Steroidogenesis in the Ovarian Follicle of Medaka (Oryzias latipes, a daily spawner) during Oocyte Maturation

The metabolism of ¹⁴C-labeled steroid precursors by cell-free homogenates of medaka ( Oryzias latipes , a daily spawner) ovarian follicles at 12 different developmental stages was examined using thin layer chromatography (TLC). The radioactive metabolites produced were identified and tested for their ability to induce germinal vesicle breakdown (GVBD) in oocytes in an in vitro homologous bioassay. When homogenates of follicles isolated during oocyte maturation were incubated with ¹⁴C-labeled 17α-hydroxyprogesterone, 13 metabolites were detected in TLC. Among these metabolites, one metabolite exhibited very high maturation inducing activity by the in vitro bioassay. This metabolite was identified as 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP) by its chromatographic mobility in TLC and recrystallization to constant specific activity. 17α,20β-DP production was high in follicles collected between 10 and 6 hr before spawning. A much less biologically active metabolite, 17α,20β-dihydroxy-5β-pregnane-3-one appeared in follicles immediately after the formation of 17α,20β-DP. A similar pattern of steroidogenesis was observed when the follicles were incubated with ¹⁴C-labeled pregnenolone and progesterone. The timely synthesis of 17α,20β-DP in medaka at the onset of oocyte maturation, together with the demonstration that this progestogen is the most potent inducer of oocyte maturation in vitro , provides further evidence that 17α,20β-DP is the naturally occurring maturation-inducing hormone in the medaka. The results also suggest that the conversion of 17α,20β-DP to its 5β-reduced metabolite may be an inactivation process.     

In Vivo Study of Vitellogenin-Gold Transport in the Ovarian Follicle and Oocyte of Xenopus laevis

The transport of injected vitellogenin (VTG)-gold in the ovarian follicle and developing oocyte in Xenopus is described. The gold particles reached the extracellular spaces of the theca and interfollicular spaces within 1 and 2 hr, respectively, after a tracer injection at 20°C. The tracers moved through channels between the constitutive cells of both the capillary endothelium and the follicle cell layer.
Compartments in the peripheral cytoplasm of vitellogenic oocytes at stage IV, which relate to yolk formation, seemed to be segregated as follows: (a) internalization compartment consisting of coated pits and vesicles of the oolemma covering the oocyte "macrovilli", (b) transport compartment of endosomes and multivesicular endosomes in the oocyte cortex, and (c) crystallization compartment of primordial yolk platelets (PYP) in the sub-cortical region. The gold particles appeared in the internalization and transport compartments at 3–6 hr after the tracer injection and in the cystallization compartment at 12–18 hr. The VTG, internalized by receptor-mediated endocytosis, was transferred from coated vesicles to multivesicular endosomes by vesicle-to-vesicle fusion. VTG crystallization took place in globular-shaped PYPs of about 1 μm. At 24 hr after the tracer injection, the gold particles appeared in completely crystallized yolk platelets, most of them clustered in the superficial layer and some integrated into the crystals.     

Di (2-ethylhexyl) Phthalate Exposure Impairs Growth of Antral Follicle in Mice

Di (2-ethylhexyl) phthalate (DEHP) is a widely used plastic additive. As an environmental endocrine disruptor, it has been shown to be harmful to the mammalian reproductive system. Previous studies indicated that DEHP inhibited the development of mouse ovarian follicles. However, the mechanisms by which DEHP affects ovarian antral follicle development during the pre-puberty stage are poorly understand. Thus, we investigated the effects of direct DEHP exposure on antral follicle growth in pre-pubescent mice by use of intraperitoneal injection. Our results demonstrated that the percentage of large antral follicles was significantly reduced when mice were exposed to 20 or 40 μg/kg DEHP every 5 days from postnatal day 0 (0 dpp) to 15 dpp. In 20 dpp, we performed microarray of these ovaries. The microarray results indicated that mRNA levels of apoptosis related genes were increased. The mRNA levels of the apoptosis and cell proliferation (negative) related genes Apoe, Agt, Glo1 and Grina were increased after DEHP exposure. DEHP induced the differential gene expression of Hsp90ab1, Rhoa, Grina and Xdh which may play an important role in this process. In addition, TUNEL staining and immunofluorescence showed that DEHP exposure significantly increased the number of TUNEL, Caspase3 and γH2AX positive ovarian somatic cells within the mouse ovaries. Flow cytometer analyses of redox-sensitive probes showed that DEHP caused the accumulation of reactive oxygen species. Moreover, the mRNA expression of ovarian somatic cell antioxidative enzymes was down-regulated both in vivo and in vitro. In conclusion, our data here demonstrated that DEHP exposure induced oxidative stress and ovarian somatic cell apoptosis, and thus may impact antral follicle enlargement during the pre-pubertal stage in mice.     

Reverse Mutational Analysis Reveals Threonine-373 as a Potentially Sufficient Phosphorylation Site for Inactivation of the Retinoblastoma Tumor Suppressor Protein (pRB)

Previous studies in our laboratory have shown that constitutive cyclin E expression can alleviate the requirement for cyclin Δ-CDK activity in the inactivation of the retinoblastoma protein (pRb). RbΔCDK, a mutant construct of pRb with 15 of the 16 CDK phosphorylation sites mutated to alanine represses activation of E2F by mitogen, despite cyclin E over-expression. However, restoration of the four cyclin E-CDK2 phosphorylation sites to RbΔCDK renders this construct sensitive to inactivation by CDK phosphorylation. In the present study, we engage a “reverse mutational analysis” by restoring cyclin E-CDK2 phosphorylation sites to RbΔCDK individually and in combinations in an attempt to discover phosphorylation sites on Rb that are critical for inactivation. Surprisingly, we report that, in both rodent and human cells, restoration of threonine-373 to RbΔCDK, alone or in combination with other phospho-resotrations, results in a loss of the constitutively repressive effect of this construct on E2F activation. Further, induction of endogenous cyclin A protein is blocked by RbΔCDK, but not by mutants of RbΔCDK containing a restored threonine-373. Finally, while S phase entry is blocked by expression of RbΔCDK, restoration of threonine-373 largely attenuates this effect. These findings reveal that phosphorylation of threonine-373 by CDK2-cyclin E represent a potentially crucial event in the inactivation of the pRb protein.     

Induction of Ovarian Leiomyosarcomas in Mice by Conditional Inactivation of Brca1 and p53

Background

Approximately one out of every ten cases of epithelial ovarian cancer (EOC) is inherited. The majority of inherited cases of EOC result from mutations in the breast cancer associated gene 1 (BRCA1). In addition to mutation of BRCA1, mutation of the p53 gene is often found in patients with inherited breast and ovarian cancer syndrome.

Methodology/Principal Findings

We investigated the role of loss of function of BRCA1 and p53 in ovarian cancer development using mouse models with conditionally expressed alleles of Brca1 and/or p53. Our results show that ovary-specific Cre-recombinase-mediated conditional inactivation of both Brca1^LoxP/LoxP and p53^LoxP/LoxP resulted in ovarian or reproductive tract tumor formation in 54% of mice, whereas conditional inactivation of either allele alone infrequently resulted in tumors (≤5% of mice). In mice with conditionally inactivated Brca1^LoxP/LoxP and p53^LoxP/LoxP, ovarian tumors arose after long latency with the majority exhibiting histological features consistent with high grade leiomyosarcomas lacking expression of epithelial, follicular or lymphocyte markers. In addition, tumors with conditional inactivation of both Brca1^LoxP/LoxP and p53^LoxP/LoxP exhibited greater genomic instability compared to an ovarian tumor with inactivation of only p53^LoxP/LoxP.

Conclusions/Significance

Although conditional inactivation of both Brca1 and p53 results in ovarian tumorigenesis, our results suggest that additional genetic alterations or alternative methods for targeting epithelial cells of the ovary or fallopian tube for conditional inactivation of Brca1 and p53 are required for the development of a mouse model of Brca1-associated inherited EOC.     

Decreased Peritoneal Ovarian Cancer Growth in Mice Lacking Expression of Lipid Phosphate Phosphohydrolase 1

Lysophosphatidic acid (LPA) is a bioactive lipid that enhances ovarian cancer cell proliferation, migration and invasion in vitro and stimulates peritoneal metastasis in vivo. LPA is generated through the action of autotaxin or phospholipases, and degradation begins with lipid phosphate phosphohydrolase (LPP)-dependent removal of the phosphate. While the effects of LPA on ovarian cancer progression are clear, the effects of LPA metabolism within the tumor microenvironment on peritoneal metastasis have not been reported. We examined the contribution of lipid phosphatase activity to ovarian cancer peritoneal metastasis using mice deficient in LPP1 expression. Homozygous deletion of LPP1 (LPP1 KO) results in elevated levels and decreased turnover of LPA in vivo. Within 2 weeks of intraperitoneal injection of syngeneic mouse ovarian cancer cells, we observed enhanced tumor seeding in the LPP1 KO mice compared to wild type. However, tumor growth plateaued in the LPP1 KO mice by 3 weeks while tumors continued to grow in wild type mice. The decreased tumor burden was accompanied by increased apoptosis and no change in proliferation or angiogenesis. Tumor growth was restored and apoptosis reversed with exogenous administration of LPA. Together, these observations demonstrate that the elevated levels of LPA per se in LPP1 KO mice do not inhibit tumor growth. Rather, the data support the notion that either elevated LPA concentration or altered LPA metabolism affects other growth-promoting contributions of the tumor microenvironment.     

LINE-1 Mediated Insertion into Poc1a (Protein of Centriole 1 A) Causes Growth Insufficiency and Male Infertility in Mice

Skeletal dysplasias are a common, genetically heterogeneous cause of short stature that can result from disruptions in many cellular processes. We report the identification of the lesion responsible for skeletal dysplasia and male infertility in the spontaneous, recessive mouse mutant chagun. We determined that Poc1a, encoding protein of the centriole 1a, is disrupted by the insertion of a processed Cenpw cDNA, which is flanked by target site duplications, suggestive of a LINE-1 retrotransposon-mediated event. Mutant fibroblasts have impaired cilia formation and multipolar spindles. Male infertility is caused by defective spermatogenesis early in meiosis and progressive germ cell loss. Spermatogonial stem cell transplantation studies revealed that Poc1a is essential for normal function of both Sertoli cells and germ cells. The proliferative zone of the growth plate is small and disorganized because chondrocytes fail to re-align after cell division and undergo increased apoptosis. Poc1a and several other genes associated with centrosome function can affect the skeleton and lead to skeletal dysplasias and primordial dwarfisms. This mouse mutant reveals how centrosome dysfunction contributes to defects in skeletal growth and male infertility.     

Shift in Steroidogenesis in the Ovarian Follicles of the Goldfish (Carassius auratus) during Gonadotropin-Induced Oocyte Maturation

Both partially purified chum salmon gonadotropin and 17α-hydroxyprogesterone stimulated in vitro production of testosterone by postvitellogenic follicles of goldfish ( Carassius auratus ). Chum salmon gonadotropin further enhanced the conversion of exogenously supplied 17α-hydroxyprogesterone to 17α, 20β-dihydroxy-4-pregnen-3-one. The increased medium concentrations of 17α, 20β-dihydroxy-4-pregnen-3-one were associated with the induction of final oocyte maturation.
The capacity of postvitellogenic follicles to produce steroids in response to exogenous 17α-hydroxyprogesterone was examined in females at various stages of final oocyte maturation following the administration of human chorionic gonadotropin in vivo combined with elevation of holding temperature. The maximum production of testosterone in response to 17α-hydroxyprogesterone was obtained in follicles from initial controls. In contrast, 17α 20β-diOHprog production was very low in initial controls and markedly increased during oocyte maturation (3–6 hr following injection), followed by a significant decrease in follicles collected at 15 hr. Estradiol-17β production by the follicles was very low at any stages of gonadotropin-induced oocyte maturation. These results suggest that gonadotropin-induced shift in the biosynthetic pathway in the follicle from the secretion of predominantly testosterone to 17α, 20β-dihydroxy-4-pregnen-3-one secretion is a prerequisite step for the induction of oocyte maturation in goldfish.     

A Neo-Sex Chromosome That Drives Postzygotic Sex Determination in the Hessian Fly (Mayetiola destructor)

Two nonoverlapping autosomal inversions defined unusual neo-sex chromosomes in the Hessian fly (Mayetiola destructor). Like other neo-sex chromosomes, these were normally heterozygous, present only in one sex, and suppressed recombination around a sex-determining master switch. Their unusual properties originated from the anomalous Hessian fly sex determination system in which postzygotic chromosome elimination is used to establish the sex-determining karyotypes. This system permitted the evolution of a master switch (Chromosome maintenance, Cm) that acts maternally. All of the offspring of females that carry Cm-associated neo-sex chromosomes attain a female-determining somatic karyotype and develop as females. Thus, the chromosomes act as maternal effect neo-W''s, or W-prime (W′) chromosomes, where ZW′ females mate with ZZ males to engender female-producing (ZW′) and male-producing (ZZ) females in equal numbers. Genetic mapping and physical mapping identified the inversions. Their distribution was determined in nine populations. Experimental matings established the association of the inversions with Cm and measured their recombination suppression. The inversions are the functional equivalent of the sciarid X-prime chromosomes. We speculate that W′ chromosomes exist in a variety of species that produce unisexual broods.SEX chromosomes are usually classified as X, Y, Z, or W on the basis of their pattern of segregation and the gender of the heterogametic sex (Ohno 1967). However, when chromosome-based sex determination occurs postzygotically, the same nomenclature confounds important distinctions and may hide interesting evolutionary phenomena. The Hessian fly (Mayetiola destructor), a gall midge (Diptera: Cecidomyiidae) and an important insect pest of wheat, presents an excellent example (Stuart and Hatchett 1988, 1991). In this insect, all of the female gametes and all of the male gametes have the same number of X chromosomes (Figure 1A); no heterogametic sex exists. Nevertheless, Hessian fly sex determination is chromosome based; postzygotic chromosome elimination produces different X chromosome to autosome ratios in somatic cells (male A1A2X1X2/A1A2OO and female A1A2X1X2/A1A2X1X2, where A1 and A2 are the autosomes, X1 and X2 are the X chromosomes, and the paternally derived chromosomes follow the slash) (Stuart and Hatchett 1991; Marin and Baker 1998). Thus, Hessian fly “X” chromosomes are defined by their haploid condition in males, rather than by their segregation in the gametes.Open in a separate windowFigure 1.—Chromosome behavior and sex determination in the Hessian fly. (A) Syngamy (1) establishes the germ-line chromosome constitution: ∼32 maternally derived E chromosomes (represented as a single white chromosome) and both maternally derived (black) and paternally derived (gray) autosomes and X chromosomes. During embryogenesis, while the E chromosomes are eliminated, the paternally derived X chromosomes are either retained (2) or excluded (3) from the presumptive somatic cells. When the paternally derived X chromosomes are retained (2), a female-determining karyotype is established. When they are eliminated (3), a male-determining karyotype is established. Thelygenic mothers carry Cm (white arrow), which conditions all of their offspring to retain the X chromosomes. Recombination occurs during oogenesis (4). All ova contain a full complement of E chromosomes and a haploid complement of autosomes and X chromosomes. Chromosome elimination occurs during spermatogenesis (5). Sperm contain only the maternally derived autosomes and X chromosomes. (B) The segregation of Cm (white dot) on a Hessian fly autosome among monogenic families. Thelygenic females produce broods composed of equal numbers of thelygenic (Cm/−) and arrhenogenic (−/−) females (box 1). Arrhenogenic females produce males (box 2). (C) Matings between monogenic and amphigenic families. Cm (white dot) is dominant to the amphigenic-derived chromosomes (gray dot) and generates all-female offspring (box 3). Amphigenic-derived chromosomes are dominant to the arrhenogenic-derived chromosomes (no dot) and generate offspring of both sexes (box 4).An autosomal, dominant, genetic factor called Chromosome maintenance (Cm) complicates Hessian fly sex determination further (Stuart and Hatchett 1991). Cm has a maternal effect that acts upstream of X chromosome elimination during embryogenesis (Figure 1A). It prevents X chromosome elimination so that all of the offspring of Cm-bearing mothers obtain a female-determining karyotype. Cm-bearing females produce only female offspring and are therefore thelygenic. The absence of Cm usually has the opposite effect; all of the offspring of most Cm-lacking females obtain a male-determining karyotype. These Cm-lacking females produce only male offspring and are therefore arrhenogenic. Like a sex-determining master switch, Cm is usually heterozygous and present in only one sex (Figure 1B). Thus, thelygenic females (Cm/−) are “heterogametic,” as their Cm-containing gametes and Cm-lacking gametes produce thelygenic (Cm/−) and arrhenogenic (−/−) females in a 1:1 ratio. Collectively, thelygenic and arrhenogenic females are called monogenic because they produce unisexual families. However, some Hessian fly females produce broods of both sexes and are called amphigenic. No mating barrier between monogenic and amphigenic families exists (Figure 1C), but amphigenic females have always been found in lower abundance (Painter 1930; Gallun et al. 1961; Stuart and Hatchett 1991). In experimental matings, the inheritance of maternal phenotype was consistent with the segregation of three Cm alleles (Figure 1C): a dominant thelygenic allele, a hypomorphic amphigenic allele, and a null arrhenogenic allele (Stuart and Hatchett 1991).Here we report the genetic and physical mapping of Cm on Hessian fly autosome 1 (A1). Two nonoverlapping inversions were identified that segregated perfectly with Cm. The most distal inversion was present in all thelygenic females examined. The more proximal inversion extended recombination suppression. These observations suggested that successive inversions evolved to suppress recombination around Cm after it arose. The inversions therefore appear to have evolved in response to the forces that shaped vertebrate Y and W chromosomes (Charlesworth 1996; Graves and Shetty 2001; Rice and Chippindale 2001; Carvalho and Clark 2005). We therefore believe the inversion-bearing chromosomes may be classified as maternal effect neo-W''s.     

Insulin-Like Growth Factor 1 (IGF-1) Enhances the Protein Expression of CFTR

Low levels of insulin-like growth factor 1 (IGF-1) have been observed in the serum of cystic fibrosis (CF) patients. However, the effects of low serum IGF-1 on the cystic fibrosis transmembrane conductance regulator (CFTR), whose defective function is the primary cause of cystic fibrosis, have not been studied. Here, we show in human cells that IGF-1 increases the steady-state levels of mature wildtype CFTR in a CFTR-associated ligand (CAL)- and TC10-dependent manner; moreover, IGF-1 increases CFTR-mediated chloride transport. Using an acceptor photobleaching fluorescence resonance energy transfer (FRET) assay, we have confirmed the binding of CAL and CFTR in the Golgi. We also show that CAL overexpression inhibits forskolin-induced increases in the cell-surface expression of CFTR. We found that IGF-1 activates TC10, and active TC10 alters the functional association between CAL and CFTR. Furthermore, IGF-1 and active TC10 can reverse the CAL-mediated reduction in the cell-surface expression of CFTR. IGF-1 does not increase the expression of ΔF508 CFTR, whose processing is arrested in the ER. This finding is consistent with our observation that IGF-1 alters the functional interaction of CAL and CFTR in the Golgi. However, when ΔF508 CFTR is rescued with low temperature or the corrector VRT-325 and proceeds to the Golgi, IGF-1 can increase the expression of the rescued ΔF508 CFTR. Our data support a model indicating that CAL-CFTR binding in the Golgi inhibits CFTR trafficking to the cell surface, leading CFTR to the degradation pathway instead. IGF-1-activated TC10 changes the interaction of CFTR and CAL, allowing CFTR to progress to the plasma membrane. These findings offer a potential strategy using a combinational treatment of IGF-1 and correctors to increase the post-Golgi expression of CFTR in cystic fibrosis patients bearing the ΔF508 mutation.     

NS1-Binding Protein (NS1-BP): a Novel Human Protein That Interacts with the Influenza A Virus Nonstructural NS1 Protein Is Relocalized in the Nuclei of Infected Cells

We used the yeast interaction trap system to identify a novel human 70-kDa protein, termed NS1-binding protein (NS1-BP), which interacts with the nonstructural NS1 protein of the influenza A virus. The genetic interaction was confirmed by the specific coprecipitation of the NS1 protein from solution by a glutathione S-transferase–NS1-BP fusion protein and glutathione-Sepharose. NS1-BP contains an N-terminal BTB/POZ domain and five kelch-like tandem repeat elements of ~50 amino acids. In noninfected cells, affinity-purified antibodies localized NS1-BP in nuclear regions enriched with the spliceosome assembly factor SC35, suggesting an association of NS1-BP with the cellular splicing apparatus. In influenza A virus-infected cells, NS1-BP relocalized throughout the nucleoplasm and appeared distinct from the SC35 domains, which suggests that NS1-BP function may be disturbed or altered. The addition of a truncated NS1-BP mutant protein to a HeLa cell nuclear extract efficiently inhibited pre-mRNA splicing but not spliceosome assembly. This result could be explained by a possible dominant-negative effect of the NS1-BP mutant protein and suggests a role of the wild-type NS1-BP in promoting pre-mRNA splicing. These data suggest that the inhibition of splicing by the NS1 protein may be mediated by binding to NS1-BP.     

Inhibiting Heat Shock Protein 90 (HSP90) Limits the Formation of Liver Cysts Induced by Conditional Deletion of Pkd1 in Mice

Polycystic liver disease (PLD) occurs in 75–90% of patients affected by autosomal dominant polycystic kidney disease (ADPKD), which affects 1∶400–1,000 adults and arises from inherited mutations in the PKD1 or PKD2 genes. PLD can lead to bile duct obstructions, infected or bleeding cysts, and hepatomegaly, which can diminish quality of life. At present, no effective, approved therapy exists for ADPKD or PLD. We recently showed that inhibition of the molecular chaperone heat shock protein 90 (HSP90) with a small molecule inhibitor, STA-2842, induced the degradation of multiple HSP90-dependent client proteins that contribute to ADPKD pathogenesis and slowed the progression of renal cystogenesis in mice with conditional deletion of Pkd1. Here, we analyzed the effects of STA-2842 on liver size and cystic burden in Pkd^-/- mice with established PLD. Using magnetic resonance imaging over time, we demonstrate that ten weeks of STA-2842 treatment significantly reduced both liver mass and cystic index suggesting selective elimination of cystic tissue. Pre-treatment cystic epithelia contain abundant HSP90; the degree of reduction in cysts was accompanied by inhibition of proliferation-associated signaling proteins EGFR and others, and induced cleavage of caspase 8 and PARP1, and correlated with degree of HSP90 inhibition and with inactivation of ERK1/2. Our results suggest that HSP90 inhibition is worth further evaluation as a therapeutic approach for patients with PLD.     

Formation of the G-quadruplex and i-motif structures in retinoblastoma susceptibility genes (Rb)

The formation of G-quadruplex and i-motif structures in the 5′ end of the retinoblastoma (Rb) gene was examined using chemical modifications, circular dichroism (CD) and fluorescence spectroscopy. It was found that substitutions of 8-methylguanine at positions that show syn conformations in antiparallel G-quadruplexes stabilize the structure in the G-rich strand. The complementary C-rich 18mer forms an i-motif structure, as suggested by CD spectroscopy. Based on the C to T mutation experiments, C bases participated in the C–C⁺ base pair of the i-motif structure were determined. Experiments of 2-aminopurine (2-AP) substitution reveal that an increase of fluorescence in the G-quadruplex relative to duplex is attributed to unstacked 2-AP within the loop of G-quadruplex. The fluorescence experiments suggest that formation of the G-quadruplex and i-motif can compete with duplex formation. Furthermore, a polymerase arrest assay indicated that formation the G-quadruplex structure in the Rb gene acts as a barrier in DNA synthesis.     

Morbid Obesity Resulting from Inactivation of the Ciliary Protein CEP19 in Humans and Mice

Obesity is a major public health concern, and complementary research strategies have been directed toward the identification of the underlying causative gene mutations that affect the normal pathways and networks that regulate energy balance. Here, we describe an autosomal-recessive morbid-obesity syndrome and identify the disease-causing gene defect. The average body mass index of affected family members was 48.7 (range = 36.7–61.0), and all had features of the metabolic syndrome. Homozygosity mapping localized the disease locus to a region in 3q29; we designated this region the morbid obesity 1 (MO1) locus. Sequence analysis identified a homozygous nonsense mutation in CEP19, the gene encoding the ciliary protein CEP19, in all affected family members. CEP19 is highly conserved in vertebrates and invertebrates, is expressed in multiple tissues, and localizes to the centrosome and primary cilia. Homozygous Cep19-knockout mice were morbidly obese, hyperphagic, glucose intolerant, and insulin resistant. Thus, loss of the ciliary protein CEP19 in humans and mice causes morbid obesity and defines a target for investigating the molecular pathogenesis of this disease and potential treatments for obesity and malnutrition.