CPEB is a sequence-specific RNA-binding protein that regulates polyadenylation-induced translation. In Cpeb knockout mice, meiotic progression is disrupted at pachytene due to inhibited translation of synaptonemal complex protein mRNAs. To assess the function of CPEB after pachytene, we used the zona pellucida 3 (Zp3) promoter to generate transgenic mice expressing siRNA that induce the destruction of Cpeb mRNA. Oocytes from these animals do not develop normally; they undergo parthenogenetic cell division in the ovary, exhibit abnormal polar bodies, are detached from the cumulus granulosa cell layer, and display spindle and nuclear anomalies. In addition, many follicles contain apoptotic granulosa cells. CPEB binds several oocyte mRNAs, including Smad1, Smad5, spindlin, Bub1b, Mos, H1foo, Obox1, Dnmt1o, TiParp, Trim61 and Gdf9, a well described oocyte-expressed growth factor that is necessary for follicle development. In Cpeb knockdown oocytes, Gdf9 RNA has a shortened poly(A) tail and reduced expression. These data indicate that CPEB controls the expression of Gdf9 mRNA, which in turn is necessary for oocyte-follicle development. Finally, several phenotypes, i.e. progressive oocyte loss and infertility, elicited by the knockdown of CPEB in oocytes resemble those of the human premature ovarian failure syndrome.

We previously discovered a germ cell-specific spermatogenesis and oogenesis basic helix-loop-helix transcription factor, Sohlh2. We generated Sohlh2-deficient mice to understand physiologic consequences of Sohlh2 deletion. We discovered that Sohlh2-knockout adult female mice are infertile due to lack of ovarian follicles. Sohlh2-deficient ovaries can form primordial follicles and, despite limited oocyte growth, do not differentiate surrounding granulosa cells into cuboidal and multilayered structures. Oocytes are rapidly lost in Sohlh2-deficient ovaries, and few are present by 14 days of postnatal life. However, the primordial oocytes are abnormal at the molecular level because they misexpress numerous germ cell- and oocyte-specific genes, including Sohlh1, Nobox, Figla, Gdf9, Pou5f1, Zp1, Zp3, Kit, Oosp1, Nlrp14, H1foo, and Stra8. Our findings show that Sohlh2 is a critical factor for maintenance and differentiation of the oocyte during early oogenesis.

The most distinctive feature of oocyte-specific linker histones is the specific timing of their expression during embryonic development. In Xenopus nuclear transfer, somatic linker histones in the donor nucleus are replaced with oocyte-specific linker histone B4, leading to the involvement of oocyte-specific linker histones in nuclear reprogramming. We recently have discovered a mouse oocyte-specific linker histone, named H1foo, and demonstrated its expression pattern in normal preimplantation embryos. The present study was undertaken to determine whether the replacement of somatic linker histones with H1foo occurs during the process of mouse nuclear transfer. H1foo was detected in the donor nucleus soon after transplantation. Thereafter, H1foo was restricted to the chromatin in up to two-cell stage embryos. After fusion of an oocyte with a cell expressing GFP (green fluorescent protein)-tagged somatic linker histone H1c, immediate release of H1c in the donor nucleus was observed. In addition, we used fluorescence recovery after photobleaching (FRAP), and found that H1foo is more mobile than H1c in living cells. The greater mobility of H1foo may contribute to its rapid replacement and decreased stability of the embryonic chromatin structure. These results suggest that rapid replacement of H1c with H1foo may play an important role in nuclear remodeling.

H1 linker histones (H1s) are key regulators of chromatin structure and function. The functions of different H1s during early embryogenesis, and mechanisms regulating their associations with chromatin are largely unknown. The developmental transitions of H1s during oocyte growth and maturation, fertilization and early embryogenesis, and in cloned embryos were examined. Oocyte-specific H1FOO, but not somatic H1s, associated with chromatin in oocytes (growing, GV-stage, and MII-arrested), pronuclei, and polar bodies. H1FOO associated with sperm or somatic cell chromatin within 5 min of intracytoplasmic sperm injection (ICSI) or somatic cell nuclear transfer (SCNT), and completely replaced somatic H1s by 60 min. The switching from somatic H1s to H1FOO following SCNT was developmentally regulated. H1FOO was replaced by somatic H1s during the late two- and four-cell stages. H1FOO association with chromatin can occur in the presence of a nuclear envelope and independently of pronucleus formation, is regulated by factors associated with the spindle, and is likely an active process. All SCNT constructs recapitulated the normal sequence of H1 transitions, indicating that this alone does not signify a high developmental potential. A paucity of all known H1s in two-cell embryos may contribute to precocious gene transcription in fertilized embryos, and the elaboration of somatic cell characteristics in cloned embryos.

The embryonic genome is formed by fusion of a maternal and a paternal genome. To accommodate the resulting diploid genome in the fertilized oocyte dramatic global genome reorganizations must occur. The higher order structure of chromatin in vivo is critically dependent on architectural chromatin proteins, with the family of linker histone proteins among the most critical structural determinants. Although somatic cells contain numerous linker histone variants, only one, H1FOO, is present in mouse oocytes. Upon fertilization H1FOO rapidly populates the introduced paternal genome and replaces sperm-specific histone-like proteins. The same dynamic replacement occurs upon introduction of a nucleus during somatic cell nuclear transfer. To understand the molecular basis of this dynamic histone replacement process, we compared the localization and binding dynamics of somatic H1 and oocyte-specific H1FOO and identified the molecular determinants of binding to either oocyte or somatic chromatin in living cells. We find that although both histones associate readily with chromatin in nuclei of somatic cells, only H1FOO is capable of correct chromatin association in the germinal vesicle stage oocyte nuclei. This specificity is generated by the N-terminal and globular domains of H1FOO. Measurement of in vivo binding properties of the H1 variants suggest that H1FOO binds chromatin more tightly than somatic linker histones. We provide evidence that both the binding properties of linker histones as well as additional, active processes contribute to the replacement of somatic histones with H1FOO during nuclear transfer. These results provide the first mechanistic insights into the crucial step of linker histone replacement as it occurs during fertilization and somatic cell nuclear transfer.

Bisphenol A (BPA) is recognized as one of several environmental estrogens. Pre-puberty is an important part of reproductive system development, and even a short-term exposure to BPA during this period may cause serious damage to the reproductive system. In this study, Pre-puberty female Wistar rats were exposed to BPA for one week. The effects of BPA on ovarian structure and function were assessed. The expression levels of follicle development-related genes were analyzed. Our study showed that BPA reduced rat ovarian weights and follicle numbers, and interferes with the constituent ratio of follicles. With increasing doses of BPA, the expression of factor in the germline alpha (FIGLA) and oocyte-specific histone H1 variant (H1FOO) genes decreased, and anti-mullerian hormone (AMH) genes expression increased, suggesting that BPA exposure during the pre-pubertal period may inhibit the development of ovaries, and follicle development-related genes may play certain roles in this process.

BACKGROUND

Improving human nuclear transfer (NT) efficiencies is paramount for the development of patient-specific stem cell lines, although the opportunities remain limited owing to difficulties in obtaining fresh mature oocytes.

METHODS

Therefore, the developmental competence of aged, failed-to-fertilize human oocytes as an alternate cytoplasmic source for NT was assessed and compared with use of fresh, ovulation-induced oocytes. To further characterize the developmental potential of aged oocytes, parthenogenetic activation, immunocytochemical analysis of essential microtubule proteins involved in meiotic and mitotic division, and RT-PCR in single oocytes (n = 6) was performed to determine expression of oocyte-specific genes [oocyte-specific histone 1 (H1FOO), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), zygote arrest 1 (ZAR1)] and microtubule markers [nuclear mitotic arrest (NuMA), minus-end directed motor protein HSET and the microtubule kinesin motor protein EG5].

RESULTS

For NT, enucleation and fusion rates of aged oocytes were significantly lower compared with fresh oocytes (P < 0.05). Cleavage rates and subsequent development were poor. In addition, parthenote cleavage was low. Immunocytochemical analysis revealed that many oocytes displayed aberrant expression of NuMA and EG5, had disrupted meiotic spindles and tetrapolar spindles. One of the six oocytes misexpressed GDF9, BMP15 and ZAR1. Two oocytes expressed EG5 messenger RNA (mRNA), and HSET and NuMA were not detectable. RT-PCR of mRNA for oocyte specific genes and microtubule markers in single aged oocytes.

CONCLUSIONS

Thus, aneuploidy and spindle defects may contribute to poor parthenogenetic development and developmental outcomes following NT.

Mammalian oocytes contain the histone H1foo, a distinct member with low sequence similarity to other members in the H1 histone family. Oocyte-specific H1foo exists until the second embryonic cell stage. H1foo is essential for oocyte maturation in mice; however, the molecular function of this H1 subtype is unclear. To explore the function of H1foo, we generated embryonic stem (ES) cells ectopically expressing H1foo fused to an EGFP (H1foo-ES). Interestingly, ectopic expression of H1foo prevented normal differentiation into embryoid bodies (EBs). The EB preparations from H1foo-ES cells maintained the expression of pluripotent marker genes, including Nanog, Myc and Klf9, and prevented the shift of the DNA methylation profile. Because the short hairpin RNA-mediated knockdown of H1foo-EGFP recovered the differentiation ability, H1foo was involved in preventing differentiation. Furthermore, ChIP analysis revealed that H1foo-EGFP bound selectively to a set of hypomethylated genomic loci in H1foo-ES, clearly indicating that these loci were targets of H1foo. Finally, nuclease sensitivity assay suggested that H1foo made these target loci decondensed. We concluded that H1foo has an impact on the genome-wide, locus-specific epigenetic status.

Oocyte-specific linker histone H1foo is localized in the oocyte nucleus, either diffusely or bound to chromatin, during the processes of meiotic maturation and fertilization. This expression pattern suggests that H1foo plays a key role in the control of gene expression and chromatin modification during oogenesis and early embryogenesis. To reveal the function of H1foo, we microinjected antisense morpholino oligonucleotides (MO) against H1foo into mouse germinal-vesicle stage oocytes. The rate of in vitro maturation of the antisense MO group was significantly lower than that of the control group. Eggs that failed to extrude a first polar body following injection of antisense MO arrested at metaphase I. Additionally, co-injection of in vitro synthesized H1foo mRNA along with antisense MO successfully rescued expression of H1foo and improved the in vitro maturation rate. There was no difference in the rate of parthenogenesis between the antisense MO and control groups. These results indicate that H1foo is essential for maturation of germinal vesicle-stage oocytes.

To identify the genes related to oocyte competence, we quantified transcripts for candidate genes in oocytes (H1Foo, H2A, H3A, GHR, GDF9, BMP15, OOSP1) and cumulus cells (FSHR, EGFR, GHR, PTX3, IGFII) using the follicle size model to select oocytes of better developmental quality. Follicles were dissected and distributed into four groups according to diameter as follows: 1.0-3.0, 3.1-6.0, 6.1-8.0 and>>or=8.1 mm. Cumulus-oocyte complexes (COCs) were released, classified morphologically, matured, fertilised and cultured in vitro or denuded for measurement of diameter and determination of gene expression. Denuded germinal vesicle oocytes and their cumulus cells were used for gene expression analysis by reverse transcription-polymerase chain reaction. The blastocyst rate was highest for oocytes recovered from follicles>6 mm in diameter. In the oocyte, expression of the H2A transcript only increased gradually according to follicle size, being greater (P<0.05) in oocytes from follicles>or=8.1 mm in diameter than in oocytes from follicles<6.0 mm in diameter. In cumulus cells, expression of FSHR, EGFR and GHR mRNA increased with follicular size. In conclusion, we confirmed the importance of H2A for developmental competence and identified important genes in cumulus cells that may be associated with oocyte competence.

We previously reported the discovery of a novel mammalian H1 linker histone termed H1FOO (formerly H1OO), a replacement H1, the expression of which is restricted to the growing/ maturing oocyte and to the zygote. The significance of this pre-embryonic H1 draws on its substantial orthologous conservation, singular structural attributes, selectivity for the germ cell lineage, prolonged nucleosomal residence, and apparent predominance among germ cell H1s. Herein, we report that the intronic, single-copy, five-exon >> or =5301 base pair) H1foo gene maps to chromosome 6 and that the corresponding primary H1foo transcript gives rise to two distinct, alternatively spliced mRNA species (H1foo(alpha) and H1foo(beta)). The expression of the oocytic H1FOO transcript and protein proved temporally coupled to the recruitment of resting primordial follicles into a developing primary follicular cohort and thus to the critical transition marking the onset of oocytic growth. The corresponding potential protein isoforms (H1FOO(alpha) and H1FOO(beta)), both nuclear localization sequence-endowed but export consensus sequence-free and possessing a significant net positive charge, localized primarily to perinucleolar heterochromatin in the oocytic germinal vesicle. Further investigation will be required to define the functional role of the H1FOO protein in the ordering of the chromatin of early mammalian development as well as its potential role in defining the primordial-to-primary follicle transition.

There is mounting evidence to suggest that the epigenetic reprogramming capacity of the oocyte is superior to that of the current factor-based reprogramming approaches and that some factor-reprogrammed induced pluripotent stem cells (iPSCs) retain a degree of epigenetic memory that can influence differentiation capacity and may be linked to the observed expression of immunogenicity genes in iPSC derivatives. One hypothesis for this differential reprogramming capacity is the "chromatin loosening/enhanced reprogramming" concept, as previously described by John Gurdon and Ian Wilmut, as well as others, which postulates that the oocyte possesses factors that loosen the somatic cell chromatin structure, providing the epigenetic and transcriptional regulatory factors more ready access to repressed genes and thereby significantly increasing epigenetic reprogramming. However, to empirically test this hypothesis a list of candidate oocyte reprogramming factors (CORFs) must be ascertained that are significantly expressed in metaphase II oocytes. Previous studies have focused on intraspecies or cross-species transcriptional analysis of up to two different species of oocytes. In this study, we have identified eight CORFs (ARID2, ASF1A, ASF1B, DPPA3, ING3, MSL3, H1FOO, and KDM6B) based on unbiased global transcriptional analysis of oocytes from three different species (human, rhesus monkey, and mouse) that both demonstrate significant (p<0.05, FC>3) expression in oocytes of all three species and have well-established roles in loosening/opening up chromatin structure. We also identified an additional 15 CORFs that fit within our proposed "chromatin opening/fate transformative" (COFT) model. These CORFs may be able to augment Shinya Yamanaka's previously identified reprogramming factors (OCT4, SOX2, KLF4, and cMYC) and potentially facilitate the removal of epigenetic memory in iPSCs and/or reduce the expression of immunogenicity genes in iPSC derivatives, and may have applications in future personalized pluripotent stem cell based therapeutics.

Embryonic stem cells (ESCs) are a hallmark of ideal pluripotent stem cells. Epigenetic reprogramming of induced pluripotent stem cells (iPSCs) has not been fully accomplished. iPSC generation is similar to somatic cell nuclear transfer (SCNT) in oocytes, and this procedure can be used to generate ESCs (SCNT-ESCs), which suggests the contribution of oocyte-specific constituents. Here, we show that the mammalian oocyte-specific linker histone H1foo has beneficial effects on iPSC generation. Induction of H1foo with Oct4, Sox2, and Klf4 significantly enhanced the efficiency of iPSC generation. H1foo promoted in vitro differentiation characteristics with low heterogeneity in iPSCs. H1foo enhanced the generation of germline-competent chimeric mice from iPSCs in a manner similar to that for ESCs. These findings indicate that H1foo contributes to the generation of higher-quality iPSCs.

Linker histones H1 are involved in various mechanisms, such as chromatin organization and gene transcription. In different organisms, a unique subtype can be found in the oocyte, however its function remains unclear. To assess the potential involvement of this oocyte linker histone (H1FOO) in chromatin modulation, we have cloned and sequenced the bovine H1FOO cDNA and followed its mRNA profile by quantitative RT-PCR in the oocyte and throughout bovine early embryo development. The highest level of mRNA was found in the germinal vesicle (GV) oocyte and diminished constantly throughout embryo development. In the 16-cell embryo and blastocyst, respectively, the mRNA levels were 200 and 2,000 times lower than in the GV oocyte. A specific antibody raised against bovine H1FOO was used to establish protein distribution in the oocyte and preimplantation embryo by immunocytochemistry. In the GV and metaphase II (MII) oocyte, as well as in the 1-, 2- and 4-cell embryo, H1FOO was localized in the cytoplasm and nucleus. The protein was uniformly spread within the cytoplasm, while it was concentrated onto the chromatin in the nucleus. In the 8- to 16-cell embryo, H1FOO's presence diminished in the cytoplasm, although it was still strongly expressed in nucleus. In the morula and blastocyst stages, the protein was totally lacking. By its position on chromatin, H1FOO could not only be involved in chromatin conformation but could also participate in activation or repression of genes during oogenesis and embryo development before embryonic genome activation.

OBJECTIVE

To confirm that oocyte-specific messenger RNAs are detectable in the polar body (PB) of metaphase II (MII) oocytes and determine the effect of age on oocyte-specific transcript levels.

METHODS

Prospective study.

METHODS

Hospital-based academic research laboratory.

METHODS

CD1 female mice.

METHODS

Aged (40-50 weeks) and young (7-9 weeks) mice were administered pregnant mare serum gonadotropin (PMSG) and hCG. Oocytes were fertilized in vitro to assess fertilization and developmental competence. The MII oocytes were obtained and first PBs were removed. Messenger RNAs from each PB and its sibling oocyte were reverse transcribed and analyzed by real-time quantitative polymerase chain reaction (PCR).

METHODS

Fertilization and developmental rates and expression of six oocyte-specific genes (Bmp15, Gdf9, H1foo, Nlrp5, Tcl1, and Zp3) in PBs and sibling oocytes from young versus aged mice.

RESULTS

Oocytes from aged mice had lower developmental competence. Four genes (H1foo, Nlrp5, Tcl1, and Zp3) were differentially expressed in aged versus young oocytes. All six transcripts were present in PBs from aged and young mice at lower levels than in the sibling oocytes; transcript levels were lower in aged PBs compared with young PBs.

CONCLUSIONS

There is a significant difference in the transcript levels of oocyte-specific genes in aged versus young PB that correlates with age-related decreases in oocyte competence. Differences in gene expression in PB may be potential biomarkers of MII oocyte competence.

Human inner cell mass (ICM) cells isolated from in vitro fertilized blastocysts are the progenitor cells used to establish in vitro stable human embryonic stem cells (hESCs) which are pluripotent and self-renew indefinitely. This long-term perpetuation of hESCs in the undifferentiated state is thought to be an in vitro adaptation of the ICM cells. To investigate at the molecular level how hESCs acquired their unique properties, transcriptional profiles of isolated ICM cells and undifferentiated hESCs were compared. We identified 33 genes enriched in the ICM compared to the trophectoderm and hESCs. These genes are involved in signaling cascades (SEMA7A and MAP3K10), cell proliferation (CUZD1 and MS4A7) and chromatin remodeling (H1FOO and HRMT1L4). Furthermore, primordial germ cell-specific genes (SGCA and TEX11) were detected as expressed in the ICM cells and not hESCs. We propose that the transcriptional differences observed between ICM cells and hESCs might be accounted for by adaptive reprogramming events induced by the in vitro culture conditions which are distinct from that of in vitro fertilized blastocysts. hESCs are a distinct cell type lacking in the human embryo but, nonetheless, resemble the ICM in their ability to differentiate into cells representative of the endodermal, ectodermal and mesodermal cell lineages.

The oocyte-specific subtype of the linker histone H1 is H1FOO, which constitutes a major part of oocyte chromatin. H1foo is expressed in growing oocytes, through fertilization, up until the two-cell embryo stage, when it is subsequently replaced by somatic H1 subtypes. To elucidate whether an epigenetic mechanism is involved in the limited expression of H1foo, we analyzed the dynamics of the DNA methylation status of the H1foo locus in germ and somatic cells. We identified a tissue-dependent and differentially methylated region (T-DMR) upstream of the H1foo gene, which was hypermethylated in sperm, somatic cells, and stem cell lines. This region was specifically unmethylated in the ovulated oocyte, where H1foo is expressed. 5-Aza-2'-deoxycytidine treatments and luciferase assays provided in vitro evidence that DNA methylation plays a role in repressing H1foo in nonexpressing cells. DNA methylation analyses of fetal germ cells revealed the T-DMR to be hypomethylated in female and male germ cells at Embryonic Day 9.5 (E9.5), whereas it was highly methylated in somatic cells at this stage. Intriguingly, the unmethylated status was continuously observed throughout oogenesis at E9.5, E12.5, E15.5, E18.5, in mature oocytes, and after fertilization, in E3.5 blastocysts. In comparison, male germ cells acquired methylation beyond E18.5. These data demonstrate a continuously unmethylated circuit at the H1foo locus in the female germline.

A major challenge in applying genomics to oocyte physiology is that many RNAs are present but will not be translated into proteins, making it difficult to draw conclusions from RNAseq and array data. Oocyte maturation and early embryo development rely on maternal storage of specific RNAs with a short poly(A) tail, which must be elongated for translation. To resolve the role of key genes during that period, we aimed to characterize both extremes of mRNA: deadenylated RNA and long polyA tails mRNA population in immature bovine oocytes. Using magnetic beads coupled to oligodT, we isolated deadenylated (A-, 20-50 adenosines) from polyadenylated (A+, up to 200 adenosines) RNAs. After transcriptomic analysis, we observed that A+ candidates are associated with short-term processes required for immediate cell survival (translation or protein transport) or meiotic resumption, while several A- candidates are involved in processes (chromatin modification, gene transcription and post-transcriptional modifications) that will be extremely important in the development of the early embryo. In addition to a list of candidates probably translated early or late, sequence analysis revealed that cytoplasmic polyadenylation element (CPE) and U(3)GU(3) were enriched in A- sequences. Moreover, a motif associated with polyadenylation signals (MAPS, U(5)CU(2)) appeared to be enriched in 3'untranslated regions (UTR) with CPE or U(3)GU(3) sequences in bovine but also in zebrafish and Xenopus tropicalis. To further validate our methodology, we measured specific tail length of known candidates (AURKA, PTTG1, H2A1) but also determined the poly(A) tail length of other candidate RNAs (H3F3A, H1FOO, DAZAP2, ATF1, ATF2, KAT5, DAZL, ELAVL2). In conclusion, we have reported a methodology to isolate deadenylated from polyadenylated RNAs in samples with small total RNA quantities such as mammals. Moreover, we identified deadenylated RNAs in bovine oocytes that may be stored for the long-term process of early embryo development and described a conserved motif enriched in the 3'UTR of deadenylated RNAs.

The effects of exogenous hormones, used for estrus synchronization and ovarian hyper stimulation, on cumulus oocyte complexes (COCs) gene expression in sexually mature rats were determined using microarrays. Gene expression in COCs collected from GnRH (G(trt)), GnRH + eCG (G + E(trt)), and GnRH + eCG + hCG (G + E + H(trt)) treatments were compared to COCs from naturally cycling (NC) rats before the preovulatory luteninizing hormone surge. There was no significant difference in gene expression among NC, G(trt), and G + E(trt); however, over 2,600 genes were significantly different between NC and G + E + H(trt) (P < 0.05). Genes upregulated in G + E + H(trt) encode for: proteins that are involved in prostaglandin synthesis (Ptgs2, Pla2g4a, and Runx1) and cholesterol biosynthesis (Hmgcr, Sc4mol, and Dhcr24); receptors that allow cholesterol uptake (Ldlr and Scarb1), regulate progesterone synthesis (Star), and inactivate estrogen (Sult1e1); and downstream effectors of LH signal (Pgr, Cebpb, Creb3l1, Areg, Ereg, and Adamts1). Conversely, G + E + H(trt) downregulated genes encoding proteins involved in: DNA replication and cell cycle progression (Ccne2, Orc5l, Rad50, and Mcm6); reproductive developmental process; and granulosa cell expansion (Gdf9, Bmp15, Amh, Amhr2, Bmpr1b, Tgfb2, Foxl2, Pde3a, Esr2, Fshr, Ybx2, Ccnd2, Ccnb1ip1, and Zp3); maternal effect genes required for embryo development (Zar1, Npm2, Nlrp5, Dnmt1, H1foo, and Zfp57); amino acid degradation; and ketogenesis (Hmgcs2, and Cpt1b). These results from the rat show that hormones used for estrus synchronization (G(trt)) and ovarian hyper stimulation (G + E(trt)) had minimal effects on gene expression, whereas induction of ovulation (G + E + H(trt)) caused major changes in gene expression of rat COCs. This study provides comprehensive information about regulated genes during late follicle development and ovulation induction.

Linker histone variants are involved in regulation of chromosome organization and gene transcription; several subtypes are expressed in the maturing oocyte and developing embryo. In Xenopus and mice, the transition between linker histone variants occurred following nuclear transfer, and apparently contributed to donor nuclear reprogramming. To determine whether such linker histone replacement occurred after bovine nuclear transfer, red fluorescent protein (RFP) tagged H1e (somatic linker histone H1e) donor cells and Venus tagged H1foo eggs were created, enucleated eggs were injected with donor cells, and embryos were created by fusion. Using fluorescence microscopy, release of H1e in the donor nucleus, acquisition of H1foo by donor chromosomes, and the H1foo-to-H1e transition were observed in live cells. Linker histone replacement occurred more slowly in bovine than murine embryos. Low levels of diffuse red fluorescence (H1e) in the donor nucleus were detected 5 h after fusion, at which time green fluorescence (H1foo) had incorporated into donor chromosomes. However, complete replacement did not occur until 8 h after fusion. We concluded that the linker histone transition was sufficiently conserved among species, which provided further evidence regarding its important role in nuclear reprogramming.

The primary function of ovarian granulosa cells (GCs) is the support of oocytes during maturation and development. Molecular analyses of granulosa cell-associated processes, leading to improvement of understanding of the cell cycle events during the formation of ovarian follicles (folliculogenesis), may be key to improve the in vitro fertilization procedures. Primary in vitro culture of porcine GCs was employed to examine the changes in the transcriptomic profile of genes belonging to "cell cycle", "cell division", "cell cycle process", "cell cycle phase transition", "cell cycle G1/S phase transition", "cell cycle G2/M phase transition" and "cell cycle checkpoint" ontology groups. During the analysis, microarrays were employed to study the transcriptome of GCs, analyzing the total RNA of cells from specific periods of in vitro cultures. This research was based on material obtained from 40 landrace gilts of similar weight, age and the same living conditions. RNA was isolated at specific timeframes: before the culture was established (0 h) and after 48 h, 96 h and 144 h in vitro. Out of 133 differentially expressed genes, we chose the 10 most up-regulated (SFRP2, PDPN, PDE3A, FGFR2, PLK2, THBS1, ETS1, LIF, ANXA1, TGFB1) and the 10 most downregulated (IGF1, NCAPD2, CABLES1, H1FOO, NEK2, PPAT, TXNIP, NUP210, RGS2 and CCNE2). Some of these genes known to play key roles in the regulation of correct cell cycle passage (up-regulated SFRP2, PDE3A, PLK2, LIF and down-regulated CCNE2, TXNIP, NEK2). The data obtained provide a potential reference for studies on the process of mammalian folliculogenesis, as well as suggests possible new genetic markers for cell cycle progress in in vitro cultured porcine granulosa cells.

The aim of this study was to investigate the expression profile of candidate genes involved in competence during oocyte growth. The candidate genes (BMP15, OOSP1, H1FOO, H2A, H3A, H4, SLBP, DNMT1, DNMT3B, HAT1, HDAC2 and SUV39H1) were selected because of their possible involvement in determining oocyte developmental competence. Pre-antral and antral follicles were isolated from the ovaries of Zebu (Bos indicus) cows, measured and classified into the following categories according to their diameter: (i) oocytes from primordial follicles: diameter <20 μm, (ii) oocytes from primary follicles: 25-35 μm, (iii) oocytes from small secondary follicles: 40-60 μm, (iv) oocytes from large secondary follicles: 65-85 μm, (v) oocytes from small antral follicles: 100-120 μm, and (vi) oocytes from large antral follicles: >128 μm. Total RNA was extracted from four pools of 25 oocytes for each category of follicles, and the genes were quantified by qPCR. Target gene expression was normalized using the gene PPIA. The results suggest that stocks of the studied transcript genes accumulate before the final phase of folliculogenesis. The HDAC2 gene was the only gene in which a differential expression was observed at stage associated with competence acquisition.

Oocyte-specific linker histone, H1foo, is localized on the oocyte chromosomes during the process of meiotic maturation, and is essential for mouse oocyte maturation. Bovine H1foo has been identified, and its expression profile throughout oocyte maturation and early embryo development has been established. However, it has not been confirmed if H1foo is indispensable during bovine oocyte maturation. Effective siRNAs against H1foo were screened in HeLa cells, and then siRNA was microinjected into bovine oocytes to down-regulate H1foo expression. H1foo overexpression was achieved via mRNA injection. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that H1foo was up-regulated by 200% and down-regulated by 70%. Based on the first polar body extrusion (PB1E) rate, H1foo overexpression apparently promoted meiotic progression. The knockdown of H1foo significantly impaired bovine oocyte maturation compared with H1foo overexpression and control groups (H1foo overexpression = 88.7%, H1foo siRNA = 41.2%, control = 71.2%; P < 0.05). This decrease can be rescued by co-injection of a modified H1foo mRNA that has escaped from the siRNA target. However, the H1e (somatic linker histone) overexpression had no effect on PB1E rate when compared with the control group. Therefore we concluded that H1foo is essential for bovine oocyte maturation and its overexpression stimulates the process.

Epigenetic and transcriptional mechanisms have been shown to contribute to long-lasting functional changes in adult neurons. The purpose of this study was to identify any such modifications in diseased retinal tissues from a mouse model of rhodopsin mutation-associated autosomal dominant retinitis pigmentosa (ADRP), Q344X, relative to age-matched wild-type (WT) controls.

We performed RNA sequencing (RNA-seq) at poly(A) selected RNA to profile the transcriptional patterns in 3-week-old ADRP mouse model rhodopsin Q344X compared to WT controls. Differentially expressed genes were determined by DESeq2 using the Benjamini & Hochberg p value adjustment and an absolute log2 fold change cutoff. Quantitative western blots were conducted to evaluate protein expression levels of histone H3 phosphorylated at serine 10 and histone H4. qRT-PCR was performed to validate the expression patterns of differentially expressed genes.

We observed significant differential expression in 2151 genes in the retina of Q344X mice compared to WT controls, including downregulation in the potassium channel gene, Kcnv2, and differential expression of histone genes, including the H1 family histone member, H1foo; the H3 histone family 3B, H3f3b; and the histone deacetylase 9, Hdac9. Quantitative western blots revealed statistically significant decreased protein expression of both histone H3 phosphorylated at serine 10 and histone H4 in 3-week-old Q344X retinas. Furthermore, qRT-PCR performed on select differentially expressed genes based on our RNA-seq results revealed matched expression patterns of up or downregulation.

These findings provide evidence that transcriptomic alterations occur in the ADRP mouse model rhodopsin Q344X retina and that these processes may contribute to the dysfunction and neurodegeneration seen in this animal model.