Spermatocyte apoptosis, which involves both intrinsic and extrinsic pathways, explains the sterility of Graomys griseoflavus Graomys centralis male hybrids

Spermatogenic impairment and the apoptotic pathways involved in establishing sterility of male hybrids obtained from crossing Graomys griseoflavus females with Graomys centralis males were studied. Testes from G. centralis, G. griseoflavus and hybrids were compared at different ages. Terminal transferase-mediated dUTP nick-end labelling assay (TUNEL), Fas, Bax and cytochrome c labelling were used for apoptosis evaluation, and calbindin D 28k staining as an anti-apoptotic molecule. In 1-month-old animals, spermatocytes were positive for all apoptotic markers, but moderate TUNEL (+) spermatocyte frequency was only found in G. centralis. At subsequent ages, the apoptotic markers were downregulated in testes from parental cytotypes, but not in hybrid testes. TUNEL (+) spermatocytes were present at 78% and 44% per tubule cross-section in 2- and 3-month-old hybrid animals, respectively. Pachytene spermatocyte death in adult hybrids occurs via apoptosis, as revealed by high caspase-3 expression. Calbindin was highly expressed in spermatocytes of adult hybrids, in which massive cell death occurs via apoptosis. Calbindin co-localisation with TUNEL or Fas, Bax and cytochrome c was very limited, suggesting an inverse regulation of calbindin and apoptotic markers. Hybrid sterility is due to breakdown of spermatogenesis at the pachytene spermatocyte stage. Both extrinsic and intrinsic pathways are involved in apoptosis of spermatocytes, which are the most sensitive cell type to apoptotic stimuli.Fil: Rodriguez, Valeria. Universidad Nacional de Córdoba. Facultad de Medicina. Cátedra de Bioquímica y Biología Molecular; ArgentinaFil: Díaz de Barboza, Gabriela Edith. Universidad Nacional de Córdoba. Facultad de Medicina. Cátedra de Bioquímica y Biología Molecular; ArgentinaFil: Ponce, Rubén Hugo. Universidad Nacional de Córdoba. Facultad de Odontología; ArgentinaFil: Merico, Valeria. Universita degli Studi di Pavia; ItaliaFil: Garagna, Silvia. Universita degli Studi di Pavia; ItaliaFil: Tolosa, Nori Graciela. Universidad Nacional de Córdoba. Facultad de Medicina. Cátedra de Bioquímica y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentin

Maternal Oct-4 is a potential key regulator of the developmental competence of mouse oocytes

Background The maternal contribution of transcripts and proteins supplied to the zygote is crucial for the progression from a gametic to an embryonic control of preimplantation development. Here we compared the transcriptional profiles of two types of mouse MII oocytes, one which is developmentally competent (MIISN oocyte), the other that ceases development at the 2-cell stage (MIINSN oocyte), with the aim of identifying genes and gene expression networks whose misregulated expression would contribute to a reduced developmental competence. Results We report that: 1) the transcription factor Oct-4 is absent in MIINSN oocytes, accounting for 2) the down-regulation of Stella, a maternal-effect factor required for the oocyte-to-embryo transition and of which Oct-4 is a positive regulator; 3) eighteen Oct-4-regulated genes are up-regulated in MIINSN oocytes and are part of gene expression networks implicated in the activation of adverse biochemical pathways such as oxidative phosphorylation, mitochondrial dysfunction and apoptosis. Conclusion The down-regulation of Oct-4 plays a crucial function in a sequence of molecular processes that leads to the developmental arrest of MIINSN oocytes. The use of a model study in which the MII oocyte ceases development consistently at the 2-cell stage has allowed to attribute a role to the maternal Oct-4 that has never been described before. Oct-4 emerges as a key regulator of the molecular events that govern the establishment of the developmental competence of mouse oocytes

OCT4 and the acquisition of oocyte developmental competence during folliculogenesis

The role that the transcription factor OCT4 plays during oocyte growth is yet unknown. In this review, we summarise the data on its potential role in the acquisition of oocyte developmental competence in the mouse. These studies describe the presence in MII oocytes and 2-cell embryos of an OCT4 transcriptional network that might be part of the molecular signature of maternal origin on which the inner cell mass and the embryonic stem cell-associated pluripotency is assembled and shaped. The Oct4-gene regulatory network thus provides a connection between eggs, early preimplantation embryos and embryonic stem cells

A half-century of studies on a chromosomal hybrid zone of the house mouse

The first natural chromosomal variation in the house mouse was described nearly 50 years ago in Val Poschiavo on the Swiss side of the Swiss–Italian border in the Central Eastern Alps. Studies have extended into neighboring Valtellina, and the house mice of the Poschiavo-Valtellina area have been subject to detailed analysis, reviewed here. The maximum extent of this area is 70 km, yet it has 4 metacentric races and the standard 40-chromosome telocentric race distributed in a patchwork fashion. The metacentric races are characterized by highly reduced diploid numbers (2n = 22–26) resulting from Robertsonian fusions, perhaps modified by whole-arm reciprocal translocations. The races hybridize and the whole Poschiavo-Valtellina area can be considered a “hybrid zone.” The studies of this area have provided insights into origin of races within hybrid zones, gene flow within hybrid zones and the possibility of speciation in hybrid zones. This provides a case study of how chromosomal rearrangements may impact the genetic structure of populations and their diversification.Fil: Giménez, Mabel Dionisia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas; Argentina. University of York; Reino UnidoFil: Förster, Daniel W.. Leibniz-institute For Zoo And Wildlife Research; Alemania. University of York; Reino UnidoFil: Jones, Eleanor P.. University of York; Reino Unido. Fera Science; Reino UnidoFil: Jóhannesdóttir, Fríđa. University of York; Reino Unido. Cornell University; Estados UnidosFil: Gabriel, Sofia I.. Universidade de Lisboa; Portugal. University of York; Reino UnidoFil: Panithanarak, Thadsin. Burapha University; Tailandia. University of York; Reino UnidoFil: Scascitelli, Moira. University of York; Reino UnidoFil: Merico, Valeria. Universita Di Pavia; ItaliaFil: Garagna, Silvia. Universita Di Pavia; ItaliaFil: Searle, Jeremy B.. University of York; Reino Unido. Cornell University; Estados UnidosFil: Hauffe, Heidi C.. Instituto Agrario San Michele All'adige Fondazione Edmund Mach. Centro Ricerca E Innovazione; Italia. University of York; Reino Unid

Transcriptome based identification of mouse cumulus cell markers that predict the developmental competence of their enclosed antral oocytes

BACKGROUND: The cumulus cells (CCs) enveloping antral and ovulated oocytes have been regarded as putative source of non-invasive markers of the oocyte developmental competence. A number of studies have indeed observed a correlation between CCs gene expression, embryo quality, and final pregnancy outcome. Here, we isolated CCs from antral mouse oocytes of known developmental incompetence (NSN-CCs) or competence (SN-CCs) and compared their transcriptomes with the aim of identifying distinct marker transcripts. RESULTS: Global gene expression analysis highlighted that both types of CCs share similar transcriptomes, with the exception of 422 genes, 97.6% of which were down-regulated in NSN-CCs vs. SN-CCs. This transcriptional down-regulation in NSN-CCs was confirmed by qRT-PCR analysis of CC-related genes (Has2, Ptx3, Tnfaip6 and Ptgs2). Only ten of the 422 genes were up-regulated with Amh being the most up-regulated in NSN-CCs, with an average 4-fold higher expression when analysed by qRT-PCR. CONCLUSIONS: The developmental incompetence (NSN) or competence (SN) of antral oocytes can be predicted using transcript markers expressed by their surrounding CCs (i.e., Has2, Ptx3, Tnfaip6, Ptgs2 and Amh). Overall, the regulated nature of the group of genes brought out by whole transcriptome analysis constitutes the molecular signature of CCs associated either with developmentally incompetent or competent oocytes and may represent a valuable resource for developing new molecular tools for the assessment of oocyte quality and to further investigate the complex bi-directional interaction occurring between CCs and oocyte

A Brief Incubation of Cumulus-Enclosed Mouse Eggs in a Calcium-Free Medium Containing a High Concentration of Calcium-Chelator Markedly Improves Preimplantation Development

The presence of cumulus cells (CCs) surrounding ovulated eggs is beneficial to in vitro fertilization and preimplantation development outcomes in several mammalian species. In the mouse, this contribution has a negligible effect on the fertilization rate; however, it is not yet clear whether it has positive effects on preimplantation development. Here, we compared the rates of in vitro fertilization and preimplantation development of ovulated B6C3F1 CC-enclosed vs. CC-free eggs, the latter obtained either after a 5 min treatment in M2 medium containing hyaluronidase or after 5–25 min in M2 medium supplemented with 34.2 mM EDTA (M2-EDTA). We found that, although the maintenance of CCs around ovulated eggs does not increment their developmental rate to blastocyst, the quality of the latter is significantly enhanced. Most importantly, for the first time, we describe a further quantitative and qualitative improvement, on preimplantation development, when CC-enclosed eggs are isolated from the oviducts in M2-EDTA and left in this medium for a total of 5 min prior to sperm insemination. Altogether, our results establish an important advancement in mouse IVF procedures that would be now interesting to test on other mammalian species

An epigenomic biography of the mammalian oocyte

Successful fertilisation and early development depend on the quality of the ovulated oocyte. Even though this is endorsed by both the experimental and clinical practice, our still scarce knowledge of the biology of the mammalian oocyte makes it difficult to identify parameters that define the quality or 'developmental competence' of an oocyte. The many attempts to establish markers of the oocyte's developmental competence have produced divergent results or have worked only in specific experimental contexts. To this respect, the oocyte morphology, the concentration of various factors in the follicular fluid, the role of the oocyte's mitochondria, the telomere length and the transcription profile of cumulus cell-specific genes are some of the most studied aspects of the oocyte and of the ovarian follicle biology. The analysis of the level of cumulus cell-specific transcripts has identified groups of genes that are directly (PTGS2, HAS2, GREM1 and PTX3) [1-3] or inversely (GPX3, CXCR4, CCND2 and CTNND1) [4-5] correlated to human embryo preimplantation quality and pregnancy outcome. Recent studies analysed the whole cumulus cells transcriptome in human [6]and bovine [7] cumulus cell-oocyte complexes bringing up a new set of putative marker transcripts. The concentration in the follicular fluid of myo-inositol (a serum trophic factor), inhibin B [8-9] or AMH have been used for their predictive value of human preimplantation embryonic development, with the latter suggested as a better predictor of oocyte fertilisability [10] and pregnancy rate [11]. Some authors have proposed the presence of a high level of estradiol on the day of hCG administration as a candidate marker of low pregnancy rate [12-13]), but these data are conflicting with others that describe no correlations with the final pregnancy outcome [14-15]. Similarly, a reduction at the time of oocyte collection in the level of progesterone receptor in human cumulus cells was associated with morphologically good oocytes [16]. Mitochondria have been the subject of a large number of studies, but how and whether they contribute to the determination of the oocyte developmental competence is still unclear. The whole preimplantation period is sustained by mitochondria produced during oogenesis and only when the embryo begins implantation, their production is resumed. Therefore, an unbalanced number of these organelles, an incorrect distribution or an altered function may have negative effects on the early stages of development [for a review see 17]. The number of mitochondria in mouse primordial germ cells (PGCs) is very small, being approximately 10-100 per cell, then, by the mature oocyte they sum up to ~ 90.000 [18]. The total number of mitochondria seems to be critical to the developmental competence of an oocyte, since subnormal levels of these organelles correlate with premature maturation arrest of the oocyte and early death of the preimplantation embryo [19-20]. A low mitochondrial complement may determine a bioenergetic/metabolic shortage with consequences on the oocyte’s ability of meiotic resumption, fertilisation and to sustain the early phases of development [reviewed in 20-23]. Along with these studies, ATP values have also been associated with the oocyte’s developmental competence; an ATP content of > 2 pmol seems a threshold to distinguish between developmentally competent and incompetent human oocytes [24]. During folliculogenesis, mitochondria are located in different regions of the oocyte [25-28] and by the mature oocyte they will have an asymmetric polar distribution that will be maintained through segmentation resulting in blastomeres that will own a different number of mitochondria with a different spatial patterning. A number of observations substantiate the involvement of these organelles not only as powerhouse, producing most of the ATP in the cell, but they may also regulate development by modulating Ca2+ signalling, reactive oxygen species (ROS) and intermediary metabolites and through their control of apoptosis. Oxidative stress and intracellular redox potential (IRP) have been shown to regulate the function of a number of transcription factors important in early development. For example, NF-KB (nuclear factor kappa-light-chain-enhancer of activated B cells) and GSK3β (glycogen synthase kinase-3β), expressed during preimplantation development, are activated by mitochondrial ROS production [29-30]. S-glutathionylation of many transcription factors [31] occurs after the oxidation of the IRP, thus, the ability of mitochondria to modulate the IRP will possibly change the activity of these proteins. Altered mitochondrial Ca2+ cycling and ROS production are determinant for the oocyte to enter and accomplish the apoptotic programme [32]. Oxidative stress induces mitochondrial dysfunction that triggers apoptosis in the mouse oocyte and zygote [33]. Specific oocyte morphological features are used to select female gametes of good quality. Oocytes are graded as good, when they posses a cytoplasm with fine granules, a single polar body, a narrow perivitelline space and a ring shaped zona pellucida [34]. On the contrary, they are correlated with low fertilisability and developmental competence, when they own vacuoles and refractile bodies within the ooplasm. Although used in some laboratories that practice human assisted reproduction, these morphological markers have been the subject of many criticisms and are not widely accepted [34-40]. As for nuclear morphological features, a large literature as demonstrated the possibility to select between developmentally competent and incompetent antral oocyte, depending on their chromatin configuration (see below). This selection, however, relies on the use of fluorochromes whose use, for obvious reasons, is not advisable in our species. Up to date, our knowledge lacks of links between the morphological aspects described and the genomic and epigenomic features that work in the backstage. Bringing to light these links would permit the identification of molecular markers of the oocyte developmental competence. In this article, we will review our current understanding of the changes that occur to the oocyte epigenetic signature during folliculogenesis and in mature oocytes