Margaret Mobberley

The Transcriptional Corepressor RIP140 Regulates Oxidative Metabolism in Skeletal Muscle

Summary Nuclear receptor signaling plays an important role in energy metabolism. In this study we demonstrate that the nuclear receptor corepressor RIP140 is a key regulator of metabolism in skeletal muscle. RIP140 is expressed in a fiber type-specific manner, and manipulation of its levels in null, heterozygous, and transgenic mice demonstrate that low levels promote while increased expression suppresses the formation of oxidative fibers. Expression profiling reveals global changes in the expression of genes implicated in both myofiber phenotype and metabolic functions. Genes involved in fatty-acid oxidation, oxidative phosphorylation, and mitochondrial biogenesis are upregulated in the absence of RIP140. Analysis of cultured myofibers demonstrates that the changes in expression are intrinsic to muscle cells and that nuclear receptor-regulated genes are direct targets for repression by RIP140. Therefore RIP140 is an important signaling factor in the regulation of skeletal muscle function and physiology.

Mechanical isolation and in vitro growth of preantral and small antral human follicles.

OBJECTIVE To develop a procedure for isolating small human follicles and to determine their growth requirements. DESIGN Preantral and early antral follicles were isolated manually, allocated randomly to experimental groups, and cultured for a few weeks. SETTING Patients giving informed consent in hospitals. PATIENT(S) Women undergoing laparotomy or oophorectomy. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Follicular size, E2, histology. RESULT(S) Human FSH (at a dose of 1.5 U/mL) induced antral growth of follicles, and the addition of human LH (2.5 ng/mL) to human FSH stimulated growth and antral development. Histologic studies showed that most of the early antral follicles did not contain an oocyte and already had begun to undergo atresia before culturing. Levels of E2 increased in the incubation medium as the follicles increased in size, but those levels were significantly greater when the follicles contained oocytes. CONCLUSION(S) It is possible to grow small human follicles after they have been isolated manually. To develop successfully, they require a low concentration of human LH in addition to human FSH. The rate of atresia between the preantral and early antral stages in vivo is very high; therefore, it is worthwhile to develop techniques for isolating and culturing the follicles before the antral stages.

Effect of cell shape and packing density on granulosa cell proliferation and formation of multiple layers during early follicle development in the ovary

The postnatal mouse ovary is rich in quiescent and early-growing oocytes, each one surrounded by a layer of somatic granulosa cells (GCs) on a basal lamina. As oocytes start to grow the GCs change shape from flattened to cuboidal, increase their proliferation and form multiple layers, providing a unique model for studying the relationship between cell shape, proliferation and multilayering within the context of two different intercommunicating cell types: somatic and germ cells. Proliferation of GCs was quantified using immunohistochemistry for Ki67 and demonstrated that, unusually, cuboidal cells divided more than flat cells. As a second layer of GCs started to appear, cells on the basal lamina reached maximum packing density and the axes of their mitoses became perpendicular to the basal lamina, resulting in cells dividing inwards to form second and subsequent layers. Proliferation of basal GCs was less than that of inner cells. Ultrastructurally, collagen fibrils outside the basal lamina became more numerous as follicles developed. We propose that the basement membrane and/or theca cells that surround the follicle provide an important confinement for rapidly dividing columnar cells so that they attain maximum packing density, which restricts lateral mitosis and promotes inwardly oriented cell divisions and subsequent multilayering.

Characterization and significance of adhesion and junction-related proteins in mouse ovarian follicles.

ABSTRACT In the ovary, initiation of follicle growth is marked by cuboidalization of flattened granulosa cells (GCs). The regulation and cell biology of this shape change remains poorly understood. We propose that characterization of intercellular junctions and associated proteins is key to identifying as yet unknown regulators of this important transition. As GCs are conventionally described as epithelial cells, this study used mouse ovaries and isolated follicles to investigate epithelial junctional complexes (tight junctions [TJ], adherens junctions [AJ], and desmosomes) and associated molecules, as well as classic epithelial markers, by quantitative PCR and immunofluorescence. These junctions were further characterized using ultrastructural, calcium depletion and biotin tracer studies. Junctions observed by transmission electron microscopy between GCs and between GCs and oocyte were identified as AJs by expression of N-cadherin and nectin 2 and by the lack of TJ and desmosome-associated proteins. Follicles were also permeable to biotin, confirming a lack of functional TJs. Surprisingly, GCs lacked all epithelial markers analyzed, including E-cadherin, cytokeratin 8, and zonula occludens (ZO)-1alpha+. Furthermore, vimentin was expressed by GCs, suggesting a more mesenchymal phenotype. Under calcium-free conditions, small follicles maintained oocyte-GC contact, confirming the importance of calcium-independent nectin at this stage. However, in primary and multilayered follicles, lack of calcium resulted in loss of contact between GCs and oocyte, showing that nectin alone cannot maintain attachment between these two cell types. Lack of classic markers suggests that GCs are not epithelial. Identification of AJs during GC cuboidalization highlights the importance of AJs in regulating initiation of follicle growth.

LKB1 is required for hepatic bile acid transport and canalicular membrane integrity in mice

LKB1 is a ‘master’ protein kinase implicated in the regulation of metabolism, cell proliferation, cell polarity and tumorigenesis. However, the long-term role of LKB1 in hepatic function is unknown. In the present study, it is shown that hepatic LKB1 plays a key role in liver cellular architecture and metabolism. We report that liver-specific deletion of LKB1 in mice leads to defective canaliculi and bile duct formation, causing impaired bile acid clearance and subsequent accumulation of bile acids in serum and liver. Concomitant with this, it was found that the majority of BSEP (bile salt export pump) was retained in intracellular pools rather than localized to the canalicular membrane in hepatocytes from LLKB1KO (liver-specific Lkb1-knockout) mice. Together, these changes resulted in toxic accumulation of bile salts, reduced liver function and failure to thrive. Additionally, circulating LDL (low-density lipoprotein)-cholesterol and non-esterified cholesterol levels were increased in LLKB1KO mice with an associated alteration in red blood cell morphology and development of hyperbilirubinaemia. These results indicate that LKB1 plays a critical role in bile acid homoeostasis and that lack of LKB1 in the liver results in cholestasis. These findings indicate a novel key role for LKB1 in the development of hepatic morphology and membrane targeting of canalicular proteins.

Two-sided culture of human placental trophoblast. Morphology, immunocytochemistry and permeability properties*

We describe the culture of human term placental trophoblast cells on cell-free amniotic membrane, with medium on both sides. Over the course of 2 days, the isolated cells, initially simple, mononucleated and probably cytotrophoblast, form a confluent layer of multinucleated syncytial cells with morphological and immunocytochemical properties of syncytiotrophoblast. This layer becomes polarized with respect to morphology, alkaline phosphatase distribution and hCG secretion. Contamination with amnion cells, and with other cell types that are present in placental tissue, was less than 1 per cent. A preliminary investigation of the permeability properties of the preparation showed that the trophoblast cell layer, rather than the amniotic membrane, was rate-limiting to transtrophoblast transfer, but that possible effects of the supporting membrane should be considered. The transtrophoblast transfer of D-glucose and the non-metabolisable analogue, 3-O-methyl-D-glucose (3OMG), had saturable and non-saturable/leak components in both directions, indicating that carrier-mediated processes were involved. The non-metabolisable amino acid 2-aminoisobutyrate (AIB) was both accumulated within the trophoblast cells, and transferred by saturable and non-saturable processes from the microvillous side, but no saturable accumulation or transfer was observed from the basal side, at the concentrations tried. The results suggest that this model may prove suitable for studies of transtrophoblast transfer.

A survey of the ultrastructural defects associated with absent or impaired human sperm motility

The sperm tails of 400 patients having absent or impaired sperm motility were examined by electron microscopy. A wide variety of fine-structural defects were observed although all of the patients fell into clearly defined groups. Total or partial dynein arm deficiency was observed in 12 patients (3%). Ninety-one patients (23%) had sperm with a spectrum of fine-structural defects, whereas 90 patients (23%) were necrospermic. Subjects with low motility, but with at least a few tails of normal structure, had a 5% pregnancy rate, whereas those patients with similar overall motility, but in whom no normal sperm were seen, produced no pregnancies. The results confirm the importance of making an electron microscopical examination of the sperm of patients with asthenozoospermia.

The endometrial nucleolar channel system as an indicator of progestin potency in HRT

A quantitative assessment has been made of nucleolar channel systems (NCS) in the endometrial glands of postmenopausal women receiving hormone replacement therapy. The women were taking conjugated equine oestrogen and one of five progestins. The number of NCS induced was related to the dose of progestin administered. The minimum doses of progestin inducing a comparable response to premenopausal secretory phase endometria were found to be 1-2.5 mg norethindrone, 150 micrograms norgestrel and 20 mg dydrogesterone. Progesterone and medroxyprogesterone acetate were inadequate at the doses tested. The results show that the quantification of endometrial gland NCS would be a useful addition to the biochemical and morphological assessments made of any new progestin treatment.

The inhibitory activity of a peptide derivative against the growth of simian immunodeficiency virus in C8166 cells

The peptide derivative Ro 31-8959 is a potent and selective inhibitor of the aspartic proteinases encoded by HIV-1 and HIV-2 and it arrests the growth of both viruses in cell culture. We have demonstrated similar effects against the simian immunodeficiency virus SIVmac251 in the human T-cell line, C8166 (ED50 = 6nM) with a therapeutic index of 4,500. The antiviral activity of Ro 31-8959 was 250 and 22 times greater than that of ddI and ddC, respectively. The mode of action was confirmed by accumulation of the polyprotein p55 with concomitant reduction of the cleavage product, p27, and by the production of immature virions.

BEHAVIOR OF A CELL LINE DERIVED FROM NORMAL HUMAN HEPATOCYTES ON NON- PHYSIOLOGICAL AND PHYSIOLOGICAL-TYPE SUBSTRATES: EVIDENCE FOR ENHANCEMENT OF SECRETION OF LIVER-SPECIFIC PROTEINS BY A THREE-DIMENSIONAL GROWTH PATTERN

SummaryThe behavior of a recently described cell line, HH25, derived from normal human hepatocytes, has been investigated on several different substrates—tissue-culture plastic, glass, a thin layer of rat-tail collagen I, and thin layers or thick gels of extracellular matrix derived from the Engelbreth-Holm-Swarm murine sarcoma (EHS matrix). Cellular morphology, proliferation, and secretion of three hepatocyte-specific proteins (albumin, α1 acid glycoprotein, and α1 antitrypsin) have been examined. There were no differences in morphology, proliferation, or differentiated function in the cells on either plastic, glass, collagen, I, or a thin layer of EHS matrix, but on a thick EHS matrix gel the cells altered their morphology (forming three-dimensional colonies with canalicular-like structures) and their production of albumin and α1 acid glycoprotein was enhanced. This suggests that the enhanced differentiated function is associated with the morphological change (occurring only on the thick EHS gel) rather than with receptor-mediated cell-matrix interactions (which can also occur on the thin layer of EHS matrix). This cell line is therefore a good in vitro cellular model for the investigation of the roles of morphological changes and of cell-cell and cell-matrix interactions in the control of human hepatocyte behavior without the need for an extensive source of primary tissue.