ABSTRACT
PF20 was first identified in Chlamydomonas rheinhardtii as an essential component of the axoneme central apparatus. We discovered that the mouse Pf20 gene encodes two major transcripts (2.5 and 1.4 kb), which are expressed in different patterns during spermatogenesis, yielding proteins of 71 and 35 kDa, respectively. Both proteins contain contiguous WD repeats in their C termini. The meiotically expressed 71-kDa protein is incorporated into the central apparatus, whereas the 35-kDa protein, which accumulates in postmeiotic male germ cells, is abundant in the nucleus. We disrupted the Pf20 gene domains that encode the C-terminal WD repeats in embryonic stem cells. Highly chimeric mice carrying the mutant Pf20 allele had impaired spermatogenesis with a significant loss of germ cells at the round spermatid stage, in association with disorganization of sperm axoneme structure. The mutated Pf20 allele was never transmitted, indicating that Pf20 haploinsufficiency caused the defects in spermatogenesis. The 35-kDa PF20 protein was shown to bind to meiosis-expressed gene 1 (MEIG1), a chromosome/chromatin-binding protein initially expressed during meiosis but retained in the germ cell nucleus throughout later stages of spermatogenesis. Our findings reveal an essential role for Pf20 in mouse spermatogenesis, sustaining postmeiotic germ cell viability. The different patterns of expression of the two PF20 proteins suggest the possibility that the Pf20 gene has multiple functions during spermatogenesis.
Subject(s)
Microtubule-Associated Proteins/genetics , Protozoan Proteins/genetics , Spermatogenesis/genetics , Animals , Cell Cycle Proteins , Cell Nucleus/metabolism , Chimera/genetics , Chimera/metabolism , GRB10 Adaptor Protein , Gene Targeting , Gonadal Steroid Hormones/blood , Male , Mice , Microtubule-Associated Proteins/deficiency , Microtubule-Associated Proteins/metabolism , Nuclear Proteins , Phosphoproteins , Protein Isoforms/genetics , Protein Isoforms/metabolism , Proteins/metabolism , Protozoan Proteins/metabolism , Spermatogenesis/physiologyABSTRACT
The StAR-related lipid transfer (START) domain, first identified in the steroidogenic acute regulatory protein (StAR), is involved in the intracellular trafficking of lipids. Sixteen mammalian START domain-containing proteins have been identified to date. StAR, a protein targeted to mitochondria, stimulates the movement of cholesterol from the outer to the inner mitochondrial membranes, where it is metabolized into pregnenolone in steroidogenic cells. MLN64, the START domain protein most closely related to StAR, is localized to late endosomes along with other proteins involved in sterol trafficking, including NPC1 and NPC2, where it has been postulated to participate in sterol distribution to intracellular membranes. To investigate the role of MLN64 in sterol metabolism, we created mice with a targeted mutation in the Mln64 START domain, expecting to find a phenotype similar to that in humans and mice lacking NPC1 or NPC2 (progressive neurodegenerative symptoms, free cholesterol accumulation in lysosomes). Unexpectedly, mice homozygous for the Mln64 mutant allele were viable, neurologically intact, and fertile. No significant alterations in plasma lipid levels, liver lipid content and distribution, and expression of genes involved in sterol metabolism were observed, except for an increase in sterol ester storage in mutant mice fed a high fat diet. Embryonic fibroblast cells transfected with the cholesterol side-chain cleavage system and primary cultures of granulosa cells from Mln64 mutant mice showed defects in sterol trafficking as reflected in reduced conversion of endogenous cholesterol to steroid hormones. These observations suggest that the Mln64 START domain is largely dispensable for sterol metabolism in mice.
Subject(s)
Mutation , Phosphoproteins/physiology , Sterols/metabolism , Animals , Biological Transport , Cholesterol/metabolism , Female , Fertility , Gene Expression Profiling , Lipids/analysis , Lipids/blood , Liver/metabolism , Mice , Mice, Knockout , Phenotype , Phosphoproteins/genetics , Protein Structure, Tertiary , RNA, Messenger/analysisABSTRACT
The intracellular trafficking of cholesterol in steroidogenic cells plays an important role in the regulation of hormone synthesis. Recent evidence indicates that a family of proteins related to the steroidogenic acute regulatory protein (StAR) perform critical functions in moving the sterol substrate to the mitochondrial inner membrane where the first committed step in steroid hormone synthesis occurs. StAR, the prototype of the family, is known to promote the translocation of cholesterol from the outer to the inner mitochondrial membrane. Mutations in StAR cause congenital lipoid adrenal hyperplasia, a cholesterol storage disorder in which synthesis of all gonadal and adrenocortical steroid hormones is severely impaired, and the cholesterol that is not efficiently moved into the mitochondria accumulates in cytoplasmic lipid droplets. The StAR-related lipid transfer (START) domain consists of an approximately 210 amino acid residue sequence that forms a compact alpha/beta structure, a helix-grip fold, with a hydrophobic tunnel that can accommodate a sterol molecule. START domains can bind sterol, facilitate the transfer of cholesterol from sterol-rich unilammelar liposomes to acceptor membranes, and stimulate steroidogenesis when expressed in cells co-expressing the cholesterol side-chain cleavage system or when added to isolated steroidogenic mitochondria. Sixteen human START domain proteins have been identified to date. Of these, StAR and MLN64 consist of one subfamily and newly described proteins named StarD4, StarD5, and StarD6 represent a closely related second subfamily. MLN64 is incorporated into the late endosomal compartment and is involved in the movement of cholesterol acquired from endocytosed LDL out of these vesicles. Expression of a dominant negative form of MLN64 causes accumulation of free cholesterol in lysosomes. The roles of StarD4, StarD5, and StarD6 in sterol movement remain to be determined. These genes have tissue-specific patterns of expression that may predict specialized roles.
Subject(s)
Cholesterol/metabolism , Membrane Transport Proteins/metabolism , Steroids/biosynthesis , Animals , Biological Transport, Active , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Female , Gene Expression Regulation , Humans , Male , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/genetics , Mice , Models, Biological , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphorylation , Tissue DistributionABSTRACT
OBJECTIVE: The objective of this study was to investigate the predictive factors of premature rupture of the membranes (preterm PROM). METHODS: The study was undertaken with cervical secretions collected from 72 consenting singleton pregnant women between 20 and 33 weeks of gestation. The levels of interleukin (IL) 1alpha, IL-1beta, IL-6, IL-8, matrix metalloproteinase (MMP) 1, MMP-2, MMP-9, tissue inhibitors of matrix metalloproteinase (TIMP) 1, TIMP-2, granulocyte elastase, and fetal fibronectin in cervical diluted specimens were measured by immunoassay, and the uterine cervix was assessed by transvaginal ultrasonography. Demographic, obstetric, clinical, neonatal, and laboratory data were analyzed by univariate analysis, multiple logistic regression, and receiver operator characteristic curve analysis. RESULTS: Preterm PROM occurred in 6 women, and 63 women delivered at term. Multiple logistic regression analysis indicated a significant independent association with preterm PROM for the cervical IL-6 levels and cervical length. The receiver operator characteristic curve analysis revealed that an IL-6 level of >/=240 pg/ml in cervical secretions and a cervical length of
