A germination-specific endo-beta-mannanase gene is expressed in the micropylar endosperm cap of tomato seeds.

Endo-beta-mannanase (EC 3.2.1.78) is involved in hydrolysis of the mannan-rich cell walls of the tomato (Lycopersicon esculentum Mill.) endosperm during germination and post-germinative seedling growth. Different electrophoretic isoforms of endo-beta-mannanase are expressed sequentially in different parts of the endosperm, initially in the micropylar endosperm cap covering the radicle tip and subsequently in the remaining lateral endosperm surrounding the rest of the embryo. We have isolated a cDNA from imbibed tomato seeds (LeMAN2) that shares 77% deduced amino acid sequence similarity with a post-germinative tomato mannanase (LeMAN1). When expressed in Escherichia coli, the protein encoded by LeMAN2 cDNA was recognized by anti-mannanase antibody and exhibited endo-beta-mannanase activity, confirming the identity of the gene. LeMAN2 was expressed exclusively in the endosperm cap tissue of tomato seeds prior to radicle emergence, whereas LeMAN1 was expressed only in the lateral endosperm after radicle emergence. LeMAN2 mRNA accumulation and mannanase activity were induced by gibberellin in gibberellin-deficient gib-1 mutant seeds but were not inhibited by abscisic acid in wild-type seeds. Distinct mannanases are involved in germination and post-germinative growth, with LeMAN2 being associated with endosperm cap weakening prior to radicle emergence, whereas LeMAN1 mobilizes galactomannan reserves in the lateral endosperm.

An Endo-[beta]-Mannanase Develops Exclusively in the Micropylar Endosperm of Tomato Seeds Prior to Radicle Emergence.

A galactomannan-hydrolyzing enzyme that develops pregerminatively in the micropylar region of the endosperm of the tomato (Lycopersicon esculentum [L.] Mill.) seed was characterized. The enzyme was endo-[beta]-mannanase (EC 3.2.1.78), since it hydrolyzed galactomannan into oligosaccharides with no release of galactose and mannose. The mobility of this pregerminative enzyme in sodium dodecyl sulfate and native polyacrylamide gel electrophoresis was not identical to that of any of the three endo-[beta]-mannanases that develop in the same tissue (endosperm) after germination (H. Nonogaki, M. Nomaguchi, Y. Morohashi [1995] Physiol Plant 94: 328-334). There were also some differences in the products of galactomannan hydrolysis between the pregerminative and the postgerminative enzymes, indicating that the action pattern is different between the two types of enzymes. The pregerminative enzyme began to develop in the micropylar region of the endosperm at about 18 h postimbibition and increased up to the time immediately before radicle protrusion (24 h postimbibition). This enzyme was not present in the lateral part of the endosperm at any stage before or after germination. It is proposed that the enzyme develops prior to germination specifically at the micropylar region of the endosperm.

Expression of adult T-cell leukaemia-derived factor, a human thioredoxin homologue, in the human ovary throughout the menstrual cycle.

An immunohistochemical study of the expression of adult T-cell leukaemia-derived factor (ADF), a human thioredoxin homologue, was performed in the normal human ovary throughout the menstrual cycle. Primordial follicles were negative for ADF. Both granulosa cells and theca interna cells at the stages of preantral and antral follicles contained ADF. The staining intensity of these cells was very strong in the preovulatory dominant follicle. After ovulation, both granulo-lutein and theca-lutein cells were positive for ADF. During pregnancy, the theca-lutein cells revealed very intense ADF staining. The theca interna cells of the atretic follicles showed ADF staining, while the granulosa cells of such follicles did not. These results suggest that ADF localizes in the ovarian steroidogenic cells which have the binding sites of either luteinizing hormone or follicle-stimulating hormone, and that ADF expression is closely associated with the activity of the ovarian steroidogenic cells.

Immunohistochemical localization of c-erbB-2 protein and epidermal growth factor receptor in normal surface epithelium, surface inclusion cysts, and common epithelial tumours of the ovary.

The c-erbB-2 (HER-2/neu) protein is a membrane glycoprotein growth factor receptor showing molecular homology with the epidermal growth factor receptor (EGFR). We examined the immunohistochemical reactivity of monoclonal antibodies against both of these proteins in normal surface epithelium, surface inclusion cysts, and common epithelial tumours of the ovary. The ovarian tumours were classified as benign (16), borderline malignant (2), and malignant (19). Normal surface ovarian epithelium was weakly positive for both c-erbB-2 protein and EGFR. In surface inclusion cysts, however, the epithelial cells lining the lumen exhibited stronger staining for c-erbB-2 protein, but no staining for EGFR. All 16 benign ovarian tumours and the 2 borderline malignant ovarian tumours were positive for c-erbB-2 protein and negative for EGFR. Of the ovarian carcinomas, 13 of the 19 (68.4%) were positive for c-erbB-2 protein and negative for EGFR, while 4 showed positivity for both c-erbB-2 protein and EGFR. Two cases were negative for both proteins. Expression of both c-erbB-2 protein and EGFR was found in endometrioid carcinoma with squamous differentiation and in clinically advanced poorly differentiated serous carcinomas. Expression of c-erbB-2 protein appears to be increased and that of EGFR is reduced in the early stage of epithelial ovarian oncogenesis. The expression of EGFR with c-erbB-2 protein in ovarian carcinoma is related both to histological differentiation and/or advanced clinical stage.

Expression of c-erbB-2 protein and epidermal growth factor receptor in normal tissues of the female genital tract and in the placenta.

The c-erbB-2 (HER-2/neu) protein is a membrane glycoprotein growth factor receptor that has molecular homology with the epidermal growth factor receptor (EGFR). To investigate the relationship between the expression of c-erbB-2 protein and EGFR in the tissues of the human female genital tract and in the placenta, we examined the immunohistochemical reactivity of monoclonal antibodies against both of these proteins. In the müllerian-derived genital tract, epithelial cells of the fallopian tube, endometrium, and endocervix showed reactivity for c-erbB-2 protein, whereas reactivity for EGFR was distributed mainly in the stromal cells throughout the menstrual cycle and during pregnancy. In addition, the staining intensity for EGFR in the endometrial stroma increased with its decidualization. In the exocervical squamous epithelium, basal cells were c-erbB-2 protein-negative and EGFR-positive, but the more differentiated squamous cells of the intermediate layer were c-erbB-2 protein-positive and EGFR-negative. In the placental tissues, cytotrophoblasts and syncytiotrophoblasts of the chorionic villi were c-erbB-2 protein-negative and EGFR-positive. In contrast, intermediate trophoblasts in the extravillous space were c-erbB-2 protein-positive and EGFR-negative. Thus, there is an inverse relationship between the expression of c-erbB-2 protein and EGFR in the tissues of the female genital tract and in the placenta. This suggests that there may be a regulatory mechanism(s) for the expression of both proteins that is associated with the differentiation and/or function of cells in the female genital tract and the placenta.

Serial changes of serum CA125 levels during menstrual cycles.

Serial changes of serum CA125 levels during 32 menstrual cycles were examined in 32 healthy young women (19-21 years of age) charting basal body temperature (BBT) and measuring serum estradiol and progesterone levels. Analysis of BBT charts and serum progesterone levels revealed that the 32 menstrual cycles could be classified into either an ovulatory cycle with a sustained BBT temperature increase for at least 10 days (type I: 9 cycles), an ovulatory cycle with a sustained BBT temperature increase for less than 10 days (type II: 17 cycles), or anovulation (type III: 6 cycles). All 32 cycles exhibited basal CA125 levels of less than 35 u/ml throughout the cycle, except during the period of menstruation. At menstruation, 7 of the 9 cycles of type I showed a marked, transient elevation of CA125 levels from previous basal levels (a mean net increase). On the other hand, 12 of 17 cycles of type II showed only a slight elevation of CA125 levels during the period of menstruation. None of the 6 cycles of type III showed any apparent increase in CA125 levels during the period of menstruation. The mean net increase of CA125 levels during the period of menstruation in the cycles of type I was significantly higher than that of the cycles of type II (p less than 0.05) and type III (p less than 0.05). These results imply that a transient elevation of serum CA125 levels during the period of menstruation occurs in cycles with ovulation, and the levels of elevation seem to be closely associated with the duration of the sustained BBT temperature increase.

An extraordinarily high CA 125 level in a woman without apparent pathologic foci of CA 125 production: dissociation between serum levels of CA 125 and CA 130.

We describe a patient with high serum CA 125 levels before and after surgery (greater than 403 U/ml) who had no apparent pathologic foci of CA 125 production. Serum levels of CA 130, which exists on the same glycoprotein as CA 125, were within normal ranges (less than 35 U/ml) before and after surgery.

Effect of human chorionic gonadotropin on the expression of progesterone receptors and estrogen receptors in rabbit ovarian granulosa cells and the uterus.

To investigate whether LH/human CG (hCG) or progesterone acts as a regulator of estrogen receptors (ER) and progesterone receptors (PR) in granulosa cells, we studied the immunohistochemical expression of both ER and PR in the ovary and the uterus of mature rabbits, during the induction of ovulation by FSH followed by administration of hCG, progesterone, or a progesterone antagonist (RU486) and hCG. Granulosa cells pretreated with FSH for 3 days showed ER staining, but negligible PR staining. The staining pattern for ER and PR changed in animals pretreated with FSH followed by hCG injection; by 6 h after hCG injection, we observed the disappearance of ER and the appearance of PR, and by 3 days after hCG injection, we observed the reappearance of ER and the disappearance of PR. However, the expression of ER and PR in the granulosa cells of animals pretreated with FSH followed by progesterone administration instead of hCG was almost the same as that of animals pretreated with FSH alone. In addition, the expression of ER and PR in the granulosa cells of animals pretreated with FSH followed by RU486 and hCG was almost the same as that of animals pretreated with FSH followed by hCG administration. The uterine glandular epithelium, in contrast, began to show decreased appearance of ER and PR by 48 h after hCG injection, and we observed the disappearance of both receptors by 3 days after hCG administration. These results suggest that the expression of ER and PR in granulosa cells is not regulated by the action of progesterone, but by that of LH/hCG.

Coexpression of adult T-cell leukemia-derived factor, a human thioredoxin homologue, and human papillomavirus DNA in neoplastic cervical squamous epithelium.

Adult T-cell leukemia-derived factor (ADF) is an autocrine interleukin-2 receptor-inducing factor produced by human T-lymphotropic virus-1 (HTLV-1)-transformed lymphocytes, which has a high structural homology with an endogenous dithiol reducing coenzyme, thioredoxin. Its localization was investigated immunohistochemically in the cervix, using normal tissue (27 samples) and squamous neoplastic tissue (three condylomas, 42 cervical intraepithelial neoplasia [CIN] samples, 34 invasive squamous cell carcinoma samples). The expression of human papillomavirus (HPV) DNA was also studied in serial sections of the same subjects. Normal squamous cells and glandular cells of the cervix were negative for ADF. However, intracytoplasmic and/or intranuclear ADF-positive cells were usually found in the intermediate and superficial layers of the neoplastic squamous epithelium of condylomas (three of three cases) and CIN (35/42 cases). HPV DNA was detected in all condylomas and in 27 of 42 CIN specimens. HPV DNA-positive cells were usually localized in the intermediate and superficial layers of the neoplastic squamous epithelium. These HPV DNA-positive cells were also positive for ADF. Invasive squamous cell carcinoma was also positive for ADF (24/34 cases) and HPV DNA (11/34 cases). The coexpression of HPV DNA and ADF was observed in all HPV DNA-positive cases. Coexistence of HPV DNA and ADF immunopositivity in neoplastic squamous cells of the cervix suggests that ADF expression closely reflects the intracellular event on HPV DNA replication.

Immunohistochemical analysis of estrogen receptors, progesterone receptors, Ki-67 antigen, and human papillomavirus DNA in normal and neoplastic epithelium of the uterine cervix.

To investigate the relationship between the sex steroid receptor (estrogen receptor [ER] and progesterone receptor [PR]) status and the cell proliferation kinetics during the menstrual cycle in normal and neoplastic epithelium of the uterine cervix, immunohistochemical localization of ER, PR, and cell proliferation-associated antigen, Ki-67, was investigated in 35 normal cervical specimens, 3 condylomas, 26 cervical intraepithelial neoplasia (CIN) samples, and 22 invasive squamous carcinoma samples. The presence of human papillomavirus (HPV) DNA was also studied. In the normal cervix, basal cells were usually ER positive, PR negative, and Ki-67 negative throughout the menstrual cycle. Parabasal cells were ER positive and PR negative in the follicular phase, but ER negative and PR positive, and Ki-67 positive in the luteal phase, and Ki-67-positive cells increased in number in the luteal phase. In contrast, PR positivity was observed in the cells of condyloma (2 of 2 cases), CIN (19 of 26 cases), and invasive squamous carcinoma (13 of 22 cases) irrespective of the menstrual phase. Moreover, most neoplastic cells containing HPV DNA type 16/18 were ER negative, whereas several lesions containing HPV DNA type 31/33/35 were weakly ER positive. Many Ki-67-labeled cells were observed in the neoplastic lesions. These results suggest that reduced ER expression and increased PR expression are associated with the proliferation of normal cervical squamous epithelium, and this proliferation-related receptor status, which is probably induced by HPV infection, is usually expressed in neoplastic cervical squamous cells.

[Clinico-pathological study of cervical carcinoma with massive infiltration of eosinophils].

Eight patients with cervical carcinoma with massive stromal infiltration of eosinophils were selected for a clinicopathological study among 474 patients with cervical carcinoma at stages Ib-IIIb who underwent radical hysterectomy between 1971 and 1989 at Kyoto University Hospital. The 8 patients ranged in age from 28 to 48 years and had menstrual cycles. The peripheral leucocyte count in 5 patients showed eosinophilia of the peripheral blood. Histologically, the cervical carcinoma was a locally advanced tumor with invasion of more than 2/3rds the depth of the cervical wall in 6 cases, and with parametrial involvement in 4 cases. Massive infiltration of eosinophils was observed not only around the cancer nests but also within the regional lymph nodes. Mast cells expressing immunoreactivity for histamine were scarce around the tumor. Consequently, the production of an eosinophil chemotactic factor by the tumor cells was strongly suggested. PAS and Alcian-blue staining as well as electron microscopy revealed that the tumor cells in most cases had the histological characteristics of both squamous carcinoma and adenocarcinoma. Collectively, these clinicopathological characteristics of cervical carcinoma with massive infiltration of eosinophils distinguish this tumor from conventional cervical carcinomas.

Immunohistochemical analysis of oestrogen receptors, progesterone receptors and Ki-67 in leiomyoma and myometrium during the menstrual cycle and pregnancy.

Immunohistochemical distribution of oestrogen receptors (ER), progesterone receptors (PR), and the cell proliferation-associated antigen Ki-67 was investigated in leiomyomas and the myometrium during the menstrual cycle and pregnancy. In the myometrium, ER expression was observed in the proliferative phase, but was suppressed in the secretory phase and during pregnancy. In leiomyomas, ER expression was observed throughout the menstrual cycle, but was suppressed during pregnancy. However, PR was expressed both in the myometrium and leiomyomas throughout the menstrual cycle and pregnancy. In both the myometrium and leiomyomas, a higher number of Ki-67-positive cells was observed during pregnancy than in the secretory phase, and Ki-67 was negative during menopause. The Ki-67-positive cell count in leiomyomas was significantly higher than that in the myometrium throughout the menstrual cycle and pregnancy. Thus both myometrium and leiomyomas have high growth activity under the hormonal milieu of high progesterone levels. The growth potential of leiomyomas is apparently higher than that of myometrium throughout the menstrual cycle and during pregnancy.

Estrogen receptor localization in normal and neoplastic epithelium of the uterine cervix.

To investigate the estrogen receptor (ER) status of cells during carcinogenesis of the uterine cervix, the immunohistochemical reactivity for a monoclonal anti-ER antibody (H 222) was studied in 26 normal cervical specimens, 21 cases of cervical intraepithelial neoplasia (CIN), and 21 cases of invasive cervical carcinoma. In addition, the presence of human papillomavirus (HPV) DNA (types 6/11, 16/18, or 31/33/35) was analyzed by in situ hybridization. In the normal cervix, basal cells of the squamous epithelium, metaplastic cells, and endocervical glandular cells were ER positive. In contrast, neoplastic cells of CIN (17 of 21 cases) and invasive carcinoma (19 of 21 cases) were ER negative. The remaining four cases of CIN and two cases of invasive carcinoma were focally ER positive. The HPV DNA analysis revealed that HPV DNA in ER-negative cases was either types 16/18 or undetectable, but all ER-positive neoplasms contained HPV DNA types 31/33/35. These results suggest that most neoplastic cells in CIN and invasive cervical carcinoma lose their ER expression and that this may be related to the HPV DNA types which they possess.

Immunohistochemical localization of CA130 in fetal tissues, and in normal and neoplastic tissues of the female genital tract.

A murine monoclonal antibody, 130-22, produced against a human lung adenocarcinoma cell line (PC-9) has been suggested as recognizing an antigenic determinant (CA130) which is different from an epitope recognized by OC125 on CA125 glycoprotein molecules. The immunohistochemical reactivity with the 130-22 antibody (anti-CA130) was examined in human fetal tissues, and normal and neoplastic tissues of female genital tracts, and compared to those using OC125. Among the fetal tissues, the amnion and the cells of coelomic epithelium and mullerian-derived epithelia reacted with anti-CA130. In normal adult tissues, cervical and endometrial gland cells, tubal epithelial cells, and ovarian surface cells reacted with anti-CA130. In addition, predecidual cells in the late secretory endometrium and decidual cells during gestation were positive. Among the neoplastic tissues, tubal and endometrial adenocarcinomas and epithelial ovarian tumors were positive for anti-CA130. There were no differences in the respective specimens between the immunohistochemical localization of anti-CA130 and of OC125. Therefore, anti-CA130 is considered to be useful in the immunohistochemical detection of CA125 glycoprotein molecules as well as OC125.

Primary ovarian malignant amelanotic melanoma arising in cystic teratoma.

A case of primary ovarian malignant amelanotic melanoma arising in a cystic teratoma is presented with the histology, immunohistochemistry, and ultrastructure of the tumor.

Construction of an immunoradiometric assay for ovarian cancer associated antigen CA125 recognizing different antigenic determinant.

We generated five murine monoclonal antibodies reactive with ovarian cancer-associated antigen CA125. These monoclonal antibodies seemed to bind to separate epitopes from OC125 antibody, known to recognize CA125. A series of immunoradiometric assays for measuring serum CA125 values rapidly and sensitively were devised using these monoclonal antibodies. The antigenic determinant of a new immunoradiometric assay was different from that of a currently used CA125 kit employing OC125 both as a catcher and a tracer. However, serum antigen levels were closely correlated to each other and were elevated not only in patients with ovarian cancer, but also in patients with endometriosis and in some normal females during menstruation. These results suggest that CA125 has at least two antigenic determinants close to each other and this new rapid assay is useful, although not specific for ovarian cancer, in patients with gynecological disorders.

Mucin leakage into the cervical stroma may increase lymph node metastasis in mucin-producing cervical adenocarcinomas.

The relationships between the histologic feature of mucin leakage into the cervical stroma, lymph node metastasis, and the levels of serum carcinoembryonic antigen (CEA), CA 19-9, and CA 125 were analyzed in 35 cases of cervical adenocarcinoma. Histologically, mucin leakage was identified in 14 (40%) cases as amorphorous materials dissecting the connective tissues and permeating the lymphatic channels, associated with or without cancer cells. The cases with mucin leakage showed a significantly higher incidence of lymph node involvement than those without mucin leakage (71.4% versus 23.8%; P less than 0.01). In addition, when the mucin leakage was immunohistochemically positive for CEA or CA 19-9, elevated serum levels of these antigens were frequently observed. These results suggest that the mucin leakage into the cervical stroma represents not only stromal invasion, but also a means that frequently conducts cancer cells into the lymphatic channels. Clinically, this is reflected in an elevation of serum CEA or CA 19-9 levels in cervical adenocarcinoma patients.

CA 125 in the epithelium closely related to the embryonic ectoderm: the periderm and the amnion.

The amnion is believed to be derived from either cytotrophoblastic cells or embryonic ectoderm. However, it produces and secretes CA 125, which is considered a differentiation antigen of fetal coelomic epithelium derived from the mesoderm of germ cells. To verify this, the immunohistochemical localization of CA 125 in human fetal tissues (between 7 and 23 weeks of gestation) derived from the ectoderm, endoderm, or mesoderm, and in the fetal membranes and placenta was studied. Among the mesoderm-derived tissues, only the fetal coelomic epithelium-related tissues were positive for anti-CA 125 from 15 weeks of gestation. The endoderm-derived tissues did not react with anti-CA 125. However, among the ectoderm-derived tissues, only the periderm reacted with anti-CA 125 from 7 weeks until it sloughed from the stratum intermedium by 23 weeks of gestation. Among the fetal membranes and placenta, only the amnion reacted with anti-CA 125 from 9 weeks to term. These findings indicate that the amnion and the periderm, both of which constitute the epithelia covering the amniotic cavity, in addition to the fetal coelomic epithelium-related tissues, produce CA 125.

Mitotic activity in uterine leiomyomas during the menstrual cycle.

To investigate the role of the menstrual cycle in the growth of uterine leiomyomas, the mitotic count per 100 high-power fields and the relation of this to the patient's age (30 to 54 years) were examined in tissue sections of leiomyomas from 181 surgically removed myomatous uteri. The mean mitotic count in the secretory phase (12.7 per 100 high-power fields) was significantly higher than that of the proliferative phase (3.8 per 100 high-power fields) (p less than 0.01) or menses (8.3 per 100 high-power fields) (p less than 0.05). We found the highest mitotic count (54 per 100 high-power fields) in a leiomyoma at the early secretory phase. In the secretory phase, the younger age group exhibited a significantly higher mitotic count than the older group. Increased mitotic activity in leiomyomas under the hormonal milieu of the secretory phase of the menstrual cycle suggests that the growth of these tumors is affected by progesterone level. In addition, this study defined the range of mitotic counts occurring in the tissue sections of typical leiomyomas during the menstrual cycle.