Ovarian hypercytolipidemia induced by obese (ob/ob) and diabetes (db/db) mutations: basis of female reproductive tract involution II

The diabetes (db/db) and obese (ob/ob) genotype mutations induce a progressive, hypercytolipidemic condition within the ovarian compartments of the female reproductive tract that results in sterility and premature organ involution in C57BL/KsJ mice. The current studies focus on the ultrastructural changes that occur within the ovarian interstitial, thecal, and follicular granulosa cell layers during the progressive expression of these mutations which promote tissue cytolipidemia-induced organoinvolution. Control (normal: +/?), diabetes (db/db), and obese (ob/ob) genotype groups were prepared for high resolution light (HRLM) and transmission electron microscopic (TEM) analysis of ovarian tissue samples collected from 4 (young)- to 20 (aged)-week-old mice, allowing for the progressive influences of the mutational aberrations on tissue structure to be evaluated. Compared to controls, both (ob/ob) and (db/db) mutations induced a dramatic increase in ovarian interstitial, thecal and follicular granulosa cytolipid vacuole accumulations, which increased in density between 4 and 20 weeks of age. Initially, lipid vacuoles aggregated in the interstitial and thecal regions of ovarian follicles in response to the hyperglycemic-hypertriglyceridemic metabolic conditions typical of both (ob/ob) and (db/db) groups. Progressive cytoplasmic movement of the lipid pools established a perinuclear isolation from associated cytoplasmic organelles. Progressive lipid accumulations forced cytoplasmic organelles to peripheral cell compartments and altered the follicular cell profile towards that of adipocyte-like entities relative to controls. The progressive hypercytolipidemia-induced alterations in cell structure disrupted normal tissue continuity, which culminated in premature ovarian organo-involution and female reproductive sterility.     

Structural,metabolic and endocrine analysis of the diabetes (db/db) hypogonadal syndrome: relationship to hypophyseal hypercytolipidemia

Expression of the diabetes (db/db) mutation in C57BL/KsJ mice results in functional suppression of the female pituitary-gonadal axis accompanied by premature utero-ovarian cytolipoatrophy. Cellular gluco- and lipo-metabolic disturbances promoted by the db/db systemic hyperglycemic-hyperinsulinemic state suppress pituitary gonadotropin release in response to gonadotropin-releasing hormone and gonadal steroid stimulation and results in a hypogonadal-infertility syndrome. Adult female C57BL/KsJ control (+/+ and +/? genotypes) and db/db littermates were monitored for associations in systemic and cellular alterations in luteinizing hormone (LH), follicle-stimulating hormone (FSH), gonadal steroid (binding) levels, and pituitary glucometabolic indices associated with db/db-enhanced lipid imbibition and cytostructural disruption. Obesity, hyperglycemia, and hyperinsulinemia characterized all db/db mutants relative to controls. Serum and pituitary progesterone and estradiol concentrations were suppressed in db/db mutants, in association with serum LH and FSH levels, but not with pituitary LH and FSH concentrations, which were comparable between groups. Pituitary insulin receptor binding and glucose utilization rates were suppressed in db/db groups relative to +/? indices. Structural and cytochemical analysis of anterior (AP), intermediate (IL), and neuro-(NP) hypophyseal lobes demonstrated prominent hypercytolipidemia in db/db mutants relative to controls. Prominent cytolipidemia was localized within well-granulated basophilic gonadotrophs and within IL and NP pituicytes. Vasolipidemia and interstitial cytoadiposity were prominent throughout all db/db pituitary lobes. Thus, disturbances associated with pituitary hypercytolipidemia are functional components of the expressed diabetes-associated hypogonadal syndrome in db/db mutants. Progressive alterations in hypophyseal cytoarchitecture are correlated with suppression of pituitary metabolic and endocrine indices, alterations that contribute to functional disruption of the pituitary-hypogonadal axis in C57BL/KsJ-db/db mice.

     

Hypercytolipidemia-induced cellular lipoapoptosis: cytostructural and endometabolic basis of progressive organo-involution following expression of diabetes (db/db) and obese (ob/ob) mutation syndromes

Onset expression of Type 2 (NIDDM) diabetes and obesity metabolic syndromes (DOS) are characterized by premature, progressive cytoatrophy and organo-involution induced by dysregulated cellular gluco- and lipo-metabolic cascades. The consequential systemic, interstitial and intracellular hyperlipidemia disrupts normal cytointegrity and metabolic responsivity to the established hypercaloric pericellular environments. The sequential cytostructural, metabolic and endocrine disturbances associated with the development of progressive DOS-associated hypercytolipidemia compromises cellular metabolic response cascades and promotes cytochemical disturbances which culminate with nuclear lipoapoptosis and cytoatrophy. The dramatic alterations in interstitial glucose and lipid (free fatty acids/triglycerides) concentrations are recognized to influence interstitial and cytoplasmic microchemical environments, which markedly alter cellular nutrient diffusion and active trans-membrane flux rates. The progressive exacerbation of interstitial and cytoplasmic lipid imbibition has been demonstrated to be associated with DNA fragmentation by lipo-infiltration into the chromatin matrix, inducing structural disruption and physical dissolution, indexed as nuclear lipoapoptosis. Therapeutic reduction of the severity of hypercytolipidemia-induced structural and cytochemical compromise promotes the restoration of homeostatic metabolic support for normalized cytostructural indices and supportive cellular gluco- and lipo-metabolic cascades. The re-establishment of a homeostatic interstitial microenvironment moderates the severity of cytolipidemic compromise within affected cell types, reduces nuclear lipo-infiltration and DNA lipo-dissolution, resulting in the preservation of cytostructural integrity. Through the therapeutic restoration of extra- and intra-cellular microchemical environments in genetically dysregulated metabolic syndrome models, the coincident cytochemical, endocrine and metabolic disturbances associated with progressive hypercytolipidemia, resulting from the expressed systemic hypercaloric environmental and hepato-pancreatic endometabolic disturbances which characterize Type 2 (NIDDM) diabetes–obesity and metabolic (X) syndromes, may be ameliorated.     

Estrogenic restoration of functional pancreatic islet cytoarchitecture in diabetes (db/db) mutant C57BL/KsJ mice: relationship to estradiol localization,systemic glycemia,and persistent hyperinsulinemia

Cell and Tissue Research - The diabetes (db/db) genotype mutation induces a hyperglycemic–hyperinsulinemic endometabolic state in C57BL/KsJ mice, manifesting a type 2 NIDDM diabetes-obesity...     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

Callus induction and <Emphasis Type="Italic">de novo</Emphasis> regeneration from callus in Guar (<Emphasis Type="Italic">Cyamopsis tetragonoloba</Emphasis>)

Guar (Cyamopsis tetragonoloba L. Taub) is a drought tolerant and multipurpose grain legume cash crop grown primarily under rainfed conditions in several countries. The effect of various growth regulators and their combinations on a variety of explants, namely the embryo, cotyledons, cotyledonary nodes, shoot tip and hypocotyle, has been studied and an efficient system for callus induction and regeneration from callus has been developed. It was established that Murashige and Skoogs culture medium containing 2,4-dichlorophenoxyacetic acid (10.0M) in combination with 6-benzylaminopurine (5.0M) with embryo or cotyledon explants is most suitable for induction of green and friable morphogenic callus, with a range of 82.5–95% of cultured explants responding to callus induction. Efficient de novo shoot regeneration was achieved by culturing the callus obtained on this medium on Murashige and Skoogs medium containing 1-naphthlenacetic acid (13.0M) in combination with 6-benzylaminopurine (5.0M) with a range of 82.1–88.4% of callus clumps producing 20–25 shoots. In vitro rooting of cultured shoots was obtained on half-salt concentration of Murashige and Skoogs culture medium supplied with indole-3-butyric acid (5.0M) on which 82–90% of cultured shoots produced healthy roots. The in vitro regenerated plants were grown to pod setting and subsequent maturity under greenhouse conditions.     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

The ontogenetic basis for floral diversity in <Emphasis Type="Italic">Agonis</Emphasis>, <Emphasis Type="Italic">Leptospermum</Emphasis> and <Emphasis Type="Italic">Kunzea</Emphasis> (Myrtaceae)

Floral development in three species each of Leptospermum and Kunzea, and one species of Agonis, is described and compared. Differences in the number of stamens and their arrangement in the flower at anthesis are determined by the growth dynamics of the bud. In Leptospermum, early expansion of the bud is predominantly in the axial direction and causes the stamen primordia to be initiated in antepetalous chevrons. In Kunzea, early expansion occurs predominantly in the lateral direction and successive iterations of stamen primordia are inserted alternately at more or less the same level. In both genera, further expansion in the lateral plane spreads the stamens into a ring around the hypanthium. Agonis flexuosa is similar to Leptospermum. Other variable factors include the timing at which stamen initiation commences (earlier in Leptospermum than Kunzea), the duration of stamen initiation (hence the total number of stamens produced – varies within genera), and very late differential expansion that forces stamens into secondary antesepalous groups in A. flexuosa and L. myrsinoides.The authors thank Dr H. Toelken for kindly providing some material and the impetus for this project. This research was supported by Australian Research Council grant AS19131815.     

The Madagascan genus <Emphasis Type="Italic">Vaughania</Emphasis> is reduced to synonymy under <Emphasis Type="Italic">Indigofera</Emphasis> (<Emphasis Type="Italic">Leguminosae-Papilionoideae-Indigofereae</Emphasis>)

Summary  Eleven species comprising the Madagascan genus Vaughania are subsumed within the large pantropical genus Indigofera. Six new combinations are made; the remaining species were originally described in Indigofera.     

Hermes copper (<Emphasis Type="Italic">Lycaena</Emphasis> [<Emphasis Type="Italic">Hermelycaena</Emphasis>] <Emphasis Type="Italic">hermes</Emphasis>: Lycaenidae): life history and population estimation of a rare butterfly

Hermes copper (Lycaena [Hermelycaena] hermes: Lycaenidae) is a rare species endemic to the coastal sage scrub in and around San Diego, CA, USA. This species has experienced substantial habitat loss due to urbanization and recent wildfires. We present data collected from field surveys conducted in 2003 and 2004. The flight season lasted 1–2 months with densities varying among sites and years. We observed adults most often near California buckwheat (Eriogonum fasciculatum) plants and significantly more often on north and west sides of trails or roads. We compared the robustness and statistical power of three indices of population size from the modified Pollard Walk surveys. We recorded the largest single-day count (Max Count), the cumulative number observed throughout the flight season (Pollard) and an estimate based on a four-parameter model (INCA: Insect Count Analyzer). The Pollard estimate was the most robust to sampling error and the most powerful at detecting population changes in simulated data. Improved monitoring techniques, both field methods and statistical estimation, are critical to determine the conservation status of rare butterflies like Hermes copper.     

<Emphasis Type="Italic">Phuphanochloa,</Emphasis> a new bamboo genus (<Emphasis Type="Italic">Poaceae</Emphasis>: <Emphasis Type="Italic">Bambusoideae</Emphasis>) from Thailand

Summary  A new monotypic bamboo genus Phuphanochloa (Poaceae: Bambusoideae) from north-eastern Thailand is described, together with a new species, P. speciosa.     

Sociality in <Emphasis Type="Italic">Euglossa</Emphasis> (<Emphasis Type="Italic">Euglossa</Emphasis>) <Emphasis Type="Italic">viridissima</Emphasis> Friese (Hymenoptera,Apidae, Euglossini)

Euglossa viridissima is an orchid bee that forms both solitary and multiple female nests, making it a suitable species for the study of factors leading to diverse degrees of sociality in Euglossines. We conducted observations in eight reused nests (where a first generation of bees had been produced) kept in artificial boxes from the Yucatan Peninsula, Mexico. Five nests were reused (reactivated) by a single female (SFN), two nests reused by a mother and one daughter (MFN1) and one nest reused by the mother and two daughters (MFN2). No single nest was reactivated by unrelated females. The number of foraging trips, their duration and the duration of cell provisioning was not different between SFN and MFN. The overall production of cells per female was not different either between both types of nest. However, in MFN although all females did lay eggs, there was a reproductive skew in favor of the mother (95 and 45% of the brood produced in MFN1 and MFN2 respectively). She showed reproductive control of her daughters through oophagy and displaying threatening behavior when the daughters tried to open a cell where she had laid an egg. Brood losses to parasites (Anthrax sp. (Bom-byliidae) and Hoplostelis bivittata (Megachilidae)) were only found in SFN which possibly reflects and advantage of MFN in this respect. Our results coupled with other studies in Euglossa, reveal that a wide range of social behaviors occur in this genus, from solitary and communal to primitive reproductive division of labor. Multiple factors involving different levels of pressure imposed by food availability and parasites may favor such a diverse range of nesting behaviors. Interestingly, female associations in E. viridissima seem a result of kin selection that is enforced by coercion from mother females on their daughters. More studies are needed to shed light upon the social organization of Euglossa and other Euglossines and on their phylogenetic relationships in order to trace the origins of eusociality in Apidae. Received 12 February 2008; revised 25 June 2008; accepted 17 July 2008.     

Highly selective biotransformation of ginsenoside Rb<Subscript>1</Subscript> to Rd by the phytopathogenic fungus<Emphasis Type="Italic"> Cladosporium fulvum</Emphasis> (<Emphasis Type="Italic">syn</Emphasis>.<Emphasis Type="Italic"> Fulvia fulva</Emphasis>)

Fourteen phytopathogenic fungi were tested for their ability to transform the major ginsenosides to the active minor ginsenoside Rd. The transformation products were identified by TLC and HPLC, and their structures were assigned by NMR analysis. Cladosporium fulvum, a tomato pathogen, was found to transform major ginsenoside Rb₁ to Rd as the sole product. The following optimum conditions for transforming Rd by C. fulvum were determined: the time of substrate addition, 24 h; substrate concentration, 0.25 mg ml⁻¹; temperature, 37°C; pH 5.0; and biotransformation period, 8 days. At these optimum conditions, the maximum yield was 86% (molar ratio). Further, a preparative scale transformation with C. fulvum was performed at a dose of 100 mg of Rb₁ by a yield of 80%. This fungus has potential to be applied on the preparation for Rd in pharmaceutical industry.     

Morphological and immunohistochemical study of testicular capsule and peritubular tissue of emu (<Emphasis Type="Italic">Dromaius novaehollandiae)</Emphasis> and ostrich (<Emphasis Type="Italic">Struthio camelus</Emphasis>)

The testicular capsule and peritubular boundary tissue of the emu and ostrich, as typical representatives of ratite birds, were studied in sexually mature and active birds. The testicular capsule was much thicker (578.1±73.4 μm for the free surface of the ostrich testis, and 176.2±57.5 μm for the emu) than those of members of the Galloanserae. The cellular composition of both testicular capsule and peritubular tissue was similar generally to that of members of the previously studied Galloanserae and of mammals. The tunica albuginea of the testicular capsule mainly comprised smooth-muscle-like or myoid cells mostly running in one direction and occurring in one main mass. Unlike the Galloanserae, the tunica albuginea contained more collagen fibres than smooth muscle cells, especially in the ostrich. Peritubular tissue was similarly composed of smooth-muscle-like cells distributed in several layers. Actin microfilaments and desmin and vimentin intermediate filaments were variably immunoexpressed in these two tissue types in both birds, with a clear dichotomy in the peritubular tissue. Thus, taken together with studies of some members of the Galloanserae, avian testes clearly contain a morphological mechanism that is represented partly by the smooth muscle cells of the testicular capsule and peritubular tissue for transporting the testicular fluid, which is usually copious in birds, and its cellular content from the testis into the excurrent duct system; this mechanism is similar to that found in mammals. The authors are grateful for a University of Pretoria research grant, which aided this work. Dr Peter Ozegbe of the Department of Veterinary Anatomy, University of Ibadan is a recipient of the University of Pretoria Foreign Post-Doctoral Fellowship.     

Isolation and characterization of a <Emphasis Type="Italic">GAI/RGA</Emphasis>-like gene from <Emphasis Type="Italic">Gossypium hirsutum</Emphasis>

The aim of the investigation reported here was to assess the role of gibberellin in cotton fiber development. The results of experiments in which the gibberellin (GA) biosynthesis inhibitor paclobutrazol (PAC) was tested on in vitro cultured cotton ovules revealed that GA is critical in promoting cotton fiber development. Plant responses to GA are mediated by DELLA proteins. A cotton nucleotide with high sequence homology to Arabidopsis thaliana GAI (AtGAI) was identified from the GenBank database and analyzed with the BLAST program. The full-length cDNA was cloned from upland cotton (Gossypium hirsutum, Gh) and sequenced. A comparison of the putative protein sequence of this cDNA with all Arabidopsis DELLA proteins indicated that GhRGL is a putative ortholog of AtRGL. Over-expression of this cDNA in Arabidopsis plants resulted in the dwarfed phenotype, and the degrees of dwarfism were related to the expression levels of GhRGL. The deletion of 17 amino acids, including the DELLA domain, resulted in the dominant dwarf phenotype, demonstrating that GhRGL is a functional protein that affects plant growth. Real-time quantitative PCR results showed that GhRGL mRNA is highly expressed in the cotton ovule at the elongation stage, suggesting that GhRGL may play a regulatory role in cotton fiber elongation.     

Evaluation of the anti-<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp <Emphasis Type="Italic">cicer</Emphasis> and anti-<Emphasis Type="Italic">Alternaria porri</Emphasis> effects of some essential oils

The anti-Fusarium oxysporum f. sp cicer (FOC) and anti-Alternaria porri (A. porri) effects were evaluated for 75 different essential oils. The most active essential oils found were those of lemongrass, clove, cinnamon bark, cinnamon leaf, cassia, fennel, basil and evening primrose. However, the effectiveness of these essential oils with both the tested fungi showed different responses. The level of inhibition was compared with Hexaconazole. GC–MS analysis for five oils amongst the 75 essential oils tested was performed. The potential of these essential oils as an ecofriendly and economic approach as a fungicide for FOC and A. porri is discussed.     

Expression patterns of <Emphasis Type="Italic">engrailed</Emphasis> and <Emphasis Type="Italic">dpp</Emphasis> in the gastropod <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis>

We isolated the full-length cDNAs of engrailed and dpp-BMP2/4 orthologues from the pond snail Lymnaea stagnalis and examined their expression patterns during development by the whole mount in situ hybridization. At the gastrula and trochophore stages, engrailed is expressed in the peripheral ectoderm of the presumptive and invaginating shell gland, corroborating its role in the shell formation that is widely conserved among molluscs. At the same stages, dpp-BMP2/4 is expressed in the right-hand side ectoderm of the shell gland and in the invaginating stomodaeum. Unlike in the gastropod Patella vulgata, our results suggested that dpp-BMP2/4 has a role in the shell formation, rather than in the regional specification and that it could be involved in the specification pathway of the left–right asymmetry of the developing shell in L. stagnalis.     

Inhibition of connexin 43 alters <Emphasis Type="Italic">Shh</Emphasis> and <Emphasis Type="Italic">Bmp-2</Emphasis> expression patterns in embryonic mouse tongue

The morphogenesis of fungiform papillae occurs in a stereotyped pattern on the dorsal surface of the mammalian tongue via epithelial–mesenchymal interactions. These interactions are thought to be achieved via intercellular communication. Gap junctions can be observed in many developing tissues and have been suggested to participate in a variety of functions, including the regulation of cell proliferation, differentiation, and apoptosis. Here, we demonstrate that the expression of Connexin 43 (Cx43), a gap junction protein, is correlated significantly with the development of fungiform papillae, which exhibit a pattern formation and morphogenesis similar to the development of other epithelial appendages. Antisense-oligodeoxynucleotide (AS-ODN) against Cx43 was used to assess the developmental functions of Cx43. The expression patterns of the signaling molecules were disrupted by Cx43 inhibition. Interestingly, the expression patterns of Shh, a key molecule in the determination of the spacing patterns of fungiform papillae, were disturbed after treatment with Cx43 AS-ODN. We have also attempted to determine the functions of Bmp-2 by applying NOGGIN protein to tongue cultures. Our results indicate that upstream regulation via Cx43 controls the Shh and Bmp-2 pathways for the morphogenesis and pattern formation of fungiform papillae.This work was supported by grant no. R01-2003-000-11649 from the Korea Science and Engineering Foundation.