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In brief RNA granules travel through the cumulus cell network of transzonal projections which is associated with oocyte developmental competence, and RNA packaging involves RNA-binding proteins of the Fragile X protein family. Abstract The determinants of oocyte developmental competence have puzzled scientists for decades. It is known that follicular conditions can nurture the production of a high-quality oocyte, but the underlying mechanisms remain unknown. Somatic cumulus cells most proximal to the oocyte are known to have cellular extensions that reach across the zona pellucida and contact with the oocyte plasma membrane. Herein, it was found that transzonal projections (TZPs) network quality is associated with developmental competence. Knowing that ribonucleoparticles are abundant within TZPs, the distribution of RNA-binding proteins was studied. The Fragile X-related proteins (FXR1P and FXR2P) and two partnering protein families, namely cytoplasmic FMRP-interacting protein and nuclear FMRP-interacting protein, exhibited distinctive patterns consistent with roles in regulating mRNA packaging, transport, and translation. The expression of green fluorescent protein (GFP)–FMRP fusion protein in cumulus cells showed active granule formation and their transport and transfer through filipodia connecting with neighboring cells. Near the projections’ ends was found the cytoskeletal anchoring protein Filamin A and active protein synthesis sites. This study highlights key proteins involved in delivering mRNA to the oocyte. Thus, cumulus cells appear to indeed support the development of high-quality oocytes via the transzonal network.

In brief The proposed culture system improves the current state of in vitro culture of growing oocytes in the bovine species and allows access to the untapped gamete reserve, thus improving reproductive efficiency. Abstract The present study aimed to improve the in vitro culture of bovine oocytes collected from early antral follicles (EAFs) to support the progressive acquisition of meiotic and developmental competence. The rationale that drove the development of such a culture system was to maintain as much as possible the physiological conditions that support the oocyte growth and differentiation in vivo. To this extent, oocytes were cultured for 5 days, which parallels the transition from early to medium antral follicles (MAFs) in the bovine, and supports promoting a 3D-like structure were provided. Additionally, the main hormones (follicle-stimulating hormone, estradiol, progesterone, and testosterone) were added in concentrations similar to the ones previously observed in bovine EAFs. The meiotic arrest was imposed using cilostamide. The cultured cumulus–oocyte complexes (COCs) reached a mean diameter of 113.4 ± 0.75 µm and showed a progressive condensation of the chromatin enclosed in the germinal vesicle (GV), together with a gradual decrease in the global transcriptional activity, measured by 5-ethynyl uridine incorporation. The described morpho-functional changes were accompanied by an increased ability to mature and develop to the blastocyst stage in vitro, although not matching the rates obtained by MAF-retrieved oocytes. The described system improves the current state of in vitro culture of growing oocytes in the bovine species, and it can be used to increase the number of gametes usable for in vitro embryo production in animals of high genetic merit or with specific desirable traits.

In the past four decades, the bovine model has been highly informative and inspiring to assisted reproductive technologies (ART) in other species. Most of the recent advances in ART have come from studies in cattle, particularly those unveiling the importance of several processes that must be recapitulated in vitro to ensure the proper development of the oocyte. The maintenance of structural and functional communications between the cumulus cells and the oocyte and a well-orchestrated chromatin remodeling with the gradual silencing of transcriptional activity represent essential processes for the progressive acquisition of oocyte developmental competence. These markers are now considered the milestones of physiological approaches to increase the efficiency of reproductive technologies. Different in vitro approaches have been proposed. In particular, the so-called "pre-IVM" or "prematuration" is a culture step performed before in vitro maturation (IVM) to support the completion of the oocyte differentiation process. Although these attempts only partially improved the embryo quality and yield, they currently represent a proof of principle that oocytes retrieved from an ovary or an ovarian batch shouldn't be treated as a whole and that tailored approaches can be developed for culturing competent oocytes in several species, including humans. An advancement in ART's efficiency would be desirable in carnivores, where the success is still limited. Since the progress in reproductive medicine has often come from comparative studies, this review highlights aspects that have been critical in other species and how they may be extended to carnivores.

Programmed cell death (PCD) has been discussed and categorized endlessly over the years. Herein, we comment on the chaotic classification of PCD in the mammalian ovary and the compartmentalized ovarian follicle based on an unfiltered overview of the extent of publication about some of the different modes of PCD on PubMed.

The definition of new reliable markers for neonatal maturity evaluation is crucial in canine clinical practice. Concerns about the safety of amniotic sampling in pregnant dogs have prevented its collection for diagnostic purposes. Moreover, amniotic fluid had been considered waste material until the latest studies reported amniocentesis as a reliable and safe procedure, even in the canine species. In our study, amniotic fluid (n = 63) collected at birth from ten dogs undergoing elective Caesarean sections at term was analysed to discover new potential indices of canine neonatal maturity. Based on gestational age, mothers and puppies were divided into two groups: the early group (≤65 days from luteinizing hormone (LH) surge, n = 5) and the late group (65 days from LH surge, n = 5). Amniotic parameters of the lightest and heaviest puppy in individual/each litter, with a birth weight difference of at least 20% among littermates, were also compared. In particular, the content of lecithin, sphingomyelin, surfactant protein A (SP-A), cortisol, and pentraxin 3 (PTX3) in amniotic fluid, which is considered predictive of foetal development in humans, were investigated. Maternal serum SP-A and cortisol were also measured simultaneously. All amniotic parameters were detectable in canine amniotic fluid. Interestingly, the concentrations of different amniotic parameters correlated with each other. Lecithin was positively correlated with sphingomyelin (p 0.0001), maternal SP-A (p 0.0005), and the ratio of amniotic and maternal cortisol (p 0.004). Amniotic SP-A was inversely correlated to maternal SP-A (p 0.05), lecithin (p 0.005), and lecithin-sphingomyelin ratio (p 0.05). A positive correlation was also recorded between amniotic and maternal cortisol (p 0.008). Considering that all puppies were born alive and mature, these data could provide a potential range of expected amniotic values in full-term new-born dogs. Furthermore, since gestational age was positively correlated with both maternal and amniotic cortisol (p 0.0001) and amniotic PTX3 (p 0.05), amniotic fluid seems to be an attractive, innovative, and minimally invasive matrix with potential diagnostic and prognostic utility for the investigation of canine maturity.

The mammalian ovary is a large source of oocytes organized into follicles at various stages of folliculogenesis. However, only a limited number of them can be used for in vitro embryo production (IVEP), while most have yet to complete growth and development to attain full meiotic and embryonic developmental competence. While the in vitro growth of primordial follicles in the ovarian cortex has the potential to produce mature oocytes, it is still at an experimental stage. The population of early antral follicles (EAFs), instead, may represent a reserve of oocytes close to completing the growth phase, which might be more easily exploited in vitro and could increase the number of female gametes dedicated to IVEP.Here we present in vitro culture strategies that have been developed utilizing physiological parameters to support the specific needs of oocytes at distinct stages of differentiation, in order to expand the source of female gametes for IVEP by maximizing the attainment of fertilizable oocytes. Furthermore, these culture systems provide powerful tools to dissect the molecular processes that direct the final differentiation of the mammalian oocyte.

Modern farming is increasingly relying on in vitro embryo production (IVP). However, in vitro culture systems are not yet capable to fully support oocyte and early embryonic development. Recent studies in mice have revealed the importance of zinc in regulating oogenesis and early embryogenesis (Bernhardt et al. 2012; Tian & Diaz, 2013). Moreover, zinc transporters are differentially expressed in different cell types throughout several steps of oogenesis and embryogenesis in cattle (Dieci et al. 2016; http://emb-bioinfo.fsaa.ulaval.ca/IMAGE/). Nevertheless, zinc modulation during culture of oocytes and early embryos has not been yet implemented in cattle. Thus, we assessed whether zinc is one of the essential players in the process of bovine oocyte competence acquisition and embryo quality by supplementing the media used to 1) grow oocytes collected from small antral follicle (SAF), 2) mature oocytes collected from middle-size antral follicles (MAF) and 3) culture in vitro produced zygotes. In each experiment, oocytes cultured under standard conditions served as control group. Zinc supplementation during oocytes in vitro growth significantly improved the proportion of oocytes reaching the metaphase-II stage of meiosis after subsequent standard in vitro maturation (IVM). On the contrary supplementation of IVM or embryo culture media had no effects on blastocysts rates and embryo quality, as assessed by cell number per embryo. In conclusion, zinc supplementation can greatly improve the exploitation of the ovarian reserve since it increases the meiotic competence of growing oocytes, which are usually discarded in standard IVP settings. These results are in accordance with in vivo studies in mouse, showing the detrimental effects of zinc deficiency before ovulation on subsequent maturation and embryonic development (Tian & Diaz, 2013). On the contrary, bovine fully-grown oocyte from MAF seems able to compensate for zinc absence in the in vitro culture medium., International Journal of Health, Animal Science and Food Safety, Vol 5, No 1s (2018)

Background: Polycystic-ovary syndrome (PCOS) is a reproductive illness characterized by hyperandrogenism and anovulation. Using hyperandrogenized mice, it was demonstrated that the oral administration of incremental dose of follicle stimulating hormone (FSH) attenuated some of PCOS characteristics. This work aimed to study the effect of ultra-low doses of combined FSH and progesterone orally administered on PCOS murine model. Moreover, the effect of sequential kinetic activation of administered hormones was tested. Methods: Thirty-two female mice were used as animal model (four groups of eight animals each). Mice were hyperandrogenized by injection of dehyidroepiandrosterone diluted in sesame oil. Control group received only oil. Simultaneously, each animal daily received per os an activated or a not-activated combination of FSH (0.44 pg) plus progesterone (0.44 pg) or saline solution as control. Serum testosterone, estradiol, progesterone and luteinizing hormone were analyzed as endocrine markers and a morphological study of antral follicle was conducted. Data were analyzed by one-way ANOVA, followed by multiple comparison test. The p0.05 vs. control group) and reduced cystic signs in the follicles (p

Folliculogenesis is the fundamental process leading to oocyte maturation and its developmental competence, which are determined by oocyte and follicular cells interplay (Luciano et al., 2004). Recent studies in cattle describe Progesterone (P4) as a key molecule acting during follicle development through different signaling pathways involving different receptors (Aparicio et al., 2011, Nilsson et al., 2009). The aim of this study is to evaluate the effect on oocyte meiotic maturation of inhibiting two P4 receptors: Progesterone Receptor Membrane Component 1 (PGRMC1) and the classic nuclear Progesterone Receptor (nPR) respectively using the specific inhibitors AG205 and Aglepristone. Bovine cumulus cell-oocyte complexes (COCs) and denuded oocytes (DOs) were in vitro matured with different concentrations of AG205. Our results showed a decrease both in first polar body (PBI) extrusion and in the percentage of oocytes reaching MII stage in treated oocytes compared to controls (one way ANOVA, P, International Journal of Health, Animal Science and Food Safety, V. 4, N. 1s (2017)

Progesterone Receptor Membrane Component-1 (PGRMC1) is a multifunctional protein that is found in multiple subcellular compartments of several organs, suggesting a specific function at each localization site. Yet, its mechanism of action and putative role in different places is not clear (Cahill 2007; Rohe et al. 2009). Among the several subcellular sites of expression, PGRMC1 was found in the nucleolus of human cells (Ahmad et al. 2009) as well as in nucleolar precursors bodies of bovine zygotes (Luciano et al. 2010). The aim of this study is to test the hypothesis that PGRMC1 might modulate nucleolar function. Immunofluorescence experiments confirmed nucleolar localization in rat spontaneously immortalized granulosa cells (SIGCs), bovine granulosa cells (bGC) and bovine oocytes. Moreover, additional immune localization experiments conducted in bGC revealed that PGRMC1 co-localizes with nucleolin, a well-known nucleolar marker that exerts an important function within the nucleolus (Tajrishi et al. 2011). Additionally, siRNA mediated gene knockdown experiments, showed that when PGRMC1 expression is silenced, nucleolin localization shifts from the nucleolus to the nucleoplasm. This suggests a PGRMC1/nucleolin functional association. However, in situ proximity ligation assay did not detect a direct interaction between these two proteins, suggesting the involvement of additional molecules that could mediate PGRMC1/nucleolin interaction. In conclusion, these studies suggest a function for PGRMC1 in nucleolar activity and set the stage for further investigations aimed at dissecting PGRMC1’s molecular mechanisms of action in the nuclear compartment., International Journal of Health, Animal Science and Food Safety, Vol 3, No 1s (2016)

The present studies were aimed to assess Progesterone Receptor Membrane Component-1 (PGRMC1) role in regulating bovine granulosa cells (bGC) mitosis. First, we performed immunofluorescence studies on in vitro cultured bGC collected from antral follicles, which showed that PGRMC1 localizes to the spindle apparatus in mitotic cells. Then, to evaluate PGRMC1 effect on cell proliferation we silenced its expression with RNA interference technique (RNAi). Quantitative RT-PCR and immunoblotting confirmed down-regulation of PGRMC1 expression, when compared to CTRL-RNAi treated bGC (p, International Journal of Health, Animal Science and Food Safety, Vol 2, No 1s (2015): Proceeding of Veterinary and Animal Science Days

Oocytes isolated from cows of reproductive age with reduced antral follicle counts (AFC) have a diminished capacity of embryonic development, which may be related to alterations in the mechanism that directs the proper segregation of chromosomes. Because we demonstrated that progesterone receptor membrane component 1 (PGRMC1) is involved in chromosome congression and metaphase II (MII) plate formation, the present study was designed to determine 1) if the decrease in oocyte developmental competence observed in dairy cows with a reduced AFC is due to a higher incidence of aneuploidy and 2) whether alterations in PGRMC1 contributes to the incidence of aneuploidy. Oocytes from ovaries with reduced AFC and age-matched controls were matured in vitro and the occurrence of aneuploidy determined as well as the mRNA level and localization of PGRMC1. Although oocytes from ovaries with reduced AFC were capable of undergoing meiosis in vitro, these oocytes showed a 3-fold increase in aneuploidy compared to oocytes isolated from control ovaries (P < 0.05). Although Pgrmc1 mRNA levels were not altered, PGRMC1 and aurora kinase B (AURKB) failed to localize to precise focal points on MII chromosomes of oocytes from ovaries with reduced AFC. Furthermore, when oocytes of control ovaries were cultured with an inhibitor of AURKB activity, their MII plate was disrupted and PGRMC1 was not properly localized to the chromosomes. These results suggest that alterations in PGRMC1 and/or AURKB localization account in part for the increased aneuploidy and low development competence of oocytes from ovaries with reduced AFC.

In the last few years, several works suggest that Growth Hormone (GH) is involved in follicular development and oocyte maturation. These actions may reflect endocrine roles of pituitary GH and also account for local autocrine or paracrine activities of GH produced in reproductive tissue. This study was aimed to verify whether the developmental competence of bovine female gametes might be related to ovarian GH. We evaluated the localisation and distribution of GH in the cumulus oocytes complexes (COCs) and the concentration of GH in the oocytes and in the follicular fluids (FF) from ovaries classified on the basis of the follicles number. Oocytes retrieved from ovaries with more than 10 follicles of 2 to 5 mm in diameter (High ovaries, Hi) show higher rate of maturation and blastocyst formation than those retrieved from ovaries with less than 10 follicles (Low ovaries, Lo). At the same time we measured Estrogen (E2) and Progesterone (P4) concentrations in FF, to relate oocytes quality, GH concentration and follicle health. GH localization in COCs and oocytes was performed by indirect immunofluorescence and its concentration within the ooplasm was evaluated by microspectrophotometer analysis. GH, E2 and P4 concentrations in FF were measured by an Enzyme Linked ImmunoSorbent assay (ELISA). We observed a positive, diffuse signal at cytoplasmic level in most of the cumulus cells, with no differences between COCs collected from Hi and Lo ovaries. On the contrary, GH level was significantly higher in the oocytes collected from Lo ovaries than in those recovered from Hi ovaries. Finally we found that also GH level in the FF was inversely related to the oocytes developmental capability. We suggest that the increase of GH in the oocytes and in the FF derived from Lo ovaries might be interpreted as attempt of the follicular environment to improve ovarian activity and in turn oocytes developmental competence in a autocrine-paracrine manner. Moreover, E2, and P4 levels in FF suggest that, in our model, atresia processes are also involved in oocyte developmental capability and that the highest level of GH may represent a local reaction to these phenomena.