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Mutation within the Mitf gene causes, in microphthalmic Mitfi/Mitfi' (mi/mi) mice, multiple defects, including white coat color and functional defects in macrophages and osteoclasts. Our previous mating experiments have demonstrated that the mi mutation reduces the numbers of newborns and induces uterine inversion at delivery. The present study was designed to determine the causes of these pregnancy defects. The histology and number of F4/80-positive macrophages were not different between the ovaries of 23-day-old mi/mi and +/+ mice given eCG 48 h earlier. When ovulation was induced in these mice by hCG, the number of ovulated ova was significantly smaller in mi/mi mice than in wild-type (+/+) mice (p < 0.05). When bone marrow cells from +/+ mice were transplanted i.p. into 42 mi/mi female newborns, successful transplantation was observed in 16 of them at 20 days after birth. In one of these, the upper incisors had erupted. The mean number of tubal ova in mi/mi mice significantly increased after transplantation (p < 0.05) and was almost equal to that of +/+ mice. No uterine inversion occurred at 6 deliveries in 5 mi/mi females after bone marrow transplantation, while it occurred at 4 of 5 deliveries in mi/mi females during the same observation period (p < 0.05). These results indicate that bone marrow-derived cells, defective in mi/ mi mice, are necessary for normal ovulation and delivery; the findings are consistent with the notion that macrophages play major roles in ovulation.

Differentiation of endometrial stromal cells (decidualization) is essential for embryo implantation and maintenance of pregnancy. By sequential complementary DNA subtractive hybridization, one of the messenger RNAs (mRNA) induced by progesterone in human endometrial stromal cells decidualized in vitro was identified as that of a tissue transglutaminase type II (TGase). TGase mRNA was induced within 6 h after the addition of progesterone to the culture, and the effect was dose dependent. Both the TGase inhibitor monodansylcadaverine and oligodeoxynucleotide complementary to the TGase mRNA inhibited the decidualization, as assessed by PRL production and morphological transformation. Expression of TGase mRNA in human decidua and endometria exposed to high levels of progesterone in vivo was demonstrated by Northern blotting and in situ hybridization. These data suggest that TGase is necessary for the decidualization of human endometrial stromal cells and that clarification of the mechanism of action of TGase will facilitate further insight into the diagnosis and treatment of infertility.

Endometrial stromal differentiation (decidualization) is essential for implantation of the developing blastocyst. To investigate the process of progesterone (P)-induced decidualization of human endometrial stromal cells (ESC), a complementary DNA library enriched with P-induced genes was constructed from cultured human ESC by subtractive hybridization and the polymerase chain reaction. One of the isolated clones was the complementary DNA for the tissue inhibitor of metalloproteinase-3 (TIMP-3), a recently identified member of the human TIMP family. When human ESC were cultured in the presence of P for 6 days, the induction of TIMP-3 messenger RNA (mRNA) expression was observed by Northern blotting. In contrast, the marked induction of PRL mRNA expression and morphological changes were observed after 9 days of culture. P-induced TIMP-3 mRNA expression was dose dependent, and this induction was inhibited by the antiprogestin RU486. Estrogen did not induce TIMP-3 mRNA expression under similar conditions. In situ hybridization analysis of endometria from nonpregnant women revealed that the TIMP-3 mRNA expression was restricted to predecidualized stromal cells. At the feto-maternal interface, TIMP-3 expression was observed in fetal extravillous trophoblasts that had invaded the maternal decidual tissues as well as in the maternal decidual cells. These findings suggest that TIMP-3 is a sensitive indicator of ESC decidualization, and that the induction of TIMP-3 expression in decidual cells and trophoblasts may be important in the regulation of trophoblast invasion.

Leukaemia inhibitory factor (LIF) is a cytokine that displays multiple activities in various tissues and is essential for blastocyst implantation in mice. In the human uterus, LIF is expressed in endometrial tissue and the decidua. To elucidate the role it plays, the mRNA levels for two LIF receptor (R) subunits, LIF-R and gp130, were examined in human endometrium, placenta and decidua by Northern blot hybridization. The expression of LIF-R gene was detected in the chorionic villus during the first trimester, in term placenta, and at lower levels in the decidua. The expression of LIF-R gene was not detectable in non-pregnant endometrium. The expression of the gp130 gene was detected in all tissues examined. During pregnancy, there was no significant change in the mRNA concentration of LIF-R in the placenta, while that of gp130 increased after the second trimester. The human choriocarcinoma cell line, BeWo, was found to express LIF-R and gp130. LIF inhibited forskolin-induced human chorionic gonadotrophin (HCG)-beta production by BeWo in a dose-dependent manner, and it ameliorated forskolin-induced growth suppression. These findings suggest that LIF plays a regulatory role in trophoblast growth and differentiation during pregnancy in human placenta.

Objective To investigate the expression of messenger RNA (mRNA) for gonadal steroid hormone receptors in the human pelvic peritoneum. Design Analysis of estrogen receptor (ER), progesterone receptor (PR), and androgen receptor (AR) mRNA expressions in the pelvic peritoneum was carried out using the quantitative reverse transcription-polymerase chain reaction (PCR) method. Setting Department of Gynecology and Obstetrics, Kyoto University Hospital, Kyoto, Japan. Patients Pelvic peritoneal tissues from patients with (n = 10) and without (n = 10) endometriosis who had undergone gynecological surgery were studied. Results Estrogen receptor, PR, and AR mRNAs were detected in all pelvic peritoneal samples analyzed. In the pelvic peritoneum of patients without endometriosis, ER mRNA levels were significantly lower in the luteal phase than in the follicular phase. This cyclic profile of ER mRNA expression was not observed in the pelvic peritoneum of patients with endometriosis. During the follicular phase, ER mRNA levels in the pelvic peritoneum of patients with endometriosis were significantly lower than those of patients with endometriosis. Neither PR nor AR mRNA levels in the pelvic peritoneum of either patient group showed significant cyclic variations throughout the menstrual cycle. A comparison of PR and AR mRNA levels in the pelvic peritoneum of the endometriosis and the nonendometriosis groups revealed no significant differences. Conclusions These data indicate a decrease in ER gene expression in the pelvic peritoneum of patients with endometriosis during the follicular phase. This suggests that the possible responsiveness of peritoneal cells to estrogen may be related to the occurrence and/or development of endometriosis.

Interleukin-1 (IL-1) has been reported previously to inhibit the in-vitro decidualization of human endometrial stromal cells as assessed by progesterone-induced prolactin production and morphological transformation. In this study we examined whether other cytokines, such as tumour necrosis factor-alpha (TNF alpha), interferon-beta (IFN beta), IFN gamma or granulocyte-macrophage colony-stimulating factor (GM-CSF), could affect the decidualization of human endometrial stromal cells in vitro. Of these cytokines, TNF alpha significantly suppressed prolactin production in a dose-dependent manner, with no apparent effect on cell number. The morphological transformation of endometrial stromal cells was also inhibited by TNF alpha. TNF alpha and IL-1 significantly suppressed cAMP-stimulated prolactin production by endometrial stromal cells. Neither the progesterone concentration in the supernatant of the endometrial stromal cell culture system nor intracellular calcium concentration of the endometrial stromal cells were affected by the addition of TNF alpha or IL-1. These results indicated that TNF alpha and IL-1 suppress both progesterone-induced and cAMP-mediated prolactin production in endometrial stromal cells, and that this inhibition was not attributable to direct effects on progesterone metabolism or related to Ca(2+)-mediated signal transduction. These experiments suggested that a local increase of TNF alpha and IL-1 under certain pathological conditions in vivo may disturb blastocyst implantation and/or the maintenance of pregnancy by inhibiting the decidualization of endometrial stromal cells.

Mutations at either W or mi (microphthalmia) loci in the mouse can lead to a deficiency in melanocytes and mast cells. In addition, W mutants can be anemic and sterile, whereas mi mice are osteopetrotic because of a monocyte/macrophage/osteoclast defect. Since c-kit receptor tyrosine kinase is the gene product of the W locus and mi mutation has been suggested to affect the transduction of signals from the c-kit and c-fms receptors, we here examined the effect of mi mutation on fertility. Testes and ovaries from mi/mi mice were histologically normal, and the pattern of c-kit protein expression was not different from that of +/+ mice. Homozygous mutant crosses (mi/mi x mi/mi) were fertile, but inversion of the uterus occurred in 86% of the deliveries. In some cases, the placenta was found still attached to the inverted uterus after delivery. Decidual cells were present and expressed c-kit protein normally in the placenta of mi/mi mice. The inversion was also observed in mi/mi females mated to +/+ males. No uterine inversion was noted when +/mi females were crossed with mi/mi or +/mi males, suggesting that the genotype of the mother but not of the father or fetus is important for the pathogenesis. The numbers and body weights of mi/mi newborns were less than those of +/mi littermates. Mast cells were absent, but c-kit-positive cells were present, in the uterine muscle layers of pregnant mi/mi mice. These results indicate that the normal mi gene product is necessary, directly or indirectly, for the normal growth and delivery of the fetus, and that the mi/mi mouse will be useful as an animal model of the

The c-kit proto-oncogene encodes a tyrosine kinase receptor and is allelic with the dominant white-spotting (W) locus of the mouse. In this study we investigated the expression of human c-kit protein in various adult and fetal human tissues immunohistochemically using anti-human c-kit monoclonal antibody. To discriminate c-kit+ cells from mast cells expressing c-kit, mast cells were identified by staining with Toluidine blue. In oogonia, spermatogonia and skin melanocytes of the fetus and in oocytes of adult ovary, c-kit expression was detected. In adult uterus, c-kit+ cells were widely distributed in the basal layer of the endometrium, myometrium and cervix, the number and distribution being almost identical to those of mast cells. In fetal uterus, c-kit+ non-mast cells clustered beneath the epithelium and a few mast cells were observed in the myometrium and subserosal layer. In both adult and fetus, c-kit+ non-mast cells were detected within smooth muscle layers of the intestine, colon and oesophagus, while mast cells were observed in the mucosal and submucosal layers of these organs. In contrast to mice, no expression of c-kit protein was detected in the human placenta and decidua. Thus, the distribution of c-kit+ cells in various tissues is similar but not identical between adult and fetus and between human and mouse.

Rat mRNAs encoding two subtypes of the endothelin (ET) receptor (ET(A) and ETB) were studied in the rat ovary and fallopian tube by means of Northern blotting and in situ hybridization. The mRNA transcripts for the endothelin-1-specific type receptor (ET(A)) in pooled RNA from the ovary and fallopian tube were 4.2 and 5.2 kilonucleotides, and that for the nonselective type receptor (ETB) was 4.7 kilonucleotides; these were similar to transcripts for endothelin receptors from other tissues. ET(A) mRNA expression was abundant in the muscle cell layer of the fallopian tube, but low in the ovary. On the other hand, ETB mRNA was abundant in the granulosa cells in the developing follicles, but low in atretic follicles and absent in the fallopian tube. These results demonstrated that the mRNAs for the two subtypes of the rat endothelin receptor have different expression profiles in the ovary and fallopian tube. ETs may mainly affect the granulosa cells in the dominant follicles as well as the muscle cells of the fallopian tube through ETB and ET(A), respectively.

The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor and is shown to be allelic with the white-spotting locus (W) of the mouse. In order to elucidate the role of c-kit protein during placental development, we have examined the expression of c-kit protein in the uterus and placenta of mice at pre- and post-implantation stages by the avidin-biotin-peroxidase (ABC) method using rat anti-mouse c-kit monoclonal antibody. At Days 3 and 5 of pregnancy and pseudo-pregnancy, c-kit protein was detected in the glandular epithelium, but little expression was observed in the luminal epithelium. At Day 7 of pregnancy, expression was detected in the stromal cells around the uterine crypts of the mesometrial portion, but not in the vigorously proliferating decidual cells around the developing embryo. At Days 9 and 10 of pregnancy, the decidua basalis facing invading trophoblasts gradually expressed c-kit protein. In the mature placenta, c-kit protein was detected in the labyrinthine and decidual layers, but in neither the giant trophoblastic nor the spongiotrophoblastic layer. By Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR), c-kit mRNA was detected at the stages of periimplantation and placental development. These results suggested that the c-kit protein might be involved in the proliferation and differentiation of placenta.

Endothelin (ET), a novel vasoconstrictor peptide containing three isopeptides [ET-1, ET-2, and ET-3 (ETs)], has various biological effects including vasoconstriction, mitogenesis, and steroidogenesis. We examined the ET-1-like immunoreactivity level in porcine follicular fluid and culture medium of porcine granulosa cells by RIA. The ET level in the follicular fluid was 9.4-14.2 pg/ml. These levels were within 0.42 to 0.62-fold of the porcine plasma level (22.7 +/- 3.1 pg/ml) (mean +/- SE). ET was detected in the culture medium of granulosa cells with and without LH treatment at the concentration of 56 +/- 9.3 and 4.9 +/- 1.2 pg/10(6) cells.h, respectively. We also examined whether ETs affect the luteinization of granulosa cells. ETs inhibited the LH-stimulated progesterone and cAMP accumulation in cultured porcine granulosa cells in a dose-dependent manner with an EC50 of 5 x 10(-11) M. ET-1, ET-2, and ET-3 (5 x 10(-8)M inhibited progesterone accumulation by 62.3 +/- 1.8, 59.8 +/- 4.0, and 63.3 +/- 5.7% in 6-day cultures, respectively, and significant inhibition was observed within 24 h of culture. ET-1, ET-2, and ET-3 (5 x 10(-8) M) inhibited the LH-stimulated cAMP accumulation in granulosa cells by 54.8 +/- 2.3, 55.4 +/- 7.1, and 55.5 +/- 6.2%, respectively, whereas they did not affect basal cAMP levels. As well as progesterone accumulation, ETs partially inhibited LH-stimulated morphological transformation of granulosa cells. In this study, we demonstrated that ET exists in follicular fluid and in the culture medium of granulosa cells, and that ET inhibited LH-induced progesterone accumulation, morphological transformation, and cAMP accumulation in cultured porcine granulosa cells. These findings suggest that ET acts as a modulator of steroid metabolism in preovulatory follicles.

We have examined the effect of LH on the regulation of the progesterone receptor (PR) in cultured porcine granulosa cells. In this study we used the RNase protection assay to evaluate the PR mRNA levels with a porcine cDNA clone isolated by the polymerase chain reaction (PCR) method. This clone was regarded as part of the porcine PR cDNA because of its 98.3% and 95.7% homology to the hormone-binding domain of human PR cDNA in amino acid and nucleotide sequences, respectively. Treatment with LH (500 ng/ml) increased porcine PR mRNA to a maximum level of 8.6 +/- 1.1-fold (mean +/- SE) after 3-h exposure. This induction was mimicked by (Bu)2cAMP as well as by FSH and hCG, and the increased PR caused by LH and (Bu)2cAMP occurred in a dose-dependent manner. Basal and LH-induced PR mRNA levels were not affected by progesterone (100 ng/ml), estrogen (100 ng/ml), and RU 486 (10 ng/ml) at 3 h. The mechanism of the increased PR mRNA levels was studied in the presence of actinomycin-D and cycloheximide. While inhibition of RNA synthesis with actinomycin-D blocked LH-induced PR mRNA expression, inhibition of protein synthesis with cycloheximide increased basal and LH-induced PR mRNA levels. These results indicate that the expression of PR mRNA is positively regulated by LH, and this induction does not require ongoing protein synthesis. There may be a cycloheximide-sensitive mechanism that modulates PR mRNA stability. From our results we suspect that progesterone modulates ovarian function through LH-induced PR in granulosa cells.

The ISK protein is a novel, probably epithelial potassium channel which differs from conventional potassium channels in its structure, electrophysiology, and tissue distribution. In this investigation, we isolated and analyzed genomic and cDNA clones coding for the rat ISK protein to characterize the structural organization and expression pattern of the ISK protein gene. This analysis, together with primer extension and RNase protection experiments, indicated that the ISK protein mRNA is initiated from two different upstream exons and then encoded by an uninterrupted downstream exon covering the protein-coding and the 3'-untranslated regions of the mRNA. RNA blot hybridization analysis showed additional generation of several large species of mRNAs which result from inclusion of a part of the intron sequence and the 3'-flanking region of the ISK protein gene. Thus, the single ISK protein gene is involved in the production of multiple species of mRNAs through a variety of cellular mechanisms including transcription initiation at different sites, alternative RNA splicing, and polyadenylation at different sites. The heterogeneity of the ISK protein mRNAs may be associated with the emergence of the functional and regulatory diversity observed for potassium ion permeation in epithelial cells.

We previously reported a novel rat membrane protein that exhibits a voltage-dependent potassium channel activity on the basis of molecular cloning combined with an electrophysiological assay. This protein, termed IsK protein, is small and different from the conventional potassium channel proteins but induces selective permeation of potassium ions on its expression in Xenopus oocytes. In this investigation, we examined cellular localization of rat IsK protein by preparing three different types of antibody that specifically reacts with a distinct part of rat IsK protein. Immunohistochemical analysis using these antibody preparations demonstrated that rat IsK protein is confined to the apical membrane portion of epithelial cells in the proximal tubule of the kidney, the submandibular duct and the uterine endometrium. The observed tissue distribution of rat IsK protein was consistent with that of the IsK protein mRNA determined by blot hybridization analysis. In epithelial cells, the sodium, potassium-ATPase pump in the basolateral membrane generates a sodium gradient across the epithelial cell and allows sodium ions to enter the cell through the apical membrane. Thus, taking into account the cellular localization of the IsK protein, together with its electrophysiological properties, we discussed a possible function of the IsK protein, namely that this protein is involved in potassium permeation in the apical membrane of epithelial cells through the depolarizing effect of sodium entry.

Leukaemia inhibitory factor (LIF) is a cytokine that displays multiple activities in various tissues and is essential for blastocyst implantation in mice. In the human uterus, LIF is expressed in endometrial tissue and the decidua. To elucidate the role it plays, the mRNA levels for two LIF receptor (R) subunits, LIF-R and gp130, were examined in human endometrium, placenta and decidua by Northern blot hybridization. The expression of LIF-R gene was detected in the chorionic villus during the first trimester, in term placenta, and at lower levels in the decidua. The expression of LIF-R gene was not detectable in non-pregnant endometrium. The expression of the gp130 gene was detected in all tissues examined. During pregnancy, there was no significant change in the mRNA concentration of LIF-R in the placenta, while that of gp130 increased after the second trimester. The human choriocarcinoma cell line, BeWo, was found to express LIF-R and gp130. LIF inhibited forskolin-induced human chorionic gonadotrophin (HCG)-beta production by BeWo in a dose-dependent manner, and it ameliorated forskolin-induced growth suppression. These findings suggest that LIF plays a regulatory role in trophoblast growth and differentiation during pregnancy in human placenta.

Progesterone (P) is known to regulate sex steroid receptors in uterine cells. However, its precise regulation at the messenger ribonucleic acid (mRNA) level is unclear. In this study we examined the effects of P and testosterone (T) on the regulation of sex steroid receptors in cultured human endometrial stromal cells (ESC), using the quantitative reverse transcriptase polymerase chain reaction method. We isolated ESC from human endometrial tissues and cultured them with or without P (10(-6) mol/L) or T (10(-8) mol/L) for 9 days. Incubation with P decreased progesterone receptor (PR), estrogen receptor, and androgen receptor mRNA levels in cultured human ESC to 0.56 +/- 0.04-, 0.53 +/- 0.08-, and 0.84 +/- 0.04-fold (mean +/- SE), respectively. T also decreased PR, estrogen receptor, and androgen receptor mRNA levels in cultured human ESC to 0.48 +/- 0.06-, 0.52 +/- 0.05-, and 0.82 +/- 0.04-fold (mean +/- SE), respectively. These decreases by P and T occurred in a dose-dependent manner. We also examined the sex steroid receptor levels in human ESC cultured for 0, 3, 6, and 9 days. The PR mRNA level in ESC without P was increased in a time-dependent manner. This increase was also inhibited by P, and the mRNA level in the presence of P was almost constant throughout the culture period. Our results demonstrated that P or T is a regulator of sex steroid receptors in ESC and that this regulation may influence the responsiveness to P of decidual change in ESC.

We developed a non-radioisotopic quantitative RT-PCR method with high sensitivity and reproducibility. The results of this RT-PCR were in agreement with those of the Northern blot analysis. We measured the mRNA levels of beta-actin, transferrin receptor, and two cell cycle-related genes, cyclin B and cdc25, in early mouse embryos by the RT-PCR. In late two-cell stage embryos, beta-actin, transferrin receptor and cyclin B mRNA levels were 10-20% of those in MII stage oocytes. In contrast, the cdc25 mRNA levels were not different between these stages. When we cultured mouse embryos, the presence of an RNA polymerase inhibitor, alpha-amanitin, in the medium did not affect the mRNA levels at the two-cell stage, indicating that most of the detected mRNAs in two-cell embryos were maternally derived. These results suggest that the rate of mRNA degradation is different between cyclin B and cdc25 during early embryogenesis.

To clarify the possible roles of cytokines in the regulation of luteal cell function, we examined the effects of interferon (IFN), interleukin-1 (IL-1), and tumor necrosis factor (TNF) on progesterone and estradiol secretion in cultures of luteinized human granulosa cells. IFN gamma reduced hCG-stimulated progesterone secretion in a concentration-dependent manner; at its maximal inhibitory concentration (10 ng/mL), IFN gamma reduced progesterone secretion to 20% of that in the hCG-stimulated controls. Whereas other IFN (alpha and beta) reproduced the inhibitory effect of IFN gamma, IL-1 and TNF had no effect on hCG-stimulated progesterone secretion at concentrations of 1 and 10 ng/mL. IFN gamma also markedly reduced FSH-stimulated estradiol secretion. Unlike their effects on hCG-stimulated progesterone secretion, IL-1 and TNF reproduced the inhibitory effect of IFN gamma on FSH-stimulated estradiol secretion. IFN gamma significantly reduced both hCG- and FSH-stimulated cAMP generation in granulosa cells. IL-1 and TNF inhibited FSH-stimulated cAMP generation, but they did not inhibit hCG-stimulated cAMP generation. None of these cytokines reduced forskolin-stimulated cAMP generation, thus suggesting that these cytokines affect steps proximal to cAMP generation without affecting cAMP generation itself. IFN gamma also reduced progesterone secretion in response to (Bu)2cAMP, suggesting that it also affects steps distal to cAMP generation. This study has demonstrated that cytokines modulate the steroidogenesis of luteinized human granulosa cells in vitro; the results suggest that cytokines may play permissive roles in regulating luteal cell function.

The decidua constitutes an anatomical compartment of the placenta in deciduate mammals including humans. By virtue of its anatomical location at the interface between maternal and fetal tissues, it has long been recognized that the decidua possesses a particular function which leads to the formation of an immunologically privileged site to accommodate the semi-allograft implant and its subsequent development.

The role of the renin-angiotensin system in blood pressure control and in the development of hypertension was investigated by generating transgenic mice carrying the rat renin or angiotensinogen gene or both genes under the control of the mouse metallothionein I promoter. The systolic blood pressure was significantly elevated in transgenic mice carrying both transgenes but was maintained normally in those bearing either of the transgenes. The transgene was effectively and properly transcribed to form the mature mRNA in the transgenic mice. The production of rat renin and angiotensinogen in the transgenic mice carrying the corresponding transgene was also verified by immunoanalyses of these proteins. Furthermore, the specific angiotensin-converting enzyme inhibitor captopril was effective in reducing the elevated blood pressure of the hypertensive transgenic mice. These results indicate that the combined action of the exogenous rat renin and angiotensinogen is responsible and necessary for elevation of blood pressure in the hypertensive transgenic mice.

Immunohistochemistry was used to determine the distribution of oestrogen receptors (ER) and progesterone receptors (PR) in the human ovary during folliculogenesis. Primordial and preantral follicles did not contain ER or PR. The granulosa cells of antral follicles had ER, but negligible PR, before the LH surge. In contrast, at the time of LH surge, these cells of the dominant follicle contained PR, but not ER. On the other hand, granulosa cells of the non-dominant follicles had ER, but not PR. After ovulation, the PR persisted in the luteinized granulosa cells and in the corpus luteum during early pregnancy. The theca interna and surrounding stromal cells were ER-negative and PR-positive throughout the menstrual cycle. Thus, the results show that ER and PR are not expressed simultaneously in the granulosa cells, the thecal cells, or the stromal cells during folliculogenesis. Mechanisms controlling the expression of steroid receptors during the normal menstrual cycle and in early pregnancy are discussed.