syndrome (PCOS) who underwent ovarian wedge resection (8, 9) or laparoscopic laser drilling (10) as infertility treatments. Our recent studies demonstrated that fragmentation of rodent ovaries promotes follicle development by increasing actin polymerization, leading to Hippo signaling disruption and nuclear localization of the Hippo signaling effector YAP, followed by increases in downstream CCN growth factors (11). To provide an alternative to the use of ovarian damaging procedures for the promotion of follicle growth, we tested if actin polymerization-promoting agents could increase nuclear localization of YAP, stimulate connective tissue growth factor (CCN2) expression, and enhance follicle growth using rodent and human ovarian tissues.

MATERIALS AND METHODS

Animals

Female CD-1 and severe combined immunodeficient (SCID) mice were purchased from Charles River Laboratories (Wilmington, MA, USA). Animals were housed in the animal facility of Stanford University under 12 hour dark/light with free access to food and water. By using site-specific recombination in embryonic stem cells, a doxycycline-dependent allele of a constitutive active (CA)-YAP mutant was targeted downstream of the collagen 1a1 locus by site-specific integration in a transgenic mouse strain (12, 13). The mutated YAP gene contains a S127A mutation; ablation of this phosphorylation site increases the nuclear localization and transcriptional activity of YAP by escaping 14-3-3-mediated inhibition (14).

Ovarian incubation and grafting

For testing the effects of actin polymerization drugs, ovaries from 10-day-old CD-1 mice were excised in L-15 medium before transferring to culture plate inserts (Millipore, Bedford, MA, USA). Ovaries were cultured in minimum essential media α with 3 mg/ml bovine serum albumin (BSA), 0.23 mM sodium pyruvate, 50 μg/ml vitamin C, 30 mIU/ml follicle-stimulating hormone (FSH), 50 mg/L streptomycin sulfate, and 75 mg/L penicillin G. Paired ovaries were incubated with or without Jasplakinolide (JASP) or sphingosine-1-phosphate (S1P) for different times, followed by measurement of F- and G-actin levels, nuclear localization of YAP, and CCN2 transcript levels. The control groups were incubated with respective solvent alone. For grafting studies, paired ovaries (intact and treated) from the same donor were inserted under the kidney capsule of the same adult ovariectomized hosts (9 to 10 wk of age). Hosts were injected with 1 IU FSH daily starting from the day after transplantation for 5 days. For studies using transgenic mice with inducible expression of the CA-YAP, ovaries from wild-type or transgenic mice were collected at day 10 of age, then transplanted under kidney capsules of adult ovariectomized SCID mice for 5 days with daily FSH (1 IU) and doxycycline (50 μg/g) injections. At the end of the transplant procedure, grafts were collected for fixation before weight determination and histologic analyses.

Measurement of F-actin levels

Ratios of F-actin to G-actin in ovaries were determined by F-actin/G-actin in vivo assay kit (Cytoskeleton, Denver, CO, USA). After treatment of ovaries with JASP or S1P for different times, ovaries were homogenized in the F-actin stabilization buffer containing 50 mM PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid)], pH 6.9, 50 mM KCl, 5 mM MgCl₂, 5 mM EGTA, 5% glycerol, 0.1% Nonidet P-40, 0.1% Triton X-100, 0.1% Tween-20, 0.1% 2-mecraptoethanol, 0.0001% Antifoam C, 1 mM ATP, 0.4 μM tosyl arginine methyl ester, 15 μM leupeptin, 1 μM pepstatin A, and 1 mM bezamidine. After incubation at 37°C for 10 minutes, the lysate was centrifuged at 350 g for 5 minutes at 37°C to remove tissue debris. After further centrifugation at 100,000 g for 1 hour at 37°C, the supernatant was collected. Pellets were resuspended in ice-cold molecular-grade water containing 8 M urea and incubated on ice for 1 hour with gentle mixing every 15 minutes. To measure F-/G-actin ratios, equal amounts of supernatant (G-actin) and resuspended pellets (F-actin) were subjected to immunoblotting analysis using pan-actin antibody (Cytoskeleton).

Real-time RT-PCR and immunostaining analyses

Murine ovaries were treated with JASP (Invitrogen, Carlsbad, CA, USA) or S1P (Avanti Polar Lipids, Alabaster, AL, USA) for different durations before measurement of CCN2 transcripts, whereas human ovarian strips (1 × 1 cm) containing secondary and smaller follicles were thawed before treatment with or without JASP (10 μM) for 30 minutes or S1P (12 μM) for 3 hours. Total RNA was extracted using an RNeasy Micro Kit (Qiagen, Germantown, MD, USA), and cDNA was synthesized using a Sensicript RT Kit (Qiagen). Real-time RT-PCR was performed using iTaq SYBR Green SuperMix (Bio-Rad, Hercules, CA, USA) on a Smart Cycler TD System (Cepheid, Sunnyvale, CA, USA) as follows: 15 minutes at 95°C, followed by 45 cycles of 15 s at 95°C and 60 seconds at 60°C. The relative abundance of CCN2 was normalized to that of glyceraldehyde phosphate dehydrogenase (GAPDH). Immunohistochemical (IHC) staining was performed using a Histostain-SP IHC kit (Invitrogen). Briefly, paraffin-embedded sections were deparaffinization and rehydrated, and the sections were treated with 10 mM sodium citrate buffer (pH 6.0; 5 minutes for 4 times in a 700 W microwave). The sections were then washed with Tris-buffered saline 5 min for 3 times and incubated with 0.3% H₂O₂ for 30 minutes followed by 10% bovine serum albumin (BSA; catalog no. A4503; Sigma-Aldrich, St. Louis, MO, USA) for 1 hour at room temperature. The sections were incubated with anti-YAP (1:200 dilution; Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. Sections were stained using the Histostain-SP IHC kit (catalog no. 95-6143; Invitrogen) according to the manufacturer’s instructions. Sections were counterstained by using Mayer hematoxylin histologic staining (Dako, Glostrup, Denmark) and examined using a Leica DMIRB microscope (Leica, Wetzlar, Germany) under bright-field optics. For negative controls, nonimmune IgG (Dako) was used.

Ovarian explant cultures and follicle counting

Ovaries from day 10 mice were placed on culture plate inserts (Millipore) and cultured in 400 μl DMEM/F12 containing 0.1% BSA (Sigma-Aldrich), 0.1% Albumax II (Invitrogen), insulin–transferrin–selenium (Invitrogen), 0.05 mg/ml l-ascorbic acid, and penicillin-streptomycin under the membrane insert to cover ovaries with a thin layer of medium. Ovaries were treated with 1 to 10 μM JASP for 30 minutes and washed; cultures continued for 4 days with medium changes after 2 days of culture. For S1P treatment, 12 μM S1P was added every 12 hours for 4 days. Medium was changed after 2 days of culture. At the end of culture, ovaries were fixed in 10% formalin before weighing. Some ovaries were then embedded with paraffin, then cut into continuous sections before staining with hematoxylin and eosin. Every third section from each ovary was used for counting. Follicles with 1 oocyte surrounded by a single layer of flattened granulosa cells were scored as primordial follicles; follicles with an oocyte surrounded by 1 layer of cubical granulosa cells were considered primary; follicles with an oocyte surrounded by 2 layers of granulosa cells were considered as early secondary; and follicles with 3 or more layers of granulosa cells but without an antrum were considered late secondary. Only follicles with clearly stained oocyte nuclei were counted to prevent recounting of the same follicle (15).

Animal and human subject approval

Mice were treated in accordance with established guidelines and after receipt of approval of local animal research committees. Human ovarian strips from patients who had undergone a caesarean section were obtained after receiving informed consent from patients and approval from the human subjects committee of Akita University.

Statistical analyses

Results are presented as the mean ± sem of 3 or more independent assays. Statistical significance was determined by Student’s t test, with P < 0.05 considered statistically significant.

RESULTS

Treatment with JASP enhanced actin polymerization, increased nuclear localization of YAP, stimulated CCN2 expression, and promoted follicle growth

We treated paired ovaries from day 10 mice with JASP, a cyclic peptide known to induce actin polymerization (16), or with medium alone. As shown in Fig. 1A, treatment with JASP for 10 to 30 minutes increased ovarian F-/G-actin ratios compared to controls. After JASP exposure, IHC staining revealed increased nuclear localization of YAP in granulosa and theca cells as well as oocytes of follicles of different sizes (Fig. 1B). Also, time-dependent increases in ovarian CCN2 transcripts (Fig. 1C) were found after JASP treatment. Peak increases in CCN2 transcript levels were detected at 9 hours after initial exposure to JASP for 30 minutes.

Figure 1.

Treatment with JASP enhanced actin polymerization, increased nuclear localization of YAP, and stimulated CCN2 expression in the ovary. A) Paired ovaries from day 10 mice were treated with or without JASP (10 µM) for 10 to 30 minutes before analyses of F- and G-actin content. Top, representative immunoblots with 2 exposure times; bottom, quantitative analyses of immunoblotting data. Numbers inside parentheses represent number of replicates used; mean ± sem; *P < 0.05. B) Immunostaining of YAP in paired ovaries with or without JASP treatment. Paired ovaries were treated with or without JASP for 30 minutes. Media were changed and samples incubated for another 3 hours before IHC staining with YAP antibodies. Oo, oocyte; GC, granulosa cells; TC, theca cells. Upper, lower magnification (×200); middle, higher magnification (×800); scale bars, 50 μm. For negative controls, nonimmune IgG (Dako) was used. C) Real-time RT-PCR analyses of CCN2 transcripts after exposure to JASP for 30 minutes, followed by incubation for up to 12 hours; n = 6–8; mean ± sem; *P < 0.05.

We further used explant cultures to test the effect of JASP treatment on follicle growth in vitro. Ovarian explants from mice at day 10 of age were treated for 30 minutes with different doses of JASP, washed, and cultured for 4 days with media changes at the end of day 2. As shown in Fig. 2A, transient exposure of ovaries to JASP increased follicle growth as reflected by increased explant weights, with 10 μM JASP showing highest increases. After histologic analyses (Fig. 2B) and follicle counting of ovarian sections (Fig. 2C), increases in the percentage of secondary follicles were found in JASP-treated group compared with controls. In vivo development of ovaries after transient exposure to JASP was also investigated. After incubating paired ovaries from day 10 mice without or with different concentrations of JASP for 30 minutes, ovaries were grafted into kidney capsules of adult ovariectomized hosts for 5 days, and hosts were injected daily with FSH to promote follicle growth. As shown in Fig. 2D, major increases in graft weights were evident after treatment with 5 to 20 μM JASP. Histologic analyses (Fig. 2E) and follicle counting (Fig. 2F) of grafts showed increases in antral/preovulatory follicles accompanied by apparent decreases in primordial and primary follicles.

Figure 2.

Treatment with JASP stimulated follicle growth in cultured ovarian explants and in ovarian grafts in vivo. A) Ovaries from day 10 mice were treated with different doses of JASP (1 to 10 µM) for 30 minutes, washed twice, and then incubated for 4 days with medium changes after 2 days of culture. Ovarian explant weights were determined at the end of culture; n = 8–10; mean ± sem; *P < 0.05. B) Representative histologic sections of explants. Ovarian explants after JASP (10 μM) treatment for 30 minutes were sectioned serially. Higher-magnification images are available in Supplemental Fig. 1A, B. C) Numbers of follicles at different developmental stages in explants were determined; n = 4; mean ± sem; *P < 0.05. D) Ovaries were treated with or without 5 to 20 μM JASP for 30 minutes before grafting into adult hosts treated daily with FSH for 5 days. After graft removal, graft weights were determined; mean ± sem; *P < 0.05; n = 8. E) Representative histologic sections of grafts. Higher-magnification images are available in Supplemental Fig. 1C, D. F) Follicles dynamics of untreated grafts or paired grafts exposed to JASP (10 μM) for 30 minutes; n = 4; mean ± sem; *P < 0.05.

Treatment with S1P enhanced actin polymerization, increased nuclear localization of YAP, stimulated CCN2 expression, and promoted follicle growth

On the basis of recent data showing the ability of S1P to increase nuclear localization of YAP in cultured cells (17, 18), we tested if treatment with S1P could enhance actin polymerization in the ovary and promote follicle growth. Paired ovaries from day 10 mice were incubated with S1P or medium alone. As shown in Fig. 3A, a transient increase in the conversion of G-actin to F-actin was detected at 2 hours after S1P treatment, followed by a decline. Also, IHC staining at 6 h after S1P treatment showed increased nuclear localization of YAP in granulosa and theca cells as well as oocytes of follicles at different sizes (Fig. 3B). Measurement of CCN2 transcript levels indicated time-dependent increases of CCN2 transcripts with a peak at 3 h after S1P treatment (Fig. 3C).

Figure 3.

Treatment with S1P enhanced actin polymerization, increased nuclear localization of YAP, and stimulated CCN2 expression in the ovary. A) Paired ovaries were treated with or without S1P (12 µM) for 1 to 3 hours before analyses of F- and G-actin content. Top, representative immunoblots with 2 exposure times; bottom, quantitative analyses of blotting data. Numbers inside parentheses represent number of replicates used; mean ± sem; *P < 0.05. B) Immunostaining of YAP in paired ovaries with or without S1P treatment for 6 hours. Upper, lower magnification (×200); bottom, higher magnification (×800). Scale bars, 50 μm. Oo, oocyte; GC, granulosa cells; TC, theca cells. C) Real-time RT-PCR analyses of CCN2 transcript levels after incubation of ovaries with S1P (12 μM); n = 6–8; means ± sem; *P < 0.05.

Ovarian explants from day 10 mice were treated continuously with 12 and 24 μM S1P for 4 days. As shown in Fig. 4A, S1P treatment increased ovarian weights. Histologic analyses (Fig. 4B) and follicle counting (Fig. 4C) indicated increases in late secondary follicles. When paired ovaries were treated with or without increasing doses of S1P for 18 hours before grafting into adult hosts, increases in graft weights were evident at 5 days after transplantation (Fig. 4D). Histologic analyses (Fig. 4E) and follicle counting (Fig. 4F) indicated increases in primary and antral/preovulatory follicles, accompanied by decreases in primordial follicles.

Figure 4.

Treatment with S1P-stimulated follicle growth in cultured ovarian explants and ovarian grafts in vivo. A) Ovaries from day 10 mice were cultured with S1P (12 µM) for 4 days with medium changes after 2 days of culture. To avoid culture-related instability, freshly prepared S1P was added daily at 12 h intervals. Ovarian explant weights were determined at the end of culture. Numbers inside parentheses represent number of replicates used; mean ± sem; *P < 0.05. B) Representative histologic sections of explants. Higher-magnification images are available in Supplemental Fig. 1E, F. C) Follicle dynamics of ovarian explants; n = 4; mean ± sem; *P < 0.05. D) Ovaries were treated with or without different concentrations (6 to 24 μM) of S1P for 18 hours before grafting into adult hosts treated daily with FSH for 5 days. After removal of grafts, ovarian graft weights were determined. Numbers inside parentheses represent number of replicates; mean ± sem; *P < 0.05. E) Representative histologic sections of grafts. Higher-magnification images are available in Supplemental Fig. 1G, H. F) Follicle dynamics of grafts; n = 6; mean ± sem; *P < 0.05.

Induction of a CA-YAP transgene promoted follicle growth in ovarian grafts

To further demonstrate the essential role of YAP in promoting follicle growth, we obtained transgenic mice carrying an inducible form of a constitutively active (CA)-YAP transgene (12). After genotyping, ovaries from 10-day-old wild-type or CA-YAP animals were paired and grafted into each side of kidney capsules of adult ovariectomized immunodeficient hosts. Hosts were injected daily with doxycycline to induce transgene expression in grafts, together with daily FSH administrations to promote follicle growth. As shown in Fig. 5A, major increases in graft weight were evident in CA-YAP grafts compared to wild-type controls. Histologic analyses (Fig. 5B) and follicle counting (Fig. 5C) indicated the promotion of follicles to the late secondary stage, accompanied by an apparent but not statistically significant decrease in primary follicles.

Figure 5.

Overexpression of a CA-YAP transgene increased ovarian weights and promoted follicle growth in grafts. Ovaries from wild-type mice or mice expressing an inducible CA form of YAP (CA-YAP) were collected at 10 days of age. One ovary each from wild-type and CA-YAP animals was grafted into one side of the kidney capsule of 10-week-old recipients. Recipients were injected daily with 50 μg/kg i.p. doxycycline to induce transgene expression and 1 IU FSH per day to promote follicle growth. After 5 days, ovarian grafts were retrieved from kidney capsules before histologic analyses. A) Ovarian weights from wild-type and CA-YAP grafts. Higher-magnification images are available in Supplemental Fig. 1I, J. Numbers inside parentheses represent number of replicates; mean ± sem; *P < 0.05. B) Representative histology of ovarian grafts. Scale bar = 100 μm. C) Follicle dynamics following counting serial sections of grafts; n = 4; mean ± sem; *P < 0.05.

Treatment with JASP and S1P increased CCN2 expression in human cortical strips

To test the potential clinical application of JASP and S1P treatments in promoting follicle growth in patients containing preantral follicles, we obtained human ovarian strips containing primordial, primary, and secondary follicles from patients who had undergone caesarean section (19). As shown in Fig. 6, treatment with JASP and S1P increased CCN2 transcript levels by 2.8- and 1.9-fold, respectively. These findings suggest the possibility of using actin polymerization–promoting agents to enhance nuclear YAP localization in human ovarian cells with the potential to promote follicle growth in infertile patients.

Figure 6.

Treatment with JASP and S1P increased ovarian CCN2 transcript levels in human cortical fragments. Vitrified human ovarian strips (1 × 1 cm) containing secondary and smaller follicles were thawed and cultured with or without JASP (10 μM) for 30 minutes, washed, and incubated in medium alone until 9 h later based on optimal conditions derived from murine studies. Some strips were treated with S1P (12 μM) for 3 h. At the end of incubation, CCN2 transcript levels were measured by real-time RT-PCR (N = 4). For all samples, GAPDH transcript levels were also measured. After normalizing using GAPDH levels, relative abundance of CCN2 transcripts was expressed as fold changes relative to the controls expressed as 1.0.

DISCUSSION

Our results demonstrated that both the natural product JASP and the follicular fluid component S1P are capable of inducing actin polymerization in the ovary, leading to Hippo signaling disruption, nuclear localization of YAP, CCN2 expression, and follicle growth. This is the first demonstration of the ability of actin polymerization-promoting drugs in suppressing Hippo signaling pathway and promoting organ growth.

JASP is a cyclic peptide from the marine sponge, Jaspis johnstoni, and has fungicidal activity. JASP acts by direct binding to F-actin, leading to its stabilization (16). However, long-term exposure to JASP suppresses prostate cancer growth (20). In our model, we exposed ovaries to JASP for only 30 minutes, and JASP promoted follicle growth to an extent comparable to the ovarian fragmentation procedure (11). S1P is an sphingolipid, acting both as an intracellular second messenger and extracellular mediator (21). In contrast to JASP, a longer exposure to S1P is needed to promote follicle growth. In cultured cells, F-actin formation in the stress fiber is required for the disruption of Hippo signaling and nuclear YAP accumulation (22). F-actin probably functions as a scaffold for Hippo pathway components because LATS1, MST1/2, and upstream Hippo signaling genes (merlin and Amot) all bind to actin (5, 23); conversion of G-actin to F-actin suppresses activities of serine/threonine kinases in the Hippo signaling pathway. Although earlier cell culture studies demonstrated the importance of actin polymerization in regulating cell proliferation in vitro (17, 24), our studies provide in vivo evidence to highlight the potential role of actin polymerization–promoting agents in tissue growth.

Extracellular S1P is known to regulate angiogenesis, vascular maturation, and immunity (25) by acting through a family of 5 related GPCR, S1PR1-5 (26). Serum-derived S1P has recently been shown to act through G12/13-coupled S1P receptors to inhibit Hippo pathway kinases LATS1/2 in different cell lines, thereby activating YAP and TAZ transcription coactivators and promoting cell proliferation (17, 24). In the ovary, S1P is a normal constituent of the follicular fluid (27), and ovarian granulosa cells express 4 of the 5 S1P receptors. A recent study demonstrated that ovarian S1P is associated with high-density lipoprotein in the follicular fluid and promotes actin polymerization and migration of human granulosa lutein cells by acting through the S1P type 3 receptor (27). Furthermore, earlier studies demonstrated that oocyte apoptosis induced by X-ray irradiation is suppressed by one-time intrabursal treatment with S1P (28), consistent with an earlier observation showing increases in BIRC apoptosis inhibitors following ovarian fragmentation and Hippo signaling disruption (11). In addition to the promotion of cell proliferation and suppression of apoptosis downstream of Hippo signaling disruption, S1P treatment promotes neoangiogenesis of human ovarian grafts and reduces ischemic reperfusion injury in a xenografting model (29), and thus could further augment graft growth in the present in vivo model.

The essential role of YAP in promoting follicle growth was further demonstrated using transgenic mice expressing the 5SA-YAP transgene under an inducible promoter. Earlier studies indicated that mutation of the 5 key phosphorylation sites of YAP leads to constitutive activity (30). Because this transgene is ubiquitously expressed after induction and could lead to unwanted side effects in whole animals, we took advantage of the ovarian grafting model and found major enhancement in follicle growth in grafts. These findings conclusively demonstrated the essential role of YAP in ovarian follicle growth. Of interest, a recent study demonstrated that S1P-YAP-CCN2 signaling is important for endoderm formation required for cardiac precursor cell migration in zebrafish (31).

In addition to studies using murine models, our use of human cortical strips extends the murine findings to human follicles by showing increases in CCN2 transcripts after JASP and S1P treatment. Our findings suggest a potential use of JASP and S1P as tools to promote ovarian follicle growth in patients with PCOS, with the potential to replace the present use of ovarian wedge resection (9) or laparoscopic laser drilling (10). Although future studies are needed to evaluate oocyte functions after JASP and S1P exposure, intraovarian administration of JASP or S1P, used at safe doses, could minimize follicle loss associated with the present damaging procedures. Although JASP has to be administered locally at ovarian sites because of its toxicity, S1P has the advantage of being a component of the follicular fluid. Indeed, FTY720 (fingolimod; trade name Gilenya; Novartis, Basel, Switzerland), an S1P agonist and immunomodulating drug, has been approved for treating multiple sclerosis (32). In vivo delivery of FTY720 using intraovarian cannulation has been shown to prevent radiation-induced ovarian failure and infertility in nonhuman primates (33). Our demonstration of S1P induction of YAP nuclear localization, stimulation of CCN growth factor expression, and promotion of follicle growth provide the basis for the use of this sphingolipid and its agonists as therapeutic agents in the treatment of prevalent PCOS (34).

Glossary

BIRC

baculoviral inhibitors of apoptosis repeat containing

BSA

bovine serum albumin

CA

constitutive active

CCN2

connective tissue growth factor

F-actin

filamentous actin

FSH

follicle-stimulating hormone

G-actin

globular actin

GAPDH

glyceraldehyde phosphate dehydrogenase

GC

granulosa cells

IHC

immunohistochemistry

JASP

Jasplakinolide

LATS

large tumor-suppressor homolog

Oo

oocyte

PCOS

polycystic ovarian syndrome

PIPES

piperazine-N,N′-bis(2-ethanesulfonic acid)

S1P

sphingosine-1-phosphate

SAV

Salvador

SCID

severe combined immunodeficient

TAZ

transcriptional coactivator with PDZ binding motif

TC

theca cells

YAP

Yes-associated protein