BIOLOGY OF REPRODUCTION (2014) 91(3):61, 1–9 Published online before print 30 July 2014. DOI 10.1095/biolreprod.114.118869

Natriuretic Peptide Precursor C Delays Meiotic Resumption and Sustains Gap Junction-Mediated Communication in Bovine Cumulus-Enclosed Oocytes1 Federica Franciosi,3,4 Giovanni Coticchio,3,5 Valentina Lodde,4 Irene Tessaro,4 Silvia C. Modina,4 Rubens Fadini,5 Mariabeatrice Dal Canto,5 Mario Mignini Renzini,5 David F. Albertini,6 and Alberto M. Luciano2,4 4

Reproductive and Developmental Biology Laboratory, Department of Health, Animal Science and Food Safety, University of Milan, Milan, Italy 5 Biogenesi Reproductive Medicine Centre, Istituti Clinici Zucchi, Monza, Italy 6 Institute for Reproductive Health and Regenerative Medicine, Center for Reproductive Sciences, University of Kansas Medical Center, Kansas City, Kansas INTRODUCTION

Oocyte in vitro maturation (IVM) has become a valuable technological tool for animal breeding and cloning and the treatment of human infertility because it does not require the administration of exogenous gonadotropin to obtain fertilizable oocytes. However, embryo development after IVM is lower compared to in vivo maturation, most likely because oocytes collected for IVM are heterogeneous with respect to their developmental competencies. Attempts to improve IVM outcome have relied upon either prematuration culture (PMC) or two-step maturation strategies in the hope of normalizing variations in developmental competence. Such culture systems invoke the pharmacological arrest of meiosis, in theory providing oocytes sufficient time to complete the acquisition of developmental competence after cumulus-enclosed oocytes isolation from the follicle. The present study was designed to test the efficiency of natriuretic peptide precursor C (NPPC) as a nonpharmacologic meiosis-arresting agent during IVM in a monoovulatory species. NPPC has been shown to maintain meiotic arrest in vivo and in vitro in mice and pigs; however, the use of this molecule for PMC has yet to have been explored. Toward this end, meiotic cell cycle reentry, gap-junction functionality, and chromatin configuration changes were investigated in bovine cumulus-enclosed oocytes cultured in the presence of NPPC. Moreover, oocyte developmental competence was investigated after IVM, in vitro fertilization, and embryo culture and compared to standard IVMin vitro fertilization protocol without PMC. Our results suggest that NPPC can be used to delay meiotic resumption and increase the developmental competence of bovine oocytes when used in PMC protocols.

Oocyte development takes place in ovarian follicles, where oocytes and somatic follicular cells communicate via paracrine and juxtacrine mechanisms [1]. During folliculogenesis, oocytes grow and sequentially acquire the ability to complete meiosis I, or meiotic competence, and the ability to sustain fertilization and embryo development, termed developmental competence [2]. When the oocyte acquires the cell cycle machinery to restart meiosis, it becomes imperative that the follicular environment assumes responsibility for the maintenance of meiotic arrest. During this phase of folliculogenesis, which in the bovine spans from the middle antral to periovulatory stages [3], a series of metabolic and structural modifications takes place. These modifications, though still poorly defined, encompass accumulation of proteins and energy substrates, organelles reallocation, and changes in the structure of chromatin that are essential for the onset of the developmental competence (reviewed in [4]). The signaling pathway between follicular cells and oocytes responsible for meiotic arrest has been extensively investigated. Meiotic arrest depends on the second messenger cyclic adenosine 3 0 ,5 0 -monophosphate (cAMP), which is synthesized by the oocyte through a constitutively active G-coupled receptor protein [5, 6], and is also supplied to the ooplasm by adjacent cumulus cells via gap junction-mediated communication (GJC) [7, 8]. Intraoocyte cAMP concentration is regulated by the activity of phosphodiesterase (PDE) enzymes that degrade cAMP into 5 0 AMP. The main PDE in the oocyte is represented by PDE3A [9], whose activity is inhibited by cyclic guanosine 3 0 ,5 0 -monophosphate (cGMP) [10, 11]. Recent studies have shown that cGMP is produced in cumulus cells upon the activation of the guanylyl cyclasecoupled natriuretic peptide receptor type-2 (NPR2) [12]. NPR2 activity is induced by its ligand natriuretic peptide precursor C (NPPC), which is mainly synthesized by mural granulosa cells [13, 14]. Cyclic GMP is then transferred via GJC [15] to the oocyte where it inhibits PDE3A, thus providing it with a role in maintaining meiotic arrest [10, 11]. Meiotic arrest is maintained in periovulatory oocytes until a surge in luteinizing hormone (LH) induces the expression of epidermal growth factor (EGF)-like peptides, restarting meiosis [16, 17]. EGF-like peptides propagate the LH stimulus from mural granulosa cells to cumulus cells, which are insensitive to direct LH stimulation because they express few or no LH receptors [18]. The isolation of a meiotically competent oocyte from its follicular environment triggers meiotic resumption in the absence of the ovulatory stimulus represented by the LH surge

bovine, chromatin configuration, cilostamide, cumulus cells, developmental competence, gap junctions, meiotic arrest, natriuretic peptide precursor C, oocyte 1 Supported in part by CIG-Marie Curie Actions FP7-ReintegrationGrants within the 7th European Community Framework Programme (Contract: 303640,"Pro-Ovum,’’ V.L., A.M.L.). F.F. was granted by ‘‘L’Oreal Italia per le Donne e la Scienza 2012’’ fellowship; I.T. was supported by ‘‘Dote Ricercatori’’ FSE, Regione Lombardia, Italy. 2 Correspondence: Alberto M. Luciano, Dipartimento di Scienze Veterinarie per la Salute, la Produzione Animale e la Sicurezza Alimentare, Universita` degli Studi di Milano, Via Celoria, 10 - 20133 Milano, Italy. E-mail: [email protected] 3 These authors contributed equally to this work.

Received: 26 February 2014. First decision: 20 March 2014. Accepted: 9 July 2014. Ó 2014 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363

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ABSTRACT

FRANCIOSI ET AL. oocyte with finely granulated homogenous ooplasm were used. Groups of 20– 30 CEOs were cultured in M199 supplemented with 0.68 mM L-glutamine, 25 mM NaHCO3, 0.4% BSA (fatty acid-free), 0.2 mM sodium pyruvate, 100 nM estradiol, and 104 international units/ml of recombinant human FSH (Gonal-F; Serono) in humidified air under 5% CO2 at 38.58C [34]. The basic PMC medium was then supplemented either with 100 nM NPPC, 10 lM cilostamide [28, 34], or neither (NPPC, cilostamide, and control, respectively). In the first set of experiments, at the end of the PMC, oocytes were freed of cumulus cells by vortexing for 2 min at 35 Hz as previously described [30]. The presence or absence of an intact GV was observed by differential interference contrast (DIC) microscopy and confirmed by nuclear staining.

[19]. This so-called spontaneous maturation has found several applications in basic research and assisted reproductive technologies because it allows an oocyte harvested from an antral follicle to be matured in vitro and subsequently fertilized. However, embryo production through in vitro maturation (IVM)-in vitro fertilization (IVF) approaches remains inefficient compared to the developmental competence observed with oocytes from hormonally induced ovulation. It is commonly accepted that the lower efficiency of IVM is due to the use of a heterogeneous population of oocytes collected from antral follicles at different stages of their growth. Therefore, the process by which the oocytes acquire all the essential components for their developmental program might be not complete, and their ability to properly mature, fertilize, and develop to term might be compromised (reviewed in [20]). Attempts have been made in order to coordinate the acquisition of meiotic and developmental competence in vitro using prematuration culture (PMC) or two-step maturation systems [21–27]. Among the systems that have been developed, the most promising are those that pharmacologically inhibit or retard meiotic resumption by elevating the intraoocyte content of cAMP while sustaining GJC functionality [24, 28]. In the bovine and swine species, it has been demonstrated that delaying meiotic resumption permits the completion of oocyte differentiation, during which time a progressive remodeling of the chromatin occurs within the germinal vesicle (GV) [21, 22]. Importantly, these changes in chromatin organization are related to oocyte metabolic properties [29], with the progressive heterochromatinization considered a feature of developmental competence acquisition [22, 30–32]. The aim of the present study was to test the efficacy of a PMC system based on the use of NPPC as an alternative to the pharmacologically induced meiotic arrest. The fact that NPPC can maintain meiotic arrest in vitro in cumulus-enclosed oocytes (CEOs) isolated from antral follicles has been already proved in mice and pigs [13, 14, 33]; however, the use of this molecule as a natural inducer of the oocyte’s last differentiation steps has never been explored. To this end, the meiotic arrest, GJC functionality, and chromatin configuration of bovine CEOs cultured in the presence of NPPC have been compared to CEOs cultured with the pharmacological PDE3 specific inhibitor, cilostamide. Furthermore the developmental competence after IVM, IVF, and embryo culture has been assessed in the two groups and compared to standard IVM-IVF protocol without PMC. Because IVM is a widespread practice in cattle breeding, the study has been conducted on bovine oocytes as a wellestablished IVM model for monoovulatory species.

Cumulus Cells RNA Isolation and RT-PCR

Immunofluorescence Staining Indirect immunofluorescence was carried out to evaluate the pattern of mitosis-associated ser/thr phospho-proteins (MPM2) by using the monoclonal antibody MPM2 (Merck, Millipore). This antibody is known to detect epitopes of many relevant substrates for M-phase specific kinases and is therefore considered a biomarker for G2/M cell cycle transition [21, 36–39]. Oocytes were freed of cumulus cells, fixed in 4% paraformaldehyde in DPBS for 15 min at 378C, followed by 45 min at 48C, washed in DPBS with 0.1% polyvinyl-alcohol, and permeabilized in 0.3% Triton X-100 for 10 min at room temperature (RT). Nonspecific binding was blocked by incubation in 10% donkey serum, 1% BSA for 30 min at RT, and then the samples were incubated with MPM2 antibody (1:100) overnight at 48C. After extensive washing in DPBS with 0.05% Tween-20, oocytes were incubated with AlexaFluor-488-labeled donkey anti-mouse (1:500; Invitrogen, Life Technologies) for 1 h at RT. Samples were extensively washed and mounted on slides with the anti-fade medium Vectashield (Vector Laboratories) supplemented with 1 lg/ml 4 0 ,6-diamidino-2-phenylindole. Negative controls were performed by omitting the primary antibody. Samples were analyzed on an epifluorescence microscope (Eclipse E600; Nikon Corp.) equipped with a digital camera (DS-Fi2; Nikon Corp.). Images were captured using identical exposure times and gain settings. The mean fluorescence intensity was calculated using NIH ImageJ software [40] by averaging the fluorescent intensity in five different areas of the cytoplasm of each oocyte.

MATERIALS AND METHODS The chemicals used in this study were purchased from Sigma Chemical Company except for those specifically mentioned.

CEOs Collection and PMC

Assessment of Functional GJC Between the Oocyte and the Cumulus Cells

Bovine ovaries were recovered at the abattoir (INALCA Spa., Ospedaletto Lodigiano, Italy) from pubertal females (4–8 yr old) subjected to routine veterinary inspection and in accordance to the specific health requirements stated in Council Directive 89/556/ECC and subsequent modifications. Ovaries were transported to the laboratory within 2 h in sterile saline at 268C. All the subsequent procedures, unless differently specified, were performed at 358C– 388C and carried out as previously described [30]. CEOs were retrieved from midsized antral follicles (2–6 mm) with a 16-gauge needle mounted on an aspiration pump (COOK-IVF; Brisbane QLD) in M199 supplemented with 20 mM HEPES, 1790 units/L heparin, and 0.4% bovine serum albumin (BSA). After examination under a stereomicroscope, only CEOs that are mediumbrown in color with five or more complete layers of cumulus cells enclosing an

The functionality of GJC between the oocyte and the cumulus cells was assessed as previously described [24, 30, 34]. Briefly, by means of a microinjection apparatus (Narishige Co. Ltd.), a 3% solution of the fluorescent dye Lucifer yellow in 5 mM lithium chloride was pressure injected into the ooplasm of oocytes surrounded by several complete layers of cumulus cells. After 10 min incubation, the spread into the surrounding cumulus cells was monitored on an inverted fluorescence microscope (Nikon Diaphot; Nikon Corp.) and the CEOs were classified as open, partially open, or closed according to the pattern of Lucifer yellow spreading from the oocyte to the cumulus cells as previously described [21, 24, 41] (see Fig. 2A). In particular, GJC were classified as partially open when only a limited number of cells

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Expression of NPR2 was examined by standard RT-PCR. CEOs were collected and cumulus cells were stripped as described above. Cumulus cells were harvested and washed in cold Dulbecco phosphate buffered saline (DPBS), pelleted by centrifuging at 10 000 rpm for 1 min at 48C, and snap frozen. Total RNA was extracted from cumulus cells isolated from groups of 30 CEOs using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s instruction. Total RNA was then retrotranscribed with oligodT using the SuperScript First-Strand Synthesis System (Invitrogen). A total amount of cDNA equivalent to cumulus cells of five CEOs was used in each amplification reaction. Amplification was performed with specific primers for NPR2. b-Actin (ACTB) was used as the internal control as described in [35] (see Fig. 1 for primer and reference sequences). ACTB primers were designed to span introns and evaluate the presence of contaminating DNA in each RNA sample. PCR amplification products were analyzed by electrophoresis on a 2% agarose gel. RT-PCR experiments were repeated on three RNA samples from independently collected and extracted cumulus cells. Negative controls were performed by omitting the cDNA template in the PCR reaction. NPR2 primers specificity was confirmed by sequencing analysis that was performed by an external sequencing facility (http://www.primmbiotech.com/).

NPPC AND MEIOTIC ARREST IN BOVINE OOCYTES

showed signs of dye diffusion between ooplasm and the surrounding corona radiata cells.

the telophase I and metaphase II stages were classified as mature; oocytes that could not be identified as being at any of the previous stages were classified as degenerate.

Assessment of Chromatin Configuration and Meiotic Progression

IVM, IVF, and Embryo Culture

The patterns of large-scale chromatin configuration within the GV and the meiotic progression after GV breakdown were assessed by nuclear staining and fluorescence microscopy analysis under an inverted fluorescence microscope as previously described (Olympus IX50; Olympus) [34]. After cumulus cells removal, denuded oocytes were fixed in 60% methanol in DPBS at 48C for 30 min and stained either with 1 lg/ml 4 0 ,6-diamidino-2phenylindole or 0.5 mg/ml propidium iodide. Oocytes were classified as GV1, GV2, or GV3 according to the degree of chromatin mass condensation within the GV as previously described [29, 30] (see Fig. 2B). Oocytes from the GV breakdown to the anaphase I stage were classified as intermediate; oocytes at

After PMC, CEOs were washed and in vitro matured for 18–22 h in M199 added with 0.68 mM L-glutamine, 25 mM NaHCO3, 0.4% BSA (fatty acidfree), 0.2 mM sodium pyruvate, and 0.1 international units/ml of recombinant human FSH at 38.58C in 5% CO2 as previously described [22, 34]. A standard IVM treatment in which CEOs were directly in vitro matured for 24 h without PMC served as the control for this set of experiments. According to the experimental design, at the end of the IVM, oocytes were either fixed to assess their meiotic stage as described above or fertilized. IVF was carried out as previously described [30]. The content of a straw of cryopreserved bull spermatozoa (CIZ) was thawed and the cells separated on a

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FIG. 1. NPR2-NPPC pathway participates in the maintenance of meiotic arrest in bovine cumulus-enclosed oocytes (CEOs). A) Representative RT-PCR reactions showing ACTB (internal control) and NPR2 expression in bovine cumulus cells (CC). Neg represents RT-PCR reactions run without cDNA template. Sequences of primers used are listed in the table. B) The line graph represents the percentage (mean 6 SEM) of oocytes arrested at the GV stage after 0, 6, or 8 h of culture in presence or absence of either NPPC or cilostamide. The symbols a, b, c, and d indicate significant differences in the distribution of GV oocytes between different culture treatments and time in three independent experiments (one-way ANOVA followed by Tukey multiple comparison test, P , 0.05). C) Representative images showing the pattern of ser/thr phospho-protein MPM2 staining in bovine oocytes at the GV, prometaphase I (pro-MI), and metaphase II (MII) stages. Green: MPM2; blue: DNA. Bar ¼ 10 lm. D) The bar graph represents the fluorescence intensity (mean 6 SEM) of MPM2 within the cytoplasm of oocytes cultured for 0, 6, or 8 h in presence or absence of either NPPC or cilostamide and for 24 h in standard in vitro maturation (IVM) conditions. The symbols a, b, and c indicate significant differences in the relative fluorescence of MPM2 between different culture treatments and time (one-way ANOVA followed by Tukey multiple comparison test, P , 0.05).

FRANCIOSI ET AL.

and the cell types were not distinguishable were classified as not expanded. Blastocysts were classified as expanded if the blastocoel was fully formed and the trophectoderm and inner cell mass clearly distinguishable but still contained within a thinned zona pellucida. Hatched blastocysts were those outside the zona pellucida. The embryos were then fixed in 60% methanol in DPBS, and the cell nuclei were counted under a fluorescence microscope after staining with 0.5 mg/ ml propidium iodide.

45%–90% Percoll gradient. Sperms were counted and diluted to a final concentration of 0.75 3 106 spermatozoa/ml in a modified Tyrode solution supplemented with 0.6% BSA (fatty acid-free), 10 lg/ml heparin, 20 lM penicillamine, 1 lM epinephrine, and 100 lM hypotaurine. CEOs and sperms were incubated for 18 h at 38.58C under 5% CO2 in humidified air. Presumptive zygotes were then washed, and cumulus cells were removed by vortexing for 2 min in 500 ll of a modified synthetic oviduct fluid supplemented with 0.3% (w/ v) BSA fraction V (fatty acid-free), MEM essential and nonessential amino acids, 0.72 mM of sodium pyruvate, and buffered with 10 mM of HEPES and 5 mM of NaHCO3. Presumptive zygotes were transferred in embryo culture medium, which was synthetic oviduct fluid buffered with 25 mM of NaHCO3 supplemented with MEM essential and nonessential amino acids, 0.72 mM of sodium pyruvate, 2.74 mM of myo-inositol, 0.34 mM of sodium citrate, and 5% calf serum (Gibco, Invitrogen). Incubation was performed at 38.58C under 5% CO2, 5% O2, and 90% N2 in humidified air for 8 days. At the end of culture period (d þ9), the blastocyst rate was assessed under a stereomicroscope, and blastocysts were morphologically classified as not expanded, expanded, and hatched [42]. Briefly, blastocysts in which the blastocoel has just begun to form

Statistical Analysis Experiments were repeated three to four times. Data were analyzed by oneway ANOVA followed by the Tukey multiple comparison test or by the Fisher exact test, where appropriate. P , 0.05 was considered statistically significant.

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FIG. 2. NPPC promotes GJC between the oocyte and surrounding cumulus cells and chromatin remodeling in the GV. A) After 0, 6, or 8 h culture in presence or absence of either NPPC or cilostamide, the fluorescent dye Lucifer yellow was injected into the ooplasm of CEOs. CEOs were then classified as open, partial, or close, according to the Lucifer yellow spreading through the functionally open gap junctions, as represented in the left panel and described in [24] (original magnification 3200). The graph in the right panel represents the percentage of CEOs with open GJC after 0, 6, or 8 h of culture in presence or absence of either NPPC or cilostamide. The symbols a and b indicate significant differences in open GJC between different culture treatments and time in four independent experiments (one-way ANOVA followed by Tukey multiple comparison test, P , 0.05). B) After 0, 6, or 8 h culture in presence or absence of either NPPC or cilostamide (CILO), the oocytes that were still at the GV stage were classified according to their chromatin configuration in GV1, GV2, and GV3 as represented at the left of each graph and described in [30] (original magnification 3400). The graphs represent the distribution of GV1, GV2, and GV3 configuration (mean 6 SEM) on the total number of oocytes after 6 or 8 h of culture in presence or absence of either NPPC or cilostamide in four independent experiments. At the time of isolation, GV1 oocytes represented 20% 6 4.3% of the total oocytes, GV2 represented 51% 6 6.62% of the total oocytes, and GV3 represented 29% 6 5.09% of the total oocytes. The symbols a, b indicate significant differences in the distribution of GV1-, GV2-, and GV3-stage oocytes in the different culture treatments after 6 and 8 h (one-way ANOVA followed by Tukey multiple comparison test, P , 0.05). CTRL, control.

NPPC AND MEIOTIC ARREST IN BOVINE OOCYTES

RESULTS NPR2-NPPC Pathway Participates in the Maintenance of Meiotic Arrest in Bovine CEOs NPPC has been described to physiologically inhibit meiotic resumption in CEOs in several mammalian species [14, 33]. Its action is mediated through the guanylyl cyclase-coupled receptor NPR2, expressed by the cumulus cells. Therefore, the first set of experiments was designed to assess whether NPR2 is expressed by cumulus cells in the bovine model and verify if the administration of NPPC during the in vitro culture of bovine CEOs inhibits spontaneous meiotic resumption. RT-PCR experiments confirmed the presence of NPR2 in bovine cumulus cells (Fig. 1A). Preliminary experiments (Supplemental Fig. S1A; Supplemental Data are available online at www.biolreprod.org) were conducted on a total of 220 oocytes with NPPC concentrations ranging from 0 to 500 nM for 14 h, and no differences were observed between 100 nM and higher concentrations (43% and 44% meiotic arrest with 100 and 500 nM NPPC, respectively). Therefore, all the following experiments were conducted with 100 nM NPPC in order to minimize off-target effects. The meiotic stage of 675 oocytes was analyzed in four independent experiments. After 6–8 h of culture in presence of NPPC, 87% 6 2% and 74% 6 5.14%, respectively, of the oocytes were arrested at the GV stage (Fig. 1B), a significantly higher proportion compared to control oocytes, cultured without NPPC (61% 6 7.41% and 39% 6 6.11% at 6 or 8 h, respectively). However, the efficiency of NPPC in maintaining the meiotic arrest was transient when compared to the arrest induced by the PDE3A inhibitor, cilostamide (96% 6 1% and 97% 6 1.3% after 6–8 h, respectively). Immunofluorescence staining experiments with anti-MPM2 antibody were conducted to confirm cell cycle status in conjunction with the observations made by DIC microscopy. The MPM2 antigen in fact has been previously used as an early marker of meiotic progression [21, 36]. As shown in Figure 1C, GV oocytes were weakly positive for MPM2 staining, while the intensity of the fluorescent signal greatly increased with meiotic resumption (prometaphase I stage [pro-MI]) and remained high in metaphase II stage (MII). The relative MPM2 fluorescence (Fig. 1D) in the cytoplasm of oocytes cultured for 6 h in presence of either NPPC or cilostamide was significantly lower than in control oocytes, and comparable to the time of isolation (0 h). After 8 h of culture, the MPM2 fluorescence signal significantly increased in NPPC-treated oocytes at a level comparable to the control at 8 h. This intensification of MPM2 signal at 8 h in NPPC-treated oocytes temporally matched the increase in meiotic resumption, confirming the observation made by DIC microscopy.

A PMC System Based on NPPC Administration Increases the Oocyte Developmental Competence In order to finally assess whether a NPPC-based PMC improves oocyte developmental competence, CEOs cultured for 6 h in presence of either NPPC or cilostamide were in vitro matured and fertilized, and the presumptive zygotes cultured for 8 days (d þ9). In this set of experiments, a standard IVM treatment in which CEOs were directly matured for 24 h without PMC served as the control (Control-IVM). Preliminary experiments were conducted on a total of 458 oocytes in order to set the proper IVM timing after PMC. We observed that 22 h of IVM after PMC were sufficient to obtain maturation rates comparable to standard IVM protocols (NPPC, 75% 6 5.99%; cilostamide, 82% 6 3.57%; standard IVM, 84% 6 3.97%; one-way ANOVA followed by Tukey multiple comparison test, P . 0.05). After IVF and embryo culture, no significant differences were observed in the blastocyst rate in the different treatments (Fig. 3). Both NPPC- and cilostamide-based PMC had indeed an effect on the percentage of expanded and hatched blastocysts compared to the control. Also the number of cells per blastocyst, considered a marker of embryo quality, was significantly higher in the NPPC group compared to the control. Finally, the lengthening of the PMC by up to 14 h, tested in 108 oocytes, resulted in a blastocyst yield of 13%– 15%, considerably lower than expected in standard IVM-IVF protocols, independent of the presence or absence of NPPC (Supplemental Fig. S1C).

NPPC Promotes GJC Between the Oocyte and Surrounding Cumulus Cells and Chromatin Remodelling in the GV The maintenance of GJC and the orderly remodeling of large-scale chromatin configuration are considered critical factors for a successful PMC [21, 22, 28]. Therefore, the second set of experiments was designed to verify if during NPPC-mediated meiotic arrest, oocytes and surrounding cumulus cells maintained their gap junction-mediated interactions and if a progressive remodeling of the chromatin in the GV occurred before meiotic resumption. Gap junction coupling and chromatin configuration were analyzed in 406 and 675 oocytes, respectively, in four independent experiments. In the presence of NPPC or cilostamide for 6 or 8 h, the percentage of CEOs with functionally open gap junctions was

DISCUSSION The present study shows that NPPC delays meiotic resumption while sustaining GJC in bovine CEOs isolated from midsized antral follicles. Moreover, when CEOs were treated with NPPC before undergoing IVM and IVF, the rate of expanded and hatched blastocysts as well as the number of cells per blastocyst increased. These findings suggest that the 5

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significantly higher than in control CEOs (NPPC, 68% 6 6.34% and 60% 6 2.39%; cilostamide, 71% 6 1.36% and 59% 6 10%; control, 32% 6 4.74% and 30% 6 5.73% after 6 or 8 h, respectively), as measured by Lucifer yellow injection assay and similar to the percentage observed at the time of collection (57% 6 2.75%) (Fig. 2A). In another set of experiments, 73 CEOs were cultured in presence or absence of NPPC for up to 14 h, resulting in the complete loss of gap junction coupling (Supplemental Fig. S1B). The analysis of the distribution of the three classes of GV configuration (Fig. 2B) shows that in control oocytes the GV1 configuration was less represented than in cilostamide and NPPC group, both at 6 and 8 h, though statistical significance was evident only between cilostamide and control. After 6 h, GV2-stage oocytes were significantly more represented in cilostamide and NPPC group than in control. Significant differences in the distribution of the GV3 class were not observed after 6 h, while by 8 h either in presence of cilostamide or NPPC, GV3-stage oocytes were significantly more represented compared to control. The quantification of MPM2 signal in the different classes of GV condensation provided an independent validation of cell cycle status, further confirming the morphological observations (Supplemental Fig. S2). MPM2 relative fluorescence, both in the germplasm and in the ooplasm, increased along with chromatin condensation, in agreement with previous studies conducted in porcine oocytes [21].

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Downloaded from www.biolreprod.org. FIG. 3. A PMC system based on NPPC administration increases the oocyte developmental competence. The bar graphs represent the mean 6 SEM of the percentage of total blastocyst, expanded and hatched blastocysts, and the blastocyst cell number at d þ9 after fertilization in oocytes that were cultured with either NPPC (n ¼ 204) or cilostamide (n ¼ 201) for 6 h before in vitro maturation (IVM). Oocytes cultured in standard IVM conditions represent the control (Control IVM, n ¼ 184). The symbols a and b indicate significant differences between groups in three independent experiments (one-way ANOVA followed by Tukey multiple comparison test, P , 0.05). Representative images showing expanded/hatched blastocysts (left panel) and not expanded blastocysts (right panel) are given. Red: DNA. Bar ¼ 50 lm.

use of NPPC as an IVM pretreatment may improve the developmental competence of embryos produced by IVM-IVF procedures. Once isolated from their antral-follicle environment, mammalian oocytes spontaneously mature and can be used for IVF and embryo production. However, even in species characterized by a high success rate of IVM-IVF, such as cows, the competence to develop to the blastocyst stage is limited to 30%–35% [43]. Nevertheless, IVM is considered an important technology for animal breeding and cloning as well as for the treatment of human infertility. Because it does not require the administration of exogenous gonadotropin in order to obtain fertilizable oocytes, IVM techniques could reduce the cost and the risks associated with ovarian hyperstimulation. Therefore,

clinicians and scientists have been encouraged to develop more efficient IVM protocols. During the final stages of growth and differentiation, before LH surges and meiosis restarts, the oocytes undergo ultrastructural and molecular changes, collectively termed oocyte capacitation, that are critical to confer the developmental competence [44]. When oocytes are isolated from antral follicles and they spontaneously mature bypassing this step, their developmental competence is impaired (reviewed in [20]). Several attempts have been made to restore oocyte developmental competence. Pharmacological pretreatments that delayed the resumption of meiosis by increasing the intraoocyte cAMP content have been the most promising approaches [22, 24, 27, 28]. However, a stringent testing of the 6

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PDE3 in bovine oocytes. However, the partial and transient nature of the NPPC-mediated meiotic arrest can also be attributed to the fact that a proportion of CEOs already has closed GJC at the time of follicle retrieval (present report and [30]), thus preventing the transfer of cGMP. Clearly GJC closure cannot affect cilostamide-induced meiotic arrest because its mechanism of action is downstream GJC, directly on the oocyte compartment. The prolongation of functional intercellular communication between oocytes and cumulus cells is believed to be beneficial for oocyte developmental competence due to the maintenance of metabolites, ions, pyruvate, and regulatory molecules exchange (reviewed in [4]). Therefore, delaying the resumption of meiosis and, at the same time, extending the functional communication between oocytes and cumulus cells generally had a positive effect in PMC systems. The intimate association of the oocyte with its cumulus cells in the last phase of differentiation, before meiosis restarts, is critical to oocyte developmental competence acquisition and also supports the process of chromatin remodeling in the GV [21, 22, 34, 51]. This observation is confirmed in the present study. In fact, the maintenance of a functional coupling in the NPPC and cilostamide groups is followed by a gradual transition from dispersed to higher stages of chromatin condensation, GV2 and GV3. The degree of chromatin condensation in the GV is related to the oocyte metabolic properties and to developmental competence, with GV2- and GV3-stage oocytes having higher developmental competence compared to GV1 [30]. The enrichment in the population of GV2 oocytes in NPPC and cilostamide group at 6 h may correspond to higher embryo quality in these groups. In line with previous reports of PMC on humans [52], mice [28, 51], cows [22, 27, 28, 34], and pigs [21] that made use of pharmacological PDE inhibitors, in the present study the treatment with NPPC was successful in increasing the percentage of oocytes that progressively acquired features of developmental competence, such as higher order chromatin structure. Importantly, the PMC treatment used in our studies improved oocyte developmental competence as measured by the increase in the number of expanded and hatched blastocysts (both NPPC and cilostamide group) and of the cell number per blastocyst (NPPC group) when compared to standard IVM protocol, used as the control for this experimental phase. However, it should be noted that the improvement was limited to an increase in embryo quality while a higher blastocyst rate was not observed. Due to the mixed oocyte population collected from midsized antral follicles, it is likely that the PMC treatment has the potential to positively affect only a subset of the total collected oocytes, while others would benefit from different culture strategies. Also, some oocytes were likely derived from early atretic follicles [29, 30], and may instead have been negatively affected by prolongation of culture. Ideally, the development of noninvasive methods will benefit from an understanding of which subpopulation of oocytes would benefit from specific dedicated treatments. Interestingly, lengthening the NPPC-mediated PMC up to 14 h, though effective in arresting the meiotic resumption in 43% of the oocytes, did not sustain the functional GJC coupling between the oocyte and the cumulus cells. Under these conditions, only 15% of the oocytes reached the blastocyst stage, a considerably smaller proportion compared to the 30%–35% obtained with the standard IVM protocol. This result further supports the hypothesis that GJC between the oocyte and its cumulus cells supports the acquisition of

safety of these molecules is needed before they can be used for PMC in human fertility clinics. With this in mind, the present study was designed to test the efficacy of a PMC treatment based on NPPC, a molecule that is naturally present in the follicular fluid and known to physiologically participate to the maintenance of meiotic arrest in antral follicles. The mechanism of action of NPPC in the ovary has been recently described in mice [13, 14]. NPPC is mainly synthesized by mural granulosa cells and, by binding to its guanylyl cyclase-coupled receptor NPR2, it induces the production of cGMP in cumulus cells [14]. Cyclic GMP is then transferred through GJC into the oocyte where it inhibits PDE3, thus preventing meiotic resumption [10]. The NPPCNPR2 system has been studied in other mammalian species, such as humans [45] and pigs [33, 46], but information on the bovine species are still lacking. In the present study, the expression of NPR2 by the cumulus cells of bovine midsized antral follicles and the ability of NPPC in delaying meiotic resumption in bovine oocytes suggest that a role of NPPC in supporting meiotic arrest is conserved between mammals. Not surprisingly, the meiotic arrest triggered in bovine oocytes is only transient compared to mice (6 and 24 h, respectively), in agreement with the observation that bovine oocytes contain a larger amount of PDEs and that the same concentrations of PDE inhibitor only partially and transiently maintain meiotic arrest in cows compared to mice (reviewed in: [47]) and with the recent finding that multiple pathways regulate the cGMP content in the follicle [48]. The efficacy of NPPC in blocking meiotic resumption in bovine oocytes has been tested up to a concentration of 500 nM, 50 times the concentration required to completely block meiosis in mice, where more than 80% of the oocytes are blocked by 10 nM NPPC [13, 14, 45, 49]. However, increasing the NPPC concentration over 100 nM did not further improve the efficiency of meiotic arrest, possibly indicating a desensitization of NPR2 to the ligand or the induction of a scavenger receptor as NPR3 [50]. Similar to the bovine species, a partial meiotic arrest and the lack of a further significant increase in NPPC efficiency at concentrations higher than 100 nM have been reported for porcine CEOs [33]. The morphological observation of the cell cycle status, initially evaluated by DIC microscopy and nuclear staining, was confirmed by quantitative measurement of the mitosisassociated ser/thr phospho-proteins (MPM2), a biomarker for G2/M cell cycle transition [36]. In agreement with our morphological observation, a significant increase of the MPM2 signal was detected after 8 h in the NPPC group, exactly when the percentage of GV-arrested oocytes significantly decreases compared to cilostamide. We also observed an increase in the MPM2 signal along with the process of chromatin condensation in the GV as has been previously described for pig oocytes [21]. Consequently, the higher MPM2 signal observed in the NPPC group at 8 h can also be due, at least in part, to the increase in GV3-stage oocytes. In addition to the expression of NPR2, another critical requirement for NPPC-mediated meiotic arrest is the presence of open GJC between cumulus cells and the oocyte in order to allow for the transfer of cGMP. In our PMC system, the treatment with NPPC sustained the functionality of GJC for up to 8 h in 60% of the CEOs, similar to what was observed in the presence of the PDE3 selective inhibitor cilostamide. However, cilostamide was able to completely block meiotic resumption up to 8 h in 97% of the oocytes, a significantly higher percentage compared to the 74% of the NPPC group. As already mentioned, the cGMP produced in response to NPPC stimulation could be insufficient to durably inhibit

FRANCIOSI ET AL.

oocyte developmental competence. Therefore, this parameter should be considered when designing PMC strategies. Finally, no differences were observed between cilostamide and NPPC treatment in blastocyst rate and embryo quality. However, the cell number/blastocyst was significantly higher only in the NPPC group when compared to the control, possibly suggesting that the use of this peptide, naturally present in the follicular fluid, can be even more beneficial than a pharmacological treatment for the development of PMC systems aimed at enhancing the outcome of IVM protocols.

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