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Without adequate care, acute ankle trauma can result in chronic joint instability. Use of a standardized protocol enhances the management of ankle sprains. In patients with grades I or II sprains, emphasis should be placed on accurate diagnosis, early use of RICE (rest, ice, compression and elevation), maintenance of range of motion and use of an ankle support. Sprains with complete tendon tears (grade III) may require surgical intervention. Although early motion and mobility are recommended, ligamentous strength does not return until months after an ankle sprain. [ABSTRACT FROM AUTHOR]

Nitrate is a major nutrient and osmoticum for plants. To deal with fluctuating nitrate availability in soils, plants store this nutrient in their vacuoles. Chloride channel a (CLCa), a 2NO3-/1H+ exchanger localized to the vacuole in Arabidopsis (Arabidopsis thaliana), ensures this storage process. CLCa belongs to the CLC family, which includes anion/proton exchangers and anion channels. A mutation in a glutamate residue conserved across CLC exchangers is likely responsible for the conversion of exchangers to channels. Here, we show that CLCa with a mutation in glutamate 203 (E203) behaves as an anion channel in its native membrane. We introduced the CLCaE203A point mutation to investigate its physiological importance into the Arabidopsis clca knockout mutant. These CLCaE203A mutants displayed a growth deficit linked to the disruption of water homeostasis. Additionally, CLCaE203A expression failed to complement the defect in nitrate accumulation of clca and favored higher N-assimilation at the vegetative stage. Further analyses at the post-flowering stages indicated that CLCaE203A expression results in an increase in N uptake allocation to seeds, leading to a higher nitrogen use efficiency compared to the wild-type. Altogether, these results point to the critical function of the CLCa exchanger on the vacuole for plant metabolism and development., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

SATB homeobox proteins are important regulators of developmental gene expression. Among the stem cell lineages that emerge during early embryonic development, trophoblast stem (TS) cells exhibit robust SATB expression. Both SATB1 and SATB2 act to maintain the trophoblast stem-state. However, the molecular mechanisms that regulate TS-specific Satb expression are not yet known. We identified Satb1 variant 2 as the predominant transcript in trophoblasts. Histone marks, and RNA polymerase II occupancy in TS cells indicated an active state of the promoter. A novel cis-regulatory region with active histone marks was identified ∼21 kbp upstream of the variant 2 promoter. CRISPR/Cas9 mediated disruption of this sequence decreased Satb1 expression in TS cells and chromosome conformation capture analysis confirmed looping of this distant regulatory region into the proximal promoter. Scanning position weight matrices across the enhancer predicted two ELF5 binding sites in close proximity to SATB1 sites, which were confirmed by chromatin immunoprecipitation. Knockdown of ELF5 downregulated Satb1 expression in TS cells and overexpression of ELF5 increased the enhancer-reporter activity. Interestingly, ELF5 interacts with SATB1 in TS cells, and the enhancer activity was upregulated following SATB overexpression. Our findings indicate that trophoblast-specific Satb1 expression is regulated by long-range chromatin looping of an enhancer that interacts with ELF5 and SATB proteins., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yu, Chakravarthi, Borosha, Dilower, Lee, Ratri, Starks, Fields, Wolfe, Faruque, Tuteja and Rumi.)

Kisspeptin (KP) and kisspeptin receptor (KPR) are essential for the onset of puberty, development of gonads, and maintenance of gonadal function in both males and females. Hypothalamic KPs and KPR display a high degree of sexual dimorphism in expression and function. KPs act on KPR in gonadotropin releasing hormone (GnRH) neurons and induce distinct patterns of GnRH secretion in males and females. GnRH acts on the anterior pituitary to secrete gonadotropins, which are required for steroidogenesis and gametogenesis in testes and ovaries. Gonadal steroid hormones in turn regulate the KP neurons. Gonadal hormones inhibit the KP neurons within the arcuate nucleus and generate pulsatile GnRH mediated gonadotropin (GPN) secretion in both sexes. However, the numbers of KP neurons in the anteroventral periventricular nucleus and preoptic area are greater in females, which release a large amount of KPs in response to a high estrogen level and induce the preovulatory GPN surge. In addition to the hypothalamus, KPs and KPR are also expressed in various extrahypothalamic tissues including the liver, pancreas, fat, and gonads. There is a remarkable difference in circulating KP levels between males and females. An increased level of KPs in females can be linked to increased numbers of KP neurons in female hypothalamus and more KP production in the ovaries and adipose tissues. Although the sexually dimorphic features are well characterized for hypothalamic KPs, very little is known about the extrahypothalamic KPs. This review article summarizes current knowledge regarding the sexual dimorphism in hypothalamic as well as extrahypothalamic KP and KPR system in primates and rodents.

Background: Despite significant progress in the field of implementation science (IS), current training programs are inadequate to meet the global need, especially in low-and middle-income countries (LMICs). Even when training opportunities exist, there is a "knowledge-practice gap," where implementation research findings are not useful to practitioners in a field designed to bridge that gap. This is a critical challenge in LMICs where complex public health issues must be addressed. This paper describes results from a formal assessment of learning needs, priority topics, and delivery methods for LMIC stakeholders., Methods: We first reviewed a sample of articles published recently in Implementation Science to identify IS stakeholders and assigned labels and definitions for groups with similar roles. We then employed a multi-step sampling approach and a random sampling strategy to recruit participants (n = 39) for a semi-structured interview that lasted 30-60 min. Stakeholders with inputs critical to developing training curricula were prioritized and selected for interviews. We created memos from audio-recorded interviews and used a deductively created codebook to conduct thematic analysis. We calculated kappa coefficients for each memo and used validation techniques to establish rigor including incorporating feedback from reviewers and member checking., Results: Participants included program managers, researchers, and physicians working in over 20 countries, primarily LMICs. The majority had over 10 years of implementation experience but fewer than 5 years of IS experience. Three main themes emerged from the data, pertaining to past experience with IS, future IS training needs, and contextual issues. Most respondents (even with formal training) described their IS knowledge as basic or minimal. Preferences for future training were heterogeneous, but findings suggest that curricula must encompass a broader set of competencies than just IS, include mentorship/apprenticeship, and center the LMIC context., Conclusion: While this work is the first systematic assessment of IS learning needs among LMIC stakeholders, findings reflect existing research in that current training opportunities may not meet the demand, trainings are too narrowly focused to meet the heterogeneous needs of stakeholders, and there is a need for a broader set of competencies that moves beyond only IS. Our research also demonstrates the timely and unique needs of developing appropriately scoped, accessible training and mentorship support within LMIC settings. Therefore, we propose the novel approach of intelligent swarming as a solution to help build IS capacity in LMICs through the lens of sustainability and equity., (© 2021. The Author(s).)

Gonadotropins are essential for regulating ovarian development, steroidogenesis, and gametogenesis. While follicle stimulating hormone (FSH) promotes the development of ovarian follicles, luteinizing hormone (LH) regulates preovulatory maturation of oocytes, ovulation, and formation of corpus luteum. Cognate receptors of FSH and LH are G-protein coupled receptors that predominantly signal through cAMP-dependent and cAMP-independent mechanisms that activate protein kinases. Subsequent vital steps in response to gonadotropins are mediated through activation or inhibition of transcription factors required for follicular gene expression. Estrogen receptors, classical ligand-activated transcriptional regulators, play crucial roles in regulating gonadotropin secretion from the hypothalamic-pituitary axis as well as gonadotropin function in the target organs. In this review, we discuss the role of estrogen receptor β (ERβ) regulating gonadotropin response during folliculogenesis. Ovarian follicles in Erβ knockout (Erβ KO ) mutant female mice and rats cannot develop beyond the antral state, lack oocyte maturation, and fail to ovulate. Theca cells (TCs) in ovarian follicles express LH receptor, whereas granulosa cells (GCs) express both FSH receptor (FSHR) and LH receptor (LHCGR). As oocytes do not express the gonadotropin receptors, the somatic cells play a crucial role during gonadotropin induced oocyte maturation. Somatic cells also express high levels of estrogen receptors; while TCs express ERα and are involved in steroidogenesis, GCs express ERβ and are involved in both steroidogenesis and folliculogenesis. GCs are the primary site of ERβ-regulated gene expression. We observed that a subset of gonadotropin-induced genes in GCs, which are essential for ovarian follicle development, oocyte maturation and ovulation, are dependent on ERβ. Thus, ERβ plays a vital role in regulating the gonadotropin responses in ovary.

Over the entire reproductive lifespan in mammals, a fixed number of primordial follicles serve as the source of mature oocytes. Uncontrolled and excessive activation of primordial follicles can lead to depletion of the ovarian reserve. We observed that disruption of estrogen receptor β (ESR2) signaling results in increased activation of primordial follicles in Esr2-null (Esr2-/-) rats. However, follicle assembly was unaffected, and the total number of follicles remained comparable between neonatal wild-type and Esr2-/- ovaries. While the activated follicle counts were increased in Esr2-/- ovary, the number of primordial follicles were markedly decreased. Excessive recruitment of primordial follicles led to premature ovarian senescence in Esr2-/- rats and was associated with reduced levels of serum AMH and estradiol. Disruption of ESR2 signaling through administration of a selective antagonist (PHTPP) increased the number of activated follicles in wildtype rats, whereas a selective agonist (DPN) decreased follicle activation. In contrast, primordial follicle activation was not increased in the absence of ESR1, indicating that the regulation of primordial follicle activation is ESR2 specific. Follicle activation was also increased in Esr2 mutants lacking the DNA binding domain, suggesting a role for the canonical transcriptional activation function. Both primordial and activated follicles express ESR2, suggesting a direct regulatory role for ESR2 within these follicles. We also detected that loss of ESR2 augmented the activation of AKT, ERK, and mTOR pathways. Our results indicate that the lack of ESR2 upregulated both granulosa and oocyte factors, which can facilitate AKT and mTOR activation in Esr2-/- ovaries leading to increased activation of primordial follicles., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Both US and global communities lag on key health indicators. There has been limited progress in building capacity to improve health beyond the healthcare field. Yet, communities also need to engage in health improvement initiatives. A substantial body of literature describes standards and core components for quality improvement (QI) approaches in clinical settings. This study aims to determine how communities in the USA, Australia, New Zealand and Canada use QI approaches for health and well-being improvement and how such approaches compare to those in clinical settings., Methods and Analysis: We developed a study protocol based on scoping review framework by Arksey and O'Malley, methodological advancements for scoping studies (Levac et al ) and other published protocols. We developed research questions in an iterative process and used the Population, Intervention, Comparison, Outcomes strategy to determine eligibility criteria. Electronic databases deemed appropriate (Web of Science, Scopus, and Proquest Health Management) will be searched for studies that meet inclusion criteria. References of included studies will be included when relevant. Two reviewers will independently screen all abstracts and full-text studies for inclusion. A third reviewer will adjudicate disagreements that arise. An instrument will be developed to extract data from included studies. Quantitative and qualitative results will be reported., Ethics and Dissemination: We developed this protocol to systematically conduct a scoping review of how US communities use QI approaches to address community health and well-being. Results will benefit multiple stakeholders by informing how to better support, design and evaluate community well-being improvement interventions. Results will be distributed through peer-reviewed journals, conferences, presentations and a public health graduate course., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Estrogen signaling plays an important role in the pathophysiology of prostatic hyperplasia. While signaling through estrogen receptor alpha (ESR1) increases proliferation of stromal cells, estrogen receptor beta (ESR2) plays an anti-proliferative and differentiating role in glandular epithelium. Disruption of ESR2 signaling resulted in prostatic glandular hyperplasia in the rat. To identify the ESR2-target genes, and the molecular mechanisms involved, we performed RNA-seq analyses in prostate glands of Esr2 knockout ( Esr2 -/-) and age matched wildtype rats. The raw data were analyzed using CLC genomics workbench. High quality RNA-seq reads were aligned to the Rattus norvegicus genome. Differentially expressed genes were identified based on an absolute fold change of 2 with p Value ≤0.05. Of the total 32,623 genes detected, 824 were differentially expressed in Esr2 -/- prostate glands, 550 downregulated and 274 upregulated. Pathway analyses identified altered expression of genes involved in epithelial proliferation and benign tumor formation.

Estrogens are traditionally considered to be female sex steroid hormones and most of the studies examining estrogen regulation of metabolic function in the liver have been conducted in females. However, the liver expresses high levels of estrogen receptor alpha (ESR1) in both males and females, which mediates the hepatic response to estrogens. In this data article, we investigated whether metabolic disorders in Esr1 knockout ( Esr1-/- ) male rats were linked with loss of transcriptional regulation by ESR1 in liver. To identify the ESR1 regulated genes in the mutant liver, RNA-sequencing was performed on liver RNAs purified from young male rats. The raw data were analyzed using the CLC Genomics Workbench and high-quality RNA-sequencing reads were aligned to the Rattus norvegicus genome. Transcriptome data obtained from Esr1-/- liver RNAs were compared to that of wild type rats. Based on an absolute fold change of 2 with a p -value ≤ 0.05, a total of 618 differentially expressed genes were identified in the Esr1-/- male liver. Pathway analyses demonstrated that the majority of differentially expressed genes are regulators of carbohydrate and lipid metabolism in the liver. These differentially expressed genes and their potential roles were further examined in a companion manuscript, "Disruption of ESR1 alters the expression of genes regulating hepatic lipid and carbohydrate metabolism in male rats" (Khristi et al., 2018).

Hypothalamic expression of Kiss1 plays an essential role in the onset of puberty, gonadal development, and ovulation. Estrogens regulate the expression of Kiss1 in the hypothalamus through estrogen receptor-α. Kiss1 is also expressed in the ovary, where its expression correlates with the onset of puberty and progression of the estrous cycle. To date, estrogen regulation of Kiss1 expression in the ovary has not been investigated. We recently observed that gonadotropin-induced Kiss1 expression was absent in Esr2-null rat ovaries even though Esr1 was present. Wild-type granulosa cells abundantly expressed Kiss1 and oocytes expressed the Kiss1 receptor. We characterized estrogen receptor-β (ESR2) regulation of Kiss1 expression in granulosa cells by identifying granulosa cell-specific transcript variants and potential regulatory regions. The Kiss1 promoter, an upstream enhancer, and a downstream enhancer all possessed conserved estrogen response elements (EREs) and showed active histone marks in gonadotropin-stimulated granulosa cells. The transcriptionally active Kiss1 promoter, as well as the enhancers, also revealed enrichment for ESR2 binding. Furthermore, activity of a Kiss1 promoter construct was induced after overexpression of ESR2 and was blocked upon mutation of an ERE within the promoter. Finally, pregnant mare serum gonadotropin and human chorionic gonadotropin administration induced phosphorylation of ESR2 and upregulated the AP-1 proteins FOSL2 and JUNB in granulosa cells. Activated MAPK ERK2 was associated with the ESR2 phosphorylation in granulosa cells, and AP-1 factors could synergistically activate the Kiss1 promoter activity. These gonadotropin-induced changes paralleled Kiss1 expression in granulosa cells. We conclude that gonadotropin-stimulated Kiss1 expression in granulosa cells is dependent on both the activation of ESR2 and the upregulation of AP-1.

RNA seq analyses were performed in granulosa cells (GCs) collected from gonadotropin treated ESR2 mutant rats. Data obtained from a null mutant with Esr2 exon 3 deletion (∆3) and another DNA binding domain (DBD) mutant with exon 4 deletion (∆4) were compared to that of wildtype (WT) rats. The raw data were analyzed using CLC genomics workbench. High quality RNA-sequencing reads were aligned to the Rattus norvegicus genome. Differentially expressed genes in ∆3 or ∆4 Esr2 -mutant GCs were identified based on the following criteria: FDR p-Value ≤0.05 and an absolute fold change of 2. Fewer differentially expressed genes were identified in ∆3 compared to the ∆4 mutant group. As both mutant groups demonstrated a common phenotype of ovulation failure, differentially expressed genes common to both in ∆3 and ∆4 mutant rats were emphasized and further analyzed in the companion article "ESR2 regulates granulosa cell genes essential for follicle maturation and ovulation" [1].

Estrogens are essential hormones for the regulation of fertility. Cellular responses to estrogens are mediated by estrogen receptor α (ESR1) and estrogen receptor β (ESR2). In mouse and rat models, disruption of Esr1 causes infertility in both males and females. However, the role of ESR2 in reproductive function remains undecided because of a wide variation in phenotypic observations among Esr2-mutant mouse strains. Regulatory pathways independent of ESR2 binding to its cognate DNA response element have also been implicated in ESR2 signaling. To clarify the regulatory roles of ESR2, we generated two mutant rat models: one with a null mutation (exon 3 deletion, Esr2ΔE3) and the other with an inframe deletion selectively disrupting the DNA binding domain (exon 4 deletion, Esr2ΔE4). In both models, we observed that ESR2-mutant males were fertile. ESR2-mutant females exhibited regular estrous cycles and could be inseminated by wild-type (WT) males but did not become pregnant or pseudopregnant. Esr2-mutant ovaries were small and differed from WT ovaries by their absence of corpora lutea, despite the presence of follicles at various stages of development. Esr2ΔE3- and Esr2ΔE4-mutant females exhibited attenuated preovulatory gonadotropin surges and did not ovulate in response to a gonadotropin regimen effective in WT rats. Similarities of reproductive deficits in Esr2ΔE3 and Esr2ΔE4 mutants suggest that DNA binding-dependent transcriptional function of ESR2 is critical for preovulatory follicle maturation and ovulation. Overall, the findings indicate that neuroendocrine and ovarian deficits are linked to infertility observed in Esr2-mutant rats., (Copyright © 2017 Endocrine Society.)

"Too much of a good thing" perfectly describes the dilemma that living organisms face with metals. The tight control of metal homeostasis in cells depends on the trafficking of metal transporters between membranes of different compartments. However, the mechanisms regulating the location of transport proteins are still largely unknown. Developing Arabidopsis thaliana seedlings require the natural resistance-associated macrophage proteins (NRAMP3 and NRAMP4) transporters to remobilize iron from seed vacuolar stores and thereby acquire photosynthetic competence. Here, we report that mutations in the pleckstrin homology (PH) domain-containing protein AtPH1 rescue the iron-deficient phenotype of nramp3nramp4 Our results indicate that AtPH1 binds phosphatidylinositol 3-phosphate (PI3P) in vivo and acts in the late endosome compartment. We further show that loss of AtPH1 function leads to the mislocalization of the metal uptake transporter NRAMP1 to the vacuole, providing a rationale for the reversion of nramp3nramp4 phenotypes. This work identifies a PH domain protein as a regulator of plant metal transporter localization, providing evidence that PH domain proteins may be effectors of PI3P for protein sorting., Competing Interests: The authors declare no conflict of interest.

The progesterone receptor (PGR) is a ligand-activated transcription factor with key roles in the regulation of female fertility. Much has been learned of the actions of PGR signaling through the use of pharmacologic inhibitors and genetic manipulation, using mouse mutagenesis. Characterization of rats with a null mutation at the Pgr locus has forced a reexamination of the role of progesterone in the regulation of the female reproductive cycle. We generated two Pgr mutant rat models, using genome editing. In both cases, deletions yielded a null mutation resulting from a nonsense frame-shift and the emergence of a stop codon. Similar to Pgr null mice, Pgr null rats were infertile because of deficits in sexual behavior, ovulation, and uterine endometrial differentiation. However, in contrast to the reported phenotype of female mice with disruptions in Pgr signaling, Pgr null female rats exhibit robust estrous cycles. Cyclic changes in vaginal cytology, uterine histology, serum hormone levels, and wheel running activity were evident in Pgr null female rats, similar to wild-type controls. Furthermore, exogenous progesterone treatment inhibited estrous cycles in wild-type female rats but not in Pgr-null female rats. As previously reported, pharmacologic antagonism supports a role for PGR signaling in the regulation of the ovulatory gonadotropin surge, a result at variance with experimentation using genetic ablation of PGR signaling. To conclude, our findings in the Pgr null rat challenge current assumptions and prompt a reevaluation of the hormonal control of reproductive cyclicity.

Autophagosomes arise in yeast and animals from the sealing of a cup-shaped double-membrane precursor, the phagophore. The concerted action of about 30 evolutionarily conserved autophagy related (ATG) proteins lies at the core of this process. However, the mechanisms allowing phagophore generation and its differentiation into a sealed autophagosome are still not clear in detail, and very little is known in plants. This is due in part to the scarcity of structurally informative, real-time imaging data of ATG proteins at the phagophore site. Among these, the ATG5 complex directs anchoring of ATG8 to the phagophore, an event required for membrane expansion. Detailed real-time and 3D imaging of ATG5, ATG8, and an ER marker at the expanding phagophore allowed us to propose a model for autophagosome formation in plants. This model implies tight connections of the growing phagophore with the outer face of the cortical endoplasmic reticulum and prompts new questions on the mechanism of autophagosome biogenesis.

Autophagosomes are the organelles responsible for macroautophagy and arise, in yeast and animals, from the sealing of a cup-shaped double-membrane precursor, the phagophore. How the phagophore is generated and grows into a sealed autophagosome is still not clear in detail, and unknown in plants. This is due, in part, to the scarcity of structurally informative, real-time imaging data of the required protein machinery at the phagophore formation site. Here we find that in intact living Arabidopsis tissue, autophagy-related protein ATG5, which is essential for autophagosome formation, is present at the phagophore site from early, sub-resolution stages and later defines a torus-shaped structure on a flat cisternal early phagophore. Movement and expansion of this structure are accompanied by the underlying endoplasmic reticulum, suggesting tight connections between the two compartments. Detailed real-time and 3D imaging of the growing phagophore are leveraged to propose a model for autophagosome formation in plants.

Estrogens play pivotal roles in development and function of many organ systems, including the reproductive system. We have generated estrogen receptor 1 (Esr1)-knockout rats using zinc finger nuclease (ZFN) genome targeting. mRNAs encoding ZFNs targeted to exon 3 of Esr1 were microinjected into single-cell rat embryos and transferred to pseudopregnant recipients. Of 17 live births, 5 had biallelic and 1 had monoallelic Esr1 mutations. A founder with monoallelic mutations was backcrossed to a wild-type rat. Offspring possessed only wild-type Esr1 alleles or wild-type alleles and Esr1 alleles containing either 482 bp (Δ482) or 223 bp (Δ223) deletions, indicating mosaicism in the founder. These heterozygous mutants were bred for colony expansion, generation of homozygous mutants, and phenotypic characterization. The Δ482 Esr1 allele yielded altered transcript processing, including the absence of exon 3, aberrant splicing of exon 2 and 4, and a frameshift that generated premature stop codons located immediately after the codon for Thr157. ESR1 protein was not detected in homozygous Δ482 mutant uteri. ESR1 disruption affected sexually dimorphic postnatal growth patterns and serum levels of gonadotropins and sex steroid hormones. Both male and female Esr1-null rats were infertile. Esr1-null males had small testes with distended and dysplastic seminiferous tubules, whereas Esr1-null females possessed large polycystic ovaries, thread-like uteri, and poorly developed mammary glands. In addition, uteri of Esr1-null rats did not effectively respond to 17β-estradiol treatment, further demonstrating that the Δ482 Esr1 mutation created a null allele. This rat model provides a new experimental tool for investigating the pathophysiology of estrogen action.

The intricate molecular mechanisms that regulate ESC pluripotency are incompletely understood. Prior research indicated that activation of the Janus kinase-signal transducer and activator of transcription (STAT3) pathway or inhibition of extracellular signal-regulated kinase/glycogen synthase kinase 3 (ERK/GSK3) signaling maintains mouse ESC (mESC) pluripotency. Here, we demonstrate that inhibition of protein kinase C (PKC) isoforms maintains mESC pluripotency without the activation of STAT3 or inhibition of ERK/GSK3 signaling pathways. Our analyses revealed that the atypical PKC isoform, PKCζ plays an important role in inducing lineage commitment in mESCs through a PKCζ-nuclear factor kappa-light-chain-enhancer of activated B cells signaling axis. Furthermore, inhibition of PKC isoforms permits derivation of germline-competent ESCs from mouse blastocysts and also facilitates reprogramming of mouse embryonic fibroblasts toward induced pluripotent stem cells. Our results indicate that PKC signaling is critical to balancing ESC self-renewal and lineage commitment., (Copyright © 2011 AlphaMed Press.)

Tudor-SN (TSN) copurifies with the RNA-induced silencing complex in animal cells where, among other functions, it is thought to act on mRNA stability via the degradation of specific dsRNA templates. In plants, TSN has been identified biochemically as a cytoskeleton-associated RNA binding activity. In eukaryotes, it has recently been identified as a conserved primary target of programmed cell death-associated proteolysis. We have investigated the physiological role of TSN by isolating null mutations for two homologous genes in Arabidopsis thaliana. The double mutant tsn1 tsn2 displays only mild growth phenotypes under nonstress conditions, but germination, growth, and survival are severely affected under high salinity stress. Either TSN1 or TSN2 alone can complement the double mutant, indicating their functional redundancy. TSN accumulates heterogeneously in the cytosol and relocates transiently to a diffuse pattern in response to salt stress. Unexpectedly, stress-regulated mRNAs encoding secreted proteins are significantly enriched among the transcripts that are underrepresented in tsn1 tsn2. Our data also reveal that TSN is important for RNA stability of its targets. These findings show that TSN is essential for stress tolerance in plants and implicate TSN in new, potentially conserved mechanisms acting on mRNAs entering the secretory pathway.

The hormone adiponectin has been shown to be important in maintaining insulin sensitivity throughout the body, whereas potential effects on the placenta have not been assessed. Pregnancy constitutes a unique physiological environment in which metabolism has a profound effect on the health of both the mother and the developing fetus. It is imperative that a delicate balance in glucose delivery be maintained between maternal tissues and the fetal/placental unit. Adiponectin's role in regulating peripheral insulin responsiveness suggests it may be a factor in maintaining this balance during gestation as well. Examination of human cytotrophoblast cells revealed that mRNA for both adiponectin receptors, adipoR1 and adipoR2, are abundantly expressed at term. We were, however, unable to reliably detect mRNA for adiponectin in primary cytotrophoblasts. Expression of both receptors was maintained after induction of syncytium formation by exogenous epidermal growth factor treatment. Treatment of cytotrophoblasts with adiponectin resulted in a significant drop, as assessed by quantitative RT-PCR, in expression for a number of genes involved in the endocrine function of the placenta, including the chorionic gonadotropin subunits, placental lactogen, and some steroidogenic enzymes. Immunofluorescent staining for connexin 43 and desmoplakin in primary trophoblasts revealed that adiponectin does not inhibit syncytialization of trophoblast cells in culture. Taken together, these data describe a novel role for maternal adiponectin in regulating the placental environment. Determination of the effects of such adipokines on the maternal-fetal interface is increasingly important, because the incidence of pregnancies complicated by gestational diabetes remains a significant health problem in developed countries.

Objective: Spontaneous differentiation of human embryonic stem cell (hESC) cultures is a major concern in stem cell research. Physical removal of differentiated areas in a stem cell colony is the current approach used to keep the cultures in a pluripotent state for a prolonged period of time. All hESCs available for research require unidentified soluble factors secreted from feeder layers to maintain the undifferentiated state and pluripotency. Under experimental conditions, stem cells are grown on various matrices, the most commonly used being Matrigel., Materials and Methods: We propose an alternative method to prevent spontaneous differentiation of hESCs grown on Matrigel that uses low amounts of recombinant noggin. We make use of the porosity of Matrigel to serve as a matrix that traps noggin and gradually releases it into the culture to antagonize bone morphogenetic proteins (BMP). BMPs are known to initiate differentiation of hESCs and are either present in the conditioned medium or are secreted by hESCs themselves., Results: hESCs grown on Matrigel supplemented with noggin in conditioned medium from feeder layers (irradiated mouse embryonic fibroblasts) retained both normal karyotype and markers of hESC pluripotency for 14 days. In addition, these cultures were found to have increased cell proliferation of stem cells as compared to hESCs grown on Matrigel alone., Conclusion: Noggin can be utilized for short term prevention of spontaneous differentiation of stem cells grown on Matrigel.

Our previous studies demonstrate that GnRH-induced ERK activation required influx of extracellular Ca2+ in alphaT3-1 and rat pituitary cells. In the present studies, we examined the hypothesis that calmodulin (Cam) plays a fundamental role in mediating the effects of Ca2+ on ERK activation. Cam inhibition using W7 was sufficient to block GnRH-induced reporter gene activity for the c-Fos, murine glycoprotein hormone alpha-subunit, and MAPK phosphatase (MKP)-2 promoters, all shown to require ERK activation. Inhibition of Cam (using a dominant negative) was sufficient to block GnRH-induced ERK but not c-Jun N-terminal kinase activity activation. The Cam-dependent protein kinase (CamK) II inhibitor KN62 did not recapitulate these findings. GnRH-induced phosphorylation of MAPK/ERK kinase 1 and c-Raf kinase was blocked by Cam inhibition, whereas activity of phospholipase C was unaffected, suggesting that Ca2+/Cam modulation of the ERK cascade potentially at the level of c-Raf kinase. Enrichment of Cam-interacting proteins using a Cam agarose column revealed that c-Raf kinase forms a complex with Cam. Reconstitution studies reveal that recombinant c-Raf kinase can associate directly with Cam in a Ca2+-dependent manner and this interaction is reduced in vitro by addition of W7. Cam was localized in lipid rafts consistent with the formation of a Ca2+-sensitive signaling platform including the GnRH receptor and c-Raf kinase. These data support the conclusion that Cam may have a critical role as a Ca2+ sensor in specifically linking Ca2+ flux with ERK activation within the GnRH signaling pathway.

Early studies of glycogen synthase kinase 3 (GSK-3) in mammalian systems focused on its pivotal role in glycogen metabolism and insulin-mediated signaling. It is now recognized that GSK-3 is central to a number of diverse signaling systems. Here, we show that the major form of the kinase Shaggy (Sgg), the GSK-3 fly ortholog, is negatively regulated during insulin-like/phosphatidylinositol 3-kinase (PI3K) signaling in vivo. Since genetic studies of Drosophila melanogaster had previously shown that Wingless (Wg) signaling also acts to antagonize Sgg, we investigate how the kinase might integrate, or else discriminate, signaling inputs by Wg and insulin. Using Drosophila cell line assays, we found, in contrast to previous reports, that Wg induces accumulation of its transducer Armadillo (Arm)/beta-catenin without significant alteration of global Sgg-specific activity. In agreement with a previous study using human GSK-3beta, Wg did not cause phosphorylation changes of the Ser9 or Tyr214 regulatory phosphorylated sites of Sgg. Conversely, as shown in mammalian systems, insulin-induced inhibition of Sgg-specific activity by phosphorylation at the N-terminal pseudosubstrate site (Ser9) did not induce Arm/beta-catenin accumulation, showing selectivity in response to the different signaling pathways. Interestingly, a minigene bearing a Ser9-to-Ala change rescued mutant sgg without causing abnormal development, suggesting that the regulation of Sgg via the inhibitory pseudosubstrate domain is dispensable for many aspects of its function. Our studies of Drosophila show that Wg and insulin or PI3K pathways do not converge on Sgg but that they exhibit cross-regulatory interactions.

Equids and primates are the only species known to express the placental hormone chorionic gonadotropin (CG). CG is a member of the heterodimeric glycoprotein family and is composed of an alpha subunit linked to a hormone-specific beta subunit. Previously, we have reported that epidermal growth factor (EGF) regulates the equine glycoprotein hormone alpha subunit promoter through a protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) signal transduction pathway in trophoblasts. The current study investigates the regulatory element/factors involved in the induction of equine glycoprotein alpha subunit gene expression by EGF. Using 5' deletion mutagenesis, we have delineated the primary EGF/PKC responsive region of the equine alpha subunit gene to be located between -2039 to -2032 base pairs upstream of the transcriptional start site. The sequence within this region contains an activator protein 1 (AP-1)-like response element (TGAATCA) and is similar to a consensus AP-1 (TGAC/GTCA) response element. This element appeared to preferentially interact with a c-fos/JunD heterodimer. Stimulation by EGF induced the binding of c-fos and JunD to this element and subsequently elevated promoter activity. In conclusion, an EGF/PKC/MAPK signal transduction pathway regulates equine glycoprotein alpha subunit gene expression through a distinct regulatory element(s) that lies between -2039 to -2032 of the equine glycoprotein alpha subunit promoter in trophoblasts and involves an AP-1 complex.

In pituitary gonadotropes, gonadotropin-releasing hormone (GnRH) activates all three major mitogen-activated protein kinase (MAPK) cascades. The MAPKs play key roles in transcriptional activation of GnRH-responsive genes. MAPK phosphatases (MKPs) are dual specificity protein phosphatases involved in feedback regulation of MAPK activity. Previous studies indicate that GnRH activates MKP-2 expression in gonadotropes, dependent upon activation of multiple MAPKs and discrete Ca(2+) signals. To further understand the transcriptional mechanism(s) of MKP-2 induction by GnRH, we studied the activity of a 198-nucleotide MKP-2 proximal promoter region that supports GnRH responsiveness in reporter gene assays. Functional analysis of the MKP-2 promoter confirmed a requirement for the protein kinase C-extracellular signal-regulated kinase (ERK) pathway and VGCC-derived Ca(2+) signals in transcriptional activation of the MKP-2 gene. However, the inhibitory effect of thapsigargin on MKP-2 protein expression previously identified was not mediated at the level of promoter activation, suggesting a distinct mechanism for the action of thapsigargin-sensitive Ca(2+) signals. MGRE (MKP-2 GnRH response element) within the MKP-2 promoter mediated promoter activation through the protein kinase C-ERK pathway. The zinc finger transcription factor Egr-1 was identified in the MGRE-binding complex. Egr-1/MGRE binding was induced by GnRH in an ERK-dependent manner. Transcriptional activity of Egr-1 protein was enhanced by GnRH treatment. In addition, overexpression of the Egr-interacting protein, NAB1, resulted in increased GnRH-stimulated MKP-2 gene transcription. Consistent with the putative role of Egr-1 in MKP-2 promoter regulation, Egr-1 protein expression closely correlated with the expression of MKP-2 protein in alpha T3-1 cells. Together, these data suggest that Egr-1 may be a key factor in mediating GnRH-dependent transcriptional activation of the MKP-2 gene.

Primates and equids are the only species known to express the placental glycoprotein hormone, chorionic gonadotropin (CG), a heterodimeric glycoprotein composed of an alpha subunit linked to a hormone-specific beta subunit. The regulatory mechanisms involved in the induction of equine glycoprotein alpha subunit gene expression have not been identified. Epidermal growth factor (EGF) receptor is known to transduce signals that alter a number of different cellular functions (cell proliferation, differentiation, hormone secretion, and gene regulation). In the present study, we investigated the regulation of the equine alpha subunit gene by EGF in trophoblasts. We found that 2800 base pairs of 5' flanking sequence from the equine alpha subunit promoter is sufficient for basal expression in human choriocarcinoma cells. Epidermal growth factor and phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), increased transcriptional activity of the equine alpha subunit promoter (-2800/+21). These responses were blocked by pretreatment with bisindolylmaleimide-I, an inhibitor of PKC, suggesting an involvement of this pathway downstream of EGF. In addition, PD98059, an inhibitor of the extracellular signal-regulated kinase (ERK) pathway, completely blocked activation of the equine alpha promoter by PMA, suggesting that mitogen-activated protein kinase (MAPK) cascade was involved downstream of the PKC pathway. In conclusion, the EGF/PKC/MAPK pathway regulates equine glycoprotein alpha subunit gene expression through a distinct regulatory region (-2300 to -1900) in trophoblasts, while essential elements for basal expression appear to exist within the -2800 to -1900 region of the promoter.

PRL-like protein C variant (PLP-Cv) is a newly identified member of the PRL family. PLP-Cv is specifically expressed in the chorioallantoic placenta by two distinct cell populations: trophoblast giant cells and spongiotrophoblast cells. To gain some insight regarding the control of PLP-Cv gene expression and the regulatory factors controlling trophoblast giant cell and spongiotrophoblast cell lineages, we have initiated a structural and functional analysis of the PLP-Cv promoter. The activities of a series of PLP-Cv promoter constructs, ranging in size from 4.5 kb to 50 bp, ligated to a luciferase reporter have been assessed in the Rcho-1 trophoblast cell line (restricted to trophoblast giant cell differentiation) and in a primary spongiotrophoblast cell culture system after transient transfection. PLP-Cv promoter constructs containing 4.5 kb to 149 bp of 5'-flanking DNA possessed full activity in the trophoblast giant cell model. A region located between -149 and -124 bp upstream of the PLP-Cv transcription start site was found to be essential for activation of the PLP-Cv promoter. Spongiotrophoblast cells required additional PLP-Cv 5'-flanking DNA for full activity. A region located between -2518 and -2242 bp upstream of the PLP-Cv transcription start site significantly enhanced PLP-Cv promoter in spongiotrophoblast cells. In conclusion, mechanisms underlying the activation of the PLP-Cv promoter are different in trophoblast giant cells vs. spongiotrophoblast cells.

GnRH regulation of LH secretion is well understood and involves Ca(2+) mobilization. However, the mechanism by which GnRH activates transcription of the LHbeta gene is controversial. GnRH is known to elevate intracellular calcium and activate the protein kinase C (PKC) pathway. The present study evaluated the pathway(s) involved in GnRH induction of LHbeta transcription. We have previously reported that the equine LHbeta (eLHbeta -448/+60) promoter is active in alphaT3-1 cells. Therefore, we created a clonal, stably transfected alphaT3-1 gonadotroph cell line harboring the eLHbeta promoter (-448/+60) fused to the luciferase reporter gene. Administration of a GnRH agonist resulted in induction of promoter activity that was completely inhibited by the antagonist antide. Various calcium-affecting drugs had no effect on the promoter. Administration of phorbol 12-myristate 13-acetate (PMA) elicited an activation similar to, albeit lower than, that with GnRH. Down-regulation or pharmacological inhibition of PKC completely blocked PMA's induction of the promoter, while GnRH induction was only partly attenuated. Treatment with the mitogen-activated protein kinase (MAPK) kinase inhibitor, PD98059, completely inhibited the activation of eLHbeta by PMA but only partly diminished GnRH's induction. Expression of the transcription factor, early growth response protein 1 (Egr1), correlated completely with activation of MAPK, suggesting that Egr1 is the factor through which PKC/MAPK acts. Our data suggest that GnRH induces activity of the eLHbeta promoter by activating a signal transduction cascade involving PKC-MAPK-Egr1 but that has no significant requirement for calcium.

The requirements for basal expression of the LH beta-subunit promoter in pituitary gonadotropes are largely unknown. We have used the equine (e) LHbeta subunit promoter as a model to unravel the combinatorial code required for gonadotrope expression. Through the use of 5'-deletion mutagenesis, a region between -185 and -100 of the eLHbeta promoter was shown to play a critical role in maintaining basal promoter activity in alphaT3-1 and LbetaT2 cells. This region encompasses the steroidogenic factor-1 (SF-1) binding site that has been reported to have a functional role in expression of the LHbeta promoter in other species. We have also identified an additional SF-1 site at -55 to -48. Binding of SF-1 to both sites was confirmed by electrophoretic mobility shift assays. Mutations within these sites, either individually or in combination, did not attenuate basal activity of the eLHbeta promoter in alphaT3-1 cells, but did diminish promoter activity in LbetaT2 cells. Interestingly, cotransfection with an expression vector encoding SF-1 induced eLHbeta promoter activity, and this induction was abrogated by mutations within the SF-1 sites in alphaT3-1 cells. Block replacement mutagenesis was performed on the -185/-100 region of the eLHbeta promoter to identify DNA response elements responsible for maintaining basal promoter activity. From this analysis, two regions emerged as being important: a distal 31-bp segment (-181 to -150) and an element located immediately 3' to the distal SF-1 site (-119 to -106). It is hypothesized that these two regions as well as the SF-1 sites represent regulatory elements that contribute to a combinatorial code involved in targeting expression of the eLHbeta promoter to gonadotropes.

The hypothalamic neuropeptide, GnRH, regulates the synthesis and secretion of LH from pituitary gonadotropes. Furthermore, it has been shown that the LH beta-subunit gene is regulated by the transcription factors steroidogenic factor-1 (SF-1) and early growth response protein 1 (Egr1) in vitro and in vivo. The present study investigated the roles played by Egr1 and SF-1 in regulating activity of the equine LH beta-subunit promoter in the gonadotrope cell line, alpha T3-1, and the importance of these factors and cis-acting elements in regulation of the promoter by GnRH. All four members of the Egr family were found to induce activity of the equine promoter. The region responsible for induction by Egr was localized to the proximal 185 bp of the promoter, which contained two Egr response elements. Coexpression of Egr1 and SF-1 led to a synergistic activation of the equine (e)LH beta promoter. Mutation of any of the Egr or SF-1 response elements attenuated this synergism. Endogenous expression of Egr1 in alpha T3-1 cells was not detectable under basal conditions, but was rapidly induced after GnRH stimulation. Reexamination of the promoter constructs harboring mutant Egr or SF-1 sites indicated that these sites were required for GnRH induction. In fact, mutation of both Egr sites within the eLH beta promoter completely attenuated its induction by GnRH. Thus, GnRH induces expression of Egr1, which subsequently activates the eLH beta promoter. Finally, GnRH not only induced expression of Egr1, but also its corepressor, NGFI-A (Egr1) binding protein (Nab1), which can repress Egr1- induced transcription of the eLH beta promoter.

The equine glycoprotein hormone alpha-subunit gene is expressed in both pituitary and placenta, unlike that of all other nonprimate mammals studied, in which expression is limited to pituitary. Previous studies of the 5'-flanking region of the equine alpha-subunit promoter have revealed unique characteristics as well as similarities with the human alpha-subunit promoter, which demonstrates a similar pattern of tissue-specific expression. We have cloned and sequenced the equine alpha-subunit gene and have used tissue culture systems and transgenic mice to characterize its expression. Unlike the human promoter, the cloned equine alpha-subunit promoter failed to direct trophoblast-specific expression in either tissue culture or transgenic mouse models, suggesting an entirely different mechanism for expression. In contrast, the equine alpha-subunit promoter was able to direct gonadotroph expression in both tissue culture and transgenic mouse models. In alphaT3-1 cells, 550 base pair (bp) was sufficient for expression. This expression involves promoter elements identified in other species as playing a role in gonadotroph expression, but mutation of these elements reveals differences in their relative contributions to promoter activity. In mice, 2800 bp of 5'-flanking sequence allowed specific expression in gonadotrophs but not in thyrotrophs or placenta. The pattern of estrogen regulation observed in transgenic mice matched neither the repression that has been observed with human and bovine alpha-subunit promoters in transgenic mice nor the stimulation in mRNA levels reported in mares, suggesting a unique mechanism that is not recapitulated in the transgenic model. Thus the equine alpha-subunit promoter uses a combination of conserved and unique features of gene regulation to direct its pattern of tissue-specific expression.

Background: Medical consumers are increasingly requesting methods to discriminate among the results of different providers. Standards for appropriate modeling, risk adjustment, and evaluation ("scorecarding") in this setting are not well developed, although such evaluation is being performed by the medical insurance industry and by several states in the United States. Our objectives were to develop and examine clinically meaningful methodology for assessing the operator-specific results for percutaneous coronary revascularization., Methods and Results: From a multicenter database of patients treated since January 1, 1990, we used training and validation samples (n = 4860) to develop several models for risk adjustment and applied them to 38 providers performing 25 to 523 procedures in the database. Models were developed using multivariable logistic regression techniques for combinations of the end points of death, myocardial infarction, bypass surgery, and procedural success. Models were evaluated for predictive accuracy by using receiver operating characteristic (ROC) analysis, for the capacity to discriminate between superior and inferior provider outcomes, and for subjectivity and concordance. Major complications occurred in 3.6% of patients. The area under the ROC curve (with perfect discriminatory accuracy, area = 1.0; with no apparent accuracy, area = 0.5) in the validation sample, and frequency of identification of operators with outcomes outside the 95% CI for the outcome in question for the models were for death, 0.85 and 7.9%; for death, Q-wave infarction, and bypass surgery, 0.77 and 13.2%; for death, all infarction, and bypass surgery, 0.66 and 10.5%; and for procedural success, 0.76 and 23.7%. For the models as a group, identification of outliers was inversely related to provider volume (P = .05). Models evaluating non-Q-wave infarction or requiring measurement of percent diameter stenosis were identified as being most susceptible to provider manipulation., Conclusions: For percutaneous coronary revascularization, modeling to discriminate between provider outcomes is limited by the low incidence of major adverse events, subjectivity or susceptibility to manipulation of more frequently occurring adverse events, the generally modest predictive capacity of the models, and the low volume of individual provider treatments. Modeling will be most useful in the identification of providers with extremely poor outcomes and for discrimination between providers with very large procedural volume. Until improved understanding of the biological and mechanical correlates of major complications allows the development of more predictive models, interpretation of the results of scorecarding, particularly for low-volume providers, should be made with caution.

Background: Although several studies have established that the complications of percutaneous transluminal coronary angioplasty (PTCA) are related to clinical and angiographic variables such as advanced age and lesion complexity, it is uncertain whether the use of hospital resources after PTCA also depends on the same baseline variables. The purpose of this study was to identify the factors responsible for prolonged hospital stay after PTCA., Methods and Results: The study cohort included 591 consecutive patients undergoing conventional balloon angioplasty at nine medical centers in North America. Major or minor complications occurred in 91 patients (15.4%) and were observed to be related to several baseline characteristics, including unstable angina, multivessel coronary artery disease, patient age, and lesion complexity. Compared with a median length of hospital stay of 2.0 days after PTCA (25th, 75th percentiles: 2.0, 4.0) for the entire cohort of patients, the length of stay was increased in patients with unstable angina (3.0 days [2.0, 5.0]; P = .002), multivessel coronary artery disease (3.0 [2.0, 5.5]; P = .001), age > 65 years (3.0 [2.0, 5.5]; P = .02), complex lesions (3.0 [2.0, 6.0]; P = .001), and filling defects (6.0 [2.0, 11.0]; P < .001). The length of stay was more strikingly increased, however, in patients who experienced major or minor PTCA complications, such as emergency bypass surgery (9.0 days [8.0, 18.0]; P < .001), Q-wave or non-Q-wave myocardial infarction (8.0 [6.0, 15.5]; P < .001), transfusion unrelated to bypass surgery (8.0 [4.0, 12.0]; P < .001), or abrupt vessel closure (6.0 [3.0, 10.5]; P < .001). On stepwise multiple linear regression, PTCA complications appeared to be the strongest predictors of length of hospital stay (all P < .001) and overwhelmed the weaker relation between length of stay and several individual baseline variables. Inclusion of a composite clinical risk score (reflecting the presence of unstable angina, multivessel disease, advanced age, complex lesions, or filling defects) in the regression model confirmed that patients with several high-risk baseline variables had a significant increase in length of stay after PTCA (P = .003), but PTCA complications remained the strongest predictors of length of stay., Conclusions: Although PTCA complications were correlated with baseline variables such as unstable angina, multivessel disease, advanced age, complex lesions, and filling defects, excess length of stay after PTCA was most strongly influenced by the development of minor and major PTCA complications. Because patients with several baseline risk factors experienced significantly prolonged hospitalizations, improved selection of patients may contribute to reductions in length of stay after PTCA. A greater reduction in resource use after PTCA, however, would be expected from developing new treatments to decrease PTCA complications rather than limiting the access of patients with unstable angina, advanced age, or complex lesions to PTCA.

The hypothesis tested was that 17 beta-estradiol (E2) would increase amounts of mRNA for alpha, LH beta, and FSH beta subunits during the follicular phase of the estrous cycle prior to the preovulatory surge of gonadotropins in cows. On Day 16 (Day 0 = estrus) of the estrous cycle, all cows were treated with prostaglandin F2 alpha (PGF2 alpha). Cows served as intact controls (CONT, n = 4) were ovariectomized (OVX, n = 5), or were ovariectomized and administered E2 (OVXE, n = 6) in increasing doses starting at the time of treatment with PGF2 alpha. Cows were bled for 6 h before and for 40 h after PGF2 alpha treatment to characterize pulsatile secretion of LH and FSH. Forty hours after PGF2 alpha treatment, pituitaries were collected for evaluation of amounts of mRNA for alpha, LH beta, and FSH beta subunits. Amplitude of LH pulses was greater (p < 0.05) in cows from the OVXE than from the CONT group. Concentrations of FSH were greater in cows from both the OVXE and OVX (p < 0.01) groups than from the CONT group. Amounts of mRNA for alpha and FSH beta subunits were greater (p < 0.01) in pituitaries of cows from the OVX than from the CONT or OVXE groups. Amounts of mRNA for LH beta subunit in pituitaries of cows from the OVX group tended to be greater (p < 0.08) than from the CONT group. Cows in the OVXE group tended (p < 0.08) to have greater amounts of mRNA for FSH beta subunit than did CONT cows.(ABSTRACT TRUNCATED AT 250 WORDS)

The objective of this study was to examine the influence of 17 beta-estradiol (E2) on distribution of LH and FSH isoforms during the follicular phase of the bovine estrous cycle prior to the preovulatory surges of LH and FSH. On Day 16 of the estrous cycle (Day 0 = estrus), intact controls (CONT; n = 4) were treated with prostaglandin F2 alpha (PGF2 alpha) to induce luteal regression and initiation of the follicular phase. Other cows were also treated with PGF2 alpha and either ovariectomized (OVX; n = 5) or ovariectomized and given E2 implants (OVXE; n = 6) to mimic the pattern of increasing E2 concentrations during the follicular phase of the estrous cycle. Pituitaries were collected 40 h after treatment with PGF 2 alpha or ovariectomy (0 h). Aliquots of pituitary extracts were chromatofocused on pH 10.5-4.0 gradients. The LH resolved into thirteen isoforms (designated A-L and S, beginning with the most basic form) while FSH resolved into nine isoforms (designated I-IX, beginning with the most basic form). The percentage of LH as isoform F (elution pH = 9.32 +/- 0.01) was greater (p < 0.05) in the OVX group (48.5%) than in the OVXE group (45.0%). LH isoforms I (elution pH = 6.98 +/- 0.01) and J (elution pH = 6.48 +/- 0.01) were more abundant (p < 0.05) in cows from the OVXE (2.3 and 5.8%, respectively) than the OVX group (1.4 and 3.7%, respectively). Distribution of LH isoforms in cows from the three groups did not differ (p > 0.10).(ABSTRACT TRUNCATED AT 250 WORDS)

The working hypothesis for this study was that 17 beta-estradiol (E2), progesterone (P4), and opioid neuropeptides modulate frequency of pulsatile release of LH during the luteal phase of the bovine estrous cycle. On Day 8 of the estrous cycle (Day 0 = estrus), 20 cows were ovariectomized (OVX) and immediately received one of three steroid replacement treatments: P4 alone (n = 6), E2 alone (n = 7), or P4 and E2 (n = 7). To characterize the pattern of LH in peripheral circulation, serial blood samples were collected (12-min intervals for 22 h) on the fifth day following OVX. LHRH was administered after the 22-h sample was taken. Naloxone (opioid neuropeptide antagonist; 0.5 mg/kg) was administered on the sixth day after OVX, and change of concentration of LH in peripheral circulation was monitored. Pulse frequency of LH and mean concentration of LH were lower (p < 0.01) in cows treated with P4 and E2 compared to cows treated with P4 alone. Moreover, cows treated with E2 alone had a greater (p < 0.01) frequency of LH pulses and mean concentration of LH compared to cows treated with P4 alone. The greatest amplitude (p < 0.01) of LH release after LHRH was in cows treated with E2 alone. The greatest percentage increase (p < 0.01) in LH after administration of naloxone occurred in cows treated with P4 alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum concentrations of LH vary with season of the year in ovariectomized beef and dairy cows. The objective of the present study was to determine whether concentrations and profile of the gonadotropins and testosterone (T) in circulation vary with season of year in bovine males of a composite breed type (beef). Five INTACT and five gonadectomized (GNX) males that were 22 mo of age at the initiation of the study were used. All of the males utilized were maintained on pasture throughout the year at the latitude of 41 degrees N. Blood samples were collected in a serial regimen (10-min intervals for 24 h) at the spring and fall equinox and the summer and winter solstice. Concentrations of LH were quantified in all samples, and concentrations of T were determined in all samples of the INTACT group. Concentrations of FSH and 17 beta-estradiol (E2) were quantified in pooled samples for all animals, while concentrations of T were determined in pooled samples for males from the GNX group. Pulse frequency of LH or T and mean concentration of FSH did not vary with season of the year. A seasonal effect on mean concentration of LH (p < 0.01) occurred in males of the GNX group, with mean concentrations of LH being greatest during the spring equinox and lowest during the winter solstice. Season had an effect on pulse amplitude of LH (p < 0.01), mean concentration of T (p < 0.01), and pulse amplitude of T (p < 0.05) in males from the INTACT group.(ABSTRACT TRUNCATED AT 250 WORDS)

The objectives of this study were to determine the effects of dose of exogenous progesterone (P4) prior to artificial insemination on concentrations of 17 beta-estradiol (E2) and on conception rates in bovine females. Heifers (n = 100) and cows (n = 100) received P4-releasing intravaginal devices (PRIDs) to produce two different circulating concentrations of P4. All animals received a single PRID 10 days before (Day-10) the start of the breeding season (Day 0). In animals that received the low dose of P4 (1 PRID, target concentration of 2-3 ng/ml of plasma), the original PRID remained in place for 10 days. In animals that received the larger dose of P4 (2 PRIDs, target concentration of 5-8 ng/ml of plasma), an additional PRID was inserted on Day -9. To maintain concentrations of P4 in the 2-PRID group, the PRIDs inserted on Days -10 and -9 were replaced with new PRIDs on Days -5 and -4, respectively. Prostaglandin F2 alpha (25 mg) was administered to all animals on Days -9 and -3 to remove the endogenous source of P4. Following PRID removal, animals were artificially inseminated 12 h after signs of behavioral estrus were observed. A treatment-by-day interaction (p < 0.0001) was observed for concentrations of P4 in circulation of both heifers and cows. Animals that received 2 PRIDs had greater (p < 0.001) concentrations of P4 by Day-8 of treatment than animals that received 1 PRID. In cows that received 1 PRID, concentrations of E2 increased 2.4-fold from Day-10 (6.8 +/- 1.0 pg/ml) to Day-2 (16.7 +/- 1.4 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Our working hypothesis was that the low concentrations of progesterone (P4) and synthetic progestins administered in hormonal regimens to control estrous cycles of cows would have similar effects on secretion of LH and 17 beta-estradiol (E2). In addition, we hypothesized that concentrations of exogenous P4 typical of the midluteal phase of the estrous cycle and the corpus luteum (CL) would have similar effects on LH and E2, and the effects would be different from those of synthetic progestins and low concentrations of P4. Cows (n = 29) were randomly assigned to one of five treatment groups: 1) one Progesterone Releasing Intravaginal Device (1PRID; n = 6); 2) two PRIDs (2PRID; n = 6); 3) norgestomet, as in Syncro-Mate-B regimen (SMB; n = 6); 4) melengestrol acetate (MGA; 0.5 mg/day; n = 5); and 5) control (CONT; n = 6). Treatments were administered for 9 days (Day 0 = initiation of treatment). All cows from 1PRID, 2PRID, SMB, and MGA groups were injected with prostaglandin F2 alpha (PGF2 alpha) on Days 2 and 5 of the treatment period to regress CL. Cows in the 1PRID and SMB groups were also administered exogenous estrogen according to the respective estrous synchronization protocol for these products. Daily blood samples were collected from Day 0 to 35 to determine concentrations of P4. On Day 8, blood samples were collected at 15-min intervals for 24 h to determine pattern of LH secretion. On Day 9, all treatments ceased and cows in the CONT group received injections of PGF2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Beef heifers, 13 months old, were fed to achieve high (7.6 +/- 0.2 units) or low (3.9 +/- 0.1 units) body condition by feeding them one of two diets for 20 weeks. During week 17 of the growth phase, all heifers were ovariectomized. From week 20 to week 27 (restriction phase), all heifers were fed a daily diet containing 0.071 MJ metabolizable energy kg-1 body weight. At weekly intervals throughout the restriction phase, blood samples were collected at 10-min intervals for 11 h to determine the pattern of secretion of luteinizing hormone (LH), the amount of LH released in response to 750 ng (pituitary responsiveness) and 50 micrograms LH-releasing hormone (LHRH, releasable stores) and mean concentrations of follicle-stimulating hormone (FSH) in the circulation. Body weight declined during the restriction phase in a similar fashion in heifers with high and low body condition and changes in body weight were unrelated to mean concentrations of LH and FSH and frequency of LH pulses. Amplitude of LH pulses and responsiveness to 750 ng LHRH increased in a linear fashion with weight loss in heifers with low but not in those with high body condition. The amount of LH released in response to 50 micrograms LHRH decreased with increasing weight loss in heifers with high but not with low body condition, indicating that releasable pools of LH declined with increased weight loss in heifers with high body condition.(ABSTRACT TRUNCATED AT 250 WORDS)

Ovariectomized beef cows were actively immunized against LHRH to test the hypothesis that decreased stimulation of gonadotropes would alter the distribution of LH isoforms and amounts of mRNA for subunits of LH in the pituitary. Eight long-term (3 yr) ovariectomized beef cows were randomly assigned to one of two treatments: immunization against LHRH conjugated to human serum globulin (n = 4) and nonimmunization (control, n = 4). Mean concentration of serum LH in cows immunized against LHRH (1.0 +/- .83 ng/ml) was less (p = 0.01) than in control cows (5.0 +/- 0.83 ng/ml). Amounts of alpha- (p = 0.13) and LH beta-subunit (p = 0.10) mRNA tended to be reduced in cows immunized against LHRH compared to control cows. However, weight of the anterior pituitary and concentrations of LH in this gland did not differ (p = 0.90) among cows from the two groups. Pituitary extracts were chromatofocused on pH 10.5-7.0 gradients, and concentrations of LH in eluent fractions were determined by RIA. Extracts of all pituitaries resolved into nine isoforms (designated A through H and Z beginning with the most basic form). Only isoform F (mid-alkaline elution, pH = 8.8) was influenced by treatment (p = 0.05). Cows immunized against LHRH had a greater relative amount of isoform F (42.1 +/- 1.4%) than controls (37.2 +/- 1.4%). In summary, immunization of cows against LHRH altered 1) circulating concentrations of LH, 2) amounts of mRNA for the subunits of LH, and 3) distribution of intrapituitary LH isoforms without changing the concentrations of LH in the anterior pituitary.

The aim of this study was to determine whether the decline in oestradiol inhibition of circulating luteinizing hormone (LH) and follicle-stimulating hormone (FSH) during the peripubertal period of heifers is associated with a change in opioid modulation of LH and FSH secretion. Opioid inhibition of LH secretion was determined by response to administration of the opioid antagonist naloxone. Prepubertal heifers (403 days old) were left as intact controls, ovariectomized or ovariectomized and chronically administered oestradiol. Control heifers were used to determine time of puberty. Three weeks after ovariectomy, four doses of naloxone (0.13-0.75 mg kg-1 body weight) or saline were administered to heifers in the treatment groups in a latin square design (one dose per day). Blood samples were collected at intervals of 10 min for 2 h before and 2 h after administration of naloxone. This procedure was repeated four times at intervals of 3 weeks during the time intact control heifers were attaining puberty. All doses of naloxone induced a similar increase in concentration of serum LH within a bleeding period. During the initial bleeding period (before puberty in control heifers), administration of naloxone induced an increase in LH concentration, but the response was greater for heifers in the ovariectomized and oestradiol treated than in the ovariectomized group. At the end of the study when control heifers had attained puberty (high concentrations of progesterone indicated corpus luteum function), only heifers in the ovariectomized and oestradiol treated group responded to naloxone. Opioid inhibition of LH appeared to decline in heifers during the time control heifers were attaining puberty. Heifers in the ovariectomized group responded to naloxone at the time of administration with an increase in FSH, but FSH did not respond to naloxone at any other time. Administration of naloxone did not alter secretion of FSH in ovariectomized heifers. These results suggest that opioid neuropeptides and oestradiol are involved in regulating circulating concentrations of LH and possibly FSH during the peripubertal period. Opioid inhibition of gonadotrophin secretion appeared to decline during the peripubertal period but was still present in ovariectomized heifers treated with oestradiol after the time when age-matched control heifers had attained puberty. We conclude that opioid inhibition is important in regulating LH and FSH in circulation in heifers during the peripubertal period. However, opioids continue to be involved in regulation of circulating concentrations of LH after puberty.

The characteristics of circulating LH during sexual maturation in cattle were assessed by examining bioactive and immunoreactive LH concentrations, as well as their ratio (B/I ratio). Male and female intact control (CONT), gonadectomized (GNX; at 241 +/- 3 days of age, Day 0 of the study), and gonadectomized animals administered 17 beta-estradiol (GNXE) were evaluated. Serum samples were collected at 15-min intervals for 24 h at 1, 7, 13, 17, 21, 25, 29, 33, 37, and 43 wk subsequent to Day 0. Bioactive LH was assessed with an in vitro bioassay using mouse testicular interstitial cells. In initial experiments, immunoreactive LH was quantified in RIAs using three different antibodies. The two RIAs employing polyclonal antibodies overestimated low LH concentrations, but the absolute values obtained in each of the three assays were highly correlated. Hence, immunoreactive LH was measured in an RIA using monoclonal anti-bovine LH (bLH) (JR-518B7). No significant changes in the B/I ratios were observed during individual pulses of LH secretion. Accordingly, pools consisting of equal volumes of the serial blood samples collected during the 24-h period for each animal at each stage of maturation (pools) were compared. LH B/I ratios for GNX females increased significantly with time (p less than 0.01) and the B/I ratios for GNX males were significantly higher than for GNX females (p less than 0.05). Concentrations of LH in most of the pools for GNXE and CONT animals were extremely low or nondetectable until the later bleeding periods.(ABSTRACT TRUNCATED AT 250 WORDS)

Our primary objective for this study was to determine whether steady-state amounts of alpha- and LH beta-subunit mRNAs in the anterior pituitary are altered during sexual maturation in the bovine female. A secondary objective was to determine whether 17 beta-estradiol (E2) alters amounts of LH subunit mRNAs before onset of puberty. Heifers (7 mo old) were assigned to one of three treatments: 1) ovariectomized (OVX, n = 16); 2) OVX and administered E2 (OVXE, n = 16); or 3) ovary-intact (INTACT, n = 20). Pituitaries were collected at an estimated 120 days before onset of puberty (prepuberty) or 25 days before onset of puberty (peripuberty). Six INTACT heifers were used to determine time of puberty during the experimental period, and their pituitaries were collected 40 h after administration of prostaglandin F2 alpha (postpubertal INTACT group). Relative amounts of mRNAs for LH subunits in each pituitary were determined by Northern analysis and scanning densitometry. Amounts of alpha- and LH beta-subunit mRNAs were lower in pituitaries of INTACT heifers and OVXE heifers, regardless of stage of sexual maturation, than in those of OVX heifers. Amounts of alpha-subunit mRNA were similar in OVXE and INTACT heifers regardless of stage of sexual maturation. Amounts of LH beta-subunit mRNA did not change during sexual maturation in heifers in the INTACT group. Concentrations of E2 were higher and LH beta-subunit mRNA were lower in heifers from the prepubertal OVXE group than in heifers in all other treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)