Relative Abundance Of Gene Transcripts Research Articles

Food deprivation reduces sensitivity of liver Igf1 synthesis pathways to growth hormone in juvenile gopher rockfish (Sebastes carnatus)

Growth hormone (Gh) regulates growth in part by stimulating the liver to synthesize and release insulin-like growth factor-1 (Igf1), which then promotes somatic growth. However, for fish experiencing food limitation, elevated blood Gh can occur even with low circulating Igf1 and slow growth, suggesting that nutritional stress can alter the sensitivity of liver Igf1 synthesis pathways to Gh. Here, we examined how recent feeding experience affected Gh regulation of liver Igf1 synthesis pathways in juvenile gopher rockfish (Sebastes carnatus) to illuminate mechanisms underlying the nutritional modulation of Igf1 production. Juvenile gopher rockfish were maintained under conditions of feeding or complete food deprivation (fasting) for 14 d and then treated with recombinant sea bream (Sparus aurata) Gh or saline control. Gh upregulated hepatic igf1 mRNA levels in fed fish but not in fasted fish. The liver of fasted rockfish also showed a lower relative abundance of gene transcripts encoding teleost Gh receptors 1 (ghr1) and 2 (ghr2), as well as reduced protein levels of phosphorylated janus tyrosine kinase 2 (pJak2) and signal transducer and activator of transcription 5 (pStat5), which function to induce igf1 gene transcription following Gh binding to Gh receptors. Relative hepatic mRNA levels for suppressors of cytokine signaling (Socs) genes socs2, socs3a, and socs3b were also lower in fasted rockfish. Socs2 can suppress Gh activation of Jak2/Stat5, and fasting-related variation in socs expression may reflect modulated inhibitory control of igf1 gene transcription. Fasted rockfish also had elevated liver mRNA abundances for lipolytic hormone-sensitive lipase 1 (hsl1) and Igf binding proteins igfbp1a, -1b and –3a, reduced liver mRNAs encoding igfbp2b and an Igfbp acid labile subunit-like (igfals) gene, and higher transcript abundances for Igf1 receptors igf1ra and igf1rb in skeletal muscle. Together, these findings suggest that food deprivation impacts liver Igf1 responsiveness to Gh via multiple mechanisms that include a downregulation of hepatic Gh receptors, modulation of the intracellular Jak2/Stat5 transduction pathway, and possible shifts in Socs-inhibitory control of igf1 gene transcription, while also demonstrating that these changes occur in concert with shifts in liver Igfbp expression and muscle Gh/Igf1 signaling pathway components.

Iron uptake of etioplasts is independent from photosynthesis but applies the reduction-based strategy.

Iron (Fe) is one of themost important cofactors in the photosynthetic apparatus, and its uptake by chloroplasts has also been associated with the operation of the photosynthetic electron transport chain during reduction-based plastidial Fe uptake. Therefore, plastidial Fe uptake was considered not to be operational in the absence of the photosynthetic activity. Nevertheless, Fe is also required for enzymatic functions unrelated to photosynthesis, highlighting the importance of Fe acquisition by non-photosynthetic plastids. Yet, it remains unclear how these plastids acquire Fe in the absence of photosynthetic function. Furthermore, plastids of etiolated tissues should already possess the ability to acquire Fe, since the biosynthesis of thylakoid membrane complexes requires a massive amount of readily available Fe. Thus, we aimed to investigate whether the reduction-based plastidial Fe uptake solely relies on the functioning photosynthetic apparatus. In our combined structure, iron content and transcript amount analysis studies, we used Savoy cabbage plant as a model, which develops natural etiolation in the inner leaves of the heads due to the shading of the outer leaf layers. Foliar and plastidial Fe content of Savoy cabbage leaves decreased towards the inner leaf layers. The leaves of the innermost leaf layers proved to be etiolated, containing etioplasts that lacked the photosynthetic machinery and thus were photosynthetically inactive. However, we discovered that these etioplasts contained, and were able to take up, Fe. Although the relative transcript abundance of genes associated with plastidial Fe uptake and homeostasis decreased towards the inner leaf layers, both ferric chelate reductase FRO7 transcripts and activity were detected in the innermost leaf layer. Additionally, a significant NADP(H) pool and NAD(P)H dehydrogenase activity was detected in the etioplasts of the innermost leaf layer, indicating the presence of the reducing capacity that likely supports the reduction-based Fe uptake of etioplasts. Based on these findings, the reduction-based plastidial Fe acquisition should not be considered exclusively dependent on the photosynthetic functions.

Evaluation of the molecular response of corpora lutea to manganese-amino acid complex supplementation in gilts.

Porcine pregnancy establishment and maintenance are dependent on the formation of functional corpora lutea (CL). Manganese (Mn) is critical for CL function as it is a cofactor for Mn superoxide dismutase and enzymes involved in cholesterol synthesis. Previously, we have shown that luteal Mn content increased and luteal progesterone (P4) concentration decreased in the CL of gilts fed diets supplemented with an Mn–amino acid complex (Availa-Mn; Zinpro Corporation) compared with controls fed Mn sulfate. Importantly, serum P4 increased from 0 (estrus onset) to 12 d post estrus (dpe), as expected, but P4 abundance in circulation was not affected by dietary Mn source (P = 0.15). We hypothesized that a more bioavailable Mn source (which results in increased luteal Mn content) would alter the luteal proteome and abundance of mRNA associated with steroid biogenesis during the mid-luteal phase of the estrous cycle. Postpubertal gilts (n = 32) were assigned to one of the four gestation diets. The control diet (CON) contained 20 ppm of supplemental Mn in the form of Mn sulfate. Three additional diets included 20 (TRT1), 40 (TRT2), or 60 (TRT3) ppm of supplemental Mn in the form of a Mn–amino acid complex instead of Mn sulfate. Dietary treatment began at estrus synchronization (approximately 20 d before estrus) and continued through 12 dpe when gilts were euthanized and tissues were collected. Protein and total RNA extracts from the CL were used for proteomic analysis via label-free liquid chromatography with tandem mass spectrometry to assess global protein abundance and quantitative real-time polymerase chain reaction (qRT-PCR) to assess specific mRNA abundance, respectively. Compared with CON, 188, 382, and 401 proteins were differentially abundant (P < 0.10) in TRT1, TRT2, and TRT3, respectively. Gene Ontology enrichment software revealed that proteins involved in P4 signaling and cholesterol synthesis were downregulated in CL of gilts fed Mn–amino acid complex compared with controls. Quantitative RT-PCR showed that relative transcript abundance of genes encoding steroidogenic enzymes (CYP11A1 and StAR) in CL tissue was decreased in gilts from TRT2 compared with CON (P = 0.02), but TRT1 and TRT3 were not affected (P ≥ 0.30). Collectively, these data support our hypothesis that a more bioavailable dietary Mn source may influence luteal function by altering the abundance of protein and mRNA involved in steroidogenesis.

MiRNA-34c Regulates Bovine Sertoli Cell Proliferation, Gene Expression, and Apoptosis by Targeting the AXL Gene.

Simple SummaryFertility is one of the essential reproduction traits of bulls, and accurate prediction of fertility potential using a semen sample from a donor bull for artificial insemination is crucial to achieve consistently high reproductive efficiency. Somatic cells, such as Sertoli cells and Leydig cells, are important in testis formation and provide a nurturing and regulatory environment for spermatogenesis. Furthermore, it was suggested that non-coding RNAs, such as microRNAs, long non-coding RNAs, circular RNAs, and Piwi-interacting RNA, function as important regulators of gene expression at post-transcriptional level in spermatogenesis. In this study, microRNA-34c was verified to specifically regulate the AXL gene by targeting a sequence in the 3’ UTR; miRNA-34c can also influence the proliferation, apoptosis, and relative abundance of the transcript of male-reproduction-related genes. Therefore, microRNA-34c can be considered an essential regulator in the process of bull spermatogenesis. These results identify a key microRNA and functional genes in the process of cattle male reproduction, providing useful information for future marker-assisted selection of bulls with excellent sperm quality.MicroRNAs (miRNAs) play significant roles in mammalian spermatogenesis. Sertoli cells can provide a stable microenvironment and nutritional factors for germ cells, thus playing a vital role in spermatogenesis. However, few studies elucidate the regulation of bovine testicular Sertoli cells by miRNAs. Here, we have reported that miRNA-34c (miR-34c) regulates proliferation, apoptosis, and relative transcripts abundance gene in bovine Sertoli cells. In bovine Sertoli cells, overexpression of miR-34c inhibited proliferation and relative abundance of gene transcripts while promoting apoptosis of Sertoli cells, and the effects were the opposite when miR-34c was knocked down. Receptor tyrosine kinase (AXL) was identified as a direct target gene of miR-34c in Sertoli cells, validated by analysis of the relative abundance of AXL transcript and dual-luciferase reporter assay. The relative abundance of the transcript of genes related to male reproduction in Sertoli cells was changed after the AXL gene was overexpressed, as demonstrated by the RT2 Profiler PCR Array results. In summary, miR-34c specifically regulated the AXL gene by targeting a sequence in the 3′-UTR, which could influence proliferation, apoptosis, and relative abundance of the transcript of male reproduction-related genes. Therefore, miR-34c could be considered an essential regulator in the process of bull spermatogenesis.

Effects of food deprivation on plasma insulin-like growth factor-1 (Igf1) and Igf binding protein (Igfbp) gene transcription in juvenile cabezon (Scorpaenichthys marmoratus)

The growth hormone (GH)/insulin-like growth factor (Igf) endocrine axis regulates somatic growth in the face of changing environmental conditions. In actinopterygian fishes, food availability is a key modulator of the somatotropic axis, with lower food intake generally depressing liver Igf1 release to diminish growth. Igf1 signaling, however, also involves several distinct IGF binding proteins (Igfbps), and the functional roles of many of these Igfbps in affecting growth during shifting food availability remain uncertain. Here, we tested how complete food deprivation (fasting) affected gene transcription for paralogs of all six types of Igfbps in the liver and fast-twitch skeletal muscle of cabezon (Scorpaenichthys marmoratus), a nearshore marine fish important for recreational fisheries in the eastern North Pacific Ocean. Juvenile cabezon were maintained as either fed (6% mass food⋅g fish wet mass−1⋅d−1) or fasted for 14 d. Fasted fish exhibited a lower body condition (K), a depressed mass-specific growth rate (SGR), and reduced plasma concentrations of Igf1. In the liver, fasting reduced the relative abundance of gene transcripts encoding Igfbps igfbp2a and igfbp2b, while significantly elevating mRNA levels for igfbp1a, igfbp1b, igfbp3b, and igfbp4. Fasting also reduced hepatic mRNA levels of GH receptor-1 (ghr1) – but not GH receptor-2 (ghr2) – supporting the idea that changes in liver sensitivity to GH may underlie the decline in plasma Igf1 during food deprivation. In skeletal muscle, fasting downregulated gene transcripts encoding igf1, igfbp2b, igfbp5b, and igfbp6b, while also upregulating mRNAs for igf2 and ghr2. These data demonstrate isoform-specific regulation of Igfbps in liver and skeletal muscle in cabezon experiencing food deprivation and reinforce the idea that the repertoire of duplicated Igfbp genes that evolved in actinopterygian fishes supports a diverse scope of endocrine and paracrine functions.

Interactions of long-term food ration variation and short-term fasting on insulin-like growth factor-1 (IGF-1) pathways in copper rockfish (Sebastes caurinus)

Variation in food intake affects somatic growth by altering the expression of hormones in the somatotropic endocrine axis including insulin-like growth factor-1 (IGF-1). Here, we examined IGF-1 pathway responses to long- and short-term variation in food availability in copper rockfish (Sebastes caurinus), a nearshore Pacific rockfish important for commercial and recreational fisheries. Juvenile copper rockfish were raised under differing ration amounts (3% or 9% mass feed·g−1 fish wet mass·day−1) for 140 d to simulate ‘long-term’ feeding variation, after which some fish from both rations were fasted for 12 d to generate ‘short-term’ conditions of food deprivation. Rockfish on the 9% ration treatment grew more quickly than those on the 3% ration and were larger in mass, length, and body condition (k) after 152 d. Fish on the 9% ration had higher blood glucose than those on the 3% ration, with fasting decreasing blood glucose in both ration treatments, indicating that both long-term and short-term feed treatments altered energy status. Plasma IGF-1 was higher in rockfish from the 9% ration than those in the 3% ration and was also higher in fed fish than fasted fish. Additionally, plasma IGF-1 related positively to individual variation in specific growth rate (SGR). The positive association between IGF-1 and SGR showed discordance in fish that had experienced different levels of food and growth over the long-term but not short-term, suggesting that long-term nutritional experience can influence the relationship between IGF-1 and growth in this species. Rockfish on the 3% ration showed a lower relative abundance of gene transcripts encoding igf1 in the liver, but higher hepatic mRNAs for IGF binding proteins igfbp1a and igfbp1b. Fasting similarly decreased the abundance of igf1 mRNAs in the liver of fish reared under both the 9% and 3% rations, while concurrently increasing mRNAs encoding the IGF binding proteins igfbp1a, -1b, and -3a. Hepatic mRNAs for igfbp2b, -5a, and -5b were lower with long-term ration variation (3% ration) and fasting. Fish that experienced long-term reduced rations also had higher mRNA levels for igfbp3a, -3b, and IGF receptors isoforms A (igf1rA) and B (igf1rB) in skeletal muscle, but lower mRNA levels for igf1. Fasting increased muscle mRNA abundance for igfbp3a, igf1rA, and igf1rB, and decreased levels for igfbp2a and igf1. These data show that a positive relationship between circulating IGF-1 and individual growth rate is maintained in copper rockfish even when that growth variation relates to differences in food consumption across varying time scales, but that long- and short-term variation in food quantity can shift basal concentrations of circulating IGF-1 in this species.

Seasonal variations in developmental competence and relative abundance of gene transcripts in buffalo (Bubalus bubalis) oocytes

Hot season is a major constraint to production and reproduction in buffaloes. The present work aimed to investigate the effect of season on ovarian function, developmental competence, and the relative abundance of gene expression in buffalo oocytes. Three experiments were conducted. In experiment 1, pairs of buffalo ovaries were collected during cold season (CS, autumn and winter) and hot season (HS, spring and summer), and the number of antral follicles was recorded. Cumulus oocyte complexes (COCs) were aspirated and evaluated according to their morphology into four Grades. In experiment 2, Grade A and B COCs collected during CS and HS were in vitro matured (IVM) for 24 hours under standard conditions at 38.5 °C in a humidified air of 5% CO2. After IVM, cumulus cells were removed and oocytes were fixed, stained with 1% aceto-orcein, and evaluated for nuclear configuration. In vitro matured buffalo oocytes harvested during CS or HS were in vitro fertilized (IVF) using frozen-thawed buffalo semen and cultured in vitro to the blastocyst stage. In experiment 3, buffalo COCs and in vitro matured oocytes were collected during CS and HS, and then snap frozen in liquid nitrogen for gene expression analysis. Total RNA was extracted from COCs and in vitro matured oocytes, and complementary DNA was synthesized; quantitative Reverse Transcription-Polymerase Chain Reaction was performed for eight candidate genes including GAPDH, ACTB, B2M, GDF9, BMP15, HSP70, and SOD2. The results indicated that HS significantly (P < 0.01) decreased the number of antral follicles and the number of COCs recovered per ovary. The number of Grade A, B, and C COCs was lower (P < 0.05) during HS than CS. In vitro maturation of buffalo oocytes during HS significantly (P < 0.01) reduced the number of oocytes reaching the metaphase II stage and increased the percentage of degenerated oocytes compared with CS. Oocytes collected during HS also showed signs of cytoplasmic degeneration. After IVF, cleavage rate was lower (P < 0.01) for oocytes collected during HS, and the percentage of oocytes arrested at the two-cell stage was higher (P < 0.01) than oocytes IVF during CS. Oocytes matured during CS showed a higher (P < 0.01) blastocyst rate than those matured during HS. Also, COCs recovered in HS showed significant (P < 0.05) upregulation of HSP70 mRNA expression compared with those recovered in CS. For in vitro matured oocytes, CS down regulated the transcript abundance of ACTB and upregulated GAPDH and HSP70 mRNA levels compared with HS condition. In conclusion, HS could impair buffalo fertility by reducing the number of antral follicles and oocyte quality. In vitro maturation of buffalo oocytes during HS impairs their nuclear and cytoplasmic maturation, fertilization, and subsequent embryo development to the morula and blastocyst stages. This could be in part because of the altered gene expression found in COCs and in vitro matured oocytes.

Development competence and relative transcript abundance of oocytes derived from small and medium follicles of prepubertal gilts

The objective of this study was to examine the competence of mature oocytes aspirated from small follicles (SF, <2 mm in diameter) and medium follicles (MF, 3–6 mm) of abattoir-derived prepubertal gilt ovaries. Oocytes were selected by the presence of the first polar body (1pb) after IVM in a chemically defined medium, for sperm penetration, pronuclear formation, cleavage rate, and development to the blastocyst stage. Relative transcript abundance of genes associated with regulation of oocyte maturation (AURKA, AURKB, and MOS), fertilization (ZP3 and ZP4), maternal effect (NALP9 and HSF1), and anti-apoptosis (BCL2) were also examined in oocytes at germinal vesicle (GV) and metaphase-II (MII) stages. In SF, compared with MF, the maturation rate post-IVM was lower (P < 0.05), but there were no differences in sperm penetration rate (78.2% and 68.5% at 6 hours after insemination and 90.8% and 91.9% at 9 hours after insemination, P = 0.51 and P = 0.67, respectively), the percentage of oocytes that formed both female and male pronuclei (27.9% and 25.8% at 6 hours after insemination and 79.4% and 76.1% at 9 hours after insemination), or cleavage rate at 48 hours after insemination (85.9% and 89.7%, respectively, P = 0.46), whereas blastocyst formation rate was lower (P < 0.05) in oocytes from SF versus MF (14.7% and 31.0%). Transcript abundances decreased (P < 0.05) in all genes examined between the GV and MII stages, although only transcript abundance for MOS was lower (P < 0.05) in GV oocytes from SF versus MF. In conclusion, mature oocytes from SF and MF of prepubertal gilts with a visible 1pb had similar fertilizability in vitro and relative transcript abundance of nine genes. However, follicle size affected meiotic competence, early embryonic development to the blastocyst stage, and transcript abundance of the MOS gene.

Gene-expression patterns reveal underlying biological processes in Kawasaki disease

Analysis of patterns of gene expression in peripheral blood from children with Kawasaki disease revealed dynamic and variable gene expression programs involving neutrophil activation and apoptosis.

Predicting embryo quality: mRNA expression and the preimplantation embryo

To overcome the low implantation rate (10–20%) following IVF in humans, more than two embryos are commonly replaced, potentially leading to high order multiple pregnancies with associated significantly elevated risks. Selecting the most viable embryos and transferring fewer of them could reduce this risk. Prolonged culture of embryos in vitro to the blastocyst stage may expose the embryo to hazards not normally encountered in the female reproductive tract. Recent studies comparing bovine oocyte maturation, fertilization and embryo culture in vivo and in vitro have demonstrated that the origin of the oocyte is the main factor affecting blastocyst yield, while the post-fertilization culture environment is crucial in determining blastocyst quality, measured in terms of cryotolerance and relative transcript abundance, irrespective of the origin of the oocyte. Production of embryos in vitro, particularly when using an extended period of in-vitro culture may predispose the embryo to phenomena such as ‘large offspring syndrome’, which is probably linked to altered gene expression, particularly of imprinted genes. Post-fertilization culture environment clearly has a profound effect on the relative abundance of gene transcripts within the embryo. Culture under sub-optimal conditions for even one day can lead to perturbations in the pattern of expression.

Effect of culture environment on embryo quality and gene expression – experience from animal studies

Recent studies comparing bovine oocyte maturation, fertilization and embryo culture in vivo and in vitro have demonstrated that the origin of the oocyte is the main factor affecting blastocyst yield, while the post-fertilization culture environment is critical in determining blastocyst quality, measured in terms of cryotolerance and relative transcript abundance, irrespective of the origin of the oocyte. Production of embryos in vitro, particularly when using an extended period of in-vitro culture, may predispose the embryo to phenomena such as the large offspring syndrome, which is likely to alter gene expression, particularly of imprinted genes. It is clear now that the post-fertilization culture environment has a profound effect on the relative abundance of gene transcripts within the embryo, and culture under suboptimal conditions for as little as 1 day can lead to perturbations in the pattern of expression.

Effects of oocyte maturation regimen on the relative abundance of gene transcripts in bovine blastocysts derived in vitro or in vivo

Bovine embryos produced in vitro differ substantially from embryos produced in vivo in the mRNA expression patterns of genes important for development. Several factors in the in vitro production systems have profound effects on embryonic mRNA expression patterns. The effects of the type of maturation on the expression pattern of genes important for development in blastocysts produced in vitro have not yet been investigated. The aim of the present study was to investigate the effects of various maturational protocols on the relative abundance of a panel of six marker genes, indicative of compaction and cavitation, metabolism, stress susceptibility and RNA processing, in bovine blastocysts produced in vitro. Four groups of blastocysts were analysed by a sensitive semi-quantitative RT-PCR assay. Blastocysts were produced in vitro from oocytes of different origin from: (1) 3-8 mm follicles; (2) preovulatory follicles before the LH surge; and (3) preovulatory follicles 24 h after the LH surge. The first two groups were matured in vitro, whereas the third group had undergone maturation in vivo. A fourth group comprised blastocysts developed entirely in vivo. Expression of glucose transporter 1 was significantly (P < 0.05) higher, and expression of desmocollin 2 and plakophilin tended to be higher (P < 0.1) for in vivo (group 4) compared with in vitro blastocysts (group 1), whereas no differences were found for heat shock protein 70.1, E-cadherin and poly(A) polymerase. Expression of the six transcripts did not differ among blastocysts produced in vitro from oocytes of groups 1, 2 and 3. Results indicate that alterations in the relative abundance of these transcripts in blastocysts produced in vitro cannot primarily be attributed to the origin of the oocyte, but are likely to have been induced by post-maturation or fertilization culture conditions.

Effects of oocyte maturation regimen on the relative abundance of gene transcripts in bovine blastocysts derived in vitro or in vivo

Bovine embryos produced in vitro differ substantially from embryos produced in vivo in the mRNA expression patterns of genes important for development. Several factors in the in vitro production systems have profound effects on embryonic mRNA expression patterns. The effects of the type of maturation on the expression pattern of genes important for development in blastocysts produced in vitro have not yet been investigated. The aim of the present study was to investigate the effects of various maturational protocols on the relative abundance of a panel of six marker genes, indicative of compaction and cavitation, metabolism, stress susceptibility and RNA processing, in bovine blastocysts produced in vitro. Four groups of blastocysts were analysed by a sensitive semi-quantitative RT-PCR assay. Blastocysts were produced in vitro from oocytes of different origin from: (1) 3-8 mm follicles; (2) preovulatory follicles before the LH surge; and (3) preovulatory follicles 24 h after the LH surge. The first two groups were matured in vitro, whereas the third group had undergone maturation in vivo. A fourth group comprised blastocysts developed entirely in vivo. Expression of glucose transporter 1 was significantly (P < 0.05) higher, and expression of desmocollin 2 and plakophilin tended to be higher (P < 0.1) for in vivo (group 4) compared with in vitro blastocysts (group 1), whereas no differences were found for heat shock protein 70.1, E-cadherin and poly(A) polymerase. Expression of the six transcripts did not differ among blastocysts produced in vitro from oocytes of groups 1, 2 and 3. Results indicate that alterations in the relative abundance of these transcripts in blastocysts produced in vitro cannot primarily be attributed to the origin of the oocyte, but are likely to have been induced by post-maturation or fertilization culture conditions.

Effects of culture system and protein supplementation on mRNA expression in pre-implantation bovine embryos.

Assisted reproduction technologies have made great progress during the last 15 years in most mammalian species, including humans. Growing evidence indicates that bovine pre-implantation development is a superior model for investigating early human development than the mouse. The purpose of this study was to investigate the effects of two basic culture systems [tissue culture medium (TCM) with 5% CO(2) in air or synthetic oviduct fluid (SOF) with 7% O(2), 88% N(2,) 5% CO(2)] and various protein supplements (serum, bovine serum albumin or polyvinyl alcohol) on the relative abundance of a set of developmentally important gene transcripts in bovine morulae and blastocysts and to compare the results with those for their in-vivo-derived counterparts. The basic culture system including the basic medium composition and oxygen tension had profound effects on the amounts of specific transcripts in bovine embryos, whereas the 'protein source' had only weak effects. Significant differences (P < or = 0.05) in the relative abundance of specific gene transcripts were detected between in-vivo and in-vitro-derived embryos, especially at the morula stage. More differences were found between embryos produced in the TCM system and in-vivo-derived embryos than between SOF-generated embryos and their in-vivo counterparts. No differences were found in the relative abundance of gene transcripts in embryos generated under chemically defined conditions in the two different laboratories. It is concluded that the SOF system provides an environment in which pre-implantation development of bovine embryos is more similar to that occurring in vivo than in the TCM system.

Transcript and activity levels of different Pleurotus ostreatus peroxidases are differentially affected by Mn2+.

The white-rot fungus Pleurotus ostreatus produces both manganese-dependent peroxidase (MnP) and versatile peroxidase (VP) in non-manganese-amended peptone medium (PM). We studied the effect of Mn2+ supplementation on MnPs and VPs in P. ostreatus by analysing the enzymatic and transcript abundance profiles of the peroxidases, as well as the lignin mineralization rate. The fungus was grown in PM under solid-state conditions using perlite as an inert solid support. Mn2+ amendment resulted in a 1.7-fold increase in [14C]-lignin mineralization relative to unamended medium. Anion-exchange chromatography was used to resolve the fungal peroxidase's enzymatic activity profile. Five peaks (P1-P5) of VP and one peak (P6) of MnP activity were detected in unamended medium. In Mn2+-amended medium, a reduction in the activity of the VPs was observed. On the other hand, a sharp increase in the MnP activity level of peak P6 was detected. The P6 isoenzyme was purified and showed manganese-dependent peroxidation of phenolic substrates. Internal sequence analysis of the purified enzyme revealed 100% identity with the deduced amino acid sequence of P. ostreatus MnP3 (GenBank AB016519). The effect of Mn2+ on the relative abundance of gene transcripts of three VPs and one MnP from P. ostreatus was monitored using reverse transcription-polymerase chain reaction (RT-PCR) with oligonucleotide primer sets synthesized on the basis of non-conserved sequences of the different peroxidases. The reduction in VP gene transcript abundance and the increase in mnp3 transcript level were collinear with the changes observed in the enzyme activity profiles. These results indicate that the activity of peroxidases is regulated at the transcriptional level. We suggest that the expression of MnP and VP may be differentially regulated by the presence of Mn2+.

Alterations in the relative abundance of gene transcripts in preimplantation bovine embryos cultured in medium supplemented with either serum or PVA.

In preimplantation bovine embryos, the relative abundance of various developmentally important gene transcripts was determined by a semi-quantitative RT-PCR assay to analyze the effects of two medium supplements, serum or polyvinyl alcohol (PVA). Development to morula, blastocyst, and hatched blastocyst stages was higher (P < or = 0.05) in medium supplemented with serum than in medium supplemented with PVA. Connexin43 mRNA expression virtually disappeared from the 8-16 cell stage onward, but reappeared in the hatched blastocyst in serum-supplemented medium, whereas it was detected in PVA-derived embryos throughout development. No differences were found for plakophilin mRNA between both culture groups. Desmocollin II mRNA showed a sharp increase at the blastocyst stage in both groups with a higher transcription level in PVA-generated embryos. A significant difference in desmocollin III transcripts was detectable at the 8-16-cell stage between serum- and PVA-derived embryos. Transcripts for desmoglein 1 and desmocollin I were not detected at any preimplantation stage, irrespective of medium supplementation. The relative abundance of glucose-transporter-1 mRNA was significantly increased at the 8-16-cell stage in embryos produced in medium supplemented with PVA, but not serum. Heat shock protein and poly(A)polymerase mRNA were continuously expressed during preimplantation development in both culture groups. Although poly(A)polymerase mRNA was significantly elevated in PVA- over serum-derived embryos, heat shock protein mRNA expression was significantly enhanced in serum-generated embryos over PVA-derived embryos. Interferon tau mRNA showed a significant increase at the hatched blastocyst stage only in PVA-supplemented medium. These data suggest that alterations in mRNA expression are associated with culture environment. Timing and magnitude of the alterations varied among the different transcripts and were significantly affected by the presence of exogenous protein in a stage-specific manner, predominantly at critical developmental time points.

In situ hybridization histochemistry reveals a diversity of GABA A receptor subunit mRNAs in neurons of the rat spinal cord and dorsal root ganglia

The distribution and relative abundance of gene transcripts for diverse GABA A receptor subunits (α 1–3,5, β 1–3, γ 2) in neurons of the rat cervical spinal cord and dorsal root ganglia were determined by in situ hybridization histochemistry using 35S-labeled 60mer oligonucleotide probes. The receptor proteins (mapped by benzodiazepine receptor radioautography and immunohistochemistry with [ 3H]flumazenil and a monoclonal antibody for the β 2 + β 3 subunits, respectively) were most abundant in the dorsal horn (layers II and III) and in layer X around the central canal. Although diverse receptor subunit mRNAs were detected (to varying degrees) in neurons throughout layers II-X of the spinal cord, motoneurons in layer IX were particularly strongly labeled. The γ 2 mRNA was the most ubiquitous and abundant of the subunit variants investigated. The labeling of motoneurons in layer IX was particularly strong for α 2, moderate for β 3 and γ 2 and extremely weak for α 1 and α 3. In layers VII, VIII and X the β 3 and γ 2 transcripts were moderately expressed whereas the α 1 and β 2 transcript levels differed markedly among the cells of these layers. Although the mRNAs of all subunit variants could be detected in layers IV-VI, only α 3, α 5, β 3 and γ 2 hybridization signals were observed in layers II and III. In the dorsal root ganglia, whereas α 2 transcripts were abundant in virtually all large sensory neurons and to a much lower degree in the small diameter cells, γ 2 transcripts were confined to a subpopulation of large and small neurons. Furthermore, β 2 and α 1 transcripts were even more restricted in their distribution. The findings provided a basis for the mediation of synaptic inhibition in the spinal cord by diverse GABA A receptors and further strong evidence for the long-established view that presynaptic inhibition of inter- and motoneurons, via axoaxonic synapses between GABAergic interneurons and primary afferent terminals, is mediated by GABA A receptors. The physiological roles and pharmacological implications of this receptor diversity have yet to be determined.