Oocyte-specific inactivation of Omcg1 leads to DNA damage and c-Abl/TAp63-dependent oocyte death associated with dramatic remodeling of ovarian somatic cells.

Aberrant loss of oocytes following cancer treatments or genetic mutations leads to premature ovarian insufficiency (POI) associated with endocrine-related disorders in 1% of women. Therefore, understanding the mechanisms governing oocyte death is crucial for the preservation of female fertility. Here, we report the striking reproductive features of a novel mouse model of POI obtained through oocyte-specific inactivation (ocKO) of Omcg1/Zfp830 encoding a nuclear zinc finger protein involved in pre-mRNA processing. Genetic ablation of OMCG1 in early growing oocytes leads to reduced transcription, accumulation of DNA double-strand breaks and subsequent c-Abl/TAp63-dependent oocyte death, thus uncovering the key role of OMCG1 for oocyte genomic integrity. All adult Omcg1(ocKO) females displayed complete elimination of early growing oocytes and sterility. Unexpectedly, mutant females exhibited a normal onset of puberty and sexual receptivity. Detailed studies of Omcg1(ocKO) ovaries revealed that the ovarian somatic compartment underwent a dramatic structural and functional remodeling. This allowed the cooperation between oocyte-depleted follicles and interstitial tissue to produce estradiol. Moreover, despite early folliculogenesis arrest, mutant mice exhibited sexual cyclicity as shown by cyclical changes in estrogen secretion, vaginal epithelium cytology and genital tract weight. Collectively, our findings demonstrate the key role of Omcg1 for oocyte survival and highlight the contribution of p63 pathway in damaged oocyte elimination in adulthood. Moreover, our findings challenge the prevailing view that sexual cyclicity is tightly dependent upon the pace of folliculogenesis and luteal differentiation.

Molecular mechanisms of adenylyl cyclase desensitization in pregnant rat myometrium following in vivo administration of the beta-adrenergic agonist, isoproterenol.

Beta-adrenergic agonists are widely used for preterm labor treatment, but their effectiveness may be limited by desensitization. We thus investigated the effects of a beta-agonist, isoproterenol, on the myometrial beta-adrenergic receptor (beta-AR)/adenylyl cyclase pathway after administration in vivo to late-pregnant rats (8 mg/kg, twice-daily injections). One hour after the first injection, isoproterenol-stimulated adenylyl cyclase activity was reduced by 37%. This was associated with a rapid and transient uncoupling of the beta2-ARs (53% reduction of high-affinity receptors). After prolonged isoproterenol treatment (76 h), adenylyl cyclase activity was desensitized not only to isoproterenol but also to guanosine triphosphate and forskolin. Such treatment induced 1) a selective decrease of beta2-ARs as assessed by 125I-cyanopindolol binding, which was reversed by 5'-guanylylimidodiphosphate and thus probably did not involve irreversible loss of receptors, and 2) a rapid alteration of their transcript levels. Prolonged isoproterenol treatment also led to myometrial Gi2alpha and Gi3alpha increase (44% and 70%) as assessed by Western blotting. Furthermore, pertussis toxin pretreatment of membranes abolished the decrease in isoproterenol-stimulated adenylyl cyclase activity. Thus, we demonstrated that myometrial adenylyl cyclase desensitization after beta-agonist treatment results mainly from beta2-AR uncoupling and increase in Gi activity.

The alpha1B-adrenergic receptor subtype activates the phospholipase C signaling pathway in rat myometrium at parturition.

This study demonstrates that alpha1-adrenergic receptors previously identified in the pregnant rat myometrium are heterogeneous. They can be subtyped alpha1A- and alpha1B-adrenergic receptors on the basis of their affinity for the antagonists WB4101 (alpha1A > alpha1B) and chloroethylclonidine (alpha1B selective). Between Day 21 of pregnancy and term, the proportion of [3H]prazosin binding sites with low affinity for WB4101 and sensitive to inactivation by 10(-5) M chloroethylclonidine under hypotonic conditions (alpha1B subtype) remained constant. In contrast, the number of [3H]prazosin binding sites with a high affinity for WB4101 and insensitive to chloroethylclonidine (alpha1A subtype) increased by 88% at term. The effect of 5'-guanylylimidodiphosphate (Gpp[NH]p) on competition of the agonist phenylephrine for [3H]prazosin binding in the presence of WB4101 or after chloroethylclonidine pretreatment indicates that the alpha1A-adrenergic receptor underwent uncoupling whereas the alpha1B-adrenergic receptor-G protein coupled state was increased (+ 63%). Phenylephrine consistently stimulated phospholipase C activity on membrane fractions prepared from term myometria. This stimulation was completely inhibited after 10(-5) M chloroethylclonidine but was not consistently decreased with 5-methylurapidyl, a selective alpha1A-antagonist. Furthermore QL antibody (anti-G alpha(q)/G alpha11) also specifically blocked the phenylephrine-stimulated phospholipase C activity. Altogether these results strongly suggest that activation of the alpha1B-adrenergic receptor subtype in the pregnant myometrium at term may contribute to the stimulation of the G alpha(q)/G alpha11/phospholipase C signaling pathway.

Heterogeneity of alpha 2-adrenoceptors in human and rat myometrium and differential expression during pregnancy.

1. The aim of this study was first, to characterize alpha 2-adrenoceptor subtypes in human and rat pregnant myometrium and second, to investigate the possibility of a differential expression of the putative subtypes according to the stage of pregnancy. 2. In both species, specific [3H]-rauwolscine binding was inhibited by five different compounds with an order of affinity characteristic of the one described for alpha 2-adrenoceptors (yohimbine > or = clonidine > noradrenaline > phenylephrine > propranolol). Binding affinities (pKi) for the compounds tested were, in human and rat, respectively: 7.63 and 8.93 for yohimbine, 6.91 and 8.71 for clonidine, 6.23 and 6.09 for noradrenaline, 5.37 and 5.73 for phenylephrine, 4.64 and 4.72 for propranolol. 3. By use of non-linear iterative curve fitting procedures and by fitting the data to a two-site model, analysis of [3H]-rauwolscine inhibition binding curves performed in the presence of oxymetazoline (alpha 2A-selective), ARC239, prazosin or chlorpromazine (alpha 2B- and alpha 2C-selective) indicated that pregnant human and rat myometrium contain at least two pharmacologically distinct alpha 2-adrenoceptor subtypes (alpha 2A, alpha 2B and/or alpha 2C). RNA blot analysis with probes specific for each cloned human and rat alpha 2-adrenoceptor subtype demonstrated that alpha 2A- and alpha 2B-subtypes were present in both species but alpha 2C seems to be expressed only in human tissues. 4. In the pregnant rat myometrium, subtype selective compounds competition curves revealed a predominant expression of alpha 2A-adrenoceptors at mid-pregnancy whereas, at term, alpha 2A- and alpha 2B-subtypes density reached approximately the same level (alpha 2A:alpha 2B ratio = 73:27 at mid-pregnancy and = 43:57 at term). In addition, quantification of alpha 2A- and alpha 2B-transcripts by densitometry, following data normalization with an oligo(dT)12-18 probe, showed a pattern of expression comparable to the one characterized by pharmacological studies. 5. In conclusion, these data demonstrate heterogeneity of alpha 2-adrenoceptors in pregnant human and rat myometria and an alteration of the alpha 2A-/alpha 2B-subtypes expression pattern during rat pregnancy. Such observations lead us to suggest a multiple role for alpha 2-adrenoceptors in regulating specific functions of myometrium throughout the time course of pregnancy.

Ecdysteroid-stimulated synthesis and secretion of an N-acetyl-D-glucosamine-rich glycopeptide in a lepidopteran cell line derived from imaginal discs.

Hormone-regulated processing of N-acetyl-D-glucosamine was studied in an insect cell line derived from imaginal wing discs of the Indian meal moth, Plodia interpunctella (Hübner). The cell line, IAL-PID2, responded to treatment with 20-hydroxyecdysone with increased incorporation of GlcNAc into glycoproteins. Cycloheximide and tunicamycin counteracted the action of the hormone. In particular, treatment with 20-hydroxyecdysone resulted in the secretion of a 5,000 dalton N-acetyl-D-glucosamine-rich glycopeptide by the IAL-PID2 cells. Accumulation of this peptide was prevented by the use of teflubenzuron, a potent chitin synthesis inhibitor. A glycopeptide of similar molecular weight was observed in imaginal discs of P. interpunctella treated with 20-hydroxyecdysone in vitro, under conditions that induce chitin synthesis. Although the function of the 5,000 dalton glycopeptide is not known, we believe that the PID2 cell line is a promising model for molecular analysis of ecdysteroid-regulated processing of aminosugars by epidermal cells during insect development.