The membrane protein CD9/DRAP 27 potentiates the juxtacrine growth factor activity of the membrane-anchored heparin-binding EGF-like growth factor.

The membrane-anchored heparin-binding EGF-like growth factor precursor (proHB-EGF)/diphtheria toxin receptor (DTR) belongs to a class of transmembrane growth factors and physically associates with CD9/DRAP27 which is also a transmembrane protein. To evaluate the biological activities of proHB-EGF/DTR as a juxtacrine growth factor and the biological significance of its association with CD9/DRAP27, the mitogenic activity of proHB-EGF/DTR was analyzed using stable transfectants of mouse L cells expressing both human proHB-EGF/DTR and monkey CD9/DRAP27, or either one alone. Juxtacrine activity was assayed by measuring the ability of cells in co-culture to stimulate DNA synthesis in an EGF receptor ligand dependent cell line, EP170.7. LH-2 cells expressing human proHB-EGF/DTR stimulated EP170.7 cell growth moderately. However, LCH-1 cells, a stable co-transfectant expressing both human proHB-EGF/DTR and monkey CD9/DRAP27 cDNAs, dramatically unregulated the juxtacrine growth factor activity of proHB-EGF/DTR approximately 25 times over that of LH-2 cells even though both cell types expressed similar levels of proHB-EGF/DTR on the cell surface. Anti-CD9/DRAP27 antibodies which were not able to neutralize the mitogenic activity of soluble HB-EGF suppressed LCH-1 cell juxtacrine growth activity to the same extent as did anti-HB-EGF neutralizing antibodies and CRM 197, specific inhibitors of human HG-EGF. These findings suggest that optimal expression of the juxtacrine growth activity of proHB-EGF/DTR requires co-expression of CD9/DRAP27. These studies also indicate that growth factor potentiation effects which have been observed previously for soluble growth factors also occurs at the level of cell surface associated growth factors.

Phorbol ester induces the rapid processing of cell surface heparin-binding EGF-like growth factor: conversion from juxtacrine to paracrine growth factor activity.

Vero cell heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a 20- to 30-kDa membrane-anchored HB-EGF precursor (proHB-EGF). Localization and processing of proHB-EGF, both constitutive and 12-O-tetradecanoylphorbol 13-acetate (TPA)-inducible, was examined in Vero cells overexpressing recombinant HB-EGF (Vero H cells). Flow cytometry and fluorescence immunostaining demonstrated that Vero cell proHB-EGF is cell surface-associated and localized at the interface of cell to cell contact. Cell surface biotinylation and immunoprecipitation detected a 20- to 30-kDa heterogeneous proHB-EGF species. Vero H cell surface proHB-EGF turned over constitutively with a half-life of 1.5 h. Some of the 20- to 30-kDa cell surface-associated proHB-EGF was processed and a 14-kDa species of bioactive HB-EGF was released slowly, but most of the proHB-EGF was internalized, displaying a diffuse immunofluorescent staining pattern and accumulation of proHB-EGF in endosomes. Addition of TPA induced a rapid processing of proHB-EGF at a Pro148-Val149 site with a half-life of 7min. The TPA effect was abrogated by the protein kinase C inhibitors, staurosporine and H7. Kinetic analysis showed that loss of cell surface proHB-EGF is maximal at 30 min after addition of TPA and that proHB-EGF is resynthesized and the initial cell surface levels are regained within 12-24 h. Loss of cell surface proHB-EGF was concomitant with appearance of 14- and 19-kDa soluble HB-EGF species in conditioned medium. Vero H cell-associated proHB-EGF is a juxtacrine growth factor for EP170.7 cells in coculture. Processing of proHB-EGF resulted in loss of juxtacrine activity and a simultaneous increase in soluble HB-EGF paracrine mitogenic activity. It was concluded that processing regulates HB-EGF bioactivity by converting it from a cell-surface juxtacrine growth factor to a processed, released soluble paracrine growth factor.

Bilateral hamartoma arising in axillary accessory mammary glands. Case report.

A 57-year-old Japanese woman presented with a lump, originally noticed 10 years previously, in both axillary regions. Histologically, the axillary tumors consisted of a random admixture of ducts, lobules and fibrous stroma with predominant adipose tissue. This is the first case of bilateral ectopic hamartoma arising in the axillary regions. Surgeons and pathologists should be aware that ectopic breast hamartoma can occur in both axillary regions.

Sex-determining gene(s) on distal 9p: clinical and molecular studies in six cases.

We report on clinical and molecular findings in five karyotypic males (cases 1-5) and one karyotypic female (case 6) with distal 9p monosomy. Cases 1-3 and 6 had female external genitalia, case 4 showed ambiguous external genitalia, and case 5 exhibited male external genitalia with left cryptorchidism and right intrascrotal testis. Gonadal explorations at gonadectomy in cases 3 and 4 revealed that case 3 had left streak gonad and right agonadism, and case 4 had bilateral hypoplastic testes. Endocrine studies in cases 1-4 and 6 showed that cases 1, 3, and 6 had definite primary hypogonadism, with basal FSH levels of 54, 39, and 41 IU/L, respectively, whereas case 2 with severe malnutrition was unremarkable for the baseline values, and case 4 had fairly good testicular function. Fluorescence in situ hybridization and microsatellite analyses demonstrated that all cases had hemizygosity of the 9p sex-determining region distal to D9S1779, with loss of the candidate sex-determining genes DMRT1 and DMRT2 from the abnormal chromosome 9. Sequence analysis in cases 1-4 and 6 showed that they had normal sequences of each exon of DMRT1 and the DM domain of DMRT2 on the normal chromosome 9, and that cases 1-4 had normal SRY sequence. The results provide further support for the presence of a sex-determining gene(s) on distal 9p and favor the possibility of DMRT1 and/or DMRT2 being the sex-determining gene(s). Furthermore, as hemizygosity of the 9p sex-determining region was associated with a wide spectrum of gonadogenesis from agonadism to testis formation in karyotypic males and with primary hypogonadism regardless of karyotypic sex, it is inferred that haploinsufficiency of the 9p sex-determining gene(s) primarily hinders the formation of indifferent gonad, leading to various degrees of defective testis formation in karyotypic males and impaired ovary formation in karyotypic females.

The chromosomal normality of unfertilized oocytes from patients with polycystic ovarian syndrome.

The present study was designed to compare the cycle characteristics of in-vitro fertilization (IVF) and the chromosomal normality of oocytes in patients with polycystic ovarian syndrome (PCOS) with those of patients with tubal factor infertility. In all, 28 cycles of 24 PCOS patients and 55 cycles of 31 patients with tubal factor infertility (control) were investigated. Although a significantly greater number of oocytes were retrieved from PCOS patients (mean +/- SD: 15.6 +/- 6.4 versus 9.0 +/- 4.0, PCOS versus control group, P < 0.05), the percentage of fertilized oocytes was significantly lower in the PCOS group compared with controls (40.1 versus 73.8%, P < 0.01). The pregnancy rate per embryo transfer did not differ between the two groups. Cytogenetic analysis was performed on 74 oocytes from PCOS patients and 73 oocytes from control patients. In the PCOS group, 10 of the 74 oocytes (13.5%) demonstrated aneuploidy, four (5.4%) oocytes were diploid and six (8.1%) oocytes were metaphase II with a prematurely condensed sperm chromosome (PCC). In the tubal infertility group, nine of the 73 (12.3%) oocytes showed aneuploidy, four (5.5%) oocytes were diploid and five (6.8%) oocytes were found to have PCC. There was no significant difference in the aneuploidy, diploidy and PCC rates between the two groups. These results suggest that the reduced fertilization observed in PCOS is not attributable to chromosomal aberrations or immaturity of oocytes recruited from patients with PCOS.

Expression of superoxide dismutase (SOD) in adenomyosis.

OBJECTIVE: To assess the expression of Cu,Zn-SOD and Mn-SOD in peritoneal fluid and ectopic endometrium from women with adenomyosis. STUDY DESIGN: The levels of Cu,Zn-SOD and Mn-SOD were measured in peritoneal fluid from infertile women who did not have endometriosis (n = 27), women with adenomyosis (n = 22), and women with endometriosis (n = 15). The expression of Cu,Zn-SOD and Mn-SOD messenger ribonucleic acids in ectopic endometrium was investigated by Northern blot analysis. Mn-SOD in ectopic endometrium was investigated by immunohistochemical staining. RESULTS: The levels of Cu,Zn-SOD and Mn-SOD were significantly higher in the peritoneal fluid from women with adenomyosis than in those with endometriosis or infertile women. Mn-SOD was highly expressed in ectopic endometrium, but Cu,Zn-SOD was not. Mn-SOD was detected in ectopic endometrium of women with adenomyosis. CONCLUSIONS: These data show that reactive oxygen produced in adenomyosis may induce Mn-SOD in ectopic endometrium, which results in the release of relatively large amounts of this protein into the peritoneal fluid. These results suggest that free radicals may play a role in the pathogenesis of adenomyosis, the same as they do in other inflammatory processes.

High glucose and hyperosmolarity increase heparin-binding epidermal growth factor-like growth factor (HB-EGF) production in cultured human aortic endothelial cells.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been shown to be a potent smooth muscle cell (SMC) mitogen and chemoattractant, and might be a candidate factor for the progression of atherosclerosis. We have investigated the effects of high glucose and hyperosmolarity on HB-EGF production in cultured human aortic endothelial cells. Following the culture of the cells for 2 days with high concentrations of glucose or in the hyperosmolar conditions, we measured the content of HB-EGF and the rate of production in the cells using a semi-quantitative immunofluorescent technique and a metabolic radiolabelling method. With high glucose (16.6 mmol) and hyperosmolar conditions (glucose 5.5 mmol + mannitol 11.1 mmol or glucose 5.5 mmol + raffinose 11.1 mmol), the content of HB-EGF was significantly increased and the metabolic rate was also significantly increased (more than a twofold increase, compared to that of 5.5 mmol glucose). In conclusion, conditions of high glucose or hyperosmolarity increase HB-EGF production in human aortic endothelial cells. These results suggest that diabetic macroangiopathy might be attributed at least in part to HB-EGF-related vascular changes which may be induced by glucose.

Requirement of sperm-oocyte plasma membrane fusion for establishment of the plasma membrane block to polyspermy in human pronuclear oocytes.

We investigated whether the incorporation of the sperm membrane into the oolemma contributes to the human plasma membrane block to polyspermy. We used zona pellucida-free oocytes fertilized by intracytoplasmic sperm injection (ICSI) or activated by parthenogenetic activation. Only two of the 35 pronuclear oocytes fertilized by spermatozoa (control) demonstrated one single penetrating spermatozoa. In contrast, the majority of ICSI and parthenogenetically activated pronuclear oocytes were penetrated with an average of three spermatozoa per oocyte. The number of fused and binding spermatozoa of ICSI and parthenogenetically activated oocytes were significantly higher than in control oocytes (3.5+/-0.6 and 4.3+/-0.6 for ICSI; 3.0+/-0.3 and 3.8+/-0.4 for activated and 0.2+/-0.1 and 0.6+/-0.2 for controls, respectively, P < 0.01). Furthermore, the cortical granules were released from the cortex of ICSI and calcium ionophore-puromycin-activated pronuclear oocytes to the same extent as that of pronuclear oocytes fertilized by spermatozoa. These results suggest that the establishment of the plasma membrane block to sperm penetration in the human oocyte may require a fusion process between sperm and oocyte plasma membranes.

The clinical efficacy of low-dose step-up follicle stimulating hormone administration for treatment of unexplained infertility.

The present study was designed to compare the clinical efficacy of low-dose step-up follicle stimulating hormone (FSH) administration with conventional FSH protocol (FSH was injected daily starting with a dose of 150 IU), both combined with intrauterine insemination (IUI), for the treatment of unexplained infertility. A total of 97 unexplained infertility couples was randomly assigned to one or other of the two treatment groups, either conventional FSH with IUI (48 patients) or low-dose step-up FSH with IUI (49 patients), and only the first treatment cycle was evaluated in each protocol. The difference in pregnancy rates per cycle was not statistically significant between the low-dose FSH group and the conventional group [seven of 49 (14.3%) and seven of 48 (14.6%) respectively]. A significant reduction in the incidence of ovarian hyperstimulation syndrome (OHSS) was observed in the low-dose group (8.3% versus 27.1%, P < 0.05). The incidence of moderate OHSS requiring hospitalization was reduced significantly in the low-dose group (low-dose 0% versus conventional 16.7%, P < 0.01). However, the low-dose protocol did not completely prevent multiple pregnancies. Our results suggest that the low-dose step-up FSH treatment appeared to be useful for the treatment of unexplained infertility because of the high pregnancy rates and the significant decrease in the incidence of OHSS.