Promyelocytic leukemia nuclear body (PML-NB) -free intranuclear milieu facilitates development of oocytes in mice

Promyelocytic leukemia (PML) nuclear bodies (PML-NBs), a class of membrane-less organelles in cells, are involved in multiple biological activities and are present throughout cells of adult organisms. Although the oocyte nucleus is an active region for the flux of multiple non-membranous organelles, PML-NBs have been predicted to be absent from oocytes. Here, we show that the deliberate assembly of PML-NBs during oocyte growth preferentially sequestered Small Ubiquitin-related Modifier (SUMO) protein from the nucleoplasm. SUMO not only was involved in the regulation of oocyte nuclear maturation but also was committed to the response, mediated by liquid droplet formation, to multiple stressors including nucleolar stress and proteotoxic stresses. Exogenous assembly of PML-NBs in the nucleus of oocytes affected the efficiency of the response of SUMO. These observations suggest that the PML-NB-free intranuclear milieu ensures that a reserve of SUMO remains available for emergent responses in oocyte development. This work demonstrated a benefit of the PML-NB-free intranuclear milieu, namely the ability to redirect the flux of SUMO otherwise needed to control PML-NB dynamics.

Unique issues of the species concept in molluscs of the genus Corbicula: a mismatch of mitochondrial and nuclear genomes

Molluscs of the genus Corbicula are well-known invasive bivalves found all over the world. These ecologically important clams are characterised by a wide range of habitats (both brackish waters and freshwater environments) and by contrasting modes of reproduction in native ranges (including sexual and asexual modes) and in invasive ranges (asexual mode). The asexual freshwater Corbicula reproduces through androgenetic fertilization, also termed “egg parasitism”, where the oocyte nucleus is replaced by the spermatozoon nucleus, yet the mitochondria are inherited from the mother cell. This results in a mismatch between the genetic material of the nucleus and mitochondria (and, accordingly, with the external morphology of the shell). In addition, the low nucleotide diversity in the genus Corbicula challenges the delimitation of separate species, thus it is difficult to apply the traditional taxonomic and phylogenetic species criteria to Corbicula populations.

5 Heat stress during pregnancy compromises intrauterine development and reproductive parameters of female progeny in C57BL/6J mice

Heat stress (HS) affects the reproduction of many species, causing subfertility by reducing gametogenesis. This study assessed the effect of HS at different stages of pregnancy in C57BL/6J mice on the somatic development and reproductive parameters of F1 females. A total of 40 females and 40 males aged between 5 and 6 weeks old were mated (1:1). After mating confirmation (vaginal plug presence) the females were subjected to HS during pregnancy in the first half (FP, from Day 1 to 10; n=10), the second half (SP, from Day 11 to delivery; n=10), or the total pregnancy (TP, n=10). A control group (C, n=10) was maintained in normothermic conditions (25°C, 45% relative humidity) throughout the experiment. The HS was induced (41°C for 2h daily) in an environmental chamber heated by 2 red lamps. After delivery, birthweight was recorded and somatic development of the F1 females was monitored weekly until 8 weeks of age. They were superovulated with 5IU of equine chorionic gonadotrophin (eCG) and 5IU of human chorionic gonadotrophin (hCG) 48h later and mated with control F1 males in four groups: FP×C; SP×C; TP×C; and C×C, female and male, respectively. At 72h after mating confirmation, uterine flushing was performed with 0.5mL of phosphate-buffered saline + 0.4% bovine serum albumin and embryos classified. Ovaries were collected for histological analysis of the follicular population with the formula: follicles per ovary×n section×section thickness/n section observed×average diameter of the oocyte nucleus. Pregnancy rate was analysed by chi-squared test. Data of pups born per female, birthweight, somatic development, follicular population, total and viable structures recovered by female were tested for normality by the Shapiro-Wilk test, before ANOVA and Tukey test. Values of P<0.05 were considered to indicate a difference and P<0.10a tendency. No difference (P>0.05) among groups was detected in pregnancy rate (C=80; FP=40; SP=60; TP=60%) or in the number of pups born per female (C=7.0±1.0; FP=6.0±1.5; SP=6.5±1.5; TP=5.1±1.5). Birthweight was lower (P<0.05) for FP (1.1g) and TP (1.2g) than for C (2.2g) and SP (1.8g). However, this difference disappeared (P>0.05) in the third week of development and remained similar until the eighth week (C=21.0; FP=20.4, SP=20.3, TP=20.0g). Similar (P>0.05) follicular population by ovary (total, primordial, primary, secondary, and antral) was observed between the C and HS groups. However, among HS groups, the total number of follicles and number of primordial follicles, respectively, were lower (P<0.05) in the FP (1623; 942) compared with SP (2735; 1918) and TP (2626; 2352); with no difference in primary, secondary, and antral follicles. This resulted in similar (P>0.05) total number of structures recovered by females (C=11.8±5.0, FP=7.6±2.4, SP=10.8±5.5, TP=6.9±3.1), with a tendency (P=0.06) to fewer viable embryos in TP (4.3±2.7) compared with C (9.5±4.6). Considering the increasing global temperature, it is imperative to understand the effects of HS on animal reproductive capacity. In conclusion, gestational HS impaired mice intrauterine development and changed the ovarian follicular population in the F1 generation.

87 Population estimate and morphology of ovarian preantral follicles in fetal and adult alpacas (Vicugna pacos)

Preantral follicles are the largest ovarian follicle population and represent an important source of potentially competent oocytes. During the lifespan of the female this large population becomes atretic during their growth. In alpacas, there are few studies that estimate the number of preantral follicles. Therefore, the objective of the present study was to compare the population and morphology of preantral follicles in the ovaries of fetal and adult alpacas. Ovaries from alpacas in fetal (fetus during the last third of gestation, n=5) and adult stage (3–4 years, n=5) were collected at a local slaughterhouse. The whole ovaries were individually fixed overnight at room temperature, and later dehydrated in alcohol, cleared with xylene, and embedded in paraffin. Tissue were sectioned at 7μm with a rotating microtome. Then, sections were processed and stained with periodic acid Schiff and haematoxylin. Preantral follicles were classified for their development stage as primordial, transitional, primary, or secondary, according to the layer number and form of granulosa cells. Estimation of the number of preantral follicles was made by counting all follicles in each histological section. Only follicles in which the oocyte nucleus was visible were counted. In addition, for each follicle category (n=30 per group), oocyte and follicle diameters were measured using Motic Images Plus 2.0 software. The population estimate and follicular diameter were compared using Kruskal–Wallis test with significance set at P ≤ 0.05 using SPSS v.2 2 software (IBM Corp.). A total of 2174 histologic sections were analysed. The results showed a higher (P=0.045) number of preantral follicles (80 516.1±3623.9) for fetal alpacas compared with adult alpacas (67 870.8±2267.4). Also, primordial follicles population (31 543.4±2690) and morphologically normal follicles (98.2%) were higher (P=0.04) in fetus compared with those in the adult stage (2244.7±355.37; 76.35%) respectively. On the contrary, the diameters of primordial, transitional, and primary follicles (45.34±3.76; 52.38±6.22; 59.79±5.22µm) from adult alpaca were greater (P=0.04) than those of fetal preantral follicles (33.305±7.2; 36.715±3; 77.985±15.8µm). In conclusion, the preantral follicle population declines dramatically in adult alpaca and animals of this age show an increased percentage of degenerate primordial follicles.

The prophase oocyte nucleus is a homeostatic G-actin buffer

Formation of healthy mammalian eggs from oocytes requires specialised F-actin structures. F-actin disruption produces aneuploid eggs, which are a leading cause of human embryo deaths, genetic disorders, and infertility. We found that oocytes regulate F-actin organisation and function by promptly transferring excess monomeric G-actin from the cytoplasm to the nucleus. Inside healthy oocyte nuclei, transferred monomers form dynamic F-actin structures, a conserved feature that significantly declines with maternal age. Monomer transfer must be controlled tightly. Blocked nuclear import of G-actin triggers assembly of a dense cytoplasmic F-actin network, while excess G-actin in the nucleus dramatically stabilises nuclear F-actin. Imbalances in either direction predispose oocytes to aneuploidy. The large oocyte nucleus is thus a homeostatic G-actin buffer that is used to maintain cytoplasmic F-actin form and function.One Sentence SummaryMammalian oocyte nuclei buffer cytosolic G-actin

Long-term association of a transcription factor with its chromatin binding site can stabilize gene expression and cell fate commitment

Some lineage-determining transcription factors are overwhelmingly important in directing embryonic cells to a particular differentiation pathway, such asAscl1for nerve. They also have an exceptionally strong ability to force cells to change from an unrelated pathway to one preferred by their action. Transcription factors are believed to have a very short residence time of only a few seconds on their specific DNA or chromatin-binding sites. We have developed a procedure in which DNA containing one copy of the binding site for the neural-inducing factorAscl1is injected directly into aXenopusoocyte nucleus which has been preloaded with a limiting amount of theAscl1transcription factor protein. This is followed by a further injection of DNA as a competitor, either in a plasmid or in chromosomal DNA, containing the same binding site but with a different reporter. Importantly, expression of the reporter provides a measure of the function of the transcription factor in addition to its residence time. The same long residence time and resistance to competition are seen with the estrogen receptor and its DNA response elements. We find that in this nondividing oocyte, the nerve-inducing factorAscl1can remain bound to a specific chromatin site for hours or days and thereby help to stabilize gene expression. This stability of transcription factor binding to chromatin is a necessary part of its action because removal of this factor causes discontinuation of its effect on gene expression. Stable transcription factor binding may be a characteristic of nondividing cells.

Stable intronic sequence RNAs (sisRNAs) are selected regions in introns with distinct properties

Background: Stable introns and intronic fragments make up the largest population of RNA in the oocyte nucleus of the frog Xenopus tropicalis . These stable intronic sequence RNAs (sisRNAs) persist through the onset of zygotic transcription when synchronous cell division has ended and the developing embryo consists of approximately 8000 cells. Despite their abundance, the sequence properties and biological function of sisRNAs are just beginning to be understood. Results: To characterize this population of noncoding RNA, we identified all of the sisRNAs in the X. tropicalis oocyte nucleus using published high-throughput RNA sequencing data. Our analysis revealed that sisRNAs, have an average length of ~360 bps, are widely expressed from genes with multiple introns, and are derived from specific regions of introns that are GC and TG rich, while CpG poor. They are enriched in introns at both ends of transcripts but preferentially at the 3’ end. The consensus binding sites of specific transcription factors such as Stat3 are enriched in sisRNAs, suggesting an association between sisRNAs and transcription factors involved in early development. Evolutionary conservation analysis of sisRNA sequences in seven vertebrate genomes indicates that sisRNAs are as conserved as other parts of introns, but much less conserved than exons. Conclusion: In total, our results indicate sisRNAs are selected intron regions with distinct properties, and may play a role in gene expression regulation .

Stable intronic sequence RNAs (sisRNAs) are selected regions in introns with distinct properties

Background: Stable introns and intronic fragments make up the largest population of RNA in the oocyte nucleus of the frog Xenopus tropicalis. These stable intronic sequence RNAs (sisRNAs) persist through the onset of zygotic transcription when synchronous cell division has ended and the developing embryo consists of approximately 8000 cells. Despite their abundance, the sequence properties and biological function of sisRNAs are just beginning to be understood. Results: To characterize this population of noncoding RNA, we identified all of the sisRNAs in the X. tropicalis oocyte nucleus using published high-throughput RNA sequencing data. Our analysis revealed that sisRNAs, have an average length of ~360 bps, are widely expressed from genes with multiple introns, and are derived from specific regions of introns that are GC and TG rich, while CpG poor. They are enriched in introns at both ends of transcripts but preferentially at the 3’ end. The consensus binding sites of specific transcription factors such as Stat3 are enriched in sisRNAs, suggesting an association between sisRNAs and transcription factors involved in early development. Evolutionary conservation analysis of sisRNA sequences in seven vertebrate genomes indicates that sisRNAs are as conserved as other parts of introns, but much less conserved than exons. Conclusion: In total, our results indicate sisRNAs are selected intron regions with distinct properties, and may play a role in gene expression regulation.

Stable intronic sequence RNAs (sisRNAs) are selected regions in introns with distinct properties

Background: Stable introns and intronic fragments make up the largest population of RNA in the oocyte nucleus of the frog Xenopus tropicalis. These stable intronic sequence RNAs (sisRNAs) persist through the onset of zygotic transcription when synchronous cell division has ended and the developing embryo consists of approximately 8000 cells. Despite their abundance, the sequence properties and biological function of sisRNAs are just beginning to be understood. Results: To characterize this population of noncoding RNA, we identified all of the sisRNAs in the X. tropicalis oocyte nucleus using published high-throughput RNA sequencing data. Our analysis revealed that sisRNAs, have an average length of ~360 bps, are widely expressed from genes with multiple introns, and are derived from specific regions of introns that are GC and TG rich, while CpG poor. They are enriched in introns at both ends of transcripts but preferentially at the 3’ end. The consensus binding sites of specific transcription factors such as Stat3 are enriched in sisRNAs, suggesting an association between sisRNAs and transcription factors involved in early development. Evolutionary conservation analysis of sisRNA sequences in seven vertebrate genomes indicates that sisRNAs are as conserved as other parts of introns, but much less conserved than exons. Conclusion: In total, our results indicate sisRNAs are selected intron regions with distinct properties, supporting a biological function in gene expression regulation.