Coexpression of growth arrest-specific gene 6 and receptor tyrosine kinases, Axl and Sky, in human uterine endometrium and ovarian endometriosis.

We demonstrated the expression of Gas6, the protein product of the growth arrest-specific gene 6 (gas6) and a member of the vitamin K-dependent protein family, and its receptor tyrosine kinases, Axl and Sky, in human uterine and ovarian endometriotic endometria using RT-PCR-Southern blot analysis and immunohistochemistry. Gas6, Axl and Sky mRNA were detected in all samples analysed. There was no significant difference between the levels of Sky mRNA in normal uterine and endometriotic endometria; however, the levels of Gas6 and Axl mRNA in endometriotic endometria were significantly higher than in normal endometria. These mRNA levels showed no significant alteration during the menstrual cycle. In the immunohistochemical study, Gas6, Axl and Sky were found in endometrial glandular cells and stromal cells in all samples analysed. This study demonstrates the coexpression of receptor tyrosine kinases and their ligand, Gas6, in normal uterine and ovarian endometriotic endometria, and the overexpression of Axl and Gas6 in endometriotic endometria. It is suggested that Gas6 and Axl signal transduction is aberrantly stimulated in endometriotic endometria, and is plausibly related to its growth potential.

Leiomyoma of the fallopian tube.

Leiomyomas of the fallopian tube are rare. They are typically incidental findings seen at autopsy or unrelated surgical procedures. A 32-year-old woman presented with lower abdominal pain and mass. Transvaginal sonogram and magnetic resonance imaging showed the solid mass at the outside of the uterus. At surgery, the left fallopian tube contained a firm mass with torsion in the area of the ampullary-isthmic junction. The left tube and the infundibulopelvic ligament were rolled in torsion and showed edematous change. We report a rare case in whom torsion of a pedunculated tubal leiomyoma caused abdominal pain.

Identification of exon-deleted progesterone receptor mRNAs in human uterine endometrial cancers.

We demonstrated the expression of various exon-deleted progesterone receptor (PR) variant mRNAs in human uterine endometrial cancers using the reverse transcription-polymerase chain reaction-DNA sequencing analyses. In addition to PR wild-type mRNA, exon 4-deleted, exon 6-deleted, exon 3,4-deleted, exon 5,6-deleted, exon 4,5,6-deleted and exon 3,4,5,6-deleted PR variant mRNAs were identified. The exon 6-deleted and exon 5,6-deleted PR variant mRNAs lacked encoding for the steroid-binding domain. The exon 4-deleted, exon 3,4-deleted, exon 4,5,6-deleted and exon 3,4,5,6-deleted PR variant mRNAs lacked encoding for the DNA-binding domain in addition to encoding for the steroid-binding domain. While the exon 4-deleted, exon 6-deleted and exon 3,4-deleted PR variant mRNAs were observed in all samples analyzed, the exon 5,6-deleted, exon 4,5,6-deleted and/or exon 3,4,5,6-deleted PR variant mRNAs could not be detected in some cases, especially in poorly differentiated adenocarcinoma as compared with well-differentiated and moderately differentiated adenocarcinomas. The present study demonstrates the coexpression of PR exon-deleted variant mRNAs with the wild-type in uterine endometrial cancers. All translated variant proteins might possess functional diversity and might modify the progestational action of wild-type PR, and the expression of some PR variant mRNAs may be lost as endometrial cancer cells undergo dedifferentiation.

Progestin regulation in tumor growth of female genital tract cancers.

The irregular response to progestins directly in tumor growth might be caused by dominant negative progesterone receptor (PR) mutants and the damage to PR-A expression. Progestin treatment as an anti-angiogenic therapy would be less effective in the PR-mutated tumors. Therefore, various anti-angiogenic inhibitors must be used in progestin-refractory and progestin-dependent tumors.

Evidence for the synthesis of corticosteroid-binding globulin in human placenta.

We demonstrated the expression of corticosteroid-binding globulin (CBG) in human placenta using reverse transcription-polymerase chain reaction-Southern blot analysis and immunohistochemical and immunoblotting studies. In the RT-PCR-Southern blot analysis, only one predicted PCR product was detected without nonspecific products in all samples of human placenta and 3A (tPA-30-1) human placental cells. In Western blot analysis, polyclonal anti-CBG antibodies recognized a protein of approximately 55 kD in the protein extracts prepared from 3A (tPA-30-1) cells. Additionally, CBG mRNA expression was demonstrated by in situ hybridization in the syncytiotrophoblasts. Immunohistochemical studies performed on the placenta demonstrated the presence of specific immunoreactivity in the syncytiotrophoblast layer surrounding the chorionic villi. These findings suggest that CBG is synthesized in human placenta during pregnancy in addition to its synthesis in the liver.

Levels of corticosteroid-binding globulin mRNA in human ovarian cancers.

To understand the role of corticosteroid-binding globulin (CBG) in the intracellular steroidal actions in human ovarian cancers, the level of CBG mRNA expression was evaluated in normal ovarian tissues and in ovarian cancers using competitive reverse transcription-polymerase chain reaction-Southern blot analysis. The expression of CBG mRNA was detected in all normal ovaries and ovarian cancers analyzed. There were no significant differences in the mean CBG mRNA levels between normal ovaries and ovarian cancers. The expression in normal ovaries was significantly higher (p < 0.01) in the premenopause than in the postmenopause. A high expression of CBG mRNA was observed in 11 out of 29 cases (38%) of ovarian cancer in comparison with normal ovaries. There was no difference in the expression among the histological classifications or clinical stages of ovarian cancers. These data suggest that human normal ovaries and ovarian cancers might synthesize CBG intracellularly, ovarian cancers might conserve a progesterone-associated property via CBG, and the regulation of intracelluar CBG expression might be changed in some cancers.

Levels of sex hormone-binding globulin and corticosteroid-binding globulin mRNAs in corpus luteum of human subjects: correlation with serum steroid hormone levels.

To understand regulation of the function of human ovarian corpus luteum by sex steroid-binding proteins, the levels of luteal intracellular sex hormone-binding globulin (SHBG) and corticosteroid-binding globulin (CBG) mRNAs and serum steroid hormones were simultaneously determined. The expression of SHBG and CBG mRNAs was detected in all samples analyzed. SHBG mRNA level was positively correlated with serum estradiol-17 beta level (p < 0.05), but not with serum progesterone level. There was a positive correlation between SHBG mRNA level and serum estradiol-17 beta/progesterone ratio (p < 0.01). On the other hand, CBG mRNA level was positively correlated with serum estradiol-17 beta and progesterone level (p < 0.01 and p < 0.01, respectively). There was no correlation between CBG mRNA level and serum estradiol-17 beta/progesterone ratio. SHBG and CBG mRNA levels were not correlated with the levels of serum testosterone, free testosterone or cortisol. These findings suggest that the synthesis of luteal SHBG and CBG is complexly regulated by estrogen and progesterone, and that SHBG and CBG interact with estrogen and progesterone, respectively, for luteal steroidal activity.

Expression of sex hormone-binding globulin exon 7 splicing variant mRNA in secondary spreading lesions of gynecological cancers.

This study was designed to determine the clinical implications of intracellular expression of sex hormone-binding globulin (SHBG) wild-type and exon 7 splicing variant mRNAs in secondary spreading lesions of gynecologic cancers using the reverse transcription-polymerase chain reaction-Southern blot and DNA sequencing analyses. Compared with primary cancers, a relative increase in SHBG variant mRNA to wild-type mRNA expression was observed (4/10 cases of uterine endometrial cancers, 5/10 cases of uterine cervical cancers, 6/10 cases of ovarian cancers) or the expression of SHBG wild-type and variant mRNAs could not be detected (5/10 cases of uterine endometrial cancers, 3/10 cases of uterine cervical cancers, 4/10 cases of ovarian cancers). On the other hand, alteration to a relative increase in SHBG wild-type mRNA expression in the metastatic lesions occurred in only 3 cases (1/10 cases of uterine endometrial cancers and 2/10 cases of uterine cervical cancers) analyzed. These results suggest that the transcription of SHBG mRNA and the regulation of its splicing might be altered with metastatic potential, and this status might be involved in a change in steroidal dependency of metastatic lesions.

Expression of oestrogen receptor alpha and beta mRNA in corpus luteum of human subjects.

To investigate the role of oestrogen receptor beta (ERbeta) in the function of human ovarian corpus luteum, the levels of luteal ERalpha and ERbeta mRNA were determined using competitive reverse transcription-polymerase chain reaction-Southern blot analysis. The expression of ERalpha and ERbeta mRNA was detected in all luteal samples analysed. Luteal ERalpha and ERbeta mRNA levels were significantly lower (P<0.01 and P<0.05 respectively) at the late secretory phase than those at the early and mid-secretory phases of the endometrium. The ratio of ERalpha to ERbeta mRNA levels showed no change during the secretory phase of the endometrium. This study demonstrates that ERbeta is co-expressed with ERalpha in human corpus luteum and is likely to play a biological role in the regulation of steroidal action of the corpus luteum with ERalpha.

Dominant expression of progesterone receptor form B mRNA in ovarian endometriosis.

This study was designed to examine the biological implications of progesterone receptor form A (PR-A) and B (PR-B) mRNA expressions in human ovarian endometriosis (ectopic endometrium). A high ratio of PR-B to PR-AB (PR-A+PR-B) mRNA expression was found in 8 of 14 cases of endometriosis, compared with the ratio in eutopic endometrium. The mean ratio in ectopic endometria was significantly (p < 0.01) higher than in eutopic endometria. The ratio in eutopic and ectopic endometria showed no significant change during the menstrual cycle. The mean ratio in ectopic endometria in the proliferative and secretory phases of the endometrium was significantly (p < 0.01) higher than in eutopic endometria. In conclusion, PR-B mRNA was relatively highly expressed in some endometriomas, which might lead to aberrations in the control of progestational effects involving responsiveness to sex steroidal growth regulation.

Expression of progesterone receptor isoforms in corpora lutea of human subjects: correlation with serum oestrogen and progesterone concentrations.

To understand the biological significance of progesterone receptor forms A (PR-A) and B (PR-B) in human corpus luteum, the expression of mRNA and serum steroid hormone concentrations were determined simultaneously in the luteal stages. The expression of PR-A mRNA predominated over PR-B mRNA in all samples analysed. Total PR (PR-AB) and PR-B mRNA concentrations at the late secretory phase were significantly (P < 0.01) lower than those at the early and mid secretory phases of the menstrual cycle. The ratio of PR-B to PR-AB mRNA concentration showed no significant change during the secretory phase. In the early and mid secretory phases, there was a negative correlation between PR-B mRNA concentration and serum progesterone concentration, and between the ratio of PR-B to PR-AB mRNA concentrations and serum progesterone concentration (P < 0.01). These findings suggest that human corpus luteum might intracellularly synthesize PR-A and PR-B, and thus be involved in the steroid functional regulation of the corpus luteum, especially at the early and mid secretory phases, and that progesterone might regulate the synthesis of PR-A and PR-B.

Identification of various exon-deleted progesterone receptor mRNAs in human endometrium and ovarian endometriosis.

We demonstrated the expression of various exon-deleted progesterone receptor (PR) variant mRNAs in human uterine endometrium and ovarian endometriosis using the reverse transcription-polymerase chain reaction-DNA sequencing analyses. In addition to PR wild-type mRNA, various exon-deleted PR variant mRNAs were identified in all samples analyzed. The sequence of these variants showed a perfect junction between exons surrouding the deletion area. PR wild-type, exon 6-deleted, exon 4-deleted, exon 5, 6-deleted and exon 4, 5, 6-deleted PR variant mRNAs were observed in all samples analyzed. Exon 4, 6-deleted PR mRNA was observed only in ovarian endometriosis. This is the first study to demonstrate the coexpression of various PR exon-deleted variant mRNAs with the wild-type in uterine endometrium and ovarian endometriosis. All resulting variant proteins might indicate functional diversity and modify the progestational action of wild-type PR, and thus be involved in the pathophysiology of ovarian endometriosis.

Effects of sex steroid hormones on corticosteroid-binding globulin gene expression in human endometrial cancer cell line Ishikawa.

The effect of progestins on intracellular corticosteroid-binding globulin (CBG) mRNA expression in an endometrial cancer cell line (Ishikawa) was examined in an attempt to understand the biological effects of high-dose progestins in the treatment of well-differentiated uterine endometrial cancers. Oestradiol-17 beta (E2) significantly increased CBG mRNA expression in a dose-dependent manner, while a high dose of progesterone with or without E2 suppressed it significantly. Furthermore, a high dose of progesterone or medroxyprogesterone acetate (MPA) suppressed CBG mRNA expression to a greater degree than did chlormadinone acetate or 17 alpha-hydroxyprogesterone caproate with or without E2. These findings suggest that the effects of high-dose progestins on cancer cells may be mediated via suppression of intracellular CBG.

The failed suppression of serum estradiol level by gonadotropin-releasing hormone agonist in a case of pelvic endometriosis.

We report a case in which gonadotropin-releasing hormone agonist for a patient with pelvic endometriosis did not suppress the endometrial growth or serum levels of estradiol or follicle-stimulating hormone but did that of luteinizing hormone, regardless of the therapeutic dose in blood.

Steroid receptor mRNA levels in human corpus luteum.

To understand the biology of sex steroids in the human corpus luteum, the expression of estrogen receptor alpha, progesterone receptor, and androgen receptor mRNA levels was determined by semiquantitative reverse-transcription polymerase chain reaction-Southern blot analysis. Expression of all receptor mRNAs was detected in all samples analyzed. Each steroid receptor mRNA level was significantly lower (p < 0.05) during the late secretory phase than that during the early or the mid-secretory phase of the endometrium. These findings support the concept of a local role for sex steroids in modulating the function and life span of the human corpus luteum.

Dominant expression of sex-hormone-binding-globulin exon-7 splicing variant over wild-type mRNA in human ovarian cancers.

The intracellular expression of sex-hormone-binding-globulin(SHBG) exon-7-splicing-variant mRNA in human ovarian cancers was demonstrated by reverse-transcription/polymerase-chain-reaction, Southern-blot and DNA-sequencing analyses. Analysis of the missing base pairs proved that they corresponded to the entire exon 7, which is considered to encode a portion of the steroid-binding site, suggesting that the steroid-binding affinity of this variant might be different from that of the SHBG wild type. SHBG wild-type and variant mRNA was detected in all normal ovaries and in benign and malignant ovarian tumors analyzed. There were no significant differences in mean SHBG wild-type and variant mRNA levels among the 3 types of tissue, but the ratio of SHBG exon-7-splicing-variant to wild-type mRNA level in ovarian cancers was significantly higher (p < 0.05) than that in normal ovaries, with over-expression in some benign tumors. SHBG mRNA levels and the ratio of SHBG variant to wild-type mRNA was not associated with histological classification or clinical stages of ovarian cancers. These results suggest that over-expression of SHBG-exon 7-splicing-variant mRNA to the wild type might indicate the potential for neoplastic transformation.

Effect of medroxyprogesterone acetate on sex hormone-binding globulin mRNA expression in the human endometrial cancer cell line Ishikawa.

To understand the rationale of high-dose medroxyprogesterone acetate (MPA) in the treatment of well-differentiated uterine endometrial cancers, the effect of MPA on intracellular sex hormone-binding globulin (SHBG) mRNA expression in well-differentiated uterine endometrial cancer cell line Ishikawa was determined by competitive reverse transcription-polymerase chain reaction-Southern blot analysis. Estradiol-17beta (E2, 10(-8) mol/l) did not alter SHBG mRNA expression, but the addition of 10(-10) mol/l MPA increased it, while a high concentration of MPA (10(-6) to 10(-5) mol/l) with or without E2 suppressed it. Furthermore. a high dose (10(-6) mol/l) of chlormadinone acetate or danazol with or without E2 significantly suppressed its expression, while MPA was the most effective among the hormones tested. The effect of MPA and the other steroid hormone analogs on SHBG expression was not mediated via the progesterone receptor. These findings suggest that intracellular SHBG suppression might partly contribute to the abolition of the intracellular estrogen-dominant milieu, and may be involved as one of the mechanisms of the antitumoral effects of high-dose MPA on the development and growth of some well-differentiated endometrial cancer cells.

Expression of sex hormone-binding globulin exon VII splicing variant messenger ribonucleic acid in human ovarian endometriosis.

OBJECTIVE: To investigate the expression of sex hormone-binding globulin (SHBG) exon VII splicing variant messenger RNA (mRNA) in human ovarian endometriosis. DESIGN: The expression of SHBG variant mRNA in normal uterine endometrium and endometriotic tissue was determined. SETTING: Department of Obstetrics and Gynecology, Gifu University Hospital. PATIENT(S): Fourteen women with endometriosis and 18 women without endometriosis. INTERVENTION(S): Normal uterine endometrial and ovarian endometriotic tissues from patients who had undergone gynecological surgery were studied. MAIN OUTCOME MEASURE(S): Levels of SHBG wild-type and variant mRNAs were determined using the quantitative reverse transcription-polymerase chain reaction. RESULTS(S): Analysis of the missing base pairs proved that they corresponded to the entire exon VII. There was no significant difference between the levels of SHBG wild-type mRNA in normal endometria and in endometriotic endometria, although the levels of SHBG variant mRNA in endometriotic endometria were significantly higher than that in normal endometria. The ratio of SHBG variant to wild-type mRNA levels was significantly higher in endometriotic endometria than in normal endometria. CONCLUSION(S): This study demonstrates the coexpression of SHBG exon VII splicing variant mRNA with its wild-type and the dominant expression of the variant in ovarian endometriosis. These results might be involved in the cellular estrogenic interaction, plausibly assisting in the development and growth of ovarian endometriosis.

Relatively high expression ratio of sex hormone-binding globulin exon VII splicing variant to wild-type mRNA in human uterine cervical cancers.

We have demonstrated the intracellular expression of sex hormone-binding globulin (SHBG) exon VII splicing variant mRNA in human uterine cervical cancer using reverse transcription-polymerase chain reaction-Southern blot and DNA sequencing analyses. Analysis of the missing base pairs proved they corresponded to the entire exon VII, which is considered to encode a portion of the steroid-binding site, suggesting that the steroid-binding affinity of the variant protein might be different from that of the wild-type SHBG. In uterine cervical cancers, the wild-type mRNA levels were lower (P<0.01) and the ratio of the SHBG variant to wild-type mRNA levels was higher (P<0.01) than in the normal cervix. In cervical adenocarcinomas, the wild-type mRNA levels were higher (P<0.05) and the ratio of the SHBG variant to wild-type mRNA levels was lower (P<0.05) than in cervical keratinizing squamous cell carcinomas. There was no difference in expression among the clinical stages of cervical cancers. These results suggest that a relative increase of intracellular variant SHBG protein in human uterine cervical cancers might be involved in the disruption of the normal estrogen dependence.

Expression of sex hormone-binding globulin exon VII splicing variant mRNA in human uterine endometrium.

We have demonstrated the expression of sex hormone-binding globulin (SHBG) exon VII splicing variant mRNA in human uterine endometrium, using the reverse transcription-polymerase chain reaction-Southern blot and DNA sequencing analyses. Analysis of the missing base pairs corresponded to the entire exon VII, which are considered to encode a portion of the steroid-binding site. Therefore, the steroid-binding affinity of this variant might be different from that of the SHBG wild type. In uterine endometria, the wild-type and variant mRNA levels tended to increase with the advance of the menstrual phase, but the ratio of the SHBG variant mRNA to SHBG wild-type mRNA levels showed no significant difference during the menstrual cycle. So far, there are no indications that the SHBG variant has any biological or clinical implications in human uterine endometrium.