Androgen and FSH synergistically stimulate lipoprotein degradation and utilization by ovary granulosa cells

Androgen can directly modulate the induction of steroidogenic enzymes by FSH (follicle stimulating hormone) in ovary granulosa cells. In studies of its mechanism of action, we examined the androgen effect on granulosa cell interaction with lipoproteins, the physiologic source of cholesterol. After granulosa cells were cultured for 48 hours with and without androgen and/or FSH, the cells were incubated for 24 hours with ¹²⁵I-lipoproteins [human high density lipoprotein (HDL), rat HDL, or human low density lipoprotein (LDL)]. The media were then analyzed for lipoprotein protein coat degradation products (mainly ¹²⁵I-monoiodotyrosine) and progestin [mainly 20α-dihydroprogesterone (20α-DHP)]. In the absence of FSH and androgen, 2 × 10⁵ granulosa cells degraded basal levels of all three lipoproteins, but produced no measurable 20α-DHP. The addition of 10^?7 M androstenedione (A), testosterone (T), or 5α-dihydrotestosterone (DHT) had no effect on lipoprotein protein degradation or 20α-DHP production. FSH alone stimulated lipoprotein protein degradation by 50 to 300% while the addition of androgen synergistically augmented the FSH-stimulated 20α-DHP production as well as protein coat degradation of all three lipoproteins. DHT and T were both effective, indicating that androgens themselves, and not estrogen products, were responsible for the effect on lipoprotein protein degradation and 20α-DHP production. The addition of a 10-fold excess cyproterone acetate (an anti-androgen) inhibited the effect of T, suggesting that the action of T was mediated by the granulosa cell androgen receptor. Androgen and FSH also synergistically stimulated the production of ³H-progestin when the granulosa cells were incubated with either ³H-cholesterol ester core labeled human HDL or similarly labeled human LDL. This report demonstrates that androgen, in combination with FSH, augments the steroidogenic pathway of the granulosa cell from the degradation of lipoprotein and utilization of the cholesterol ester core, to the production of progestin product.     

The relationship of male-male mounting to mate choice and sexual performance in male dairy goats

Rearing of male farm animals in unisexual groups has been implicated as a factor contributing to the failure of many males to breed as adults. The present study examines the relationship of male-male mounting in yearling dairy goats to subsequent mate preferences and sexual performance.Twenty-four sexually inexperienced male dairy goats, representing the Alpine, LaMancha, Saanen and Toggenburg breeds, were observed for male-male mounting in their home enclosure and then tested for mate choice and sexual performance when exposed to male and female (estrous and diestrous) stimulus animals. Their sexual behavior was compared with 7 adult goats with previous breeding experience.In the mate choice-sexual performance tests, 4 sexually inexperienced goats (17%) were sexually inactive, 6 (25%) mounted both male and female stimulus animals and 14 (58%) mounted only the female stimuli. Mate choice and sexual performance of the 20 sexually active males was not related to the number of male-male mounts initiated or the number of different males mounted in their home enclosure. However, the goats that received the greatest number of mounts in their home pen tended to be bisexual (would mount both male and female stimulus animals) in the mate choice tests. Males that were sexually inactive in mate choice-sexual performance tests repeatedly mounted the same male during home pen observations. Except for ejaculation frequency, the sexual performance of the sexually naive and experienced goats was similar. Goats of the Saanen breed were favored recipients of mounts from other males. There was no relationship between the number of male-male mounts performed and received.It was hypothesized that the reproductive failure of many male farm animals reared in all-male groups may be more closely related to the formation of specific sexual attachments to other males rather than the frequency with which they exhibit homosexual behaviors.     

Modulation of beta-oxidation and proton conductance pathway of brown adipose tissue in hypo- and hyperinsulinemic states

The metabolic capacity of interscapular brown adipose tissue of hypoinsulinemic (diabetic) rats is decreased and a reduced beta-oxidative capacity contributes to this metabolic alteration. It was thus of interest to compare, in diabetic and in chronically (8 days) insulin-infused rats, the beta-oxidative capacity and indices of the thermogenic state (GDP-binding and 32 000 Mr protein) in this tissue. Mitochondrial GDP-binding and 32 000 Mr protein were both decreased in diabetic rats compared to appropriate controls and markedly increased as was also the beta-oxidative capacity in hyperinsulinemic rats.     

Metabolism of gibberellins by immature barley grain

Gibberellins A₁, A₄, A₉, A₁₂-aldehyde, A₂₀ and A₅₁, each labelled with both a radioactive and stable isotope were fed to immature barley grain by injection into the endosperm. After 7 d, extensive metabolism of all substrates had occurred, and metabolites were identified by combined capillary gas chromatography-mass spectrometry. A proposed scheme of gibberellin metabolism in immature barley grain is presented.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography     

Identification, physical map location and sequence of the denV gene from bacteriophage T4

The denV gene from bacteriophage T4, which codes for endonuclease V, a small DNA repair enzyme, has been cloned and identified by an approach combining DNA sequencing and genetics, independent of the phenotypic effect of the cloned gene. Appropriate DenV+ and DenV- deletion mutants were mapped physically to define precisely a region encompassing the denV gene. This region was sequenced in order to identify a protein-coding sequence of the correct size for the denV gene (400-500 bp). Finally, identification was confirmed by sequencing the corresponding fragments cloned from four genetically and phenotypically well-characterized denV mutants. The denV gene is located at 64 kb on the T4 genome, adjacent to the ipII gene, and codes for a basic protein of 138 amino acids with a deduced molecular weight of 16,078.     

Isoelectric focusing of glutathione S-transferases from rat liver and kidney

The glutathione S-transferases that were purified to homogeneity from liver cytosol have overlapping but distinct substrate specificities and different isoelectric points. This report explores the possibility of using preparative electrofocusing to compare the composition of the transferases in liver and kidney cytosol. Hepatic cytosol from adult male Sprague–Dawley rats was resolved by isoelectric focusing on Sephadex columns into five peaks of transferase activity, each with characteristic substrate specificity. The first four peaks of transferase activity (in order of decreasing basicity) are identified as transferases AA, B, A and C respectively, on the basis of substrate specificity, but the fifth peak (pI6.6) does not correspond to a previously described transferase. Isoelectric focusing of renal cytosol resolves only three major peaks of transferase activity, each with narrow substrate specificity. In the kidney, peak 1 (pI9.0) has most of the activity toward 1-chloro-2,4-dinitrobenzene, peak 2 (pI8.5) toward p-nitrobenzyl chloride, and peak 3 (pI7.0) toward trans-4-phenylbut-3-en-2-one. Renal transferase peak 1 (pI9.0) appears to correspond to transferase B on the basis of pI, substrate specificity and antigenicity. Kidney transferase peaks 2 (pI8.5) and 3 (pI7.0) do not correspond to previously described glutathione S-transferases, although kidney transferase peak 3 is similar to the transferase peak 5 from focused hepatic cytosol. Transferases A and C were not found in kidney cytosol, and transferase AA was detected in only one out of six replicates. Thus it is important to recognize the contribution of individual transferases to total transferase activity in that each transferase may be regulated independently.     

The use of endo-β-N-acetylglucosaminidase H in characterizing the structure and function of glycoproteins

Endo-β-N-acetylglucosaminidase H from $\text{Streptomyces plicatus}$ can be useful in determining both the molecular weight of the protein moiety of glycoproteins and their inherent number of oligosaccharide chains. In the case of carboxypeptidase Y the molecular mass of the carbohydrate free protein was confirmed as 51,000 daltons. The native enzyme was shown to contain 4 oligosaccharide chains each averaging about 14 mannose residues. On treatment of mung bean nuclease I with the endoglycosidase, the molecular mass decreased from 39,000 to 31,000 daltons. The peptides produced on reduction of this enzyme with thiol were 18,700 and 12,500 daltons, indicating that carbohydrate had been present on both. Penicillium nuclease P₁ was decreased in size from 40,000 to 30,000 daltons by the endoglycosidase. Although most of the carbohydrate was removed from each of the native enzymes by the endoglycosidase, denaturation of the glycoproteins was necessary to effect complete removal. Enzyme activitywas not affected by carbohydrate depletion of these glycoproteins, a result consistent with similar studies on other oligosaccharide-containing enzymes.     

Ultrastructural observations on the cell surface of the intestinal epithelium of the nematode,Ascaris suum

Summary The intestinal epithelium of Ascaris suum consists of a single layer of tall columnar epithelial cells that rest on a thick basal membrane in contact with the pseudocoelomic cavity. Experiments were conducted on glutaraldehyde-fixed tissue to ascertain the nature of the electronegative charges associated with both the apical microvillar surface and basal membrane.A strong electronegative charge was demonstrated on the microvillar surface and basal membrane with ruthenium red and cationic ferritin staining. The ionic nature of ferritin binding was demonstrated with poly-L-lysine, a polycation that interacts with anionic groups on the membrane and thus blocks the subsequent binding of ferritin. Tissue thus treated was devoid of reaction product. Methylation with diazomethane completely abolished staining. Since the stronger acidic groups of sulfates or phosphates would not be protonated under the conditions employed in this study, and therefore susceptible to methylation, staining by ferritin is thought to be due to its interaction with carboxyl groups. Prior enzymatic treatment of tissue with neuraminidase or phospholipase C had no effect on subsequent ferritin binding. Tissue exposed to colloidal iron at various pH values showed maximal reactivity at a pH of 2.5 or above. Above pH 2.5, the dissociation of protons from free carboxyl groups of protein-bound amino-acid residues with pK's of 3.8 and 4.2 would be maximal, and the ionized carboxyl groups are then available to interact with iron micelles. These results suggest the presence of weaker acidic groups, such as the carboxyl groups of acidic amino acids or uronic acid residues. The stronger acidic groups of sialic acid and the esterified sulfate groups, if present, contribute only minimally to overall staining. These results demonstrate that a high electronegative charge density exists, despite the apparent lack of sialic acid. Staining is believed to be due to carboxyl groups of acidic amino acids and/or carboxyl groups or uronic acid residues.Part of this work was conducted at the Department of Zoology, Louisiana State University, Baton Rouge, Louisiana     

Influence of sialic acid on cell surface properties in I-cell disease fibroblasts

Summary Fibroblasts derived from patients with I-cell disease have been shown to accumulate many natural substrates including a three to fourfold increase in sialic acid content compared to that found in normal fibroblasts. This diverse accumulation of storage material is due to a massive deficiency of multiple lysosomal hydrolases as they are preferentially excreted into the culture fluid. There is evidence that the I-cell plasma membrane itself is abnormal with respect to certain transferase activities and in its sensitivity to freezing and Triton X-100. In this study, we have shown that a neuraminidase-sensitive substrate, and perhaps others in I-cell fibroblasts, contribute to an increased electronegativity of the I-cell fibroblast surface and to the cells' sensitivity to freezing. We also found that neuraminidase treatment of I-cell fibroblasts before preservative freezing in liquid nitrogen enables the cells to adapt more easily to subculture upon thawing. This project was supported in part by National Institutes of Health (NIH) BRSG Grant RR-05493, NIH Grant 1-R01-HD-11453-01-A1, National Science Foundation Grant PCM 77-05733, and Maternal and Child Health Service Project 417. Georgirene D. Vladutiu is the recipient of Research Career Development Award 1K04 HD 00312-01A1 from the National Institutes of Health.     

Further studies on the metabolism of gibberellins (GAs) A9, A20 and A29 in immature seeds of Pisum sativum cv. progress No. 9

Seed maturation of Pisum sativum cv. Progress No. 9 proceeds more slowly in winter than in summer even when the parent plants are grown in greenhouse conditions with light-and heat-supplementation. For parent plants grown under summer and winter conditions the metabolism of [³H]GA₉ in cultured seeds is qualitatively different in seeds of equivalent age and qualitatively the same in seeds of equivalent weight. 13-Hydroxylation of [³H]GA₉[³H]GA₂₀ is restricted to early stages of seed development. 2-Hydroxylation of [³H]GA₉2-OH-[³H]GA₉ has only been observed at a stage of development after endogenous GA₉ has accumulated. 2-OH-GA₉ has been shown to be endogenous to pea and is named GA₅₁. H₂-GA₃₁ and its conjugate have not been shown to be present in pea and may be induced metabolites of [³H]GA₉. The metabolism of GA₂₀GA₂₉ is used to illustrate a technique of feeding [²H][³H]GAs in order to distinguish a metabolite from the same endogenous compound. The in vitro conversion of [³H]GA₂₀[³H]GA₂₉, and the virtual non-metabolism of [³H]GA₂₉ have been confirmed for seeds in intact fruits. These results are discussed in relation to the apparent absence of conjugated GAs in mature pea seeds.Abbreviations GA_n gibberellin A_n - GC gas chromatography - GC-MS combined gas chromatography-mass spectrometry - GC-RC combined gas chromatography-radio counting - Me methyl ester - R_T etention time - SICM selected ion current monitoring - TLC thin layer chromatography - TMS trimethyl silyl ether The author is née Frydman