CYP19A1 Promoters Activity in Human Granulosa Cells: A Comparison between PCOS and Normal Subjects.

Objective: Estrogen, a female hormone maintaining several critical functions in women's physiology, e.g., folliculogenesis and fertility, is predominantly produced by ovarian granulosa cells where aromatase enzyme converts androgen to estrogen. The principal enzyme responsible for this catalytic reaction is encoded by the CYP19A1 gene, with a long regulatory region. Abnormalities in this process cause metabolic disorders in women, one of the most common of which is polycystic ovary syndrome (PCOS). The main purpose of this research was to determine the effect of the promoters on aromatase expression in cells with normal and PCOS characteristics. Materials and Methods: In this experimental study, four promoters of the CYP19A1 gene, including PII, I.3, I.4, and PII/ I .3 promoter fragments, were cloned upstream of the luciferase gene and transfected into normal and PCOS granulosa cells. Subsequently, the effect of follicle-stimulating hormone (FSH) on the activity of these regulatory regions was examined in the presence and absence of FSH. Western blotting was used to confirm aromatase expression in all groups. Data analysis was performed using ANOVA and paired sample t test, compared by post-hoc least significant difference (LSD) test. Results: Luciferase results confirmed the intense activity of PII promoter in the presence of FSH. Moreover, the study demonstrated reduced activity of PII promoter in normal granulosa cells, possibly due to the regulatory region of I.3 next to PII. Conclusion: FSH stimulates transcription of aromatase enzyme by affecting PII promoter, a process regulated by the inhibitory role of the I.3 region in PII activity in granulosa cells. Given the distinct role of these promoters in normal and PCOS granulosa cells, the importance of nuclear factors residing in these regions can be discerned.

Differences in fatty acid profiles and desaturation indices of abdominal subcutaneous adipose tissue between pregnant women with and without PCOS.

The objective was to determine the differences in fatty acid (FA) profiles in subcutaneous adipose tissue (AT) between pregnant women with polycystic ovary syndrome (PCOS) and those without PCOS. FA profiles of AT samples from 13 PCOS and 32 non-PCOS, all of whom underwent caesarean section were compared using gas chromatography. Age and BMI in the two groups were similar. Twenty-one FAs were detected and the total saturated FA percentage of experimental groups was similar. While the total monounsaturated FA (MUFA) (p < 0.0004) and desaturase index (18:1 cis-9/18:0; p < 0.03) were higher in PCOS women than non-PCOS women, total polyunsaturated FA (PUFA) was lower in PCOS than non-PCOS women (p < 0.004). Docosahexaenoic acid level of the two groups was similar while α-linolenic acid and eicosapentaenoic acid levels were significantly (p < 0.05) lower in PCOS. Total trans-FA, C18:1 t9 and C18:2t were lower in PCOS women (p < 0.05). These results indicate differences in desaturase index, MUFA and PUFA, especially n-3 FA in AT between age and BMI-matched pregnant PCOS and non-PCOS pregnant subjects. Further studies are warranted to replicate these findings and to investigate potential changes in these profiles in non-pregnant PCOS women.

Relationship between absence of annulus and asthenozoospermia in Iranian men.

PURPOSE: To find a relationship between absence of annulus and asthenozoospermia in Iranian men. METHODS: In the present study, semen samples from 100 asthenozoospermic and 20 normozospermic patients were analyzed for sperm concentration and motility. Spermatozoa were immunostained for the two septin subunits Sept4 and Sept7. The absence of the annulus structure was confirmed by transmission electron microscopy and western blot analysis for septin 4. DNA sequencing for all coding exons of SEPT12 was performed for a patient using peripheral blood sample. RESULTS: Specific antibodies for SEPT4 and SEPT7 consistently labeled the annuli in spermatozoa from all of the 20 normozospermic men, while in one of 100 patients with asthenozoospermia, 75% of sperms lacking septin 4 or septin 7 proteins at the annulus. It was shown that the structural defect in annulus formation is not caused by point mutation of SEPT12 gene. CONCLUSIONS: In conclusion, the results of this study demonstrated that the frequency of the absence of annulus in asthenozoospermic sample of Iranian population has a low frequency and could not be assume as a diagnostic marker for classifying asthenozoospermic patients.

Effect of ovarian tissue vitrification method on mice preantral follicular development and gene expression.

Vitrification is considered a viable method for cryopreservation of ovarian tissue and selection of methods that minimize follicular damage is important. The objective of the present study was to evaluate the effects of two vitrification methods on ovarian tissue morphology, preantral follicles survival rate during in vitro culture, and relative expression of genes associated with oocyte maturation and cumulus expansion. Ovaries from 12-day-old mice were vitrified in media containing ethylene glycol, dimethyl sulphoxide, and sucrose. Before plunging in liquid nitrogen, ovaries were first loaded into an acupuncture needle (needle immersion vitrification [NIV]) or placed on a cold steel surface for 10 to 20 seconds (solid surface vitrification [SSV]). The integrity of the ovarian tissue was well-preserved after vitrification and was similar controls. Follicle viability in the SSV group was lower (P < 0.05) than in the control group after 6 days of culture and the NIV group after 10 day of culture. Follicle viability after 12 day of culture was 92.8%, 82.1%, and 58.4% in control, NIV, and SSV groups, respectively. Bmp15, Gdf9, BmprII, Alk6, Alk5, Has2, and Ptgs2 gene expression patterns were similar among groups. However, the level of gene expression in the vitrification groups during Days 6 to 10 were higher compared with the control group. In conclusion, ovarian tissue morphologic integrity was well-preserved, regardless of the vitrification method. Vitrification using the needle immersion method resulted in greater follicular survival after 12 day of culture than the SSV method. Gene expression patterns during culture did not seem to explain the reduced survival rate observed in the solid surface group.

Quantitative expression of developmental genes, Pou5f1 (Oct4) and Mest (Peg1), in vitrified mouse embryos.

BACKGROUND: Embryo cryopreservation is the process that water is removed from the cell by cryoprotectant materials, and embryos are stored at temperature below zero. This process may affect the viability and developmental potential of embryos. OBJECTIVE: In this study, the effect of the vitrification cryotop method on the expression level of Oct4 and Mest developmental genes in mouse blastocysts was examined. MATERIALS AND METHODS: The collected 2-cell embryos of superovulated mouse by oviduct flushing were divided into non-vitrified and vitrified groups. These embryos were cultured to the blastocyst stage directly in the non-vitrified group and in the vitrified group, these embryos were cultured to 4-8 cell embryos, vitrified with cryotop in these stages and after 2-6 months, warmed and cultured to blastocyst embryos. Quantitative expression of two developmental genes, namely Oct4 and Mest, were performed in these groups, using RNA purification and Real-time RT-PCR. RESULTS: Quantitative PCR analysis showed that the expression level of both genes, Oct4 and Mest, was reduced significantly in the vitrified-warmed group relative to the control group (p=0.046 and p=0.001). CONCLUSION: This study revealed that morphologically normal embryos show a reduced amount of Oct4 and Mest transcripts which indicate that the vitrification method negatively effects the expression level of these two developmental genes.