Distribution and cytological properties of macrophages in human Fallopian tubes.

The macrophages in human Fallopian tubes of women in the reproductive and postmenopausal periods were examined with a focus on their morphological properties by immunohistochemical staining and transmission electron microscopy. The fine structure of the smooth muscle cells in the Fallopian tubes was also investigated during the reproductive period. For immunohistochemical staining, we used two monoclonal antibodies that were specific for human macrophages, namely PM-1K and PM-2K. PM-1K recognizes human monocytes/ macrophages corresponding to CD68, and PM-2K recognizes tissue macrophages. PM-1K-positive cells were always present and their numbers increased significantly during the menstrual and early to mid-secretory phases. In contrast, relative numbers of PM-2K-positive cells were small throughout the menstrual cycle. In the postmenopausal period, few PM-1K-positive cells were detected, but PM-2K-positive cells remained. The macrophages during the secretory phase in the endosalpingeal stroma had well-developed intracytoplasmic organelles, but relatively few cytoplasmic vacuoles and granules. In the same phase, many cells of the monocyte/macrophage lineage appeared in the vascular lumen of the endosalpingeal stroma. The macrophages during the menstrual phase had well-developed intracellular organelles, with cytoplasmic vacuoles and granules of various sizes and configurations. During the late secretory phase, just prior to menstruation, the smooth muscle cells contained few cytoplasmic filaments but electron-lucent or electron-dense lysosome-like bodies were seen. These findings suggested the presence of macrophages in human Fallopian tubes. It is possible that such macrophages might be involved in the physiological functions of the tubes during the reproductive period and moreover that they might participate in the reconstruction of the muscle layer of the tubes.

Spatio-temporal changes of prolyl 4-hydroxylase in granulosa cells during ovulation in eCG-hCG-treated immature rat ovaries.

The immunolocalization of prolyl 4-hydroxylase (PHase), a key enzyme of collagen synthesis, and the effects of anti-progesterone RU486 on PHase during the ovulatory process in eCG-hCG-treated immature rat ovaries were studied to investigate the mechanisms of tissue repair in follicle walls after follicular rupture. Immunolocalization of PHase was studied using an anti-rat PHase subunit monoclonal antibody, and the amount of immunoreactive PHase was measured by enzymeimmunoassay. No obvious immunolocalization of PHase was observed in theca cells throughout the ovulatory process except just after follicular rupture. In contrast, in granulosa cells, PHase was first observed at 9 h after the hCG injection, and the staining intensity apparently increased from 9 to 15 h, especially around the apex of preovulatory follicles and the orifice of ruptured follicles. Consistent with these observations, PHase concentration in granulosa cells isolated from the ovaries significantly increased by 9 h (0.45 +/- 0.03 pg per cell), and reached a peak at 15 h (0.66 +/- 0.06 pg per cell) after the hCG injection. This peak was inhibited when 20 mg RU486 kg-1 was administered at 8 h (0.46 +/- 0.05 pg per cell), and the RU486-inhibited PHase concentration was recovered by the concomitant administration of 10 mg progesterone kg-1 (0.65 +/- 0.02 pg per cell). The results suggest that PHase expressed in granulosa cells may play an important role in the repair of ruptured follicle walls, via progesterone-dependent PHase production.

Establishment of a new cell line, OKT1, from small cell carcinoma secreting ectopic ACTH of the uterine cervix.

OBJECTIVE: Small cell carcinoma of the uterine cervix is rare and represents a unique entity among gynecological tumors. It sometimes demonstrates neuroendocrine differentiation, including adrenocorticotropin (ACTH) secretion. In this study, we established a new cell line, OKT1, from a case of carcinoma secreting ectopic ACTH without Cushing's syndrome and determined the character of the cell line. METHODS: OKT1 was established from OKT tumor cells, derived from a biopsy specimen of small cell cervical carcinoma, and serially heterotransplanted into nude mice. To characterize OKT1, the cell morphology, growth properties, immunohistochemical properties, hormone- and tumor-associated antigen secretion, tumorigenic potential, DNA profile, and chromosomal alteration were studied. RESULTS: The population doubling time of OKT1 was approximately 27 h. The cytological properties of OKT1, including DNA ploidy pattern, were similar to those of the primary tumor. Neuroendocrine differentiation was shown in the OKT1 cells by the positive immunocytochemical staining of neuron-specific enolase (NSE) and the presence of NSE and ACTH in the culture media. The xenograft of 1 x 10(8) OKT1 cells into nude mice yielded tumor mass. Furthermore, OKT1 demonstrated HPV type 18 and absence of a p53 gene mutation from exons 5 through 8. CONCLUSION: To our knowledge, OKT1 is the first cell line established from small cell cervical carcinoma with ACTH secretion.

Distribution and fine structure of macrophages in the human ovary during the menstrual cycle, pregnancy and menopause.

We evaluated the distribution and ultrastructural characteristics of macrophages in the ovaries of women of reproductive ages, during pregnancy, and after menopause, by immunohistochemistry and transmission electron microscopy. Macrophages appeared around the ovarian follicle with its development. Their organelles were poorly developed, and no vacuoles or granules were observed in the cytoplasm. Macrophages were also present in the cavity of the atretic follicle, being larger in size than those in the developing follicle and characterized by cytoplasmic vacuoles and granules of a lysosomal nature. With the luteinization of the follicle, macrophages were seen to be distributed inside and outside the corpus luteum, but constituted only a minor population as compared with other kinds of leukocytes. The intracellular organelles were well-developed, including the lysosomal granules. In early pregnancy, the number of macrophages was noticeably increased in the corpus luteum. They were observed mainly outside the corpus luteum, and stained strongly with hCG immunohistochemically. Macrophages were present in the regressing corpus luteum and in the corpus albicans. Numerous lipid droplets and elongated cholesterol crystals were seen in the cytoplasm. Macrophages therefore appeared to be present throughout the ovarian cycle and may be involved in the development and atresia of the follicles and the progression and the regression of luteal tissues.

Follicular development and ovulation in macrophage colony-stimulating factor-deficient mice homozygous for the osteopetrosis (op) mutation.

To clarify the role of macrophages and macrophage colony-stimulating factor (M-CSF) in follicular development and ovulation, the processes of folliculogenesis and ovulation, numerical changes in macrophages, and proliferative capacity of granulosa cells were examined in op/op mice before or after daily M-CSF administration. The natural estrous cycle was determined daily by means of vaginal smears. The number of ovulated ova in both fallopian tubes was significantly smaller in op/op mice than in normal littermates. Such ova markedly increased in number after daily M-CSF administration. The numbers of both antral and mature follicles in the proestrous ovary were markedly lower in op/op mice than in the controls and increased after daily M-CSF administration. Flash-labeling with [3H]thymidine showed that the proliferative capacity of granulosa cells in antral follicles was reduced in op/op mice but elevated after daily M-CSF administration. Numbers of granulosa cells and macrophages in the antral follicles were significantly decreased in op/op mice but were increased after M-CSF treatment. All these data provide evidence that macrophages are implicated in the process of folliculogenesis and ovulation.

Role of macrophages in ovarian follicular development.

The effects of macrophages on granulosa cell proliferation were examined using gonadotropin-primed immature female rats and osteopetrotic (op/op) mice, a model defective in monocyte-macrophage lineage cells. Macrophages were found in the follicles at various developmental stages in rats and mice. The labeling index with [3H]thymidine of cultured rat granulosa cells was maximal when they were cultured with peritoneal macrophages at a macrophages:granulosa cell ratio of 0.01. This ratio was similar to those in rat preantral and antral follicles in vivo. In op/op mice, the number of developing follicles was markedly reduced, but increased after daily macrophage-colony-stimulating factor (M-CSF) administration. In the antral follicles of op/op mice, both granulosa cells and macrophages were significantly decreased in number but were increased after M-CSF treatment. Double immunohistochemical staining revealed that epidermal growth factor (EGF)-positive cells were macrophages in the developing rat follicles. These findings suggest that macrophages are located in the developing follicles and participate in promoting granulosa cell growth through a paracrine mechanism by secreting EGF and other cytokines.

Immunohistochemical localization of epidermal growth factor and its effect on granulosa cell proliferation in rat ovary.

The localization of epidermal growth factor (EGF) in the ovary and its effect on proliferation of granulosa cells were investigated in gonadotrophin-primed immature female rats. Immunoreactions with anti-rat EGF monoclonal antibody were observed sparsely in the granulosa layer and antrum of follicles, but not in the theca layer or stromal tissue. The EGF-positive cells were round or oval shaped and often larger than granulosa cells. The localization and morphological appearances of these cells in the follicles were in good agreement with those of macrophages. Although EGF alone did not promote granulosa cell growth in vitro, the labelling index with [3H]thymidine of granulosa cells cultured with 0.1 ng/ml EGF and 0.1 ng/ml basic fibroblast growth factor was significantly greater than that without the growth factors (18.4% vs. 15.8%, P < 0.01). These results suggest that macrophages in follicles may modulate follicular development through a paracrine mechanism by secreting EGF and other growth factors.

Effects of macrophage colony-stimulating factor on folliculogenesis in gonadotrophin-primed immature rats.

The effect of macrophage colony-stimulating factor (M-CSF) on folliculogenesis and ovulation was studied. Folliculogenesis and ovulation were induced in immature female rats with a s.c. injection of equine chorionic gonadotrophin (eCG), followed 48 h later by human chorionic gonadotrophin (hCG). The ovulation rate was measured after the following treatments. (1) Graded doses of human M-CSF (1-300 x 10(3) iu per rat) were administered i.p. daily for 3 consecutive days. (2) M-CSF (100 x 10(3) iu) was administered i.p. at designated times between 96 h before and 10 h after hCG injection. (3) Rabbit anti-human M-CSF polyclonal antibody (5 micrograms) was administered into the left ovarian bursa at designated times between 49 h before and 10 h after hCG injection. In addition, the effect of M-CSF on ovarian macrophages was investigated using immunohistochemistry with mouse anti-rat macrophage monoclonal antibody, TRPM-3. The treatment with M-CSF (> 30 x 10(3) iu per rat) significantly increased the ovulation compared with controls in a dose-dependent manner. This stimulatory effect of M-CSF was observed when it was administered between 96 h and 49 h before hCG injection. The ovarian intrabursal administration of anti-M-CSF antibody significantly inhibited the number of ovulated ova from the treated ovaries compared with either those from control rats or from the contralateral untreated ovaries between 24 h before and 3 h after hCG injection. The immunohistochemistry revealed that M-CSF increased the number of ovarian macrophages in growing follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

In-vitro enhancement of mouse embryonic development by co-culture with peritoneal macrophages.

The effects of peritoneal macrophages on mouse fertilization and embryonic development in vitro were examined by a co-culture method. In experiment 1, oocytes from F1 strain mice were inseminated and cultured with a monolayer of peritoneal macrophages to determine the effects of the macrophages on fertilization. In experiment 2, 1-cell embryos, fertilized in vivo from either a non-blocking or a blocking strain, were also incubated with peritoneal macrophages to determine whether these cells overcame the developmental arrest. Although the peritoneal macrophages had an adverse effect on fertilization in the F1 strain, embryonic development was significantly improved by the co-culture. This beneficial effect on embryonic development was also observed in the embryos fertilized in vivo from the strain that exhibited a 2-cell block. These results suggest that peritoneal macrophages provide an effective co-culture system for the in-vitro development of mouse embryos.

Isolation, growth and characteristics of human ovarian surface epithelium.

The ovarian surface epithelium (OSE) is a key tissue in the pathogenesis of ovarian surface epithelial-stromal tumours and ovarian endometriosis, commonly encountered gynaecological diseases. Despite the high incidence of these diseases, experimental in vitro studies of OSE are few and so we used the scraping method with an enzymatic procedure to isolate human OSE and studied its characteristics in vitro. Nineteen normal ovaries were used. After incubation of the ovary for 40 min in collagenase type 1 solution (300 U/ml), the surface cells were removed by gentle scraping with a surgical blade. Cells obtained as a cluster after unit gravity sedimentation with 5% bovine serum albumin in medium 199 were cultured in medium 199 containing 15% fetal bovine serum. The viable cell number in a single ovary was 0.1-2.7 x 10(6). The outgrowth of cells started from a homogeneous population of single cells, and the cell population doubling time was between 7 and 10 days. Confluent monolayers were formed after 13-20 days and subcultured from one to three times. The monolayers mostly had a cobblestone appearance, and fusiform or polygonal cells were also observed. By cytochemistry, immunocytochemistry and scanning and transmission electron microscopy, the cells were shown to have characteristics of mesothelial OSE cells in short-term culture. This experimental approach was efficient in providing cultured human OSE, which can be utilized to investigate pathobiology and carcinogenesis.

Scanning electron microscopic and immunohistochemical studies of pelvic endometriosis.

The pathogenesis of pelvic endometriosis has been studied by using scanning electron and light microscopy, observing the surface structure of bluish lesions obtained from 26 patients during laparotomy. Paraffin sections included another 17 tissue samples of endometriosis, based on immunohistochemical responses to epithelial membrane antigen, keratin and vimentin. Ultrastructurally, the surface epithelial cells could not be detected in 13 out of 17 pelvic peritoneal endometriosis samples. In one case in which the surface peritoneal cells were seen histologically to dip into the subperitoneal stroma, many surface peritoneal infoldings were observed, and ciliated cells were detected at the edge of these infoldings. Ovarian endometriosis was composed of three types of cells, none of which had any cilia. These findings were observed in continuity with adjacent normal mesothelial cells. No characteristic structure of the endometrial surface was observed for the bluish lesion, but the gland surface of endometriosis located in the subperitoneal stroma initially had ciliated cells. The immunoreactions in both the columnar mesothelial cells with surface peritoneal infoldings and the glands of endometriotic tissues were similar to those of normal endometrial glands, but different from those of normal mesothelial cells. Pelvic endometriosis might originate by a process of metaplasia from the pelvic peritoneum.

Effect of macrophages on proliferation of granulosa cells in the ovary in rats.

The effect of macrophages on proliferation of granulosa cells was examined in gonadotrophin-primed immature female rats. The mouse anti-rat macrophage monoclonal antibodies TRPM-2 and TRPM-3 were used and macrophages were observed in the granulosa layer and antrum of follicles and in corpora lutea and stroma around follicles. There was no difference in distribution between TRPM-2-positive cells and TRPM-3-positive cells. Macrophages with some cytoplasmic vacuoles of various sizes were also demonstrated in growing follicles. The average ratios of macrophages to granulosa cells in preantral, antral and mature follicles were 0.008, 0.007 and 0.002, respectively. Labelling with [3H]thymidine of granulosa cells cultured with peritoneal macrophages was significantly greater and the labelling index peaked to 25.0% when the ratio of macrophages to granulosa cells was 0.01, compared with the value of 14.2% when the granulosa cells were cultured alone. This ratio of macrophages to granulosa cells was similar to that in the preantral and antral follicles in vivo. These results suggest that macrophages participate in promoting proliferation of granulosa cells as local mediators in growing follicles.

[Activated macrophages in the peritoneal fluid of women with endometriosis: examination of the intracytoplasmic localization of endogenous peroxidase and interleukin-1].

In order to study the effect of peritoneal macrophages (M phi) on conception in patients with endometriosis, the total numbers of peritoneal M phi and the proportion of the exudate type, defined on the basis of ultracytochemical localization of endogenous peroxidase (PO) activity, were investigated in 21 patients with endometriosis, five with uterine leiomyoma, three with tubal obstruction and three with carcinomatous peritonitis. An immunocytochemical observation of interleukin-1 (IL-1) was also performed in three patients with endometriosis, one with tubal obstruction, and one male patient with cholelithiasis. The total numbers of peritoneal M phi in patients with endometriosis were significantly higher than in uterine leiomyoma (2.11 x 10(7) v.s. 0.68 x 10(7), p less than 0.025). The total numbers of peritoneal M phi in tubal obstruction (0.96 x 10(7)) were not statistically different from those in uterine leiomyoma. The peritoneal M phi were remarkably increased in number in patients with carcinomatous peritonitis. On ultracytochemical observation of endogenous PO activity, the proportion of exudate M phi to whole M phi was significantly larger in endometriosis than that in uterine leiomyoma (13.3% v.s. 3.5%, p less than 0.025). This type of M phi increased even in stage I endometriosis (p less than 0.005). These data suggest that the abdominal cavity in women with endometriosis is in the stimulated conditions which may lead to infertility. A positive reaction to anti-IL-1 antibody on the cell membrane of all M phi examined in each patient suggests that an immunocytochemical study of IL-1 in M phi is not suitable for evaluating the degree of activation of M phi.

[Endosalpingosis in the pelvic peritoneum and pelvic lymph nodes].

The pelvic peritoneum with macroscopic findings characteristic of endometriosis was biopsied in 18 cases at conservative surgery for endometriosis and examined. Pelvic lymph nodes removed in 76 cases at radical surgery for uterine cervical, corpus cancer or ovarian cancer patients were examined. In 3 of the 18 cases, endosalpingiosis was encountered in the pelvic peritoneum. In 2 of these 3 cases combined lesions of endometriosis and endosalpingiosis were observed. On the other hand, in 4 of the 76 patients with gynecological malignancies, benign glandular inclusions were found in pelvic lymph nodes. In 3 of these 4 patients, there was endosalpingiosis, and another there was endometriosis. Endosalpingiosis is an interesting lesion offering some clues to the histogenesis of endometriosis, and important in the differential diagnosis of malignant tumors.