Mutations of t-complex testis expressed gene 5 transcripts in the testis of sterile t-haplotype mutant mouse.

AIM: To determine the possible roles of the t-complex testis expressed gene 5 (Tctex5) on sperm functions, the full-length sequence of mRNA was studied and compared in the testis between the normal wild-type and the sterile t-haplotype mutant mice. METHODS: We applied rapid amplification of cDNA ends, Northern blot and reverse transcription polymerase chain reaction to analyze the full length of Tctex5 mRNAs isolated from testes of the wild-type and the t-haplotype mice. Reverse transcription polymerase chain reaction was used to semi-quantitatively compare expression of Tctex5 transcripts in the 16 tissues and 9.5 day stage embryos in the wild-type mice. E-translation was applied to estimate the amino acid sequences. RESULTS: One long and one short transcript of Tctex5 mRNA were discovered in mouse testis of wild-type (Tctex5(long-+) and Tctex5(short-+)) and t-haplotype (Tctex5(long-t) and Tctex5(short-t)) mice, respectively. Being enhanced only in the testis, Tctex5(long-t) had 17 point mutations and one 15-bp-deletion in the exon 1 region, comparing with the Tctex5(long-+), whereas the Tctex5(short-t) was similar to the Tctex5(short-+). The short isoforms of Tctex5 mRNAs in the two models encoded exactly the same peptides, but the long isoforms did not. The estimated peptide encoded by Tctex5(long-t) had significant mutations on putative sites of phosphorylation and PP1 binding. CONCLUSION: We established that mutations that occur in the Tctex5 long transcript of the t-haplotype mice are important for normal sperm function, whereas the short transcript of Tctex5 might have a conserved function among different tissues.

Changes in the ratio of Bax and Bcl-2 mRNA expression and their cellular localization throughout the ovulatory cycle in the human oviduct.

PURPOSE: To examine changes in the ratio of Bax and Bcl-2 mRNA expression throughout the ovulatory cycle in the ampullary region of the human oviduct. METHODS: The mucosal layer was isolated from the human oviduct tissue and semiquantitative reverse-transcriptase polymerase chain-reaction (RT-PCR) analysis of mRNA of Bax and Bcl-2 was performed. Immunohistochemistry provided the cellular localization of the Bax and Bcl-2 proteins. The ratio of expression of Bax and Bcl-2 mRNA was examined in the ampullary region of the oviduct in samples obtained in the follicular, periovulatory, and luteal phases of the ovulatory cycle. RESULTS: Bax expression was constant in the follicular and periovulatory phase but showed a significant increase in the luteal phase. The Bax protein was present in all oviduct mucosal epithelial cells and the intensity of staining increased in luteal phase samples. Bcl-2 was expressed at a relatively constant level throughout the ovulatory cycle. The Bcl-2 protein was present in some but not all mucosal epithelial cells and the proportion of positive cells remained constant throughout the ovulatory cycle. CONCLUSION: The proapoptotic gene Bax shows a significant increase in mRNA expression in the luteal phase of the ovulatory cycle while the expression level of the antiapoptotic gene Bcl-2 remains constant throughout the ovulatory cycle. The ratio of Bax:Bcl-2 increases significantly in the luteal phase consistent with cells undergoing apoptosis.

Ratio of mRNA expression of progesterone receptor isoforms AB is to B in human oviduct mucosal cells during the ovulatory cycle.

UNLABELLED: The ratio of the active progesterone receptor B isoform is higher in the ampullary region of the oviduct. PURPOSE: To examine mRNA expression of progesterone receptor isoforms AB and B in oviduct mucosal tissue during the ovulatory cycle and in the different functional regions of the human oviduct. METHODS: The mucosal layer was isolated from human oviduct tissue and semi-quantitative RT-PCR for progesterone isoforms AB and B was performed. The RT-PCR results were verified by immunohistochemistry. RESULTS: The isthmic region showed no mRNA expression of either progesterone receptor isoform while the relative ratio of the B isoform was significantly higher in the ampullary region compared to the fimbrial region. There was a significant increase in the ratio of PRB to PRAB in the ampullary region compared to the fimbrial region in all samples. CONCLUSIONS: We found an increase in the relative abundance of the progesterone receptor B isoform in the ampullary region which is the site of fertilization and early embryo cleavage. Our results indicate that progesterone responsive genes are more likely to be activated in the ampullary region of the oviduct due to the difference in PRAB to PRB ratio. Providing support for the hypothesis that progesterone may play a specific role in providing an appropriate environment for sperm capacitation, fertilization and early embryo cleavage.

Upregulation of mRNA expression of vascular endothelial growth factor and its receptors by exogenous human chorionic gonadotropin in cultured oviduct mucosal cells.

The mucosal cells were isolated from the ampullary regions of 20 human oviducts and cultured with or without hCG in five different concentrations (1-100 ng/mL). As analyzed by the semiquantitative reverse-transcriptase polymerase chain reaction, hCG treatment significantly increased mRNA expression of vascular endothelial growth factor and its receptor flt-1 in the cultured mucosal cells in a dose-dependent manner but had no effect on the expression of another receptor, KDR.

Increased messenger RNA expression of vascular endothelial growth factor and its receptors in the implantation site of the human oviduct with ectopic gestation.

OBJECTIVE: To compare the mRNA expression of vascular endothelial growth factor (VEGF) and its receptors (KDR and flt-1) in the implantation and nonimplantation sites of the human oviduct with ectopic gestation. DESIGN: Prospective observational study. SETTING: University-based Obstetrics and Gynecology Department. PATIENT(S): Ten women undergoing laparoscopic salpingectomy for tubal pregnancy. INTERVENTION(S): The mucosal layer was isolated from the implantation and nonimplantation sites of the oviduct tissue with ectopic gestation. Semiquantitative reverse transcriptase-polymerase chain reaction was performed. MAIN OUTCOME MEASURE(S): The differences in the mRNA expression of VEGF and its receptors between the implantation and nonimplantation sites of the oviduct tissue. RESULT(S): The mRNA expression of VEGF and its receptors, both KDR and flt-1, was significantly higher in the implantation site of the human oviduct with ectopic gestation compared with the nonimplantation site. CONCLUSION(S): The results suggest that VEGF may be the angiogenic factor responsible for the implantation and placentation of an ectopic pregnancy in the oviduct.

Estradiol regulation of oviductin/oviduct-specific glycoprotein messenger ribonucleic acid expression in human oviduct mucosal cells in vitro.

OBJECTIVE: To determine whether oviduct mucosal cell culture with exogenous 17beta E(2) supports the continued production of oviductin, a putative embryotrophic protein. DESIGN: Semiquantitative reverse-transcriptase polymerase chain reaction analysis of oviductin mRNA expression after oviduct mucosal cell culture in the presence of 17beta E(2). Three different culture systems were studied to investigate the response to E(2). SETTING: University-based obstetrics and gynecology department. SUBJECTS: Oviduct tissue was obtained from 18 women undergoing laparoscopy for benign gynecologic conditions. INTERVENTION(S): The mucosal layer was isolated from the oviduct tissue and exposed to three different culture systems, which contained various concentrations of 17beta E(2), or vehicle-only control. MAIN OUTCOME MEASURE(S): The relationship between exposure to 17beta E(2) and expression of oviductin messenger (m)RNA by cultured oviduct mucosal cells. RESULT(S): There was a significant increase in oviductin mRNA expression after the addition of 17beta E(2) to the culture system in which the in vivo cell-to-cell and cell-to-basement-membrane contacts of the oviduct had been maintained. CONCLUSION(S): Estradiol failed to alter oviductin mRNA expression in oviduct mucosal cells cultured under conditions in which the ciliated mucosal cell phenotype plus the cell-to-cell and cell-to-basement-membrane contacts of the oviduct were lost. However, with a culture system that maintained the cell architecture, E(2) initiated and significantly increased oviductin mRNA expression.

In vivo regulation of mRNA expression of vascular endothelial growth factor receptors (KDR and flt-1) in the human oviduct.

OBJECTIVE: To examine the localization of vascular endothelial growth factor receptors (VEGF-R) and the changes in VEGF-R messenger ribonucleic acid (mRNA) expression in various regions of the oviduct from fertile women throughout the ovulatory cycle. DESIGN: Prospective observational study. SETTING: University-based obstetrics and gynecology department. PATIENT(S): Twenty-two women who underwent laparoscopic tubal sterilization or hysterectomy for a benign gynecological condition. INTERVENTION(S): The mucosal layer was isolated from the oviduct tissue. Immunohistochemistry and a semiquantitative reverse-transcriptase-polymerase chain reaction (RT-PCR) was performed. MAIN OUTCOME MEASURE(S): Immunohistochemical localization of VEGF-R proteins in oviduct tissue, and the differences of VEGF-R mRNA expression in the various regions of the oviduct and in the various stages of the ovulatory cycle. RESULT(S): Immunohistochemical study localized VEGF-R, both KDR and flt-1, in the oviduct luminal epithelium, smooth muscle cells as well as blood vessels within the oviduct. Messenger RNA expression of KDR, but not flt-1, was significantly higher in the ampullary and infundibular regions than in the isthmus. Messenger RNA expression of flt-1, but not KDR, varied significantly in the oviduct along the course of an ovulatory cycle, with the highest level in the periovulatory stage. CONCLUSION(S): These results suggest that the two VEGF receptors may have different roles in the oviduct. Our data support a role for KDR in oviduct angiogenesis whereas flt-1 appears to be important in the temporal regulation of oviductal secretion.

Increased mRNA expression of vascular endothelial growth factor and its receptor (flt-1) in the hydrosalpinx.

BACKGROUND: The adverse effects of hydrosalpinx on the outcomes of IVF have been well documented, but the mechanisms of hydrosalpinx fluid formation remain unclear. This study compares the mRNA expression of vascular endothelial growth factor (VEGF) and its receptors (KDR and flt-1) in the hydrosalpinx with that in the healthy oviduct. METHODS: Oviduct tissue was collected from 10 infertile women with hydrosalpinx and 10 parous women with healthy oviduct. The mRNA expression of VEGF and its receptors in isolated oviduct epithelial cells were analysed using semi-quantitative reverse-transcriptase PCR. RESULTS: mRNA expression of VEGF and its receptor flt-1 in the hydrosalpinx was significantly higher than that in the healthy oviduct, but no significant difference was demonstrated for the KDR receptor. CONCLUSIONS: This study supports the notion that VEGF may play an important role in the hydrosalpinx fluid formation, possibly by promoting vascular and epithelial permeability and therefore serum transudation.

Vascular endothelial growth factor in the human oviduct: localization and regulation of messenger RNA expression in vivo.

In this study, we examined the localization of vascular endothelial growth factor (VEGF) and the changes in VEGF mRNA expression in various regions of the oviduct in fertile women throughout the ovulatory cycle. Oviduct tissue was collected from 22 women undergoing laparoscopic tubal sterilization or hysterectomy for a benign gynecological condition. Oviduct sections were divided into isthmus, ampullary, and infundibular regions. Serial cross sections were analyzed for the presence of VEGF by specific immunohistochemical staining. The mucosal layer was isolated, and a semiquantitative reverse transcription polymerase chain reaction was performed. Immunohistochemical study revealed VEGF in the oviduct luminal epithelium, smooth muscle cells, and blood vessels within the oviduct. VEGF mRNA expression in oviduct was the highest during the periovulatory stage, and the expression in the ampullary and infundibular regions was higher than that in the isthmus. There was a significant positive correlation between serum FSH and LH concentrations and VEGF mRNA expression. There was no significant correlation between serum estradiol and progesterone concentrations and VEGF mRNA expression. These results suggest that VEGF in human oviduct may play an important role related the early reproductive events, which occur predominantly in the ampulla during the periovulatory phase when serum FSH and LH concentrations are high.