Valentina Lodde

Gap Junction-Mediated Communications Regulate Chromatin Remodeling During Bovine Oocyte Growth and Differentiation Through cAMP-Dependent Mechanism(s)

ABSTRACT Oocyte development is characterized by impressive changes in chromatin structure and function in the germinal vesicle (GV) that are crucial in conferring to the oocyte meiotic and developmental competence. During oogenesis, oocyte and follicular cells communicate by paracrine and junctional mechanisms. In cow, cumulus-enclosed oocytes (CEOs) isolated from early antral follicles have uncondensed chromatin (GV0), functionally open gap junction (GJ)-mediated communications, and limited meiotic competence. The aim of the present study was to analyze the role of GJ communications on the chromatin remodeling process during the specific phase of folliculogenesis that coincides with the transcriptional silencing and the sequential acquisition of meiotic and developmental capability. CEOs were cultured in a follicle-stimulating hormone-based culture system that sustained GJ coupling and promoted oocyte growth and transition from GV0 to higher stages of condensation. When GJ functionality was experimentally interrupted, chromatin rapidly condensed, and RNA synthesis suddenly ceased. These effects were prevented by the addition of cilostamide, a phosphodiesterase 3 inhibitor, indicating that the action of GJ-mediated communication on chromatin structure and function is mediated by cAMP. Prolonging GJ coupling during oocyte culture before in vitro maturation enhanced the ability of early antral oocytes to undergo meiosis and early embryonic development. Altogether, the evidence suggests that GJ-mediated communication between germinal and somatic compartments plays a fundamental role in the regulation of chromatin remodeling and transcription, which in turn are related to competence acquisition.

Progesterone Inhibits Apoptosis in part by PGRMC1-Regulated Gene Expression

Progesterone receptor membrane component-1 (PGRMC1) is present in both the cytoplasm and nucleus of spontaneously immortalized granulosa cells (SIGCs). PGRMC1 is detected as a monomer in the cytoplasm and a DTT-resistant PGRMC1 dimer in the nucleus. Transfected PGRMC1-GFP localizes mainly to the cytoplasm and does not form a DTT-resistant dimer. Moreover, forced expression of PGRMC1-GFP increases the sensitivity of the SIGCs to progesterone (P4)s anti-apoptotic action, indicating that the PGRMC1 monomer is functional. However, when endogenous PGRMC1 is depleted by siRNA treatment and replaced with PGRMC1-GFP, P4 responsiveness is not enhanced, although overall levels of PGRMC1 are increased. P4s anti-apoptotic action is also attenuated by actinomycin D, an inhibitor of RNA synthesis, and P4 activation of PGRMC1 suppresses Bad and increases Bcl2a1d expression. Taken together, the present studies suggest a genomic component to PGRMC1s anti-apoptotic mechanism of action, which requires the presence of the PGRMC1 dimer.

Natriuretic Peptide Precursor C Delays Meiotic Resumption and Sustains Gap Junction Mediated Communication in Bovine Cumulus Enclosed Oocytes

ABSTRACT 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 IVM-in 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.

Cytoplasmic changes and developmental competence of bovine oocytes cryopreserved without cumulus cells

The cryopreservation of female gametes is still an open problem because of their structural sensitivity to the cooling-and-freezing process and to the exposure to cryoprotectants. The present work was aimed to study the effect of vitrification on immature bovine oocytes freed of cumulus cell investment before freezing. To verify the feasibility and efficiency of denuded oocyte (DO) cryopreservation, the cytoplasmic alterations eventually induced either by cell removal or by the vitrification process were analyzed. In particular, the migration of cortical granules and Ca++ localization were studied. In addition, the localization and distribution of microtubules and microfilaments in immature fresh and vitrified DOs were evaluated. Finally, to establish whether the removal of cumulus cells influenced developmental competence, DOs were thawed after vitrification, matured in vitro and fertilized; then presumptive zygotes were cultured to reach the blastocyst stage. The results indicate that mechanical removal of cumulus cells from immature bovine oocytes does not affect their maturation competence but reduces the blastocyst rate when compared with intact cumulus oocyte complexes (COCs). The findings indicate further that the vitrification process induces changes of cytoplasmic components. However, the composition of the manipulation medium used to remove cumulus cells plays a crucial role in reducing the injuries caused by cryopreservation in both cytoplasmic and nuclear compartments. In fact, the presence of serum exerts a sort of protection, significantly improving both oocyte maturation and blastocyst rates. In conclusion, we demonstrate that denuded immature oocytes can be vitrified after cumulus cells removal and successfully develop up, after thawing, to the blastocyst stage, following in vitro maturation and fertilization.

Effect of different cryopreservation protocols on cytoskeleton and gap junction mediated communication integrity in feline germinal vesicle stage oocytes

Oocyte cryopreservation in carnivores can significantly improve assisted reproductive technologies in animal breeding and preservation programs for endangered species. However, the cooling process severely affects the integrity and the survival of the oocyte after thawing and may irreversibly compromise its subsequent developmental capability. In the present study, two different methods of oocyte cryopreservation, slow freezing and vitrification, were evaluated in order to assess which of them proved more suitable for preserving the functional coupling with cumulus cells as well as nuclear and cytoplasmic competence after warming of immature feline oocytes. From a total of 422 cumulus enclosed oocytes (COCs) obtained from queens after ovariectomy, 137 were stored by vitrification in open pulled straws, 147 by slow freezing and 138 untreated oocytes were used as controls. Immediately after collection and then after warming, functional coupling was assessed by lucifer yellow injection and groups of fresh and cryopreserved oocytes were fixed to analyze tubulin and actin distribution, and chromatin organization. Finally, COCs cryopreserved with both treatments were matured in vitro after warming. In most cases, oocytes cryopreserved by slow freezing showed a cytoskeletal distribution similar to control oocytes, while the process of vitrification induced a loss of organization of cytoskeletal elements. The slow freezing protocol ensured a significantly higher percentage of COCs with functionally open and partially open communications (37.2 vs. 19.0) and higher maturational capability (32.5 vs. 14.1) compared to vitrified oocytes. We conclude that although both protocols impaired intercellular junctions, slow freezing represents a suitable method of GV stage cat oocytes banking since it more efficiently preserves the functional coupling with cumulus cells after thawing as well as nuclear and cytoplasmic competence. Further studies are needed to technically overcome the damage induced by the cryopreservation procedures on immature mammalian oocytes.

Evidence for a genomic mechanism of action for progesterone receptor membrane component-1

Progesterone receptor membrane component 1 (PGRMC1) is highly expressed in the granulosa and luteal cells of rodent and primate ovaries. Interestingly, its molecular weight as assessed by Western blot is dependent on its cellular localization with a ≈27kDa form being detected in the cytoplasm and higher molecular weight forms being detected in the nucleus. The higher molecular weight forms of PGRMC1 are sumoylated suggesting that they are involved in regulating gene transcription, since sumoylation of nuclear proteins often is associated with regulation of transcriptional activity of the sumoylated protein. In order to identify a set of candidate genes that are regulated by PGRMC1, a human granulosa/luteal cell line (hGL5 cells) was treated with PGRMC1 siRNA and changes in gene expression monitored by microarray analysis. The microarray analysis revealed that PGRMC1 generally functioned as a repressor of transcription, since depletion of PGRMC1 resulted in a disproportionate increase in the number of transcripts. Moreover, a pathway analysis implicated PGRMC1 in the regulation of apoptosis, which is consistent with PGRMC1s known biological action. More importantly these results support the concept that PGRMC1 influences gene transcription. Additional studies reveal that progesterone (P4) acting through a PGRMC1-dependent mechanism suppresses the activity of the transcription factor, Tcf/Lef, thereby identifying one molecular pathway through which P4-PGRMC1 can regulate gene transcription and ultimately apoptosis.

Progesterone receptor membrane component-1 expression and putative function in bovine oocyte maturation, fertilization and early embryonic development

Although the mRNA that encodes progesterone receptor membrane component 1 (PGRMC1) is present in mammalian oocytes, nothing is known about either PGRMC1s expression pattern or function in oocytes during maturation, fertilization, and subsequent embryonic development. As PGRMC1 associates with the mitotic spindle in somatic cells, we hypothesized that PGRMC1 is involved in oocyte maturation (meiosis). Western blot analysis confirmed the presence of PGRMC1 in bovine oocytes. This study also shows that PGRMC1 is present at the germinal vesicle (GV)- and MII-stage oocytes and is associated with male and female pronucleus formation of the zygote and is highly expressed in blastocysts. A more detailed examination of PGRMC1 localization using confocal imaging demonstrated that in GV-stage oocytes, PGRMC1 was concentrated throughout the GV but did not localize to the chromatin. With the resumption of meiosis in vitro, PGRMC1 concentrated in the centromeric region of metaphase I chromosomes, while in the anaphase I/telophase I stages the majority of PGRMC1 concentrated between the separating chromosomes. At the metaphase II stage, PGRMC1 re-associated with the centromeric region of the chromosomes. A colocalization study demonstrated that PGRMC1 associated with the phosphorylated form of aurora kinase B, which localizes to the centromeres at metaphase. Finally, PGRMC1 antibody injection significantly lowered the percentage of oocytes that matured and reached the metaphase II stage after 24 h of culture. The majority of the PGRMC1 antibody-injected oocytes arrested in the prometaphase I stage of meiosis. Furthermore, in most of the PGRMC1 antibody-injected oocytes, the chromosomes were disorganized and scattered. Taken together, these data demonstrate that PGRMC1 is expressed in bovine oocytes and its localization changes at specific stages of oocyte maturation. These observations suggest an important role for PGRMC1 in oocyte maturation, which may be specifically related to the mechanism by which chromosomes segregate.

A Novel Role for Progesterone and Progesterone Receptor Membrane Component 1 in Regulating Spindle Microtubule Stability During Rat and Human Ovarian Cell Mitosis

The present studies were designed to assess the roles of progesterone (P4) and Progesterone Receptor Membrane Component 1 (PGRMC1) in regulating mitosis of spontaneously immortalized granulosa cells (SIGCs) and ovarian cancer cells, SKOV-3 cells. Because PGRMC1 has been detected among the proteins of the human mitotic spindle, we theorized that P4 and PGRMC1 could affect mitosis through a microtubule-dependent process. The present study confirms that SIGC growth is slowed by either P4 treatment or transfection of a PGRMC1 antibody. In both cases, slower cell proliferation was accompanied by an increased percentage of mitotic cells, which is consistent with a P4-induced prolongation of the M phase of the cell cycle. In addition, P4 increased the stability of the spindle microtubules, as assessed by the rate of beta-tubulin disassembly in response to cooling. Also, P4 increased spindle microtubule stability of SKOV-3 cells. This effect was mimicked by the depletion of PGRMC1 in these cells. Importantly, P4 did not increase the stability of the microtubules over that observed in PGRMC1-depleted SKOV-3 cells. Immunofluorescent analysis revealed that PGRMC1 is distributed to the spindle apparatus as well as to the centrosomes at metaphase. Further in situ proximity ligation assay revealed that PGRMC1 interacted with beta-tubulin. Taken together, these results suggest that P4 inhibits mitosis of ovarian cells by increasing the stability of the mitotic spindle. Moreover, P4s actions appear to be dependent on PGRMC1s function within the mitotic spindle.

Changes in histone H4 acetylation during in vivo versus in vitro maturation of equine oocytes

Epigenetic modifications are established during gametogenesis and preimplantation embryonic development. Any disturbance of the normal natural environment during these critical phases could cause alterations of the epigenetic signature. Histone acetylation is an important epigenetic modification involved in the regulation of chromatin organization and gene expression. The present study was aimed to determine whether the proper establishment of post-translational histone H4 acetylation at lysine 8 (AcH4K8), 12 (AcH4K12) and 16 (AcH4K16) of equine oocytes is adversely affected during in vitro maturation (IVM) when compared with in vivo matured oocytes collected from naturally cycling mares not undergoing ovarian hyperstimulation. The acetylation patterns were investigated by means of indirect immunofluorescence staining with specific antibodies directed against the acetylated lysine residues. Our results indicate that the acetylation state of H4 is dependent on the chromatin configuration in immature germinal vesicle (GV) stage oocytes and it changes in a residue-specific manner along with the increase of chromatin condensation. In particular, the levels of AcH4K8 and AcH4K12 increased significantly, while AcH4K16 decreased significantly from the fibrillar to the condensed state of chromatin configuration within the GV. Moreover, during meiosis, K8 and K12 were substantially deacetylated without any differences between in vivo and in vitro conditions, while K16 displayed a strong acetylation in oocytes matured in vivo, and in contrast, it was markedly deacetylated following IVM. Although the functional meaning of residue-specific acetylation during oocyte differentiation and meiotic resumption needs further investigation, our results support the hypothesis that IVM conditions can adversely affect oocyte ability to regulate the epigenetic reprogramming, critical for successful meiosis and subsequent embryonic development.

The Effect of Cilostamide on Gap Junction Communication Dynamics, Chromatin Remodeling, and Competence Acquisition in Pig Oocytes Following Parthenogenetic Activation and Nuclear Transfer

ABSTRACT In the pig, the efficiency of in vitro embryo production and somatic cell nuclear transfer (SCNT) procedures remains limited. It has been suggested that prematuration treatments (pre-IVM) based on the prolongation of a patent, bidirectional crosstalk between the oocyte and the cumulus cells through gap junction mediate communication (GJC), with the maintenance of a proper level of cAMP, could improve the developmental capability of oocytes. The aim of this study was to assess: 1) dose-dependent effects of cilostamide on nuclear maturation kinetics, 2) the relationship between treatments on GJC functionality and large-scale chromatin configuration changes, and 3) the impact of treatments on developmental competence acquisition after parthenogenetic activation (PA) and SCNT. Accordingly, cumulus-oocyte complexes were collected from 3- to 6-mm antral follicles and cultured for 24 h in defined culture medium with or without 1 μM cilostamide. GJC functionality was assessed by Lucifer yellow microinjection, while chromatin configuration was evaluated by fluorescence microscopy after nuclear staining. Cilostamide administration sustained functional coupling for up to 24 h of culture and delayed meiotic resumption, as only 25.6% of cilostamide-treated oocytes reached the pro-metaphase I stage compared to the control (69.7%; P < 0.05). Moreover, progressive chromatin condensation was delayed before meiotic resumption based upon G2/M biomarker phosphoprotein epitope acquisition using immunolocalization. Importantly, cilostamide treatment under these conditions improved oocyte developmental competence, as reflected in higher blastocyst quality after both parthenogenetic activation and SCNT.