A prematuration approach to equine IVM: considering cumulus morphology, seasonality, follicle of origin, gap junction coupling and large-scale chromatin configuration in the germinal vesicle.

Several studies report that a two-step culture where mammalian oocytes are first kept under meiosis-arresting conditions (prematuration) followed by IVM is beneficial to embryo development. The most promising results were obtained by stratifying the oocyte population using morphological criteria and allocating them to different culture conditions to best meet their metabolic needs. In this study, horse oocytes were characterised to identify subpopulations that may benefit from prematuration. We investigated gap-junction (GJ) coupling, large-scale chromatin configuration and meiotic competence in compact and expanded cumulus-oocyte complexes (COCs) according to follicle size (<1, 1-2, >2cm) and season. Then we tested the effect of cilostamide-based prematuration in compact COCs collected from follicles <1 and 1-2cm in diameter on embryo development. Meiotic competence was not affected by prematuration, whereas COCs from follicles 1-2cm in diameter yielded embryos with a higher number of cells per blastocyst than oocytes that underwent direct IVM (P<0.01, unpaired Mann-Whitney test), suggesting improved developmental competence. Oocytes collected from follicles <1cm in diameter were not affected by prematuration. This study represents an extensive characterisation of the functional properties of immature horse oocytes and is the first report of the effects of cilostamide-based prematuration in horse oocyte IVM on embryo development.

Steroid hormones interact with natriuretic peptide C to delay nuclear maturation, to maintain oocyte-cumulus communication and to improve the quality of in vitro-produced embryos in cattle.

In vivo, oocyte maturation is triggered by the ovulatory LH surge, whereas in vitro it is precociously induced when the cumulus-oocyte complex is removed from the follicle. Natriuretic peptide C (NPPC) delays germinal vesicle breakdown (GVBD) while increasing oocyte-cumulus communication during in vitro maturation (IVM) in cattle. In the present study we first tested the hypothesis that steroids secreted by the follicle (17ß-oestradiol, progesterone and androstenedione) interact with NPPC to delay GVBD and to maintain oocyte-cumulus communication as assessed by transfer of a dye (Lucifer Yellow) from the oocyte to cumulus cells. Then, we assessed the effects of steroid hormones and NPPC, alone and in combination in a pre-IVM culture, on embryo production. The combination of NPPC with steroids delayed GVDB, increased natriuretic peptide receptor 2 (NPR2) mRNA abundance in cumulus cells during culture, and maintained oocyte-cumulus communication at levels not different from non-cultured controls. The addition of steroids and/or NPPC to a pre-IVM culture did not alter blastocyst rates after IVF, but supplementation with steroids increased blastocyst total cell number. The present study provides evidence, for the first time in cattle, that steroids interact with NPPC to regulate oocyte nuclear maturation and oocyte-cumulus communication, and improve oocyte developmental competence.

In vitro maturation affects chromosome segregation, spindle morphology and acetylation of lysine 16 on histone H4 in horse oocytes.

Implantation failure and genetic developmental disabilities in mammals are caused by errors in chromosome segregation originating mainly in the oocyte during meiosis I. Some conditions, like maternal ageing or in vitro maturation (IVM), increase the incidence of oocyte aneuploidy. Here oocytes from adult mares were used to investigate oocyte maturation in a monovulatory species. Experiments were conducted to compare: (1) the incidence of aneuploidy, (2) the morphology of the spindle, (3) the acetylation of lysine 16 on histone H4 (H4K16) and (4) the relative amount of histone acetyltransferase 1 (HAT1), K(lysine) acetyltransferase 8 (KAT8, also known as MYST1), histone deacetylase 1 (HDAC1) and NAD-dependent protein deacetylase sirtuin 1 (SIRT1) mRNA in metaphase II stage oocytes that were in vitro matured or collected from peri-ovulatory follicles. The frequency of aneuploidy and anomalies in spindle morphology was increased following IVM, along with a decrease in H4K16 acetylation that was in agreement with our previous observations. However, differences in the amount of the transcripts investigated were not detected. These results suggest that the degradation of transcripts encoding for histone deacetylases and acetyltransferases is not involved in the changes of H4K16 acetylation observed following IVM, while translational or post-translational mechanisms might have a role. Our study also suggests that epigenetic instabilities introduced by IVM may affect the oocyte and embryo genetic stability.

Differences in cumulus cell gene expression indicate the benefit of a pre-maturation step to improve in-vitro bovine embryo production.

STUDY QUESTION: Does the gene expression profile of cumulus cells (CC) accompanying oocytes with different degrees of chromatin compaction within the germinal vesicle (GV) reflect the oocyte's quality and response in culture during in-vitro embryo production (IVP). SUMMARY ANSWER: The transcriptomic profile of the CC is related to oocyte competence, setting the stage for the development of customized pre-maturation strategies to improve IVP. WHAT IS KNOWN ALREADY: Oocytes complete the acquisition of their competence during antral follicle development. During this period, the chromatin configuration within the GV changes dynamically and is indicative of oocyte's developmental potential. The interactions between somatic and germ cells modulate chromatin morphology and function and are critical for acquisition of oocyte competence. STUDY DESIGN, SIZE, DURATION: Bovine cumulus-oocyte complexes (COC) were isolated from 0.5 to 6 mm antral follicles. Surrounding CC were separated from the oocyte and classified as GV0, GV1, GV2 and GV3 according to the degree of the oocyte's chromatin compaction. PARTICIPANTS/MATERIALS, SETTING, METHOD: RNA extracted from CC of each group was amplified and hybridized on a bovine embryo-specific 44 K Agilent slide. The CC_GV1, CC_GV2 and CC_GV3 classes were each hybridized against the CC_GV0 class, representing an early oocyte differentiation stage with poor development competence. The data were normalized and fold changes of the differentially expressed genes were determined. Microarray data were validated using quantitative RT-PCR on selected targets. Microarray data were further analyzed through: (i) between-group analysis (BGA), which classifies the samples according to their transcriptomic profiles; (ii) cluster analysis according to the expression profile of each gene; and (iii) Ingenuity Pathway Analysis (IPA) to study gene regulation patterns and predicted functions. Furthermore, CC of each GV group were cultured and apoptotic cells were assessed after 3 h by caspase analysis. Finally, based on the analysis of CC transcriptomic profiles and the relationship between morphological features of the COC and the oocyte chromatin configuration, a customized, stage-dependent oocyte pre-maturation (pre-IVM) system was used to improve oocyte developmental potential before IVM. For this, the blastocyst rate and quality were assessed after in-vitro maturation and fertilization of pre-matured oocytes. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, quantitative RT-PCR results of a subset of five selected genes were consistent with the microarray data. Clustering analysis generated 16 clusters representing the main profiles of transcription modulation. Of the 5571 significantly differentially expressed probes, the majority (25.49%) best fitted with cluster #6 (downregulation between CC_GV0 and CC_GV1 and stable low levels in successive groups). IPA identified the most relevant functions associated with each cluster. Genes included in cluster #1 were mostly related to biological processes such as 'cell cycle' and 'cell death and survival', whereas genes included in cluster #5 were mostly related to 'gene expression'. Interestingly, 'lipid metabolism' was the most significant function identified in clusters #6, #9 and #12. IPA of gene lists obtained from each contrast (i.e., CC_GV0 vs. CC_GV1; CC_GV0 vs. CC_GV2; CC_GV0 vs. CC_GV3) revealed that the main affected function in each contrast was 'cell death and survival'. Importantly, apoptosis was predicted to be inhibited in CC_GV1 and CC_GV2, but activated in CC_GV3. Caspase analysis indicated that a low percentage of CC_GV0 was prone to undergo apoptosis but apoptosis increased significantly in CC from oocytes with condensed chromatin, reaching a peak in CC_GV3 (P < 0.05). Finally, the tailored oocyte pre-maturation strategy, based on morphological features of the COC and the oocyte chromatin configuration, demonstrated that pre-IVM improved the developmental capability of oocytes at early stages of differentiation (GV1-enriched COC) but was detrimental for oocytes at more advanced stages of development (GV2 and GV3-enriched COC). LARGE SCALE DATA: The data are available through the GEO series accession number GSE79886. LIMITATIONS, REASONS FOR CAUTION: This study was conducted with bovine samples. Whether or not the results are applicable to human oocytes requests further elucidation. Embryo transfer experiments are required to determine whether the improvement in blastocyst rates in the tailored system leads to increased live birth rates. WIDER IMPLICATIONS OF THE FINDINGS: The identification of multiple non-invasive biomarkers predictive of oocyte quality can greatly strengthen the pre-IVM approach aimed to improve IVM outcomes. These results have potentially important implications in treating human infertility and in developing breeding schemes for domestic mammals. STUDY FUNDING/COMPETING INTERESTS: This work was supported in part by NSERC Strategic Network EmbryoGENE, Canada and in part by CIG-Marie Curie Actions-Reintegration Grants within the EU 7FP (n. 303640, 'Pro-Ovum'). The authors declare no potential conflict of interest.

Natriuretic peptide precursor C delays meiotic resumption and sustains gap junction-mediated communication in bovine cumulus-enclosed oocytes.

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.

The adenosine salvage pathway as an alternative to mitochondrial production of ATP in maturing mammalian oocytes.

Although the oocyte is the largest cell in the body and an unavoidable phase in life, its physiology is still poorly understood, and other cell types provide little insight into its unique nature. Even basic cellular functions in the oocyte such as energy metabolism are not yet fully understood. It is known that the mitochondria of the female gamete exhibit an immature form characterized by limited energy production from glucose and oxidative phosphorylation. We show that the bovine oocyte uses alternative means to maintain ATP production during maturation, namely, the adenosine salvage pathway. Meiosis resumption is triggered by destruction of cyclic AMP by phosphodiesterases producing adenosine monophosphate that is converted into ATP by adenylate kinases and creatine kinases. Inhibition of these enzymes decreased ATP production, and addition of their substrates restored ATP production in denuded oocytes. Addition of phosphocreatine to the oocyte maturation medium influenced the phenotype of the resulting blastocysts. We propose a model in which adenylate kinases and creatine kinases act as drivers of ATP production from added AMP during oocyte maturation.

Reductions in the number of mid-sized antral follicles are associated with markers of premature ovarian senescence in dairy cows.

High-producing dairy cows are subfertile; however, the mechanisms responsible for the decreased fertility are unknown. The aim of the present study was to test the hypothesis that culled dairy cows (4-8 years old) characterised by 'Lo' ovaries (i.e. those with <10 mid-antral follicles) are affected by premature ovarian senescence. Cows in which both ovaries were 'Lo' ovaries represented 5% of the total population analysed, and exhibited reduced ovarian size (P<0.001) and increased perifollicular stroma (P<0.05) compared with age-matched controls (i.e. cows in which both ovaries had >10 mid-antral follicles; 'Hi' ovaries). The total number of follicles, including healthy and atretic primordial, primary, secondary and small antral follicles, was lower in Lo ovaries (P<0.01). Interestingly, the primordial follicle population in Lo ovaries was lower (P<0.05) than in the control. Finally, the follicular fluid of mid-antral follicles from Lo ovaries had reduced oestradiol and anti-Müllerian hormone levels (P<0.05), but increased progesterone concentrations (P<0.05). Together, these data account for the reduced fertility of cows with Lo ovaries and are in agreement with previous observations that oocytes isolated from Lo ovaries have reduced embryonic developmental competence. Cows with a specific Lo ovary condition may represent a suitable model to address the causes of low fertility in high-yielding dairy cows, as well as the condition of premature ovarian aging in single-ovulating species.

The effect of cilostamide on gap junction communication dynamics, chromatin remodeling, and competence acquisition in pig oocytes following parthenogenetic activation and nuclear transfer.

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.

Role of gap junction-mediated communications in regulating large-scale chromatin configuration remodeling and embryonic developmental competence acquisition in fully grown bovine oocyte.

PURPOSE: This study was aimed to test the hypothesis that gap junction mediated communications (GJC) are required to allow the progressive chromatin configuration remodeling (from GV1 to GV3) process to occur in fully grown oocytes in order to gain the final step of developmental competence acquisition, and that a premature disruption of GJC can alter this process. METHODS: Bovine cumulus-oocytes complexes collected from medium antral follicles were cultured for 2, 4, 6 and 8 h in the presence of 10(-4) IU/ml of r-hFSH and with 2 mM of the non-selective PDE inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) to prevent meiotic resumption. GJC functionality and chromatin configuration were monitored during the culture period. After meiotic arrest, the developmental capability of oocytes was assessed after IVM and IVF. RESULTS: IBMX was effective in significantly sustaining GJC up to 6 h and maintaining meiotic arrest, when compared to control group. Moreover, the percentage of oocytes with less condensed chromatin (GV1) decreased within 4 h of culture, while the proportion of GV2 oocytes gradually increased up to 6 h. Interestingly, a decline in the proportion of GV2 oocytes and an increase in the proportion of GV3 oocytes were observed after 6 h of culture, when the major drop of GJC occurred. On the contrary, when GJC were uncoupled by adding 3 mM of 1-heptanol or through cumulus cells removal, chromatin condensation occurred rapidly throughout the culture period, more promptly in denuded oocytes. Moreover, the maintenance of GJC during meiotic arrest was accompanied by a significant increase of developmental competence compared to the control, as indicated by a higher percentage of hatched blastocysts and blastocyst cell number. CONCLUSIONS: Altogether, our data indicate that both paracrine and junctional mechanisms are involved in modulating large-scale chromatin structure during the final phase of oocyte differentiation.

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.

Gap junction-mediated communications regulate chromatin remodeling during bovine oocyte growth and differentiation through cAMP-dependent mechanism(s).

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 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 PGRMC1's 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.

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle.

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte's differentiation.

Progesterone receptor membrane component 1 (PGRMC1) expression in canine mammary tumors: A preliminary study.

Canine mammary tumors (CMT) represent the most common neoplasms in female dogs and their diagnosis and classification relies on histopathological examination. Recently, PGRMC1 has been considered to be a putative biomarker for diagnosis and prognosis in many human cancers as it is expressed in a wide variety of tumors. This study represents the first description of PGRMC1 expression in CMT. PGRMC1 expression was initially assessed by immunohistochemistry in healthy or hyperplastic tissues and in four major histopathological types of CMT: simple and complex adenomas and carcinomas. PGRMC1 staining was represented by a scoring system that considered the percentage of positive cells and staining intensity. PGRMC1 expression was defined as either weak, moderate or strong. In healthy and hyperplastic tissues almost 100% of the epithelial cells stained intensely for PGRMC1. Adenomas showed similar features but with a more variable intensity. In tubular areas of adenocarcinomas, a lower percentage of epithelial cells (30-60%) stained for PGRMC1 with a weak intensity. Both the percentage of cells and intensity of PGRMC1 staining became progressively negative in the solid parts of the tumor. Western blot analysis of healthy and neoplastic mammary tissue (carcinomas samples) revealed the presence of the 25 kDa PGRMC1 band in both types of tissue, while the 50 kDa form was mainly detected in the healthy counterpart. This study reveals that PGRMC1 is expressed in CMT and its expression pattern changes depending on the pattern of growth of CMT. Further studies are now needed to determine PGRMC1's putative role and usefulness for typing and prognosis of different CMT subtypes.

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.

Oocyte morphology and transcriptional silencing in relation to chromatin remodeling during the final phases of bovine oocyte growth.

The complexity of the events which orchestrate mammalian oocyte growth and ultimate acquisition of developmental competence is still unclear and under continuous investigation. Starting from the observation that germinal vesicle (GV) bovine oocytes exhibit different patterns of chromatin configuration (from GV0 to GV3), the present study aimed at analyzing the morphology of the nuclear and the cytoplasmic compartments and to determine the total transcriptional activity of immature oocytes sorted on the basis of their chromatin configuration. The oocyte morphology was analyzed by light microscopy (LM) on semi-thin sections and transmission electron microscopy, and the global transcriptional activity was analyzed by H(3)-Uridine incorporation and subsequent autoradiography on semi-thin sections. LM confirmed the increase in chromatin condensation from GV0 to GV3. Ultrastructurally, the nucleolus was fibrillo granular at GV0 while the other stages displayed an electron-dense fibrillar sphere with the remnant of a fibrillar center on the surface. Organelles were dispersed in the cytoplasm at GV0 while at GV1 and GV2 most of them were homogenously distributed in the oocyte cortex. At GV3 most organelles were found in clusters in the oocyte cortex. Typical features of completion of the oocyte growth phase, like undulation of the nuclear envelope and reduction of the size of Golgi complex were found at GV2 and GV3. Moreover, GV3 oocytes presented cortical granules that displayed varying degrees of degeneration. Autoradiographic labeling showed that oocytes with GV0 configuration exhibited high level of RNA synthesis, GV1 and GV2 stages showed a remarkable decrease of transcription, and the acquisition of GV3 configuration was associated with global repression of transcriptional activity. Our findings suggest a temporal relationship between the chromatin remodeling process and the main morpho-functional events that characterize the final growth phase in bovine oocyte.

The variable success of in vitro maturation: can we do better?

The efficiency of in vitro assisted reproductive technologies, consisting of the transfer of embryos obtained in vitro through in vitro maturation, in vitro fertilization and early embryo culture is still limited. The quality of the oocytes is pivotal for assisted reproductive efficiency and the maturation of the oocyte represents the first key limiting step of the in vitro embryo production system. At the time of removal from the antral follicles, the oocyte is still completing the final growth and differentiation steps, needed to provide the so-called developmental competence, i.e. the machinery required to sustain fertilization and embryo development. In mono-ovular species only one oocyte per cycle is available for procreation, therefore the current assisted reproduction techniques strive to overcome this natural boundary. However, the success is still limited and overall the effectiveness does not exceed the efficiency achieved in millions of years of mammalian evolution. One of the problems lies in the intrinsic heterogeneity of the oocytes that are subjected to in vitro maturation and in the lack of dedicated in vitro approaches to finalize the differentiation process. In this review we will try to overview some of the salient aspects of current practices by emphasizing the most critical and fundamental features in oocyte differentiation that should be carefully considered for improving current techniques.

Sirtuins in gamete biology and reproductive physiology: emerging roles and therapeutic potential in female and male infertility.

BACKGROUND: Sirtuins (SIRT1-7) are a family of NAD+-dependent deacetylases that catalyze post-translational modifications of proteins. Together, they respond to metabolic challenges, inflammatory signals or hypoxic/oxidative stress, and are associated with aging and longevity. The role of Sirtuins in the regulation of fertility emerged in 2003 when a defective reproductive phenotype was observed in SIRT1-null mice. Although studies on Sirtuins in reproductive biology have been increasing in the last years, a recent comprehensive update on this issue is still lacking. OBJECTIVE AND RATIONALE: This review is aimed to provide knowledge on the activation mechanism and cellular role of Sirtuins and to give an update of the rapid development of Sirtuin research in female and male reproduction under physiological and pathological conditions. The final goal is to assess whether strategies aimed to improve Sirtuin expression or activity could have therapeutic potential for infertility associated with polycystic ovarian syndrome (PCOS), endometriosis, diabetes, xenobiotic stress and aging. SEARCH METHODS: The MEDLINE database was examined for peer-reviewed original articles. The following keywords were searched: 'Sirtuin', 'ovary', 'oocyte', 'ovarian follicle', 'embryo', 'endometrium', 'sperm' and 'testis'. These keywords were combined with other search phrases relevant to the topic. OUTCOMES: Our knowledge of Sirtuins in reproductive functions has grown exponentially over the last few years. The majority of the work carried out so far has focused on SIRT1 with a prevalence of studies on female reproduction. Numerous studies have provided evidence that down-regulation of SIRT1 is associated with physiological or pathological reduction of ovarian reserve. SIRT1 has also been shown to regulate proliferation and apoptosis in granulosa cells whereas SIRT3 was found to promote luteinisation. Biochemical modulation of Sirtuin activity has led to discoveries of the roles of SIRT1, SIRT2, SIRT3 and SIRT6 in improving the competence of oocytes grown or matured in vitro in humans and animal models. Recently, SIRT1, SIRT2 and SIRT3 have emerged as protectors of oocyte against postovulatory aging. Transgenic models provide strong evidence that SIRT1 is involved in spermatogenesis by influencing specific functions of male germ cell, Sertoli cells and Leydig cells. When our attention moves to post-fertilization events, maternally derived SIRT3 appears crucial in the protecting early embryos against stress conditions. Finally, increasing SIRT1 activity may have the potential to ameliorate fertility in PCOS, diabetes, endometriosis, xenobiotic stress and aging. Overall, these effects have been ascribed to Sirtuin-mediated regulation of energy homoeostasis, mitochondrial biogenesis, chromatin remodelling and protection against oxidative stress. WIDER IMPLICATIONS: The present review provides challenges and opportunities to stimulate research and exploit Sirtuin-based signalling as diagnostic tools and potential targets for therapeutic applications in reproductive medicine.

Immunohistochemical Expression of FXR1 in Canine Normal Tissues and Melanomas.

Fragile X mental retardation-related protein 1 (FXR1) is a cytoplasmic RNA-binding protein highly conserved among vertebrates. It has been studied for its role in muscle development, inflammation, and tumorigenesis, being related, for example, to metastasizing behavior in human and canine uveal melanoma. Anti-FXR1 antibodies have never been validated in the canine species. To investigate FXR1 expression in canine melanocytic tumors, the present study tested two commercially available polyclonal anti-human FXR1 antibodies, raised in goat and rabbit, respectively. The cross-reactivity of the anti-FXR1 antibodies was assessed by Western blot analysis, and the protein was localized by IHC in a set of normal canine tissues and in canine melanocytic tumors (10 uveal and 10 oral). Western blot results demonstrated that the antibody raised in rabbit specifically recognized the canine FXR1, while the antibody raised in goat did not cross-react with this canine protein. FXR1 protein was immunodetected using rabbit anti-FXR1 antibody, in canine normal tissues with different levels of intensity and distribution. It was also detected in 10/10 uveal and 9/10 oral melanocytic tumors. The present study validated for the first time the use of anti-FXR1 antibody in dogs and highlighted different FXR1 protein expression in canine melanocytic tumors, the significance of which is undergoing further investigations.

Beta-catenin localization and timing of early development of bovine embryos obtained from oocytes matured in the presence of follicle stimulating hormone.

In mammalian species, embryos which grow more rapidly are believed to be more competent and viable than they are slower developing counterparts. Although the most important decrease in development occurs between the zygote and blastocyst stages, there is a growing amount of evidence to suggest that maturation conditions and oocyte quality have a profound influence on the developmental potential of early mammalian embryos. Gene transcripts and polypeptides stored in the oocytes, such as junctional proteins, sustain the initial development of embryos. In the present study we demonstrated a relationship between the timing of the development of in vitro-produced bovine embryos and the distribution and localization of the junctional protein beta-catenin. We further demonstrated that the presence of FSH during IVM supports cleavage and the blastocyst rate, and also has a positive effect on the speed of development, since embryos obtained from oocytes matured with the gonadotropin and observed on days 4, 5 and 6 post-insemination (p.i.) grew faster than those matured in a medium supplemented with BSA. Moreover, the majority of embryos which developed past the 16-cell stage showed a proper distribution of beta-catenin just beneath the membrane surfaces of all blastomeres and an appropriate morphology, as confirmed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analysis. In conclusion, our data suggest that supplementing FSH during in vitro maturation aids the development of bovine embryos and promotes the correct expression of beta-catenin, increasing the likelihood that embryos will develop to the blastocyst stage.