Transcriptome and gene expression profile of ovarian follicle tissue of the triatomine bug Rhodnius prolixus

Insect oocytes grow in close association with the ovarian follicular epithelium (OFE), which escorts the oocyte during oogenesis and is responsible for synthesis and secretion of the eggshell. We describe a transcriptome of OFE of the triatomine bug Rhodnius prolixus, a vector of Chagas disease, to increase our knowledge of the role of FE in egg development. Random clones were sequenced from a cDNA library of different stages of follicle development. The transcriptome showed high commitment to transcription, protein synthesis, and secretion. The most abundant cDNA was a secreted (S) small, proline-rich protein with maximal expression in the vitellogenic follicle, suggesting a role in oocyte maturation. We also found Rp45, a chorion protein already described, and a putative chitin-associated cuticle protein that was an eggshell component candidate. Six transcripts coding for proteins related to the unfolded-protein response (UPR) by were chosen and their expression analyzed. Surprisingly, transcripts related to UPR showed higher expression during early stages of development and downregulation during late stages, when transcripts coding for S proteins participating in chorion formation were highly expressed. Several transcripts with potential roles in oogenesis and embryo development are also discussed. We propose that intense protein synthesis at the FE results in reticulum stress (RS) and that lowering expression of a set of genes related to cell survival should lead to degeneration of follicular cells at oocyte maturation. This paradoxical suppression of UPR suggests that ovarian follicles may represent an interesting model for studying control of RS and cell survival in professional S cell types. (C) 2011 Elsevier Ltd. All rights reserved.

Consumption of a Flaxseed-Rich Food Is Not More Effective Than a Placebo in Alleviating the Climacteric Symptoms of Postmenopausal Women

Our objective in this work was to test the effects of daily intake of bread produced with partially defatted ground flaxseed on the climacteric symptoms and endometrial thickness of postmenopausal women. A double-blind, placebo-controlled, randomized clinical trial was performed with 38 women who had been postmenopausal for 1-10 y and consumed 2 slices of bread containing 25 g of flaxseed (46 mg lignans) or wheat bran (<1 mg lignans; control) every day for 12 consecutive weeks. The outcome variables were the daily number of hot flashes, the Kupperman Menopausal Index (KMI), and endometrial thickness. The plasma lipid profile (total cholesterol and HDL, LDL, and VLDL cholesterol fractions and triglycerides) and the hormones estradiol, follicle-stimulating hormone, thyroid-stimulating hormone, and free thyroxine also were measured. Food intake was evaluated by means of 2 24-hrecalls, before and after the treatment. Twenty patients in the study group and 18 in the control group completed the study. The general characteristics did not differ between the 2 groups at the start of the study. Both had significant, but similar, reductions in hot flashes and KMI after 3 mo of treatment. Moreover, endometrial thickness was not affected in either group. Our findings clearly show that although flaxseed is safe, its consumption at this level (46 mg lignans/d) is no more effective than placebo for reducing hot flashes and KMI. J. Nutr. 140: 293-297, 2010.

Endothelial and inducible nitric oxide synthases in oocytes of cattle

Nitric oxide (NO) is a chemical messenger generated by the activity of the nitric oxide synthases (NOS). The NOS/NO system appears to be involved in oocyte maturation, but there are few studies on gene expression and protein activity in oocytes of cattle. The present study aimed to investigate gene expression and protein activity of NOS in immature and in vitro matured oocytes of cattle. The influence of pre-maturation culture with butyrolactone I in NOS gene expression was also assessed. The following experiments were performed: (1) detection of the endothelial (eNOS) and inducible (iNOS) isoforms in the ovary by immunohistochemistry; (2) detection of eNOS and iNOS in the oocytes before and after in vitro maturation (W) by immunofluorescence; (3) eNOS and iNOS mRNA and protein in immature and in vitro matured oocytes, with or without pre-maturation, by real time PCR and Western blotting, respectively; and (4) NOS activity in immature and in vitro matured oocytes by NADPH-diaphorase. eNOS and iNOS were detected in oocytes within all follicle categories (primary, secondary and tertiary), and other compartments of the ovary and in the cytoplasm of immature and in vitro matured oocytes. Amount of mRNA for both isoforms decreased after IVM but was maintained after pre-maturation culture. The NOS protein was detected in immature (pre-mature or not) and was still detected in similar amount after pre-maturation and maturation for both isoforms. NOS activity was detected only in part of the immature oocytes. In conclusion, isoforms of NOS (eNOS and iNOS) are present in oocytes of cattle from early folliculogenesis up to maturation; in vitro maturation influences amount of mRNA and NOS activity. (C) 2009 Elsevier B.V. All rights reserved.

Embryo Mitochondrial DNA Depletion Is Reversed During Early Embryogenesis in Cattle

The extensive replication of mitochondria during oogenesis and the wide variability in mitochondrial DNA ( mtDNA) copy numbers present in fully grown oocytes indicate that mtDNA amount may play an important role during early embryogenesis. Using bovine oocytes derived from follicles of different sizes to study the influence of mtDNA content on development, we showed that oocytes obtained from small follicles, known to be less competent in developing into blastocysts, contain less mtDNA than those originating from larger follicles. However, because of the high variability in copy number, a more accurate approach was examined in which parthenogenetic one-cell embryos were biopsied to measure their mtDNA content and then cultured to assess development capacity. Contrasting with previous findings, mtDNA copy number in biopsies was not different between competent and incompetent embryos, indicating that mtDNA content is not related to early developmental competence. To further examine the importance of mtDNA on development, one-cell embryos were partially depleted of their mtDNA (64% +/- 4.1% less) by centrifugation followed by the removal of the mitochondrial-enriched cytoplasmic fraction. Surprisingly, depleted embryos developed normally into blastocysts, which contained mtDNA copy numbers similar to nonmanipulated controls. Development in depleted embryos was accompanied by an increase in the expression of genes (TFAM and NRF1) controlling mtDNA replication and transcription, indicating an intrinsic ability to restore the content of mtDNA at the blastocyst stage. Therefore, we concluded that competent bovine embryos are able to regulate their mtDNA content at the blastocyst stage regardless of the copy numbers accumulated during oogenesis.

Copy number variants on the X chromosome in women with primary ovarian insufficiency

Objective: To investigate whether submicroscopic copy number variants (CNVs) on the X chromosome can be identified in women with primary ovarian insufficiency (POI), defined as spontaneous secondary amenorrhea before 40 years of age accompanied by follicle-stimulating hormone levels above 40 IU/L on at least two occasions. Design: Analysis of intensity data of single nucleotide polymorphism (SNP) probes generated by genomewide Illumina 370k CNV BeadChips, followed by the validation of identified loci using a custom designed ultra-high-density comparative genomic hybridization array containing 48,325 probes evenly distributed over the X chromosome. Setting: Multicenter genetic cohort study in the Netherlands. Patient(s): 108 Dutch Caucasian women with POI, 97 of whom passed quality control, who had a normal karyogram and absent fragile X premutation, and 235 healthy Dutch Caucasian women as controls. Intervention(s): None. Main Outcome Measure(s): Amount and locus of X chromosomal microdeletions or duplications. Result(s): Intensity differences between SNP probes identify microdeletions and duplications. The initial analysis identified an overrepresentation of deletions in POI patients. Moreover, CNVs in two genes on the Xq21.3 locus (i.e., PCDH11X and TGIF2LX) were statistically significantly associated with the POI phenotype. Mean size of identified CNVs was 262 kb. However, in the validation study the identified putative Xq21.3 deletions samples did not show deviations in intensities in consecutive probes. Conclusion(s): X chromosomal submicroscopic CNVs do not play a major role in Caucasian POI patients. We provide guidelines on how submicroscopic cytogenetic POI research should be conducted. (Fertil Steril (R) 2011;95:1584-8. (C) 2011 by American Society for Reproductive Medicine.)

Obesity induced by high-fat diet promotes insulin resistance in the ovary

Besides the effects on peripheral energy homeostasis, insulin also has an important role in ovarian function. Obesity has a negative effect on fertility, and may play a role in the development of the polycystic ovary syndrome in susceptible women. Since insulin resistance in the ovary could contribute to the impairment of reproductive function in obese women, we evaluated insulin signaling in the ovary of high-fat diet-induced obese rats. Female Wistar rats were submitted to a high-fat diet for 120 or 180 days, and the insulin signaling pathway in the ovary was evaluated by immunoprecipitation and immunoblotting. At the end of the diet period, we observed insulin resistance, hyperinsulinemia, an increase in progesterone serum levels, an extended estrus cycle, and altered ovarian morphology in obese female rats. Moreover, in female obese rats treated for 120 days with the high-fat diet, the increase in progesterone levels occurred together with enhancement of LH levels. The ovary from high-fat-fed female rats showed a reduction in the insulin receptor substrate/phosphatidylinositol 3-kinase/AKT intracellular pathway, associated with an increase in FOXO3a, IL1B, and TNF alpha protein expression. These changes in the insulin signaling pathway may have a role in the infertile state associated with obesity. Journal of Endocrinology (2010) 206, 65-74

Lack of effects of isoflavones on the lipid profile of Brazilian postmenopausal women

Objective: To evaluate the effects of soy isoflavone supplementation on profile lipid and endogenous hormone levels. Methods: In this double-blind, placebo-controlled Study, 47 post menopausal women 47-66 v of age received 40 mg of isoflavone (n = 25) or 40 mg of casein placebo (11 = 22). Cardiovascular risk factors were assessed by evaluating lipid profile at baseline and after 6 mo of treatment. To examine the effects of this regime on endogenous hormone levels, follicle-stimulating hormone and beta-estradiol were measured. Urinary isoflavone concentrations (genistein and daidzein) were measured as markers of both compliance and absorption using high performance liquid chromatography. Baseline characteristics were compared by the unpaired Student`s t-test. Within-group changes were determined by paired Student`s t-test and comparison between the isoflavone and casein placebo groups were determined by analysis of variance. Results: Lipid levels (low-density lipoprotein and total cholesterol) similarly decreased in both,groups. High-density lipoprotein increased significantly in both groups and cannot thus be attributable to treatment: the reason for Such variation is unknown and can be attributed to chance or to bias (even that of a real placebo effect in both groups or perhaps in spontaneous changes in exercise and dietary habits of patients after their inclusion). Furthermore, in both groups very low-density lipoprotein and triacylglycerol levels increased in a non-significant manner. Conclusion: The results of the present Study do not support any biologically significant estrogenic effects of isoflavone on the parameters assessed. Further research will he necessary to definitively assess the safety and efficacy of isoflavone. (C) 2008 Elsevier Inc. All rights reserved.