Steady-state mRNA Concentrations Research Articles

Macromolecular Crowding as a Regulator of Gene Transcription

Studies of macromolecular crowding have shown its important effects on molecular transport and interactions in living cells. Less clear is the effect of crowding when its influence is incorporated into a complex network of interactions. Here, we explore the effects of crowding in the cell nucleus on a model of gene transcription as a network of reactions involving transcription factors, RNA polymerases, and DNA binding sites for these proteins. The novelty of our approach is that we determine the effects of crowding on the rates of these reactions using Brownian dynamics and Monte Carlo simulations, allowing us to integrate molecular-scale information, such as the shapes and sizes of each molecular species, into the rate equations of the model. The steady-state cytoplasmic mRNA concentration shows several regimes with qualitatively different dependences on the volume fraction, ϕ, of crowding agents in the nucleus, including a broad range of parameter values where it depends nonmonotonically on ϕ, with maximum mRNA production occurring at a physiologically relevant value. The extent of this crowding dependence can be modulated by a variety of means, suggesting that the transcriptional output of a gene can be regulated jointly by the local level of macromolecular crowding in the nucleus, together with the local concentrations of polymerases and DNA-binding proteins, as well as other properties of the gene’s physical environment.

Estrous cycle-dependent changes of Fas expression in the bovine corpus luteum: influence of keratin 8/18 intermediate filaments and cytokines.

BackgroundFas expression and Fas-induced apoptosis are mechanisms attributed to the selective destruction of cells of the corpus luteum (CL) during luteal regression. In certain cell-types, sensitivity to these death-inducing mechanisms is due to the loss or cleavage of keratin-containing intermediate filaments. Specifically, keratin 8/18 (K8/K18) filaments are hypothesized to influence cell death in part by regulating Fas expression at the cell surface.MethodsHere, Fas expression on bovine luteal cells was quantified by flow cytometry during the early (Day 5, postovulation) and late stages (Days 16–18, postovulation) of CL function, and the relationship between Fas expression, K8/K18 filament expression and cytokine-induced cell death in vitro was evaluated.ResultsBoth total and cell surface expression of Fas on luteal cells was greater for early versus late stage bovine CL (89% vs. 44% of cells for total Fas; 65% vs.18% of cells for cell surface Fas; respectively, P<0.05, n=6-9 CL/stage). A similar increase in the steady-state concentration of mRNA for Fas, as detected by quantitative real-time polymerase chain reaction, however, was not observed. Transient disruption of K8/K18 filaments in the luteal cells with acrylamide (5 mM), however, had no effect on the surface expression of Fas (P>0.05, n=4 CL/stage), despite evidence these conditions increased Fas expression on HepG2 cells (P<0.05, n= 3 expts). Exposure of the luteal cells to cytokines induced cell death (P<0.05) as expected, but there was no effect of K8/K18 filament disruption by acrylamide (P>0.05) or stage of CL (P>0.05, n= 4 CL/stage) on this outcome.ConclusionIn conclusion, we rejected our null hypothesis that the cell surface expression of Fas does not differ between luteal cells of early and late stage CL. The results also did not support the idea that K8/K18 filaments influence the expression of Fas on the surface of bovine luteal cells. Potential downstream effects of these filaments on death signaling, however, remain a possibility. Importantly, the elevated expression of Fas observed on cells of early stage bovine CL compared to late stage bovine CL raises a provocative question concerning the physiological role(s) of Fas in the corpus luteum, particularly during early luteal development.

Importance of culture conditions during the morula-to-blastocyst period on capacity of inner cell-mass cells of bovine blastocysts for establishment of self-renewing pluripotent cells

The hypothesis was tested that the pluripotency of the inner cell mass (ICM) of the bovine embryo is enhanced by the glycogen synthase kinase-3β inhibitor CHIR99021 and the MAPK1 and MAPK3 inhibitor PD032591. Treatment with the two inhibitors from Days 6 to 8 after insemination increased blastocyst steady state concentrations of mRNA for NANOG (P < 0.05) and SOX2 (P = 0.055) and tended to decrease (P = 0.09) expression of GATA6. To evaluate pluripotency, the inner cell mass was isolated by immunosurgery at Day 8, seeded on a feeder layer of bovine embryonic fibroblasts, and cultured in the presence of the inhibitors. Ten of 52 (19%) ICM from control embryos had primary outgrowth formation vs. 23 of 50 (46%) of the ICM from embryos cultured with inhibitors (P < 0.01). For ICM outgrowths from embryos cultured without inhibitors, colonies either did not persist through Passage 2 or became differentiated. In contrast, for the inhibitor group, four colonies survived beyond Passage 2, and one line persisted for 19 passages. This cell line possessed alkaline phosphatase activity, expressed several genes characteristically expressed in pluripotent cells, and differentiated into embryoid bodies when cultured in the absence of the signal transduction inhibitors and the feeder layer. Propagation of the cells was difficult due to slow growth and inefficiency in survival through each passage. In conclusion, exposure to inhibitors during the morula-blastocyst transition facilitated formation of self-renewing pluripotent cell lines from bovine blastocysts.

Gene Expression, Localization, and Regulation of Activated Leukocyte Cell Adhesion Molecule/CD166 in the Bovine Corpus Luteum.

Activated leukocyte adhesion molecule (ALCAM/CD166) is a transmembrane glycoprotein expressed on a variety of epithelial cell types in normal tissues, but it is predominantly relevant to the biology of ovarian, breast, prostate, pancreatic and colorectal cancers. The objectives of the present study were to 1) determine if the ALCAM gene and protein are expressed in the CL, 2) examine if there are temporal changes in expression during the estrous cycle and early pregnancy (day 17), 3) identify the cell types on which it is expressed, and 4) evaluate regulation of expression by paracrine factors. Gene expression was determined by qPCR in luteal tissue collected at early (day 5), midcycle (days 10-12) and late (day 18) stages of the estrous cycle, and 12hr post prostaglandin F2a injection (pPGF2a) administered at midcycle (n= 3-6). In addition to gene expression, protein ALCAM was determined in early (day 4), midcycle and regressing CL (1, 8 and 12hrs pPGF2a) using Western blot analysis (n= 3). The steady-state concentration of ALCAM mRNA was higher in early compared to midcycle and late CL (P<0.05) and lower at midcycle compared to 12hrs pPGF2a (P<0.05). In contrast, the pattern of ALCAM protein expression was inversely proportional to the mRNA (P<0.05). ALCAM protein at 12hrs pPGF2a was lower compared to midcycle, 1 and 8hrs pPGF2a, (P<0.05). Compared to day 17 of the estrous cycle, ALCAM protein was lower at day 17 of pregnancy (P<0.05).To determine the proportion of cells expressing ALCAM in functional and regressing CL, freshly isolated and cultured luteal cells from CL collected at midcycle and 8hrs pPGF2a were labeled with anti-ALCAM. Furthermore, to identify if ALCAM in luteal cells was expressed on monocytes/macrophage and activated leukocytes, cells were colabeled with either anti-CD14 or anti-CD18 antibodies. The cultured luteal cells from midcycle CL were also treated with luteinizing hormone (LH), PGF2a, interferon gamma (IFNg) and phorbolmyristate acetate (PMA) for 48hrs and later labeled with anti-ALCAM antibody. All labeled cells were analyzed by flow cytometry. Both freshly isolated and cultured cells expressed ALCAM. There was no difference in the proportion of ALCAM+ cells between midcycle and regressing luteal cells (P>0.05). The proportion of ALCAM+ cells expressing CD14 and CD18 ranged between 5-20%. The proportion of ALCAM+ cells was increased by LH, IFNg and PMA (P<0.05), but not PGF2a. To further determine the cell types expressing ALCAM, we performed fluorescence microscopy on luteal cells and frozen tissue sections labeled with anti-ALCAM and Bandeira simpilicifolia-1 lectin (labels endothelial cells). In dissociated luteal cells, ALCAM was mainly expressed on steroidogenic-like cells, but not endothelial cells. In tissue sections, ALCAM was observed on steroidogenic-like cells as well as endothelial cells lining blood vessels. In conclusion, this is the first report to show that ALCAM is expressed in the CL. It is predominantly expressed in steroidogenic cells and its abundance changes over time. Because ALCAM has been shown to activate T lymphocytes through the CD6 receptor, it is suggested that luteal cells may activate T cells via ALCAM. This project was supported by National Research Initiative Competitive Grant no.2008-35203-04617 from the USDA National Institute of Food and Agriculture.

Temporal gene expression in equine corpora lutea based on serial biopsies in vivo1

A biopsy procedure was developed to enable repeated sampling of a single equine corpus luteum (CL) over the course of an estrous cycle. The tissue collected was utilized in characterizing mRNA abundance for genes involved in luteal formation, function, and regression in the cyclic mare. Serial biopsies of CL in cyclic mares (2.7 to 27.5 mg per biopsy) were collected using an ultrasound-guided transvaginal technique. Biopsies were collected from each mare on d 2 and 5 (d 0 = ovulation) of the estrous cycle, and every other day from d 12 through luteolysis. Samples were obtained from 4 mares with normal estrous cycles and 1 mare with a retained CL. The biopsy procedure did not adversely affect luteal size or function, as measured by luteal area and serum concentrations of progesterone. Real-time reverse-transcription PCR was used to quantify steady state mRNA concentrations in each tissue sample obtained. Mean abundance of steroidogenic acute regulatory protein (StAR) mRNA was not different (P = 0.102 to 0.964) on any of the sampling dates, but a trend for mRNA encoding StAR to decrease between d 12 and 14 (P = 0.10) was observed. Values for mRNA encoding StAR were positively correlated to serum concentrations of progesterone on d 5 (R = 0.95; P = 0.05) and 14 (R > 0.99; P < 0.01). Steady-state abundance of mRNA for 3β-hydroxysteroid dehydrogenase, Δ 5-Δ 4 isomerase (3β-HSD) declined between d 12 and 14 (P = 0.15). There were positive correlations between mRNA for 3β-HSD and concentrations of progesterone on d 5 (R = 0.94; P = 0.06) and 12 (R > 0.99; P = 0.05). No difference was detected in abundance of mRNA encoding cyclooxygenase-2 (cox-2; P = 0.340 to 0.840) or caspase-3 (P = 0.517 to 0.882) between any of the sampling dates. A successful luteal biopsy procedure was developed that did not negatively affect luteal function, and abundance of mRNA encoding StAR, 3β-HSD, cox-2, and caspase-3 was characterized in luteal biopsy tissue collected on d 2, 5, 12, and 14 of the estrous cycle in the mare.

Developmental Changes in Expression of Genes Involved in Regulation of Apoptosis in the Bovine Preimplantation Embryo1

The early bovine preimplantation embryo is resistant to proapoptotic signals until around the 8- to 16-cell stage. We hypothesized that 2-cell embryos have higher amounts of antiapoptotic proteins and lower amounts of proapoptotic proteins when compared to embryos ≥16 cells. Steady-state concentrations of mRNA for the antiapoptotic genes BCL2 and HSPA1A were higher for MII oocytes, 2-cell embryos, and 2-cell embryos treated with alpha-amanitin as compared to ≥16-cell embryos. Steady-state concentrations of mRNA for the proapoptotic gene BAD increased in embryos ≥16 cells. There was no significant effect of stage of development on steady-state mRNA concentrations of BCL2L1, DFFA, or BAX. Using immunohistochemistry, it was found that BCL2 was present in greater relative concentrations for 2-cell embryos than for embryos ≥16 cells. These results were confirmed by Western blotting. Relative amounts of immunoreactive BAX detected by immunofluorescence were lower for 2-cell embryos than for embryos ≥16 cells. Using Western blotting, a high molecular weight (46 kDa) form of BAX was highest in ≥16-cell embryos, intermediate in 2-cell embryos, and lowest in MII oocytes. There were no effects of stage of development on relative amounts of immunoreactive BCL2L1, HSPA1A, or BAD, as determined by immunofluorescence. Treatment of embryos with alpha-amanitin from Day 0 to Day 5 or Day 4 to Day 5 after insemination reduced activation of group II caspases and terminal deoxynucleotidyl transferase dUTP nick end labeling after treatment with the proapoptotic signal C(2) ceramide at Day 5 after fertilization. Thus, transcription of BAX or other proteins is required for acquisition of the capacity for apoptosis. Results support the idea that changes in amounts of BCL2 family members are important for the inhibition of apoptosis in the 2-cell embryo and in the establishment of the capacity for apoptosis later in development.

Effects of glucocorticoids and interleukin-1β on expression and activity of aggrecanases in equine chondrocytes

To determine effects of interleukin (IL)-1 beta and glucocorticoids on total glycosaminoglycan (GAG) loss and aggrecanase-mediated matrix degradation in equine cartilage. Cartilage from 24 equine cadavers free of sepsis and musculoskeletal disease. Effects of IL-1 beta, IL-1 beta with glucocorticoids (dexamethasone and triamcinolone, 10(-6) and 10(-7)M), and glucocorticoids alone on degradation of equine articular and nasal cartilage explants were assessed by measuring GAG release in media and GAG content in cartilage. Aggrecanase-mediated cleavage within the interglobular domain at Glu373-Ala374 was evaluated via western blot analysis and ELISAs. Steady-state mRNA concentrations of matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4, and ADAMTS5 were assessed by use of real-time reverse transcriptase PCR assay (cartilage explants) and northern blot analysis (cell culture). IL-1 beta increased GAG release and aggrecanase activity (11-fold). The MMP-3, MMP-13, and ADAMTS4 mRNA were upregulated with IL-1 beta, whereas ADAMTS5 mRNA was increased (13-fold), but significantly less than ADAMTS4 mRNA (27-fold), suggesting a role for both ADAMTS4 and ADAMTS5 in degradation of cytokine-stimulated cartilage. Despite downregulation of MMP-3 and MMP-13 mRNA, glucocorticoids did not alter GAG degradation. A further increase in aggrecanase activity was detected with ELISAs and western blot analysis, whereas ADAMTS4 mRNA was downregulated and ADAMTS5 mRNA was maintained or upregulated. MMP-3, MMP-13, and ADAMTS4 were regulated differently than ADAMTS5. Glucocorticoids increased aggrecanase activity despite down-regulation of ADAMTS4 mRNA, suggesting a major role of ADAMTS5. Effects of glucocorticoids on aggrecanase activity have important implications in terms of treatment.

Low doses of estradiol partly inhibit release of GH in sheep without affecting basal levels

Estradiol increases basal growth hormone (GH) concentrations in sheep and cattle. This study sought to determine the effects of estradiol on GH-releasing hormone (GRH)-stimulated GH release in sheep. Growth hormone secretory characteristics, the GH response to GRH, and steady-state GH mRNA concentrations were determined in castrated male lambs treated with 2 different doses of estradiol 17-β for a 28-d experimental period. Although no differences between treatments in mean GH, basal GH, or GH pulse number were observed after 28 d of estradiol treatment, GH pulse amplitude was greater ( P < 0.05) in the 2.00-cm implant-treated animals than in the control and 0.75-cm implant group. The effect of estradiol treatment on GRH-stimulated GH release revealed differences between the control and estradiol-treated animals ( P < 0.05). The 15-min GH responses to 0.075 μg/kg hGRH in the control, 0.75-cm, and 2.00-cm implant groups, respectively, were 76 ± 10, 22.6 ± 2.1, and 43.6 ± 15.0 ng/mL. Growth hormone mRNA content was determined for pituitary glands from the different treatment groups, and no differences in steady-state GH mRNA levels were observed. There were no differences in the mean plasma concentrations of IGF-I, cortisol, T 3, or T 4 from weekly samples. Growth hormone release from cultured ovine pituitary cells from control sheep was not affected by estradiol after 72 h or in a subsequent 3-h incubation with estradiol combined with GRH. These data suggest that estradiol has differing actions on basal and GRH-stimulated GH concentrations in plasma, but the increase in pulse amplitude does not represent an increased pituitary sensitivity to GRH.

Expression and Regulation of Functional Oxytocin Receptors in Bovine T Lymphocytes1

The corpus luteum (CL) produces oxytocin (OXT), which has been proposed to regulate the pulsatile release of prostaglandin F2alpha during luteolysis in ruminants. This action of OXT is mediated via oxytocin receptors (OXTRs) present on uterine epithelial cells. It is hypothesized that luteal OXT acts as a paracrine regulator of resident immune cells. In the present study, OXTR mRNA expression in bovine lymphocytes was analyzed, as well as its regulation during the estrous cycle. OXTR transcripts were observed in freshly purified bovine peripheral blood mononuclear cells and T lymphocytes. OXTR mRNA in bovine lymphocytes on Day 3 was numerically greater than but not significantly different from that of Day 19 of the estrous cycle (P=0.091). In cultured T cells, estradiol (E2) treatment significantly increased the steady-state concentrations of OXTR mRNA, but the stimulatory effect of E2 was inhibited by the addition of progesterone (P4). Each of the major T cell subsets (CD4+, CD8+, and gamma delta+) expressed OXTR mRNA, with no significant difference in expression among them. Western blot analyses demonstrated the presence of the bovine OXTR protein at about 45 kDa in lymphocytes, as well as expression of the 14-kDa precursor of OXT. When lymphocytes were treated with OXT, intracellular concentrations of calcium ([Ca2+]i) were rapidly and dramatically increased. This study demonstrated that bovine lymphocytes express OXTRs and that this expression can be regulated in a steroid-dependent manner. Furthermore, OXT elicited a functional [Ca2+]i response in T lymphocytes, supporting the possibility that OXT within the CL could act as a paracrine or autocrine regulator of resident T lymphocytes.

EXPRESSION OF mRNA FOR MEMBRANE PROGESTERONE RECEPTORS BY BOVINE T LYMPHOCYTES

It has been previously demonstrated in this laboratory that progesterone (P4) inhibits luteal cell-induced T lymphocyte proliferation in a dose-dependent manner, yet freshly purified lymphocytes did not express the nuclear P4 receptor. The effects of P4 on immune cell functions within the corpus luteum (CL), and the exact mechanisms by which P4 might exert such effects are not fully understood. However, recent reports described the presence of membrane receptors for P4 in different tissues. The objectives of the present study were to determine whether bovine lymphocytes express membrane progestin receptors (mPRs), to study the regulation of these receptors, and to analyze their expression in the different T lymphocyte subpopulations. Bovine peripheral blood mononuclear cells (PBMC) were isolated from whole blood of four animals at different stages (days 3, 11, and 19) of the estrous cycle. The T cells (TC) were separated from PBMC, and frozen for RNA or cultured for 72 hours in RPMI-1640 containing 10 % heat-inactivated fetal bovine serum, and treated with progesterone and/or 17beta-estradiol (E2). Total RNA was extracted from fresh TC along with cultured TC, and examined for the three distinct forms of mPR (mPR alpha, mPR beta, mPR gamma) and for progesterone receptor membrane component 1 (PGRMC1) mRNA expression by RT-qPCR. The mRNA of mPR alpha, mPR beta, mPR gamma, and PGRMC1 were observed in bovine T lymphocytes throughout the estrous cycle. Steady-state concentrations of mPR beta, mPR gamma, and PGRMC1 mRNA were not different (p>0.05) among D3, D11, and D19 of the estrous cycle. In contrast, expression of the mPR alpha subunit was significantly less on D19 TC as compared to D3 TC (p<0.05). When the mRNAs of the three receptors and PGRMC1 in lymphocytes were compared, mPR gamma mRNA was greater compared to PGRMC1 (p<0.05) while the mRNA concentrations of mPR alpha, mPR beta, and mPR gamma were all similar. Treatment with P4 or E2 did not affect mPRs or PGRMC1 mRNA expression although a slight decrease was observed when T lymphocytes were treated with the combination of P4 and E2. We found that all T lymphocyte subsets (CD4+, CD8+, and gamma-delta+) expressed mPR alpha, mPR beta, and mPR gamma mRNA. No significant differences were observed for all mPRs subunits and PGRMC1 transcripts between CD4+, CD8+, and gamma-delta+ T cells. However, the steady-state concentrations of mRNA were greater in purified T cell subsets as compared to PBMC. In conclusion, we found that bovine lymphocytes express membrane progestin receptors during the estrous cycle with a down-regulation in mPR alpha at the end of the cycle. These results suggest that P4 could modulate the activity and function of T lymphocytes through membrane receptors. Therefore, the high intracellular concentrations of P4 present in midcycle CL may exert immunosuppressive actions, preventing the activation and proliferation of T lymphocytes. This may explain why lymphocytes present within the fully functional CL do not elicit a proinflammatory response. This project was supported by National Research Initiative Competitive Grant no. 2004–35203–14789 from the USDA Cooperative State Research, Education, and Extension Service. (poster)

Differential Effects of n-3 and n-6 Fatty Acids on Prostaglandin F2α Production by Bovine Endometrial Cells

Recent studies have implicated n-3 polyunsaturated fatty acids in the reduction of eicosanoid production in the bovine uterus. The objective of this study was to determine whether the effect of eicosapentaenoic acid (EPA; C20:5, n-3) on PGF2α production by bovine endometrial (BEND) cells is influenced by the quantity of linoleic acid (C18:2, n-6) in the incubation medium. Confluent BEND cells were incubated in the absence (control) or presence of 100μMof EPA for 24h. After incubation, cells were rinsed and then stimulated with phorbol 12,13-dibutyrate (PDBu; 100 ng/mL) for 6h. Additional sets of culture dishes were treated with a combination of EPA and increasing n-6/n-3 fatty acid ratios for 24h and then challenged with PDBu for 6h. The PDBu stimulated PGF2α secretion and upregulated steady-state concentrations of prostaglandin endoperoxide synthase-2 and peroxisome proliferator-activated receptor delta mRNA within 6h. Preincubation of BEND cells with EPA for 24h decreased PGF2α response to phorbol ester, but had no detectable effects on prostaglandin endoperoxide synthase-2 or peroxisome proliferator-activated receptor delta mRNA abundance in PDBu-stimulated BEND cells. The inhibitory effect of EPA on PGF2α production was reverted in BEND cells treated with an increasing n-6-to-n-3 fatty acid ratio. Findings indicate that the net inhibition of endometrial PGF2α bioynthesis by n-3 fatty acids may vary depending on the ratio of n-6 to n-3 fatty acids in the uterus.

Immuno-electron microscopic quantification of the fucoxanthin chlorophyll a/c binding polypeptides Fcp2, Fcp4, and Fcp6 of Cyclotella cryptica grown under low- and high-light intensities.

The diatom Cyclotella cryptica was grown under low- and high-intensity white light of 50 and 500 micromol photons m-2 s-1, respectively. Western immunoblotting showed that the diatom adapted its light-harvesting apparatus, giving rise to different amounts of distinct fucoxanthin chlorophyll a/c binding polypeptides (Fcp). The amount of Fcp2 was approximately two-fold higher under low-light than under high-light conditions, whereas the amount of Fcp6 increased four- to five-fold under high-light conditions. For Fcp4, no significant differences were detected in response to either light regime. Cells of Cyclotella grown under high- and low-light intensity were subjected to immunoelectron microscopy. Quantification of the gold label, expressed as gold particles per microm2, confirmed the results obtained by Western immunoblotting. Exposure to low light resulted in the detection of approximately six times more Fcp2-bound gold particles per microm2 in thylakoid membranes, whereas in cells grown under high light the number of Fcp6-bound gold particles increased ten-fold. For Fcp4, similar amounts of gold particles per microm2 were counted under the two light regimes. These immunocytochemical results confirmed molecular data derived from phylogenetic analyses of the sequences of genes encoding fucoxanthin chlorophyll a/c binding polypeptides (fcp genes) and from measurements of steady-state fcp mRNA concentrations. The results show that Fcp2 and Fcp6 accumulate under low- and high-light intensity, respectively, whereas Fcp4 seems to be constitutively synthesized.

Endurance training modulates the muscular transcriptome response to acute exercise

We hypothesized that in untrained individuals (n=6) a single bout of ergometer endurance exercise provokes a concerted response of muscle transcripts towards a slow-oxidative muscle phenotype over a 24-h period. We further hypothesized this response during recovery to be attenuated after six weeks of endurance training. We monitored the expression profile of 220 selected transcripts in muscle biopsies before as well as 1, 8, and 24 h after a 30-min near-maximal bout of exercise. The generalized gene response of untrained vastus lateralis muscle peaked after 8 h of recovery (P=0.001). It involved multiple transcripts of oxidative metabolism and glycolysis. Angiogenic and cell regulatory transcripts were transiently reduced after 1 h independent of the training state. In the trained state, the induction of most transcripts 8 h after exercise was less pronounced despite a moderately higher relative exercise intensity, partially because of increased steady-state mRNA concentration, and the level of metabolic and extracellular RNAs was reduced during recovery from exercise. Our data suggest that the general response of the transcriptome for regulatory and metabolic processes is different in the trained state. Thus, the response is specifically modified with repeated bouts of endurance exercise during which muscle adjustments are established.

Changes in steady-state concentrations of messenger ribonucleic acids in luteal tissue during prostaglandin F2α induced luteolysis in mares

Transvaginal ultrasound-guided luteal biopsy was used to evaluate the effects of prostaglandin (PG)F2α on steady-state concentrations of mRNA for specific genes that may be involved in regression of the corpus luteum (CL). Eight days after ovulation (Hour 0), mares ( n = 8/group) were randomized into three groups: control (no treatment or biopsy), saline + biopsy (saline treatment at Hour 0 and luteal biopsy at Hour 12), or PGF2α + biopsy (5 mg PGF2α at Hour 0 and luteal biopsy at Hour 12). The effects of biopsy on CL were compared between the controls (no biopsy) and saline + biopsy group. At Hour 24 (12 h after biopsy) there was a decrease in circulating progesterone in saline group to 56% of pre-biopsy values, indicating an effect of biopsy on luteal function. Mean plasma progesterone concentrations were lower ( P < 0.001) at Hour 12 in the PG group compared to the other two groups. The relative concentrations of mRNA for different genes in luteal tissue at Hour 12 was quantified by real time PCR. Compared to saline-treated mares, treatment with PGF2α increased mRNA for cyclooxygenase-2 (Cox-2, 310%, P < 0.006), but decreased mRNA for LH receptor to 44% ( P < 0.05), steroidogenic acute regulatory protein to 22% ( P < 0.001), and aromatase to 43% ( P < 0.1) of controls. There was no difference in mRNA levels for PGF2α receptor between PG and saline-treated groups. Results indicated that luteal biopsy alters subsequent luteal function. However, the biopsy approach was effective for collecting CL tissue for demonstrating dynamic changes in steady-state levels of mRNAs during PGF2α-induced luteolysis. Increased Cox-2 mRNA concentrations suggested that exogenous PGF2α induced the synthesis of intraluteal PGF2α. Thus, the findings are consistent with the concept that an intraluteal autocrine loop augments the luteolytic effect of uterine PGF2α in mares.

Both RNase E and RNase III control the stability of sodB mRNA upon translational inhibition by the small regulatory RNA RyhB

Previous work has demonstrated that iron-dependent variations in the steady-state concentration and translatability of sodB mRNA are modulated by the small regulatory RNA RyhB, the RNA chaperone Hfq and RNase E. In agreement with the proposed role of RNase E, we found that the decay of sodB mRNA is retarded upon inactivation of RNase E in vivo, and that the enzyme cleaves within the sodB 5′-untranslated region (5′-UTR) in vitro, thereby removing the 5′ stem–loop structure that facilitates Hfq and ribosome binding. Moreover, RNase E cleavage can also occur at a cryptic site that becomes available upon sodB 5′-UTR/RyhB base pairing. We show that while playing an important role in facilitating the interaction of RyhB with sodB mRNA, Hfq is not tightly retained by the RyhB–sodB mRNA complex and can be released from it through interaction with other RNAs added in trans. Unlike turnover of sodB mRNA, RyhB decay in vivo is mainly dependent on RNase III, and its cleavage by RNase III in vitro is facilitated upon base pairing with the sodB 5′-UTR. These data are discussed in terms of a model, which accounts for the observed roles of RNase E and RNase III in sodB mRNA turnover.

Dietary trans octadecenoic acids upregulate the liver gene encoding Peroxisome Proliferator-Activated Receptor-α in transition dairy cows

Effects of feeding calcium salts of conjugated linoleic acid (CLA) or trans octadecenoic acids (trans 18:1) on lipid metabolism and hepatic contents of mRNA encoding carnitine palmitoyltransferase 1 (CPT1), microsomal triglyceride transfer protein (MTP) and peroxisome proliferator-activated receptor alpha (PPARalpha) were examined in 15 early post-partum Holstein cows. Dietary treatments were initiated at approximately 4 weeks prior to expected calving dates and continued for 7 weeks post partum. Treatments prepartum consisted of 1) a basal diet (Control), 2) basal diet+150 g/d of CLA mix (CLA), or 3) basal diet+150 g/d of trans 18:1 mix (TRANS). Intakes of calcium salts of CLA and trans 18:1 mixes were adjusted to 225 g/d during the 7-week postpartum treatment period. Blood samples were collected at weeks 1, 2 and 4 post partum and plasma was harvested immediately for subsequent hormone and metabolite assays. Concentrations of insulin, insulin-like growth factor-I (IGF-I), and leptin in blood did not vary among cows fed the three diets. Plasma nonesterified fatty acid (NEFA) concentrations decreased between weeks 1 and 4 of lactation and were lower in cows fed the diet supplemented with trans 18:1 than in those fed a control diet at week 2 post partum. Periparturient fat supplementation had no detectable effects on CPT1 mRNA content in the liver. Steady-state concentration of MTP mRNA in the liver was greater in the TRANS treatment group than in the control group at week 1 postpartum. Feeding trans 18:1 supplements to transition dairy cows upregulated hepatic PPARalpha mRNA content during the first month of lactation. Under the present experimental conditions, dietary CLA had minimal effects on plasma and hepatic lipid metabolite concentrations in early lactation Holstein cows. Results indicate that dietary trans fatty acids may affect liver lipid metabolism in post-partum dairy cows through alterations in PPARalpha gene expression.

Pregnancy and Bovine Somatotropin in Nonlactating Dairy Cows: II. Endometrial Gene Expression Related to Maintenance of Pregnancy

The objective was to evaluate the effects of pregnancy and bovine somatotropin (bST) on endometrial gene and protein expression related to maintenance of pregnancy in nonlactating dairy cows at d 17. In endometrial tissues, treatment with bST increased the steady state concentration of oxytocin receptor (OTR) mRNA; bST-treated cyclic (bST-C) cows had greater OTR mRNA than bST-treated pregnant (bST-P) cows. Estradiol receptor α (ERα) mRNA was reduced in bST-P cows compared with control P and C (no bST) cows. Western blotting revealed that pregnancy decreased the abundance of ERα protein, and bST stimulated an increase in ERα protein in C and P cows. Treatment with bST increased steady state concentrations of progesterone receptor (PR) mRNA. No differences were detected in steady state mRNA concentrations of prostaglandin H synthase-2 (PGHS-2), prostaglandin E synthase, and prostaglandin F synthase due to pregnancy or bST treatment. However, PGHS-2 protein was increased in response to pregnancy and bST treatment. Immunostaining indicated that P decreased ERα protein in luminal epithelium and increased PR protein in epithelial cells of the uterine glands. The PR protein response in the glands was less in bST-P cows than in P cows. In the stromal layer of the endometrium, bST decreased PR protein abundance in C and P cows. The PGHS-2 protein was localized exclusively in the luminal epithelium cells of endometrium and was increased in P cows. In conclusion, distinctly different mRNA and protein responses were detected between C and P cows related to prostaglandin biosynthesis, and bST-induced changes may potentially impact mechanisms associated with maintenance of pregnancy in nonlactating cows.

A single Argonaute protein mediates both transcriptional and posttranscriptional silencing in Schizosaccharomyces pombe.

The Schizosaccharomyces pombe genome encodes only one of each of the three major classes of proteins implicated in RNA silencing: Dicer (Dcr1), RNA-dependent RNA polymerase (RdRP; Rdp1), and Argonaute (Ago1). These three proteins are required for silencing at centromeres and for the initiation of transcriptionally silent heterochromatin at the mating-type locus. Here, we show that the introduction of a double-stranded RNA (dsRNA) hairpin corresponding to a green fluorescent protein (GFP) transgene triggers classical RNA interference (RNAi) in S. pombe. That is, GFP silencing triggered by dsRNA reflects a change in the steady-state concentration of GFP mRNA, but not in the rate of GFP transcription. RNAi in S. pombe requires dcr1, rdp1, and ago1, but does not require chp1, tas3, or swi6, genes required for transcriptional silencing. Thus, the RNAi machinery in S. pombe can direct both transcriptional and posttranscriptional silencing using a single Dicer, RdRP, and Argonaute protein. Our findings suggest that these three proteins fulfill a common biochemical function in distinct siRNA-directed silencing pathways.

Insulin-like growth factors-I and -II and insulin-like growth factor-binding protein-2 in dominant equine follicles during spring transition and the ovulatory season.

The period between seasonal anoestrus and cyclicity is characterized in many mares by cyclical growth and regression of large dominant follicles. The insulin-like growth factor (IGF) system plays a key role in follicular growth and regression; therefore, we hypothesized that changes in the IGF system and its binding proteins would modulate onset of cyclicity in mares. Ovaries were obtained from pony mares on the day after detection of an actively growing 30 mm transitional anovulatory follicle, and also at the second or third oestrus of the breeding season on the day after the preovulatory follicle reached 30 mm in diameter. Size of dominant follicles at the time of removal was similar in transition (32 +/- 0.8 mm) and at oestrus (34 +/- 0.6 mm). IGF-I mRNA was present in granulosa cells, with low thecal expression, whereas IGF-II mRNA was confined to the theca layer. Expression of IGF-I and -II mRNAs, and intrafollicular concentrations of oestradiol, were lower (P < 0.01; paired t test) in transitional anovulatory follicles than in preovulatory follicles. Messenger RNA encoding IGFBP-2 was present in both theca and granulosa layers. Steady-state concentrations of mRNA encoding IGFBP-2 mRNA increased (P < 0.001) in theca in preovulatory follicles. Intrafollicular concentrations of IGFBP-2 were higher (P < 0.001) in transitional than in preovulatory follicles. The similarity in circulating concentrations of IGF-I in transitional and cyclic mares, suggested that the somatotrophic axis is not involved in transition from anovulatory to ovulatory cycles. The results suggest that the increased expression of IGF-I and -II mRNAs in preovulatory follicles, along with the decrease in IGFBP-2 concentrations, could increase the bioavailability of intrafollicular IGF in large follicles during the breeding season, and support our hypothesis that intrafollicular IGF bioavailability must exceed a threshold level before ovulation can occur.

Expression profiles of the DAZ gene family in human testis with and without spermatogenic failure

Objective To determine the expression profiles of the DAZ gene family in men with and without spermatogenic failure. Design Prospective case study. Setting University-based reproductive clinics and genetics laboratory. Patient(s) Thirty-four infertile men presenting with azoospermia. Intervention(s) The mRNA transcript concentrations of the DAZ family genes (BOULE, DAZL, DAZ) and the housekeeping GAPDH gene in the testes of azoospermic men were examined by quantitative competitive–reverse transcription–polymerase chain reaction (QC-RT-PCR). The steady-state concentrations of mRNA encoding for each gene in each testicular sample were normalized by the amounts of GAPDH. Main outcome measure(s) Transcript ratios (gene/GAPDH) of BOULE, DAZL, and DAZ. Result(s) The transcript ratios for BOULE, DAZL, and DAZ were significantly decreased in tissues with spermatogenic failure (hypospermatogenesis, maturation arrest, and Sertoli cell–only). However, the ratios of BOULE/DAZL and DAZ/DAZL did not reveal any significant difference in all tissues. Three patients with DAZ deletion possess lower transcripts of BOULE and DAZL. Conclusion(s) All members of the DAZ gene family play important roles in human spermatogenesis. Decreased concentrations of DAZ family members in men with spermatogenic failure may be due to the secondary effect of germ cell loss, and transcriptional control of BOULE, DAZL, and DAZ are not altered in the various degrees of spermatogenic failure. Although the sample size is limited, no compensatory increase of DAZL or BOULE transcription was found in men with DAZ deletion.