Jean C. Grammer

Follicle-stimulating hormone/cAMP regulation of aromatase gene expression requires β-catenin

Estrogens profoundly influence the physiology and pathology of reproductive and other tissues. Consequently, emphasis has been placed on delineating the mechanisms underlying regulation of estrogen levels. Circulating levels of estradiol in women are controlled by follicle-stimulating hormone (FSH), which regulates transcription of the aromatase gene (CYP19A1) in ovarian granulosa cells. Previous studies have focused on two downstream effectors of the FSH signal, cAMP and the orphan nuclear receptor steroidogenic factor-1 (NR5A1). In this report, we present evidence for β-catenin (CTNNB1) as an essential transcriptional regulator of CYP19A1. FSH induction of select steroidogenic enzyme mRNAs, including Cyp19a1, is enhanced by β-catenin. Additionally, β-catenin is present in transcription complexes assembled on the endogenous gonad-specific CYP19A1 promoter, as evidenced by chromatin immunoprecipitation assays. Transient expression and RNAi studies demonstrate that FSH- and cAMP-dependent regulation of this promoter is sensitive to alterations in the level of β-catenin. The stimulatory effect of β-catenin is mediated through functional interactions with steroidogenic factor-1 that involve four acidic residues within its ligand-binding domain, mutation of which attenuates FSH/cAMP-induced Cyp19a1 mRNA accumulation. Together, these data demonstrate that β-catenin is essential for FSH/cAMP-regulated gene expression in the ovary, identifying a central and previously unappreciated role for β-catenin in estrogen biosynthesis, and a potential broader role in other aspects of follicular maturation.

The essential light chains constitute part of the active site of smooth muscle myosin

Myosin, a major contractile protein, characteristically possesses a long coiled-coil α-helical tail and two heads. Each head contains both an actin binding site and an ATPase site and is formed from the NH2-terminal half of one of the two heavy chains (relative molecular mass, Mr, 200,000) and a pair of light chains; the so-called regulatory and essential light chains of approximately Mr 20,000 each. Recently we have identified1 Trp 130 of the myosin heavy chain from rabbit skeletal muscle as an active-site amino-acid residue after labelling with a new photoaffinity analogue of ADP, N-(4-azido-2-nitrophenyl)-2-aminoethyl diphosphate (NANDP)2. Nonspecific labelling was eliminated by first trapping NANDP at the active site with thiol crosslinking agents3. Exclusive labelling of the heavy chains with no labelling of the light chains agreed with previous findings4,5 that the heavy chains alone contain the actin-activated Mg-ATPase activity of rabbit skeletal myosin. Here we report similar photolabelling experiments with smooth muscle myosin (chicken gizzard) in which 3H-NANDP is trapped at the active site with vanadate6 and which show that both the heavy chains and the essential light chains are labelled. The results indicate that both chains contribute to the ATP binding site and represent the first direct evidence for participation of the essential light chains in the active site of any type of myosin.

GnRH-Regulated Expression of Jun and JUN Target Genes in Gonadotropes Requires a Functional Interaction between TCF/LEF Family Members and β-Catenin

GnRH regulates gonadotrope function through a complex transcriptional network that includes three members of the immediate early gene family: Egr1, Jun, and Atf3. These DNA-binding proteins act alone or in pairs to confer hormonal responsiveness to Cga, Lhb, Fshb, and Gnrhr. Herein we suggest that the transcriptional response of Jun requires a functional interaction between the T-cell factor (TCF)/lymphoid enhancer factor (LEF) family of DNA-binding proteins and beta-catenin (officially CTNNB1), a coactivator of TCF/LEF. Supporting data include demonstration that GnRH increases activity of TOPflash, a TCF/LEF-dependent luciferase reporter, in LbetaT2 cells, a gonadotrope-derived cell line. Additional cotransfection experiments indicate that a dominant-negative form of TCF7L2 (TCFDN) that binds DNA, but not beta-catenin, blocks GnRH induction of TOPflash. Overexpression of AXIN, an inhibitor of beta-catenin, also reduces GnRH stimulation of TOPflash. Transduction of LbetaT2 cells with TCFDN adenoviruses diminishes GnRH stimulation of Jun mRNA without altering expression of Egr1 and Atf3, two other immediate early genes that confer GnRH responsiveness. Reduction of beta-catenin in LbetaT2 cells, through stable expression of short hairpin RNA, also selectively compromises GnRH regulation of Jun expression and levels of JUN protein. Finally, overexpression of TCFDN attenuates GnRH regulation of Cga promoter activity, a known downstream target of JUN. Together, these results indicate that GnRH regulation of Jun transcription requires a functional interaction between TCF/LEF and beta-catenin and that alteration of either impacts expression of JUN downstream targets such as Cga.

Vanadate-mediated photocleavage of myosin

Publisher Summary Skeletal myosin is known to form a very stable transition state-like complex with MgADP and vanadate ions (V i ). During the irradiation ADP and V i are released simultaneously with a concomitant four-fold increase in the Ca 2+ -ATPase activity and an increase in the UV absorbance of myosin subfragment 1 (S1) over unirradiated controls. This modification is the result of the vanadate-promoted photooxidation of the fl-hydroxymethyl group of a serine to an aldehyde. This aldehyde can tautomerize to an enol. Photocleavage of myosin at active site yields a stable photomodified myosin, which, in the presence of excess V i and MgADP, will reform a new stable MgADP-V i complex at the active site. After purification of the new complex by removal of excess V i and MgADP by centrifugal gel filtration, irradiation leads to specific cleavage of the heavy chain. The heavy chain of S1 is cleaved at two sites when irradiated in the presence of miUimolar vanadate (in the absence of Mg 2+ or ADP. Mocz 6 has also reported a third site of vanadate-mediated photocleavage near the COOH terminus of the myosin heavy chain. An important consideration in photocleavage studies is that various polyvanadates, such as di-, tetra-, and pentavanadate, are in rapid equilibria with monovanadate at total vanadate concentrations in the millimolar range.

Protein content and volume of early porcine blastocysts

Mean protein and volume of 222 blastocysts collected on 6 to 9 days of pregnancy were measured. Embryo protein differed (P < 0.05) for each day of development studied. Protein content of embryos doubled between days 6 and 7 and days 7 and 8 (1.2 ± 0.04, 2.0 ± 0.14, and 3.7 ± 0.2 μg, respectively). A dramatic increase from 3.7 ± 0.2 to 56.0 ± 3.4 μg was observed between days 8 and 9. Blastocyst volume increased (P < 0.05) from 0.56 ± 0.03 × 10−2mm3 to 1.11 ± 0.04 × 10−2mm3 between days 6 and 7, and then increased 10-fold on day 8 and five-fold on day 9. Blastocyst volume was not correlated with protein for days of development and females studied. Approximately 20% of all blastocysts within a single female contained less protein than the average protein content of all embryos from the same uterus. The results indicate that day 6 of development marks the onset of an exponential increase in embryo protein. Also, blastocyst volume is not correlated with blastocyst protein, suggesting that embryo viability is difficult to estimate by size alone. Further, approximately 20% of the blastocysts collected from a single female may exhibit reduced viability, based on reduced protein content, as early as day 6 of development.

Protein content of porcine embryos during the first nine days of development

Little is known about the physiochemical aspects of porcine embryos prior to implantation. The purpose of this study was to quantitate the total protein content of porcine embryos from fertilization through day 9 of development. Thirty-seven gilts and two sows were hand mated and the reproductive tracts collected at slaughter 1 to 9 days after the onset of estrus. The uteri were flushed with phosphate buffered saline (PBS), and embryos were collected, washed with PBS and transferred directly to test tubes containing distilled water. Only embryos which appeared morphologically normal were used. Protein content was determined by the Bio-Rad microassay. Standard curves were constructed for each assay using. 8 to 19 microg gamma globulin (Bio-Rad assay). Protein standards were run in triplicate. Regression lines were calculated for protein standards, and the resulting line was used to determine total protein in the unknown samples. Protein content of unfertilized eggs and embryos increased steadily through days 3, 4 and 5 of development, from 273 to 334, 491 and 620 ng, respectively. This result suggests that the protein content of porcine embryos increases only slightly from fertilization through day 5 of development. A dramatic increase in embryo protein content was observed between days 6 and 9 of development, which is the time of blastocyst formation and hatching of the embryo from the zona pellucida.

Thiamine Deficiency and Glyoxylic Acid

The effect of thiamine deficiency on glyoxylic acid metabolism in mice and rats was investigated to determine whether the vitamin deficiency results in gross effects on glyoxylate levels via an alteration in the activity of alpha-ketoglutarate:glyoxylate carboligase. Thiamine-deprived or pyrithiamine-treated mice did not show a decreased oxidation of [1-14C]glyoxylate to respiratory CO2; there was some decrease in the conversion of [2-14C]glyoxylate into CO2 by pyrithiamine-treated mice, but not by thiamine-deprived animals. Dietary thiamine deprivation caused a decrease in carboligase levels in liver but no effect on levels in three brain regions. Pyrithiamine treatment had no significant effect on liver carboligase activities, but did decrease the levels in cerebrum, cerebellum and brainstem. Thiamine-deprived and pyrithiamine-treated mice showed decreased urinary glycolic acid excretion Glyoxylic acid excretion by thiamine-deprived rats was monitored in order to re-examine a previous report by another laboratory that glyoxyluria occurs under these conditions. Trace amounts of glyoxylate could be detected in the urine of rats fed thiamine-deficient diet for 3-5 weeks, but urinary glyoxylate was not detectable at later stages of thiamine deprivation. These results do not support a significant role for alpha-ketoglutarate:carboligase activity in the primary etiology of thiamine deficiency syndromes.

Lactate dehydrogenase isoenzyme patterns as a method of pregnancy detection in cattle.

The objective of this study was to evaluate serum lactate dehydrogenase isoenzyme patterns in open and pregnant Holstein and Hereford cows as a method of detecting pregnancy. Serum samples were collected from 26 Holstein and 13 Hereford cows and lactate dehydrogenase isoenzyme patterns were examined by electrophoresis and quantitated by scanning densitometry. Lactate dehydrogenase isoenzyme(4) and LDH(5) were found in higher concentration (P </= .05) in pregnant versus open Holstein cows. Twelve of 14 and 10 of 12 Holstein cows were correctly diagnosed as being pregnant and open, respectively by examining differences in serum concentrations of isoenzymes LDH(4) and LDH(5). Pregnancy was detected as early as 36 days of gestation. No differences were found in serum LDH isoenzyme patterns between pregnant and open Hereford cows. However, differences (P </= .05) in concentration of all five LDH isoenzymes were observed between Holstein and Hereford cows, which may lend insight into the metabolic significance of the elevation of LDH(4) and LDH(5) in pregnancy. The results suggest that elevation of serum LDH(4) and LDH(5) may be used as a means of pregnancy detection in the Holstein but not the Hereford cow.

Synthesis of a spin-labeled photoaffinity ATP analogue, and its use to specifically photolabel myosin cross-bridges in skeletal muscle fibers.

A spin-labeled photoaffinity ATP analogue 3′(2′)-O-{4-[4-oxo-(4-amido-2,2,6,6-tetramethyl-piperidino-1-oxyl)]-benzoyl}benzoyl adenosine 5′-triphosphate (SL-Bz2ATP) was synthesized and used to photolabel myosin in muscle fibers. Previous work has shown that 3′(2′)-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (Bz2ATP) photolabeled Ser-324 of the 50 kDa tryptic fragment of skeletal S1 heavy chain. In this work, [α-32P]SL-Bz2ATP was hydrolyzed and trapped as the diphosphate analogue with Co2+ and orthovanadate at the active site of myosin in rabbit psoas muscle fibers. After UV irradiation, the myosin heavy chain was the only protein band found to be significantly photolabeled as assayed by gel electrophoresis and radioactivity counting. The labeling was localized after brief trypsin digestion by SDS-PAGE to be on the 50 kDa tryptic fragment of the S1 heavy chain. Ca. 35% of the myosin in fibers was covalently photolabeled. The fibers photolabeled with SL-Bz2ATP had the same active tension and maximum shortening velocity as the control fibers. The resulting spin label on myosin was too mobile to report the orientation of the heads in fibers. Nonetheless, this is the first work to show the feasibility of utilizing active site binding and photoaffinity labeling to place covalent spectroscopic probes at the myosin active site in fibers with high specificity and yield without affecting mechanical function.