Extracellular vesicles secreted by cumulus cells contain microRNAs that are potential regulatory factors of mouse oocyte developmental competence.

The role of cumulus cells (CCs) in the acquisition of oocyte developmental competence is not yet fully understood. In a previous study, we matured cumulus-denuded fully-grown mouse oocytes to metaphase II (MII) on a feeder layer of CCs (FL-CCs) isolated from developmentally competent (FL-SN-CCs) or incompetent (FL-NSN-CCs) SN (surrounded nucleolus) or NSN (not surrounding nucleolus) oocytes, respectively. We observed that oocytes cultured on the former could develop into blastocysts, while those matured on the latter arrested at the 2-cell stage. To investigate the CC factors contributing to oocyte developmental competence, here we focused on the CCs' release into the medium of extracellular vesicles (EVs) and on their miRNA content. We found that, during the 15-h transition to MII, both FL-SN-CCs and FL-NSN-CCs release EVs that can be detected, by confocal microscopy, inside the zona pellucida (ZP) or the ooplasm. The majority of EVs are <200 nm in size, which is compatible with their ability to cross the ZP. Next-generation sequencing of the miRNome of FL-SN-CC versus FL-NSN-CC EVs highlighted 74 differentially expressed miRNAs, with 43 up- and 31 down-regulated. Although most of these miRNAs do not have known roles in the ovary, in silico functional analysis showed that seven of these miRNAs regulate 71 target genes with specific roles in meiosis resumption (N = 24), follicle growth (N = 23), fertilization (N = 1), and the acquisition of oocyte developmental competence (N = 23). Overall, our results indicate CC EVs as emerging candidates of the CC-to-oocyte communication axis and uncover a group of miRNAs as potential regulatory factors.

Inhibition of lysyl oxidase stimulates TGF-ß signaling and metalloproteinases-2 and -9 expression and contributes to the disruption of ascending aorta in rats: protection by propylthiouracil.

Mutations in lysyl oxidase (LOX) genes cause severe vascular anomalies in mice and humans. LOX activity can be irreversibly inhibited by the administration of ß-aminoproprionitrile (BAPN). We investigated the mechanisms underlying the damage to the ascending thoracic aorta induced by LOX deficiency and evaluated whether 6-propylthiouracil (PTU) can afford protection in rats. BAPN administration caused disruption of the ascending aortic wall, increased the number of apoptotic cells, stimulated TGF-ß signaling (increase of nuclear p-SMAD2 staining), and up-regulated the expression of metalloproteinases-2 and -9. In BAPN-treated animals, PTU reduced apoptosis, p-SMAD2 staining, MMP-2, and -9 expression, and markedly decreased the damage to the aortic wall. Our results suggest that, as in some heritable vascular diseases, enhanced TGF-ß signaling and upregulation of MMP-2 and -9 can contribute to the pathogenesis of ascending aorta damage caused by LOX deficiency. We have also shown that PTU, a drug already in clinical use, protects against the effects of LOX inhibition. MMP-2 and -9 might be potential targets of new therapeutic strategies for the treatment of vascular diseases caused by LOX deficiency.

Germ cell cysts, a fetal feature in mammals, are constitutively present in the adult armadillo.

Formation and subsequent break down of ovarian germ cell (GC) cysts is a key and an evolutionary-conserved developmental event, described in phylogenetically diverse species of invertebrates and vertebrates. In mammals, cyst break down (CBD) ends at the time of, or soon after, birth with the formation of primordial follicles enclosing single oocytes, which constitute the sole reservoir of gametes available through the whole female's reproductive life. In this study, we challenge this paradigm demonstrating the constitutive presence of a large number of cysts, enclosing two-thirty GCs, in the ovary of the adult armadillo Chaetophractus villosus, belonging to the superorder Xenarthra, one of the earliest offshoots among placentals. We also describe that (a) GCs enclosed within cysts are connected by intercellular bridges-intercellular bridges-markers of their clonal origin; (b) CBD occurs through four main phases, ending with primordial follicles containing single oocytes; (c) GCs encompass meiotic prophase I stages, from leptotene to diplotene; (d) seasonal variations in the number of GCs enclosed within cysts, suggesting the presence of a GC multiplying activity. The armadillo C. villosus''s ovary emerges as an extraordinary resource to investigate folliculogenesis and to explore the evolutionary past of the mammalian ovary.

Sertoli-immature spermatids disengagement during testis regression in the armadillo.

In nature, mammalian seasonal breeders undergo spermatogenetic arrest during the non-breeding season. In the large hairy armadillo Chaetophractus villosus, testis regression initiates with immature post-meiotic germ cells sloughing into the tubule lumen and continues with the death of the remaining spermatocytes. At the end of the regression period, only spermatogonia and Sertoli cells persist in the seminiferous epithelium. It has been suggested that cell sloughing is determined by changes in the adhesion complexes between Sertoli cells and spermatids, which are mediated by low intra-testicular testosterone levels. By immunofluorescence and Western blotting we studied key proteins of the N-cadherin/N-cadherin and A6B1-integrin/laminin interlocks that contribute to the complex Sertoli/spermatid adhesion system throughout the eight stages of the seminiferous epithelium cycle in the comparison between active and regressing testes. In active testis, B1-integrin, laminin G3, N-cadherin, B-catenin, P-B-catenin-Tyr654, FAK, P-FAK-Tyr397, SRC, P-SRC-Tyr416 proteins present a spermatogenetic cycle-dependent localisation pattern, unmaintained in regressing testes. In the latter, quantitative variations and changes in the phosphorylation state of protein FAK, SRC and B-catenin contribute to the disassembly of the N-cadherin/N-cadherin and A6B1-integrin/laminin interlocks, thus promoting the massive release of immature spermatids.

IVM of mouse fully grown germinal vesicle oocytes upon a feeder layer of selected cumulus cells enhances their developmental competence.

In the ovary, acquisition of oocyte developmental competence depends on a bidirectional exchange between the gamete and its companion cumulus cells (CCs). In this study we investigated the contribution of CCs surrounding oocytes of known developmental competence or incompetence to the acquisition of oocyte developmental competence. To this end, feeder layers of CCs (FL-CCs) were prepared using CCs isolated either from: (1) developmentally competent mouse oocytes whose nucleolus was surrounded by a chromatin ring (FL-SN-CCs); or (2) developmentally incompetent mouse oocytes whose nucleolus was not surrounded by a chromatin ring (FL-NSN-CCs). Denuded, fully grown oocytes (DOs) were matured to the MII stage on either FL-SN-CCs or FL-NSN-CCs, inseminated with spermatozoa and cultured throughout preimplantation development. FL-SN-CCs significantly improved the acquisition of oocyte developmental competence, with a blastocyst development rate equal to that for maturation of intact cumulus-oocyte-complexes. In contrast, DOs matured on FL-NSN-CCs or in the absence of CCs exhibited developmental failure, with embryos arresting at either the 4-cell or morula stage. These results set a culture platform to further improve the protocols for the maturation of DOs and to unravel the molecules involved in the cross-talk between the gamete and its companion CCs during the germinal vesicle to MII transition.

Cytoplasmic movement profiles of mouse surrounding nucleolus and not-surrounding nucleolus antral oocytes during meiotic resumption.

Full-grown mouse antral oocytes are classified as surrounding nucleolus (SN) or not-surrounding nucleolus (NSN), depending on the respective presence or absence of a ring of Hoechst-positive chromatin surrounding the nucleolus. In culture, both types of oocytes resume meiosis and reach the metaphase II (MII) stage, but following insemination, NSN oocytes arrest at the two-cell stage whereas SN oocytes may develop to term. By coupling time-lapse bright-field microscopy with image analysis based on particle image velocimetry, we provide the first systematic measure of the changes to the cytoplasmic movement velocity (CMV) occurring during the germinal vesicle-to-MII (GV-to-MII) transition of these two types of oocytes. Compared to SN oocytes, NSN oocytes display a delayed GV-to-MII transition, which can be mostly explained by retarded germinal vesicle break down and first polar body extrusion. SN and NSN oocytes also exhibit significantly different CMV profiles at four main time-lapse intervals, although this difference was not predictive of SN or NSN oocyte origin because of the high variability in CMV. When CMV profile was analyzed through a trained artificial neural network, however, each single SN or NSN oocyte was blindly identified with a probability of 92.2% and 88.7%, respectively. Thus, the CMV profile recorded during meiotic resumption may be exploited as a cytological signature for the non-invasive assessment of the oocyte developmental potential, and could be informative for the analysis of the GV-to-MII transition of oocytes of other species.

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.

Impact of the number of Robertsonian chromosomes on germ cell death in wild male house mice.

Previous studies in the house mouse have shown that the presence of Robertsonian (Rb) metacentric chromosomes in heterozygous condition affects the process of spermatogenesis. This detrimental effect mainly depends on the number of metacentrics involved and the complexity of the resulting meiotic figures. In this study, we aimed at elucidating the relationship between the chromosomal composition and spermatogenesis impairment in mice present in an area of chromosomal polymorphism (the so-called Barcelona system BRbS) in which Rb mice are surrounded by all acrocentric animals, no established metacentric races are present and the level of structural heterozygosity is relatively low. Using the terminal deoxinucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay, we report higher frequency of apoptotic spermatogenetic cells in mice carrying six pairs of metacentrics at the homozygous state than in those carrying two or three fusions at the heterozygous state. Specifically, we detected a higher frequency of TUNEL-positive (T+) tubules and of T+ cells per tubule cross section and also a lower spermatid/spermatocyte ratio. These results indicate that the number of metacentrics at the homozygous state is more influential in determining apoptotic germ cell death than that of moderate chromosome heterozygosity. The percentage of germ cell death lower than 50 % found in our samples and the geographic distribution of the set of metacentrics within the BRbS indicate that although the spermatogenic alterations detected in this area could act as a partial barrier to gene flow, they are not sufficient to prevent Rb chromosomes from spreading in nature.

Formulation and stability evaluation of 3D alginate beads potentially useful for cumulus-oocyte complexes culture.

Ovarian follicle encapsulation in synthetic or natural matrixes based on biopolymers is potentially a promising approach to in vitro maturation (IVM) process, since it maintains follicle 3D organisation by preventing its flattening and consequent disruption of gap junctions, preserving the functional relationship between oocyte and companion follicle cells. The aim of the work was to optimise physico-chemical parameters of alginate microcapsules for perspective IVM under 3D environments. On this purpose alginate and cross-linking agent concentrations were investigated. Alginate concentration between 0.75% and 0.125% w/w and Mg(2+), Ba(2+), Ca(2+ )at concentration between 100 and 20 mM were tested. Follicle encapsulation was obtained by on purpose modified diffusion setting gelation technique, and evaluated together with beads, chemical and mechanical stability in standard and stressing conditions. Beads permeability was tested towards albumin, fetuin, pyruvate, glucose, pullulan. Results demonstrated that 0.25% alginate cross-linked in 100 mM CaCl2 beads is suitable to follicle encapsulation.

FSH and LH receptors are differentially expressed in cumulus cells surrounding developmentally competent and incompetent mouse fully grown antral oocytes.

Cumulus cells (CCs) maintain strict functional relationships with the enclosed antral oocyte and are thought to reflect its developmental competence. Several studies have described a correlation between CC gene expression and oocyte quality. Herein, we tested whether CC-specific FSH and LH receptors (FSHR and LHR, respectively) are differentially expressed in CCs enclosing developmentally competent or incompetent oocytes. To this end, mouse fully grown cumulus-oocyte complexes were isolated and their CCs and oocytes analysed separately. Based on their chromatin organisation, oocytes were classified as those with a surrounded nucleolus (SN) or a non-surrounded nucleolus (NSN), the former being developmentally competent, whereas the latter arrest at the 2-cell stage. The CCs were then analysed to compare the pattern of expression of the Fshr and Lhr genes and their proteins. Quantitative reverse transcription-polymerase chain reaction analysis revealed that only Lhr is significantly differentially expressed. Immunofluorescence analysis revealed that both FSHR and LHR proteins are significantly upregulated in CCs surrounding oocytes arrested at the 2-cell stage, reflecting their developmental incompetence.

Loss of Sertoli-germ cell adhesion determines the rapid germ cell elimination during the seasonal regression of the seminiferous epithelium of the large hairy armadillo Chaetophractus villosus.

The armadillo Chaetophractus villosus is a seasonal breeder whose seminiferous epithelium undergoes rapid regression with massive germ cell loss, leaving the tubules with only Sertoli cells and spermatogonia. Here, we addressed the question of whether this regression entails 1) the disassembly of cell junctions (immunolocalization of nectin-3, Cadm1, N-cadherin, and beta-catenin, and transmission electron microscopy [TEM]); 2) apoptosis (immunolocalization of cytochrome c and caspase 3; TUNEL assay); and 3) the involvement of Sertoli cells in germ cell phagocytosis (TEM). We showed a dramatic reduction in the extension of vimentin filaments associated with desmosomelike junctions at the interface between Sertoli and germ cells, and an increased diffusion of the immunosignals of nectin-3, Cadm1, N-cadherin, and beta-catenin. Together, these results suggest loss of Sertoli-germ cell adhesion, which in turn might determine postmeiotic cell sloughing at the beginning of epithelium regression. Then, loss of Sertoli-germ cell adhesion triggers cell death. Cytochrome c is released from mitochondria, but although postmeiotic cells were negative for late apoptotic markers, at advanced regression spermatocytes were positive for all apoptotic markers. Transmission electron microscopy analysis showed cytoplasmic engulfment of cell debris and lipid droplets within Sertoli cells, a sign of their phagocytic activity, which contributes to the elimination of the residual meiocytes still present in the latest regression phases. These findings are novel and add new players to the mechanisms of seminiferous epithelium regression occurring in seasonal breeders, and they introduce the armadillo as an interesting model for studying seasonal spermatogenesis.

Chromosomal speciation in mice: a cytogenetic analysis of recombination.

Within species, populations differing by chromosomal rearrangements ("chromosomal races") may become reproductively isolated in association with reduced hybrid fertility due to meiotic aberrations. Speciation is also possible if hybridizing chromosomal races accumulate genetic differences because of reduced meiotic recombination in the heterozygous configuration in hybrids. Here, we examine recombination in pure races and hybrids within a model system for chromosomal speciation: the hybridization of the Poschiavo (CHPO) and Upper Valtellina (IUVA) chromosomal races of house mouse in Upper Valtellina, Italy. These races differ by Robertsonian fusions/whole-arm reciprocal translocations, such that hybrids produce a pentavalent meiotic configuration. We determined the number and position of the recombination points (using an antibody against the MutL homolog 1 [MLH1] protein) on synaptonemal complexes at pachytene in laboratory-reared CHPO, IUVA, and hybrid males, analyzing at least 112 spermatocytes per karyotypic group, up to a total of 534 spermatocytes. The mean ± standard deviation numbers of MLH1 foci per spermatocyte were 22.2 ± 3.2, 20.1 ± 2.9, 20.7 ± 2.3, and 21.9 ± 2.9 for CHPO, IUVA, CHPO × IUVA, and IUVA × CHPO, respectively. Altogether, 10,146 chromosome arms were examined, allowing multiple comparisons. Overall, recombination events were more frequently distal than proximal or interstitial. The average number of proximal MLH1 foci per chromosome arm decreased going from telocentric to metacentric bivalents to pentavalents (when present), which (together with other factors) influenced the average number of MLH1 foci per cell between CHPO, IUVA, and hybrid mice. The low frequency of proximal recombination in pentavalents of CHPO-IUVA hybrids may promote reproductive isolation between the CHPO and IUVA races, when coupled with reduced hybrid unfitness.

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.

Spermatogenesis is seasonal in the large hairy armadillo, Chaetophractus villosus (Dasypodidae, Xenarthra, Mammalia).

Very little is known about the distinct reproductive biology of armadillos. Very few studies have investigated armadillo spermatogenesis, with data available only for Euphractus sexcinctus and Dasypus novemcinctus. In the present study, we analysed male germ cell differentiation in the large hairy armadillo Chaetophractus villosus throughout the year, describing a cycle of the seminiferous epithelium made of eight different stages. Evaluation of the testis/body mass ratio, analysis of the architecture of the seminiferous epithelium and the frequency of defective seminiferous tubules allowed identification of a temporal interruption of spermatogenesis during the period between mid-May to July (mid-end autumn) in correlation with very low testosterone levels. Overall, these results suggest that spermatogenesis is seasonal in C. villosus.

Gatekeeper of pluripotency: a common Oct4 transcriptional network operates in mouse eggs and embryonic stem cells.

BACKGROUND: Oct4 is a key factor of an expanded transcriptional network (Oct4-TN) that governs pluripotency and self-renewal in embryonic stem cells (ESCs) and in the inner cell mass from which ESCs are derived. A pending question is whether the establishment of the Oct4-TN initiates during oogenesis or after fertilisation. To this regard, recent evidence has shown that Oct4 controls a poorly known Oct4-TN central to the acquisition of the mouse egg developmental competence. The aim of this study was to investigate the identity and extension of this maternal Oct4-TN, as much as whether its presence is circumscribed to the egg or maintained beyond fertilisation. RESULTS: By comparing the genome-wide transcriptional profile of developmentally competent eggs that express the OCT4 protein to that of developmentally incompetent eggs in which OCT4 is down-regulated, we unveiled a maternal Oct4-TN of 182 genes. Eighty of these transcripts escape post-fertilisation degradation and represent the maternal Oct4-TN inheritance that is passed on to the 2-cell embryo. Most of these 80 genes are expressed in cancer cells and 37 are notable companions of the Oct4 transcriptome in ESCs. CONCLUSIONS: These results provide, for the first time, a developmental link between eggs, early preimplantation embryos and ESCs, indicating that the molecular signature that characterises the ESCs identity is rooted in oogenesis. Also, they contribute a useful resource to further study the mechanisms of Oct4 function and regulation during the maternal-to-embryo transition and to explore the link between the regulation of pluripotency and the acquisition of de-differentiation in cancer cells.

In Vitro Maturation of Fully Grown Mouse Antral Follicles in the Presence of 1 nM 2-Hydroxyestradiol Improves Oocytes' Developmental Competence.

Cathecolestrogens are estradiol metabolites produced during folliculogenesis in the mammalian ovary. 2-Hydroxyestradiol (2-OHE2) is one of the most abundant although its role remains unknown. The aim of this study is to investigate whether the presence of 2-OHE2 during the germinal vesicle-to-metaphase II transition affects oocyte meiotic and preimplantation developmental competence. Mouse cumulus-oocyte complexes (COCs), isolated from fully grown antral follicles, were in vitro-matured (IVM) in the presence of 2-OHE2 (0.1, 1, 10 or 100 nM) for 6 or 15 h; then, their meiotic and developmental competence was evaluated using a number of cytological quality markers. With the exception of the highest dose (100 nM), the addition of 2-OHE2 to the IVM medium, did not alter, compared with untreated control, the frequency of oocytes that reached the MII stage. Instead, IVM in the presence of 1 nM 2-OHE2 highly increased the rate of preimplantation development and blastocyst quality. To understand whether this positive effect could be attributed to the events occurring during meiosis resumption, we analysed a number of specific cytological quality markers of the asymmetric division, such as PB-I volume and position, presence and extension of the cortical F-actin cap, meiotic spindle shape and area, and microtubule organisation centre localisation. The results highlighted how the presence of 1 nM 2-OHE2 significantly improved the overall cytological organisation required for a correct asymmetric division. Our results contribute a first step to acknowledge a potential role of this estradiol metabolite during the GV-to-MII transition, contributing to the acquisition of oocytes developmental competence.

Spermatocyte apoptosis, which involves both intrinsic and extrinsic pathways, explains the sterility of Graomys griseoflavus x 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.

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.

Expression of phase I and phase II genes in mouse embryonic stem cells cultured in the presence of 2,3,7,8-tetrachlorodibenzo-para-dioxin.

Embryonic stem (ES) cells have features that resemble the pluripotent cells of peri-implantation embryos and have been used as an in vitro model to assess the effects of test substances on these stages of development. Here, for the first time, we report on the effects of the xenobiotic 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) on mouse ES cells cultured with TCDD at concentrations ranging from 0.0001 to 100 nM for 15 min to 48 h. TCDD effects were determined by analysing the induction of Cyp1A1, Cyp1A2, Cyp1B1 (phase I) and Nqo1, Gsta1, Ugt1a6 (phase II) genes. Cyp1A1 was the phase I gene most rapidly induced (4 h at 1 nM); Cyp1B1 was induced at 48 h (1 nM), whereas Cyp1A2 expression was not affected. TCDD did not alter phase II gene expression, which remained at basal levels throughout the 48 h of culture. We studied more accurately the expression of Cyp1A1, the earliest gene to respond to the presence of TCDD. We found that: 1) Cyp1A1 gene induction is dependent on the duration of exposure (precisely it is first induced after 3 h of culture at 1 nM, the minimum effective-dose); 2) Cyp1A1 induction requires the continuous presence of TCDD, being interrupted 4 h after removal of the xenobiotic; and 3) induced expression of CYP1A1 protein is dependent on TCDD concentration, the higher the concentration the earlier the production of the enzyme. Furthermore, after 48 h of treatment, TCDD did not promote either apoptosis or changes to the differentiation status of the ES cells. These results are the first important step to investigate the effects of dioxin on the very early stages of mammalian development.

Oct-4 regulates the expression of Stella and Foxj2 at the Nanog locus: implications for the developmental competence of mouse oocytes.

BACKGROUND: Our knowledge of what determines the mammalian oocyte developmental competence is meagre. By comparing the transcriptional profiles of developmentally competent surrounded nucleolus (SN) and incompetent not surrounded nucleolus (NSN) mouse MII oocytes, we recently demonstrated that Oct-4 and Stella are key factors in the establishment of the oocytes' developmental competence. METHODS: Using RT-PCR, microarray and immunocytochemistry assays, we analysed expression of genes and proteins in oocytes isolated throughout folliculogenesis and classified based on their SN- or NSN-type of chromatin organization. RESULTS: We show that: (1) Oct-4 and Stella are expressed concurrently at the beginning of oocytes' growth and only in SN oocytes; (2) Germ Cell Nuclear Factor is a putative regulator of Oct-4 expression in MII oocytes; (3) the function of Oct-4 is directed at the Nanog locus, regulating the expression of Stella and Foxj2. CONCLUSIONS: (1) A number of factors that act upstream and downstream of Oct-4 emerge as candidate players in the acquisition of the oocyte's developmental competence; (2) we define molecular markers that identify a specific group of ovarian oocytes (SN) that have a potential to acquire developmental competence; (3) the presence of SN and NSN oocytes in human ovaries extends the interest of these results to the field of human reproduction.