Yoshihito Nakanishi

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.

Expression of sex hormone-binding globulin mRNA in human endometrial cancers

To more fully understand the role of sex hormone-binding globulin (SHBG) on the intracellular steroidal action in endometrial cancers, we investigated the expression of SHBG mRNA as the substitute of SHBG expression in human endometrial cancers. In the present study, the levels of SHBG mRNA were analyzed using competitive reverse transcription-polymerase chain reaction (RT-PCR)-Southern-blot analysis. The higher level of SHBG mRNA tended to be expressed in the normal secretory and late proliferative phase endometrium > early proliferative phase endometrium > well differentiated adenocarcinoma of the endometrium (G1) > moderately differentiated adenocarcinoma (G2) > poorly differentiated adenocarcinoma (G3), in the order shown. These studies indicate that endometrial cancer cells might synthesize intracellular SHBG to conserve their estrogen-dependent properties. Further, it indicates that endometrial cancer cell synthesis of SHBG mRNA is lost as these cells undergo de-differentiation.

Expression of sex hormone-binding globulin mRNA in uterine leiomyoma, myometrium and endometrium of human subjects

This study was designed to evaluate the effect of sex hormone-binding globulin (SHBG) on the estrogen-dependent growth of human uterine leiomyoma. Levels of SHBG mRNA were analyzed using competitive reverse transcription-polymerase chain reaction-Southern blot analysis (RT-PCR-SBA). In normal uterine endometria, the levels of SHBG mRNA in the early/mid and late proliferative phases of the menstrual cycle were significantly lower than in the secretory phase (p < 0.01). In uterine myometria and leiomyomas, SHBG mRNA level showed no significant differences during the menstrual cycle, while the ratio of leiomyoma SHBG level to the corresponding myometrium SHBG level was >1 in 21 out of 23 cases (91%). Our results suggest that steroidal regulation of intracellular SHBG synthesis in the myometrium and the leiomyoma might differ from that in the endometrium, and that the mechanism of SHBG expression in leiomyoma might, in the process of tumorigenesis, be altered from that of corresponding normal myometrium, contributing to SHBG overexpression and the abundant estrogen supply.

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.

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.

Expression of Sex Hormone-Binding Globulin and Corticosteroid-Binding Globulin mRNAs in Corpus luteum of Human Subjects

To understand the biology of sex steroids in human ovarian corpus luteum, the expression of intracellular sex hormone-binding globulin (SHBG) and corticosteroid-binding globulin (CBG) mRNAs as a manifestation of intracellular SHBG and CBG expression was determined. The expression of SHBG and CBG mRNAs was detected in all samples analyzed. Luteal SHBG mRNA level showed no significant change during the endometrial secretory phase of the menstrual cycle. On the other hand, luteal CBG mRNA level was significantly higher (p < 0.05) at the mid-secretory phase than that at the early and late secretory phases of the endometrium. These findings suggest that human ovarian corpus luteum synthesizes SHBG and CBG intracellularly, CBG being plausibly involved in the functional life span of corpus luteum.

Localization of sex hormone-binding globulin mRNA expression in human uterine endometrium

To identify the dominant cell of sex hormone-binding globulin (SHBG) synthesis in human uterine endometrium, we investigated the expression of endometrial SHBG mRNA using Northern blot and in situ hybridization analyses. Expression of a single dominant SHBG mRNA was detected in uterine endometrium using Northern blot analysis. Additionally, SHBG mRNA expression was demonstrated by in situ hybridization in the glandular epithelial cells of the endometrium, but not in the stromal cells. Therefore, in the endometrial glandular epithelial cells, SHBG might be involved in the intracellular steroidal action, but not in the endometrial stromal cells. The SHBG-mediated effects on the endometrium appear to be heterogeneous.

Expression of estrogen and progesterone receptors and their mRNAs in ovarian endometriosis

To discover the molecular mechanisms of estrogen-induced growth in ovarian endometriosis, the expression of estrogen and progesterone receptors and their mRNAs was investigated. The expression of estrogen and progesterone receptors and their mRNAs was significantly (p < 0.01) lower in endometriotic endometria than in normal endometria. The ratio of estrogen receptors: progesterone receptors was significantly (p < 0.01) higher in the endometriotic tissues than in normal proliferative- and secretory-phase endometria, as was the ratio of their respective mRNAs. These findings suggest that the absolute and relatively reduced number of estrogen receptors in ovarian endometriosis might cause the loss of control of estrogenic action, and that, furthermore, the relatively increased number of progesterone receptors might lead to an estrogen-dominant milieu, assisting in the development and growth of the ovarian endometriosis.

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.

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.