Darryl L. Russell

Obese Women Exhibit Differences in Ovarian Metabolites, Hormones, and Gene Expression Compared with Moderate-Weight Women

CONTEXT Obese women experience longer times to conception, even if they are young and cycling regularly, which is suggestive of alterations in ovarian function during the periconceptual period. OBJECTIVE This study sought to determine whether there are alterations in the preovulatory follicular environment that are likely to influence oocyte developmental competence. DESIGN, SETTING, AND PARTICIPANTS Women attending a private infertility clinic were categorized into body mass index (BMI) groups of moderate (n = 33; BMI 20-24.9 kg/m(2)), overweight (n = 31; BMI 25-29.9 kg/m(2)), and obese (n =32; BMI >or=30 kg/m(2)). INTERVENTION For each patient, follicular fluid was recovered from single follicles at oocyte retrieval, granulosa cells were pooled from multiple follicular aspirates and cumulus cells were pooled after separation from the oocytes. MAIN OUTCOME MEASURES Follicle fluid was assayed for hormones and metabolites. Granulosa and cumulus cells were analyzed for mRNA expression of insulin signaling components (IRS-2 and Glut4), glucose-regulated genes (ChREBP, ACC, and FAS) and insulin-regulated genes (SREBP-1, CD36, and SR-BI) associated with obesity/insulin resistance. RESULTS Increasing BMI was associated with increased follicular fluid insulin (P < 0.001), lactate (P = 0.01), triglycerides (P = 0.0003), and C-reactive protein (P < 0.0001) as well as decreased SHBG (P = 0.001). IRS-2, Glut4, ChREBP, and SREBP exhibited cell-type-specific expression but were not affected by BMI. CD36 and SRBI mRNA were modestly altered in granulosa cells of obese compared with moderate-weight women. CONCLUSIONS Obese women exhibit an altered ovarian follicular environment, particularly increased metabolite, C-reactive protein, and androgen activity levels, which may be associated with poorer reproductive outcomes typically observed in these patients.

Beta-Oxidation Is Essential for Mouse Oocyte Developmental Competence and Early Embryo Development

Oocyte and embryo metabolism are closely linked with their subsequent developmental capacity. Lipids are a potent source of cellular energy, yet little is known about lipid metabolism during oocyte maturation and early embryo development. Generation of ATP from lipids occurs within mitochondria via beta-oxidation of fatty acids, with the rate-limiting step catalyzed by carnitine palmitoyl transferase I (CPT1B), a process also requiring carnitine. We sought to investigate the regulation and role of beta-oxidation during oocyte maturation and preimplantation development. Expression of Cpt1b mRNA, assessed by real-time RT-PCR in murine cumulus-oocyte complexes (COCs), increased following hormonal induction of oocyte maturation and ovulation in vivo with human chorionic gonadotropin (5 IU) and in embryos reaching the blastocyst stage. Beta-oxidation, measured by the production of 3H2O from [3H]palmitic acid, was significantly increased over that in immature COCs following induction of maturation in vitro with epidermal growth factor (3 ng/ml) and follicle-stimulating hormone (50 mIU/ml). The importance of lipid metabolism for oocyte developmental competence and early embryo development was demonstrated by assessing the rate of embryo development following inhibition or upregulation of beta-oxidation with etomoxir (an inhibitor of CPT1B) or l-carnitine, respectively. Inhibition of beta-oxidation during oocyte maturation or zygote cleavage impaired subsequent blastocyst development. In contrast, l-carnitine supplementation during oocyte maturation significantly increased beta-oxidation, improved developmental competence, and in the absence of a carbohydrate energy supply, significantly increased 2-cell cleavage. Thus, carnitine is an important cofactor for developing oocytes, and fatty acids are an important energy source for oocyte and embryo development.

MOLECULAR MECHANISMS OF OVULATION AND LUTEINIZATION

Ovulation is a complex process initiated by the mid-cycle surge of luteinizing hormone (LH). Once initiated, a cascade of events occurs that culminates in the release of a fertilizable oocyte. The complex series of events involves specific ovarian cell types, diverse signaling pathways and temporally controlled expression of specific genes. This review will focus on several genes shown to control the ovulation process.

High-fat diet causes lipotoxicity responses in cumulus-oocyte complexes and decreased fertilization rates.

In obesity, accumulation of lipid in nonadipose tissues, or lipotoxicity, is associated with endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and ultimately apoptosis. We have previously shown that obese women have increased triglycerides in follicular fluid; thus, the present study examined whether high-fat diet-induced obesity causes lipotoxicity in granulosa cells and the cumulus-oocyte complex (COC). Oocytes of mice fed a high-fat diet had dramatically increased lipid content and reduced mitochondrial membrane potential compared to those of mice fed a control diet. COCs from mice fed a high-fat diet had increased expression of ER stress marker genes ATF4 and GRP78. Apoptosis was increased in granulosa and cumulus cells of mice fed a high-fat diet. Mice fed a high-fat diet also exhibited increased anovulation and decreased in vivo fertilization rates. Thus, lipid accumulation, ER stress, mitochondrial dysfunction, and apoptosis are markedly increased in ovarian cells of mice fed a high-fat diet. ER stress markers were also analyzed in granulosa cells and follicular fluid from women with varying body mass indices (BMI). ATF4 was increased in granulosa cells and [Ca(2+)] in follicular fluid from obese women compared to nonobese women. These results indicate that lipotoxicity may be occurring in ovarian cells of obese women and may contribute to the reduced pregnancy rates observed in response to obesity.

The biological role and regulation of versican levels in cancer

Increased expression of the proteoglycan, versican is strongly associated with poor outcome for many different cancers. Depending on the cancer type, versican is expressed by either the cancer cells themselves or by stromal cells surrounding the tumor. Versican plays diverse roles in cell adhesion, proliferation, migration and angiogenesis, all features of invasion and metastasis. These wide ranging functions have been attributed to the central glycosaminoglycan-binding region of versican, and to the N-(G1) and C-(G3) terminal globular domains which collectively interact with a large number of extracellular matrix and cell surface structural components. Here we review the recently identified mechanisms responsible for the regulation of versican expression and the biological roles that versican plays in cancer invasion and metastasis. The regulation of versican expression may represent one mechanism whereby cancer cells alter their surrounding microenvironment to facilitate the malignant growth and invasion of several tumor types. A greater understanding of the regulation of versican expression may contribute to the development of therapeutic methods to inhibit versican function and tumor invasion.

Ovulation: a multi-gene, multi-step process.

The luteinizing hormone (LH) surge initiates a cascade of proteolytic events that control ovulation. One of the genes induced by LH is the progesterone receptor (PR). Because mice with a mutant PR gene (PRKO) fail to ovulate and are infertile, we have used them as a model in which to determine PR target genes that might mediate the ovulatory process. The matrix metalloproteinases (MMPs: MMP2, MMP9, and MMP13) appear to be expressed in ovaries of PRKO mice in a manner similar to that in their wild-type littermates. However, the expression of two other types of proteases, cathepsin L (a member of the papain family) and ADAMTS-1 (A Disintegrin And Metalloproteinase with Thrombospondin-like motifs), are selectively induced in granulosa cells of preovulatory follicles by the LH surge. Maximal levels of these proteases are observed at 12-16 h after an LH surge, the time of ovulation. Furthermore, mRNAs encoding cathepsin L and ADAMTS-1 are reduced in the PRKO mice compared to their wild-type littermates. These novel observations indicate that these two proteases regulate some key step(s) controlling ovulation.

Adamts-1 Is Essential for the Development and Function of the Urogenital System

Abstract Successful ovulation and implantation processes play a crucial role in female fertility. Adamts-1, a matrix metalloproteinase with disintegrin and thrombospondin motifs, has been suggested to be regulated by the progesterone receptor in the hormonal pathway leading to ovulation. With the primary aim of investigating the role of Adamts-1 in female fertility, we generated Adamts-1 null mice. Forty-five percent of the newborn Adamts-1 null mice die, with death most likely caused by a kidney malformation that becomes apparent at birth. Surviving female null mice were subfertile, whereas males reproduced normally. Ovulation in null females was impaired because of mature oocytes remaining trapped in ovarian follicles. No uterine phenotype was apparent in Adamts-1 null animals. Embryo implantation occurred normally, the uteri were capable of undergoing decidualization, and no morphological changes were observed. These results demonstrate that a functional Adamts-1 is required for normal ovulation to occur, and hence the Adamts-1 gene plays an important role in female fertility, primarily during the tissue remodeling process of ovulation.

Ovarian Expression of a Disintegrin and Metalloproteinase with Thrombospondin Motifs During Ovulation in the Gonadotropin-Primed Immature Rat

Abstract Mammalian ovulation is a dynamic process that requires degradation of the collagenous connective tissue in the thecal layers of a mature follicle. In this reverse transcription-polymerase chain reaction differential display study, gonadotropin-primed immature rats were used to detect ovarian expression of a relatively new type of disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-1) that is known to cleave extracellular matrix in acutely inflamed tissues. Immature Wistar rats were primed with 10 IU eCG s.c., and the temporal pattern of expression of the ADAMTS-1 gene was delineated by extracting ovarian RNA at 0, 2, 4, 8, 12, and 24 h after induction of ovulation by injecting the primed animals with 10 IU hCG s.c. The differential display data, Northern analyses, and in situ hybridization micrographs all showed significant up-regulation of ADAMTS-1 gene expression by 8 h after hCG administration. The in situ data indicated that the ADAMTS-1 mRNA was in the granulosa layer of mature follicles. Expression reached a peak at 12 h and remained elevated at 24 h after hCG. ADAMTS-1 gene expression was impaired by the antiprogesterone agent epostane, but this inhibition could be overcome by exogenous progesterone. ADAMTS-1 expression was not affected when ovulation was blocked by treatment of the animals with the anti-eicosanoid agent indomethacin. In conclusion, the temporal pattern of expression of this gene, and its apparent regulation by progesterone, suggests that ADAMTS-1 has a significant role in the inflammatory events of the ovulatory process.

Formation of Hyaluronan- and Versican-rich Pericellular Matrix by Prostate Cancer Cells Promotes Cell Motility

Previous studies have demonstrated that high levels of hyaluronan (HA) and the chondroitin sulfate proteoglycan, versican in the peritumoral stroma are associated with metastatic spread of clinical prostate cancer. In vitro integration of HA and versican into a pericellular sheath is a prerequisite for proliferation and migration of vascular smooth muscle cells. In this study, a particle exclusion assay was used to determine whether human prostate cancer cell lines are capable of assembling a pericellular sheath following treatment with versican-containing medium and whether formation of a pericellular sheath modulated cell motility. PC3 and DU145, but not LNCaP cells formed prominent polarized pericellular sheaths following treatment with prostate fibroblast-conditioned medium. The capacity to assemble a pericellular sheath correlated with the ability to express membranous HA receptor, CD44. HA and versican histochemical staining were observed surrounding PC3 and DU145 cells following treatment with prostatic fibroblast-conditioned medium. The dependence on HA for integrity of the pericellular sheath was demonstrated by its removal following treatment with hyaluronidase. Purified versican or conditioned medium from Chinese hamster ovary K1 cells overexpressing versican V1, but not conditioned medium from parental cells, promoted pericellular sheath formation and motility of PC3 cells. Using time lapse microscopy, motile PC3 cells treated with versican but not non-motile cells exhibited a polar pericellular sheath. Polar pericellular sheath was particularly evident at the trailing edge but was excluded from the leading edge of PC3 cells. These studies indicate that prostate cancer cells recruit stromal components to remodel their pericellular environment and promote their motility.

Human cumulus cell gene expression as a biomarker of pregnancy outcome after single embryo transfer

OBJECTIVE To identify the cumulus cell gene expression associated with oocyte developmental competence, specifically live birth, after single ET (SET) assisted reproductive technology. DESIGN Retrospective gene expression analysis in human cumulus cells from oocytes that established a pregnancy resulting in live birth versus no pregnancy after SET. SETTING Independent IVF clinic and research institute. PATIENT(S) Women undergoing IVF/intracytoplasmic sperm injection with SET. INTERVENTION(S) Quantitative reverse-transcriptase-polymerase chain reaction analysis was performed on cumulus masses collected before insemination. Oocytes and embryos were cultured and transferred independently in 38 patients undergoing elective SET. Paired cumulus samples from oocytes that developed into high- versus low-grade embryos also were compared. MAIN OUTCOME MEASURE(S) Gene expression profiles of metabolic (ALDOA, LDHA, PFKP, PKM2), signaling (AHR, GREM1, PTGS2, STS), extracellular matrix (HAS2, PTX3, TNFAIP6, VCAN), and loading control GAPDH in individual cumulus masses. RESULT(S) VCAN and PTGS2 mRNA expression was significantly higher in cumulus cells from oocytes yielding a pregnancy resulting in a live birth, while PTX3 mRNA expression trended toward higher expression in pregnant samples. Cumulus cell levels of VCAN, GREM1, and PFKP correlated with birth weight in patients at 38 weeks of gestation. No genes correlated with clinical embryo morphology scores. CONCLUSION(S) Cumulus cell VCAN, PTGS2, GREM1, and PFKP expression may identify oocytes with high developmental potential, leading to enhanced implantation rates and greater developmental capacity throughout gestation.