Helena van Tol

X-chromosome inactivation (XCI) is a developmental process that aims to equalize the dosage of X-linked gene products between XY males and XX females in eutherian mammals. In female mouse embryos, paternal XCI is initiated at the 4-cell stage; however, the X chromosome is reactivated in the inner cell mass cells of blastocysts, and random XCI is subsequently initiated in epiblast cells. However, recent findings show that the patterns of XCI are not conserved among mammals. In this study, we used quantitative RT-PCR and RNA in situ hybridization combined with immunofluorescence to investigate the pattern of XCI during bovine embryo development. Expression of XIST (X-inactive specific transcript) RNA was significantly upregulated at the morula stage. For the first time, we demonstrate that XIST accumulation in bovine embryos starts in nuclei of female morulae, but its colocalization with histone H3 lysine 27 trimethylation was first detected in day 7 blastocysts. Both in the inner cel...

PIWIs are crucial guardians of genome integrity, particularly in germ cells. While mammalian PIWIs have been primarily studied in mouse and rat, a homologue for the human PIWIL3 gene is absent in the Muridae family, and hence the unique function of PIWIL3 in germ cells cannot be effectively modeled by mouse knockouts. Herein, we investigated the expression, distribution and interaction of PIWIL3 in bovine oocytes. We localized PIWIL3 to mitochondria, and demonstrated that PIWIL3 expression is stringently controlled both spatially and temporally before and after fertilization. Moreover, we identified PIWIL3 in a mitochondrial-recruited three-membered complex with TDRKH and PNLDC1, and demonstrated by mutagenesis that PIWIL3 N-terminal arginine modifications are required for complex assembly. Finally we sequenced the piRNAs bound to PIWIL3-TDRKH-PNLDC1 and report here that about 50% of these piRNAs map to transposable elements, recapitulating the important role of PIWIL3 in maintainin...

Intricate relationships between microorganisms structure the exchange of molecules between taxa, driving their physiology and evolution. On a global scale, this molecular trade is an integral component of biogeochemical cycling. As important microorganisms in the world's oceans, diatoms and bacteria have a large impact on marine biogeochemistry. Here, we describe antagonistic effects of the globally distributed flavobacterium Croceibacter atlanticus on a phylogenetically diverse group of diatoms. We used the model diatom Thalassiosira pseudonana to study the antagonistic impact in more detail. In co-culture, C. atlanticus attaches to T. pseudonana and inhibits cell division, inducing diatom cells to become larger and increase in chlorophyll a fluorescence. These changes could be explained by an absence of cytokinesis that causes individual T. pseudonana cells to elongate, accumulate more plastids and become polyploid. These morphological changes could benefit C. atlanticus by au...

Metabolic rich and poor conditions are both characterized by elevated free fatty acid levels and have been associated with impaired female fertility. In particular, saturated free fatty acids have a dose-dependent negative impact on oocyte developmental competence, while mono-unsaturated free fatty acids appear less harmful. Cumulus cells seem to protect the oocyte against free fatty acids and the aim of this study was to determine the mechanism behind this protection In particular the role of the enzyme stearoyl-CoA desaturase (SCD) that converts saturated into mono-unsaturated fatty acids was investigated. SCD gene and protein were abundantly expressed in cumulus cells, but expression was low in oocytes. The level of SCD protein expression in cumulus cells did not change when COCs were exposed to saturated stearic acid during maturation. SCD inhibition in the presence of stearic acid significantly reduced the developmental competence of oocytes and increased the incidence of apopt...

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The aim of this study was to investigate whether bovine cumulus oocyte complexes (COCs) obtained from 2 to 8 mm follicles synthesize growth hormone (GH) during in vitro maturation. In addition the expression of growth hormone releasing hormone receptor (GHRH-r) in the COCs before and after in vitro maturation was investigated. Therefore, COCs obtained from small and medium sized follicles were cultured in M199 supplemented with 10% FCS and gonadotropins for 24 hr. At 0, 6, 12, and 24 hr after the onset of culture, COCs were removed and were prepared for immunohistochemical staining to detect the presence of GH. In addition, sections of ovary were stained to study the differential localization of GH in the ovary. At 0 and 24 hr COCs were removed and together with samples from granulosa cells and theca cells were prepared for reverse transcriptase polymerase chain reaction (RT-PCR) to assess the expression of mRNA of GH and GHRH-r. Within COCs, cumulus cells and oocytes showed GH immunoreactivity, while expression of GH mRNA was only found in the oocyte. At the onset of culture, oocytes and cumulus cells in the majority of COCs generally showed moderate and strong staining intensity for GH, respectively. While GH staining in the cumulus cells did hardly change during 24 hr of culture, GH staining in the oocyte was absent after 24 hr of culture in 70% of COCs. Within the ovary, GH was localized in antral follicles larger than 2 mm and no staining was found in primordial, primary and secondary follicles or in the stroma. The intensity of the staining increased with the size of the follicles. Within the follicular wall the GH was persistently observed in granulosa cells, while theca cells were occasionally negative. GH mRNA in follicular compartments was only found in the oocyte and mural granulosa cells. No GHRH-r mRNA was found in the COCs nor in the granulosa or the stroma. In conclusion, the gradual increase of GH staining during follicular development and the consistent synthesis of GH in oocytes and granulosa cells, suggest a paracrine and/or autocrine action for GH in bovine follicular growth and oocyte maturation. The absence of mRNA for GHRH receptor in the COCs indicates that ovarian production of GH is not regulated by GHRH.

BACKGROUND: In the developing embryo, total RNA abundance fluctuates caused by functional RNA degradation and zygotic genome activation. These variations in the transcriptome in early development complicate the choice of good reference genes for gene expression studies by quantitative real time polymerase chain reaction. RESULTS: In order to identify stably expressed genes for normalisation of quantitative data, within early stages

Genes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm and inner cell mass (ICM) samples using microarray analysis. As MAPK inhibition can increase the NANOG/GATA6 ratio in the inner cell mass, additionally blastocysts were cultured in the presence of a MAPK inhibitor and changes in gene expression in the inner cell mass were analysed. Between morula and blastocyst 3,774 genes were differentially expressed and the largest differences were found in blastocyst up-regulated genes. Gene ontology (GO) analysis shows lipid metabolic process as the term most enriched w...

ABSTRACT Small noncoding RNAs such as microRNAs (miRNA) act as posttranscriptional regulators of numerous gene targets. The level of expression of these epigenetic factors can regulate the stability and translation of target mRNA molecules. Quantification of miRNA expression levels can therefore help us understand biological processes such as those regulating oocyte and pre-implantation embryo development. To date, most studies that have examined miRNA expression levels have used expression of either reference mRNAs or U6 spliceosomal RNA or miRNA Let-7a for normalization. We analysed the suitability of several miRNAs as potential expression normalizers in bovine oocytes and early embryos. Bovine oocytes at the germinal vesicle and metaphase II stages, and zygotes, 2-cell, 4-cell and 8-cell embryos, morulae, and blastocysts were collected during three in vitro fertilization replicates. qPCR was performed to quantify expression of miR-93, miR-103a, miR-26a, miR-191, miR-23b, Let-7a, and U6. The average starting material for each sample was determined with the help of specific standard curves made for each primer set. Subsequently, geNorm software was used to identify a set of stably transcribed miRNAs. Surprisingly, U6 spliceosomal RNA and Let-7a, which are widely used to normalise miRNA expression levels, were not stably expressed and were therefore considered unsuitable for normalization. Stepwise removal to determine the optimal number of reference miRNAs identified miR-93 and miR-103a as the most stably expressed. It is concluded that the combination of miR-93 and miR-103a can be used for normalizing miRNA expression for qPCR experiments on bovine oocytes and pre-implantation embryos.

Relatively little information is available on the local factors that regulate folliculogenesis in goats. To examine the possibility that the Kit ligand (KL) system is expressed throughout the folliculogenesis, we studied the presence and distribution of KL and its receptor, c-Kit, in goat ovaries. Ovaries of goats were collected and either fixed in paraformaldehyde for immunohistochemical localization of KL and c-Kit proteins, or used for the isolation of follicles, luteal cells, surface epithelium and medullary samples to study mRNA expression for KL and c-Kit, using the reverse transcriptase polymerase chain reaction (RT-PCR). KL protein and mRNA were found in follicles at all stages of development, i.e. primordial, primary, secondary, small and large antral follicles, as well as in corpora lutea, surface epithelium and medullary tissue. Antral follicles expressed both KL-1 and KL-2 mRNAs, while earlier staged follicles expressed KL-1 transcript only. KL protein was demonstrated in granulosa cells from the primordial follicle onward. Its mRNA could be detected in granulosa cells isolated from antral follicles and occasionally in their theca cells. c-Kit mRNA was expressed in all antral follicular compartments and at all stages of follicular development. c-Kit protein was predominantly found in oocytes from the primordial follicle stage onwards, in theca cells of antral follicles, as well as in corpora lutea, surface epithelium and medullary tissue, particularly in the wall of blood vessels, which may indicate these cells as the main sites of action of KL. It is concluded that the KL/c-Kit system, in goat ovaries, is widespread and that it may be involved in the regulation of various local processes, including folliculogenesis and luteal activity.

The aim of the present study was to investigate whether growth hormone (GH) has any effect on the development of cultured rat pre-antral follicles. Pre-antral follicles with a diameter between 120 microm and 160 microm were mechanically isolated from 10-day-old rat ovaries and cultured in groups for 6 days in serum-free medium without GH or with GH supplemented at concentrations of 1, 10 and 100 ng/ml, respectively. DNA content of the follicles before and after culture was measured to determine whether possible growth is due to proliferation of follicular cells. To investigate the quality of follicles cultured under different conditions, the ultrastructure of the cultured follicles was studied with transmission electron microscopy. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) was used to assess the expression of growth hormone receptor (GHR) in pre-antral follicles. GH, regardless of the concentration, stimulated the growth of pre-antral follicles. However, follicles cultured in medium supplemented with high-dose GH (100 ng/ml) showed a significantly lower survival rate compared with the other groups. Follicles cultured in GH-containing medium showed a better ultrastructure in comparison with those cultured in medium without GH. Remarkably, scattered cortical granules were observed in oocytes of follicles cultured in the presence of GH. With RT-PCR, the presence of the mRNA of GHR was demonstrated in pre-antral follicles. It can be concluded that GH promotes rat pre-antral follicle development in vitro and better supports the morphology of cultured pre-antral follicles. The gene expression of GHR suggests that the action of GH could be mediated by its receptors present in pre-antral follicles.

The effect of recombinant bovine activin A on the in vitro maturation of bovine oocytes was investigated. Culture of cumulus enclosed bovine oocytes in the presence of activin at the concentration of 100 or 500 ng/ml did not change the proportion of oocytes in which germinal vesicle breakdown had occurred at 4 and 7 h after the onset of culture. Activin had also no effect on the progression of maturation to the M II stage. The transient inhibition of germinal vesicle breakdown by 10 mM dibutyryl cyclic AMP was not affected by the addition of activin A at the onset of culture. Radiolabeling with 35S-methionine at 4 h and at 18 h after culture in the presence or absence of activin A did not show any effect of activin either on the total incorporation of radiolabel into acid precipitable material or on the protein synthesis patterns obtained after SDS-PAGE.

A muscle progenitor cell population, other than muscle satellite cells, can be isolated and purified from porcine muscle tissue. We show the presence of at least two types of stem cells in porcine muscle: those that express α6 integrin and those that lack expression of this integrin type. By flow cytometry, we could select for myogenic stem cell populations expressing the neural cell adhesion molecule in the presence and absence of α6 integrin. The expression of α6 integrin showed an advantage in the formation of myotubes, possibly by an improved cell fusion capacity. This notion was strengthened by qRT-PCR analysis showing sustained PAX7, MYF5 and DESMIN expression and a strong myogenic differentiation capacity of this stem cell population. Selective inhibition of α6 integrin function, both by blocking antibodies and RNA interference, showed the importance of α6 integrin in myogenic differentiation of muscle stem cells. It is concluded that α6 integrin expression can be used as biomarker to select for highly myogenic cell populations in muscle tissue.

This study investigated the role of the leptin system in human oocyte maturation and its prognostic value for IVF outcome. The protein concentrations of leptin and soluble leptin receptor in follicular fluid were determined and the free leptin index (FLI) were established. Additionally, mRNA expression levels of different leptin receptor (ObR) isoforms and of PTX3 and HAS2 in cumulus cells were quantified, mutually compared and analysed relative to FLI, body mass index, age and number of retrieved oocytes. Expression of all target genes was detected in the cumulus cells, with relatively low concentrations of ObR-Long. Strong mutual correlations were found between mRNA expression levels of leptin receptor isoforms (P < 0.001) and also between the short isoforms of the leptin receptor and PTX3 (P < 0.001). Although the mean values of the pregnant and non-pregnant groups did not differ significantly for any of the variables, the chance that treatment resulted in ongoing pregnancy was higher with leptin 0.5 ng/mg protein compared with concentrations >0.5 ng/mg protein (P < 0.05). It is concluded that the leptin system appears to play a role in the IVF protocol, whereby signal transduction in cumulus cells occurs predominantly via the short isoforms of ObR.

The effect of follicular cells and their conditioned media on the FSH-induced oocyte maturation of oocytes surrounded by cumulus cells connected to the membrana granulosa (COCGs) was investigated. COCGs and cumulus oocyte complexes (COCs) were cultured for 22 hr in M199 supplemented with 0.05 IU FSH/ml in either the presence of pieces of theca cell layer or in the presence of pieces of membrana granulosa. COCGs and COCs were also cultured for 22 hr in either theca-cell conditioned medium (CMt) or in granulosa cell conditioned medium (CMg), both supplemented with 0.05 IU FSH/ml. To investigate the importance of cell-cell contacts between granulosa cells and cumulus cells, oocytes were cultured as COCs in CMt, as COCs in CMt supplemented with pieces of membrana granulosa, or as COCGs in CMt. In all groups the medium was supplemented with 0.05 IU FSH/ml. After culture the nuclear status of the oocytes was assessed using orcein staining. Culture of COCGs in the presence of theca cells as well as in CMt resulted in a significantly decreased proportion of oocytes that had undergone germinal vesicle breakdown (GVBD) at the end of the culture period as compared to the control. Of the oocytes that resumed meiosis in the presence of theca cells or in CMt, the proportion of oocytes that progressed up to the MII stage was significantly reduced. This indicates the production of a meiosis-inhibiting factor by theca cells. Culture with COCs instead of COCGs resulted in comparable results although the effect was less pronounced. The significant effect on the progression of meiosis of oocytes cultured as COCGs or as COCs, obtained in the presence of granulosa cells or in CMg, was much weaker than the effect of theca cells or culture in CMt. Culture of COCs in CMt supplemented with layers of membrana granulosa and 0.05 IU FSH/ml, resulted in significantly less oocytes that resumed meiosis as compared to culture of COCs in CMt. Of the oocytes that showed GVBD, the proportion that progressed up to the MII stage was significantly reduced. Attachment of the COCs to the membrana granulosa enhanced this inhibiting action of CMt on the progression of meiosis. It is concluded that theca cells secrete a stable factor that inhibits the progression of FSH-mediated meiosis in oocytes of COCGs.

A factor, secreted by theca cells, inhibits FSH induced resumption of meiosis in bovine oocytes that are surrounded by cumulus cells which are attached to a piece of the membrana granulosa (COCGs). In order to characterize this factor, theca cell conditioned medium (CMt) was heat-treated, filtered through a 5 kD spin off filter, charcoal treated, chloroform extracted and protease treated. To investigate whether the meiosis inhibiting factor produced by theca cells was also present in follicular fluid (FF), the same treatments were done with 50% bovine follicular fluid (bFF). COCGs, originating from 2 to 8 mm follicles of bovine ovaries collected at a slaughterhouse, were cultured in groups of 15 per 600 microl medium supplemented with 0.05 IU ml FSH for 22 hr at 39 degrees C in a humidified atmosphere of 5% CO(2). After culture the oocytes were denuded, stained with orcein, and the nuclear status assessed. Heat treatment did not affect the meiosis arresting capacity of CMt since a similar proportion of the oocytes remained at the GV stage after 22 hr of culture in heat treated CMt as compared to the proportion of oocytes in the GV stage after culture in untreated CMt. Filtering through a 5 kD spin-off filter revealed that the meiosis inhibiting action was maintained in the <5 kD fraction, although there was a significant (P < 0.05) loss of inhibiting activity compared to nonfiltered CMt. No significant decrease was observed in the meiosis arresting capacity of the <5 kD fraction after charcoal or protease treatment. Extraction of the <5 kD fraction with chloroform also did not affect the theca cell produced factor. The effect of the theca cell factor on the progression of meiosis of the oocytes that resumed meiosis, as demonstrated by a very low percentage of the oocytes that matured up to the M2 stage, was not affected following any of the treatments. With regard to bFF, the results show a lower percentage of the oocytes in the GV stage after culture in 50% bFF as compared to culture in CMt, but progression of meiosis was clearly inhibited as demonstrated by a significant higher proportion of the oocytes blocked in the M1 stage after resumption of meiosis. In general, with regard to meiotic inhibition, bFF showed the same pattern as CMt following the various treatments. It is concluded that the theca cell secreted factor which inhibits the FSH-induced resumption of meiosis in COCGs is a small, stable, polar molecule which is not a peptide.

Nitric oxide (NO) plays a key role in the processes leading to cervical softening prior to labor. Inducible nitric oxide synthase (iNOS) contributes most to the increased production of NO during labor, as demonstrated in the rat cervix, or at term pregnancy in women. Changes in expression of iNOS during late gestation have not yet been studied longitudinally in any species, because repeatedly taking biopsies could not be performed. iNOS mRNA (n = 6) and protein expression (n = 3) in serial cervical biopsies of pregnant pluriparous cows taken around days 225, 250, and 275 of pregnancy and within 1.5 hr after calving (d225, d250, d275 and parturition biopsies, respectively) were measured using quantitative RT-PCR and Western blotting. iNOS mRNA expression decreased from the d225 biopsy onwards, differences being significant between the d250 and d275 (P < 0.05) and between the d275 and parturition biopsies (P < 0.05). iNOS protein expression decreased from d225 to d250 onwards. Immunohistochemical analysis of biopsies showed, besides positive staining in endothelium and epithelium, which remained unchanged at different time points, that iNOS expressing cells in the connective tissue cells of early biopsies were predominantly spindle shaped (mostly smooth muscle cells and some fibroblasts). In the parturition biopsies, iNOS reactivity was mainly found in mononuclear leucocytes. These results lead us to suggest that iNOS from spindle shaped cells is involved in prepartum cervical ripening, while iNOS in mononuclear inflammatory cells may be important for local tissue repair mechanisms during postpartum cervical involution.

In previous studies we demonstrated that bovine cumulus oocyte complexes (COCs) obtained from small and medium sized follicles express growth hormone receptor (GHR) mRNA and respond to growth hormone (GH) addition during in vitro maturation. The aim of this study was to investigate whether bovine zygotes and preimplantation embryos continue the expression of GHR gene after in vitro fertilization and during early embryo development and whether supplementation of GH during embryo culture affects embryo development. Therefore, COCs obtained from small and medium sized follicles were cultured in M199 supplemented with 10% FCS and gonadotropins for 24 hr. After in vitro fertilization the embryos were cultured: (a) on a monolayer of buffalo rat liver (BRL) cells in M199 supplemented with 10% FCS and 100 ng/ml bovine GH (NIH-GH-B18); (b) in droplets of serum-free BRL-conditioned medium supplemented with 100 ng/ml GH; (c) in droplets of synthetic oviductal fluid (SOF) supplemented with 100 ng/ml GH. Cultures without GH served as controls. Embryos were scored morphologically and the efficiency of the culture system was evaluated (a) as the percentage of cleaved embryos 4 days after IVF, (b) the percentage of blastocysts on Day 9 expressed on the basis of the number of oocytes at the onset of culture, and (c) the percentage of hatched blastocysts on Day 11 expressed on the basis of the total number of blastocysts present at Day 9. For gene expression, immature (GV) and mature (MII) oocytes (as positive control), embryos with less than 8 cells, 16-32 cells, and hatched blastocysts were prepared for reverse transcriptase polymerase chain reaction (RT-PCR) to assess the expression of mRNA of GHR. Messenger RNA for GHR was found in GV and MII oocytes and in all stages of embryo development. No mRNA for GH could be detected in early and expanded blastocysts produced in SOF medium. Immunoreactive GHR was found both in trophoblastic and embryonic cells of hatched blastocysts. Addition of 100 ng/ml GH during embryo culture on a monolayer of BRL cells in M199 supplemented with 10% FCS did not affect embryo development. However, GH (100 ng/ml) supplementation during embryo culture in droplets of serum-free BRL conditioned medium significantly (P < 0.05) enhanced the proportion of > 8-cell embryos. Similarly, culture of embryos in droplets of SOF medium in the presence of GH (100 ng/ml) significantly (P < 0.05) enhanced the number of > 8-cell embryos from 53.8% in control to 70.6% in GH-treated group. Day 9 blastocyst formation in SOF medium was also significantly (P < 0.01) increased in the presence of GH (33.9%) compared to the control (20.2%). Embryos cultured in SOF without GH rarely resulted in hatched blastocysts (0.7%). However, GH supplementation remarkably enhanced the proportion of the hatched blastocysts (13%). In conclusion, expression of GHR gene in preimplantation bovine embryos, presence of the receptor, and the beneficial effect of GH on cleavage, blastocyst formation and hatchability of the embryos point to the involvement of GH in early embryonic development.

In various cell types, there is increasing evidence for nongenomic steroid effects, i.e., effects that are not mediated via the classical steroid receptors. However, little is known about the involvement of the nongenomic pathway of estradiol (E2) on mammalian oocyte in vitro maturation (IVM). The aim of this study was to investigate whether the effects of E2 on bovine oocyte IVM are mediated via a plasma membrane receptor (nongenomic). First, we investigated the expression of estradiol (classical) receptor alpha (ERalpha) and beta (ERbeta) mRNA in oocytes and cumulus cells (CC). We also studied the effects of different exposure times to E2 (before and after germinal vesicle breakdown, GVBD) on nuclear maturation. To study the possible involvement of the putative estradiol plasma membrane receptor on the IVM of oocytes, we used E2 conjugated with bovine serum albumin (E2-BSA), which cannot cross the plasma membranes. Our results demonstrate that oocytes expressed ERbeta mRNA, while CC expressed both ERalpha and ERbeta mRNA. Exposure to E2 during the first 8 h of culture (before GVBD) induced a block at the metaphase I stage (MI). However, the presence of E2 after GVBD induced an increase of oocytes with nuclear aberrations. Meiotic spindle organization was severely affected by E2 during IVM and multipolar spindle was the most frequently observed aberration. Exposure of oocytes to E2-BSA did not affect nuclear maturation, blastocyst formation rate, nor embryo quality. Our results suggest that the detrimental effects of E2 on in vitro nuclear maturation of bovine oocyte are not exerted via a plasma membrane receptor.