Accelerated Barnes maze test in mice for assessment of stress effects on memory.

Repeated restraint stress in rodents impairs spatial memory in a Y-maze test and induces hippocampal neuronal changes that last up to 5 d after the stressor ends. Our goal was to implement a Barnes maze spatial memory test in mice that could be used to validate our findings of social stress induced Y-maze impairment. We measured performance of mice in 5- and 9-day test paradigms previously used in rats and mice, respectively. Selecting features from each paradigm, we implemented a 5-d test (pre-training, training (4 trials/d/3 d) and probe testing for assessment of spatial memory in mice. Stress consisted of placing each test mouse in a stainless steel perforated box (25.5 cm x 21.5 cm x 16.5 cm) within an aggressor's home cage for 6 h/d for 21 d; direct agonistic encounters occurred randomly throughout stress periods. Barnes maze pre-training (habituation) was on day 21 of the stress exposures. In a preliminary experiment, mice that habituated following their last stressor performed poorly relative to unstressed and to those not habituated prior to the last stressor, as demonstrated by a greater latency to escape and more errors. We conclude that acute stress in a chronic stress paradigm may impair spatial memory acquisition.

Vascular endothelial growth factor and interleukin-6 in peritoneal fluid of women with endometriosis.

OBJECTIVE(S): To determine [1] vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) levels in peritoneal fluid from women with endometriosis and compare them with those from oral contraceptive (OC) users and normal cycling women and [2] any correlation between VEGF and IL-6 concentrations. DESIGN: Controlled clinical study. SETTING: University medical center. PATIENT(S): Patients undergoing laparoscopy for infertility or other benign gynecologic conditions. INTERVENTION(S): Peritoneal fluid samples were collected. MAIN OUTCOME MEASURE(S): Levels of VEGF and IL-6 in peritoneal fluid were determined. RESULT(S): Compared with normal controls or women with less severe endometriosis (implant scores of 5 or less), women with more advanced endometriosis (implant scores of 6 or more) have elevated VEGF and IL-6 levels in peritoneal fluid. Compared with normal controls, markedly suppressed IL-6 but similar VEGF levels were found in peritoneal fluid from OC users. Neither VEGF nor IL-6 varied cyclically in normal women or those with endometriosis. There was no correlation between levels of VEGF and IL-6 in peritoneal fluid. There was no correlation between implant scores and VEGF or IL-6 levels. CONCLUSION: The inflammation associated with endometriosis, through increased levels of peritoneal fluid VEGF, may promote angiogenesis for the progressive growth of endometriosis. Effective treatment of endometriosis by combination estrogen-progestin pills may involve the suppression of such inflammatory responses.

Laparoscopic surgery of the fallopian tubes and ovaries.

Operative laparoscopy can be used for many surgical procedures on the fallopian tube and ovary. These include: (1) tubal sterilization; (2) salpingectomy and salpingostomy for tubal pregnancy; (3) fimbrioplasty, salpingoneostomy, and linear salpingostomy for tubal obstruction and infertility; (4) microsurgical tubal reanastomosis for reversal of tubal sterilization; (5) oophorectomy, cystectomy, cyst drainage and fulguration, and excision of ovarian tumors; (6) wedge resection and ovarian drilling for polycystic ovaries; and (7) fulguration and laser vaporization for endometriosis. Many of these procedures are conservative and involve reconstruction of the tube and ovaries to preserve fertility. Microsurgical techniques are incorporated into such fertility sparing or enhancing procedures. Comparison of similar surgical procedures on the tube and ovaries indicates better or similar surgical outcome when done through the laparoscope rather than laparotomy; less blood loss, faster recovery, and cheaper cost are the hallmarks when the procedure is done by laparoscopy. With further improvement and expansion in laparoscopy equipment, it can be expected that more surgical procedures on the adnexa can be undertaken safely and effectively.

Localization and expression of oxytocin receptor and its messenger ribonucleic acid in peri-implantation phase human endometrium during control and clomiphene-treated cycles.

OBJECTIVES: This study was undertaken to determine expression levels of oxytocin receptor and its gene in peri-implantation phase human endometrium during clomiphene-treated cycles compared with control cycles. STUDY DESIGN: Oxytocin receptor and its messenger ribonucleic acid in peri-implantation phase endometrium during control and clomiphene-treated (50 mg days 5 to 9) cycles of 5 healthy fertile women were determined by immunohistochemical methods, Western blot analysis with monoclonal antibody against amino acids 20 through 40 of the extracellular N-terminal human oxytocin receptor, and reverse transcription-polymerase chain reaction with oligonucleotide primers to amplify the 391-base pair fragment of the oxytocin receptor gene. RESULTS: Oxytocin receptor and its messenger ribonucleic acid were expressed in human peri-implantation phase endometrial samples from both control and clomiphene-treated cycles. The receptor was localized predominantly in the epithelial cells and glands, with little or none detected in the stroma. Oxytocin receptor protein was separated out as a single 70-kd band by Western blot analysis; its relative abundance was significantly reduced during clomiphene-treated cycles. The messenger ribonucleic acid was detected in all endometrium during control and clomiphene-treated cycles, with greater expression during control cycles. CONCLUSIONS: The expressions of oxytocin receptor and its gene in luteal phase human endometrium suggest a functional relevance in modulation of biochemical changes for implantation.

Comparative aspects of oxytocin in baboon (Papio hamadryus anubis) and human corpora lutea.

In spite of the importance of the corpus luteum in human reproduction, little is known about its formation after ovulation and during regression in the absence of conception. This is largely due to constraints on the availability of normal human tissue; therefore an appropriate model which could be studied and could provide information applicable to the human was sought. The baboon (Papio), a non-human primate, has been determined to be one such model. Thus, in the past several years our studies have examined the role of luteal peptides in corpus luteum function, and, when possible, we have attempted to examine corpora lutea from the human and baboon in parallel. Although a milk-ejection factor was recognized to be present in luteal tissue in 1910 (Ott and Scott, Proc. Soc. Exp. Biol. Med., Vol. 8, p. 49), the role of oxytocin in luteal physiology has not been easy to ascertain. This is in part due to the methodologies employed to assess its role. Our studies summarized below suggest that oxytocin does not directly affect luteal steroidogenesis but that it may play a role in cell to cell communication involving the expression of the gap junction proteins, the connexins. In view of the fact that oxytocin, its receptor, gap junctions and associated proteins are not unique to the human and non-human primates, the model of luteal development and demise proposed may be applicable to most species.

Oxytocin receptor and its messenger ribonucleic acid in human leiomyoma and myometrium.

OBJECTIVE: The study determined the expression of oxytocin receptor and its gene in human uterine leiomyoma compared with the adjacent myometrium. STUDY DESIGN: Paired samples of leiomyoma and the adjacent myometrium from 20 women through the menstrual cycle, menopause, and various hormone treatments were studied. Oxytocin receptor was immunohistochemically localized with use of the specific antibody (2F8) to human oxytocin receptor. Oxytocin receptor protein was determined by Western blotting, whereas reverse transcription-polymerase chain reaction was used for oxytocin receptor messenger ribonucleic acid expression. RESULTS: Immunohistochemistry showed positive staining in all tissues examined, relatively more intense in the myometrium than in the adjacent leiomyoma, and in tissues from the preovulatory than the postovulatory phase. Western blotting showed a single 70-kd band corresponding to the oxytocin receptor. The relative abundance of oxytocin receptor in both leiomyoma and myometrium was significantly higher during the preovulatory (n = 5) than the postovulatory (n = 5) phase (P = .034 and .05). In women receiving gonadotropin-releasing hormone agonist (n = 1) or oral contraceptives (n = 1), after the menopause (n = 2), and with irregular vaginal bleeding (n = 1), oxytocin receptor levels in leiomyoma and myometrium were unchanged but were reduced in anovulatory cycles (amenorrhea, n = 2). Reverse transcription-polymerase chain reaction showed messenger ribonucleic acid for oxytocin receptor as a 391-bp band in all leiomyomas and myometrium examined. CONCLUSIONS: Leiomyoma and myometrium express the gene and protein for oxytocin receptor, which is probably partially regulated by ovarian sex steroids during the menstrual cycle.

Gene transfer to cultured human endometrial stromal cells: a model to study cyclooxygenase-2 gene regulation.

OBJECTIVE: To compare and optimize conditions for gene transfer to human endometrial stromal cells derived from primary culture and to determine the effect of interleukin-1beta (IL-1beta) and phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator. on the promoter activity of the human cyclooxygenase-2 (COX-2) gene. DESIGN: Prospective controlled study. SETTING: Academic research laboratory. PATIENT(S): Women undergoing benign gynecologic surgery for indications other than endometrial diseases. INTERVENTION(S): Endometrial stromal cells were used for transient transfection study. MAIN OUTCOME MEASURE(S): Luciferase activity in transfected endometrial stromal cells. RESULT(S): Gene transfer mediated by cationic lipid was more efficient and more consistent. Lipofectamine, a polycationic lipid, yielded the highest efficiency. Phorbol 12-myristate 13-acetate (30 nM) and IL-1beta (100 ng/mL) increased COX-2 promoter activity by 2.6-fold and 2.2-fold, respectively. CONCLUSION(S): Induction of COX-2 by IL-1beta and PMA suggests that COX-2 and prostaglandin have important roles in the growth and differentiation of endometrial stromal cells. This model can be used to explore the roles of different promoter regulatory elements in COX-2 gene activation.

Hormonal regulation of connexin-43 in baboon corpora lutea.

The synthesis and secretion of progesterone in the corpus luteum are regulated by both endocrine and paracrine/ autocrine factors which affect the steroidogenic cells. Evidence suggests that these cells communicate via cell-cell junctional proteins, the connexins. Previously we have shown that connexin-43 is expressed in both human and baboon (Papio hamadryus anubis) corpora lutea, with differential expression throughout luteal development, but is not detectable in corpora albicantia. We have examined the effect of human chorionic gonadotropin (hCG), oxytocin, clomiphene citrate and the anti-progesterone onapristone on expression of connexin-43 protein in the early luteal phase 1-5 days after the mid-cycle luteinizing hormone (LH) surge (LH+ 1-5 days), the mid-luteal phase 6-10 days after the LH surge (LH+ 6-10 days), and the late luteal phase 11-15 days after the LH surge (LH+ 11-15 days) in corpora lutea obtained from normal adult cycling females. Connexin-43 was localized by immunohistochemistry in cultured cells from all the three stages. Western blot analysis of the treated cells indicated the presence of two bands at 43 and 45 kDa. The band at 45 kDa was found to be phosphorylated connexin-43, indicating the presence of functional gap junctions. hCG (10 IU/ml) stimulated the expression of connexin-43 throughout luteal development; however, maximum expression occurred in the early luteal phase with a significantly greater expression of the non-phosphorylated protein. In contrast, in the mid-luteal phase, the expression of the phosphorylated protein was predominant. Oxytocin (200 mU/ml) also stimulated connexin-43 expression throughout luteal development with similar effects on the phosphorylated and non-phosphorylated protein in the early and mid-luteal phase; however, compared with hCG, oxytocin had a greater effect on mid-luteal phase connexin-43 expression. In the presence of both hCG and oxytocin, the expression of connexin-43 was significantly higher than the control only in the late luteal phase. Both clomiphene citrate and onapristone suppressed connexin-43 expression, and concomitant addition of hCG did not counteract their effect. In the context of our previous studies, it is concluded that, together with LH/hCG and the steroid hormones, oxytocin is involved in cell-cell contact-dependent communication in the corpus luteum.

The expression of vascular endothelial growth factor isoforms in cultured human endometrial stromal cells and its regulation by 17beta-oestradiol.

Angiogenesis or formation of new blood vessels is required for regeneration of the endometrium after its breakdown during each menstruation. Vascular endothelial growth factor (VEGF), a family of recently discovered angiogenic factors, may be involved in the repair and growth of the endometrium. In this study reverse transcription-polymerase chain reaction (PCR) was used to confirm the presence of VEGF mRNA and restriction enzyme digestion to confirm the identity of PCR products generated from different VEGF isoforms in cultured human endometrial stromal cells. The shortest isoform, VEGF 121, was the most abundant in quiescent stromal cells. It was about one-and-a-half times that of VEGF 165. The longest isoform, VEGF 206, was not detected; only a relatively weak signal for VEGF 189 was detectable. The mRNA for VEGF increased 2-fold after stimulation by 17beta-oestradiol (10 nM) for 30 min. A further increase to 3-fold above baseline occurred after 2 h incubation and remained steady at 6 h incubation, but decreased to 2-fold of baseline after 15 h. There was no differential stimulation of mRNA for VEGF isoforms: the ratio of VEGF 121 to 165 remained constant at 3:2 during the course of the incubation, with the exception at 15 h incubation when the ratio was 2:1. The VEGF protein, determined by specific enzyme immunoassay, increased from undetectable at baseline to 79.8 +/- 18.9 pg/ml (n = 4, mean +/- SD, 9.6 cm2/well/ml) after 2 h, with a further significant increase to 249.5 +/- 27.3 pg/ml after 15 h and 695.0 +/- 41.4 pg/well after 39 h. At 15 h incubation, the specific oestradiol antagonist ICI 182,780 (1 microM) significantly reduced VEGF secretion by 25% from 249.5 +/- 27.3 to 189.0 +/- 26.6 pg/ml. Thus, VEGF showed specific patterns of isoform expression in the human endometrial stromal cells; oestradiol (10 microM) stimulated, but not differentially, the mRNA for VEGF isoforms.

E-cadherin and its messenger ribonucleic acid in periimplantation phase human endometrium in normal and clomiphene-treated cycles.

OBJECTIVE: The purpose of this investigation was to determine whether treatment with clomiphene citrate, which is estrogenic and antiestrogenic, affects the expression of the cell adhesion molecule E-cadherin in human periimplantation phase endometrium. STUDY DESIGN: Five healthy women were studied for two cycles each, a control and a treated (clomiphene 50 mg daily, days 5 through 9) cycle. A biopsy specimen of endometrial tissue was studied (8 to 10 days post luteinizing hormone surge) for immunohistochemical localization, Western analysis of E-cadherin with use of a highly specific monoclonal antibody to human E-cadherin, and determination of messenger ribonucleic acid for E-cadherin by reverse transcription-polymerase chain reaction by use of oligonucleotide primers specific to E-cadherin and amplifying a 432 bp fragment. RESULTS: Luteal phase plasma progesterone levels were significantly higher in clomiphene cycles. E-cadherin was immunocytochemically present in endometrium of control and treated cycles with no apparent difference in staining intensity. Western blots revealed the presence of E-cadherin. It was relatively more abundant in clomiphene-treated than control cycles but not significantly different. The message for E-cadherin gene is expressed in endometrium of control (n = 5) and clomiphene cycles (n = 4). CONCLUSIONS: E-cadherin and its gene transcripts are expressed in periimplantation phase endometrium and are not significantly affected by clomiphene treatment.

Interleukin-1 beta induces cyclooxygenase-2 gene expression in cultured endometrial stromal cells.

Increasing evidence indicates that PGs may play an obligatory role in blastocyst implantation. Cyclooxygenase (also known as PGH synthase) isozymes 1 and 2 catalyze the rate limiting steps in the biosynthesis of PGs. The ubiquitous cyclooxygenase-1 (COX-1) subserves housekeeping functions, whereas the inducible cyclooxygenase-2 (COX-2) is expressed by limited cell types and tightly controlled. Here we report the induction of COX-2 gene expression by interleukin-1 beta (IL-1 beta) in cultured human endometrial stromal cells. COX-2 activity was induced by IL-1 beta (1 ng/mL); conversion of exogenous arachidonic acid to PGF2 alpha increased from 2.6 +/- 0.6 ng/well (mean +/- SEM; n = 6) to 22.2 +/- 5.6 ng, but was completely blocked (2.8 +/- 0.7 ng/well) by NS-398, a specific COX-2 inhibitor. Undetectable in quiescent stromal cells, messenger ribonucleic acid for COX-2 was induced 30 min after IL-1 beta treatment, reached a maximum at 4 h, and decreased after 15 h. Protein synthesis was not required for induction of the COX-2 gene, as it was blocked by actinomycin D but not by cycloheximide. The 70-kDa COX-2 protein was not detected in quiescent cells, became detectable 6 h after IL-1 beta treatment, and remained detectable even after 15 h. IL-1 beta (0.1-100 ng/mL) increased the luciferase activity in promoterless luciferase reporter containing the 900-bp 5'-flanking sequence (-891 to +9) of the COX-2 gene in a dose-dependent manner, with an ED50 of 0.1-1 ng/mL.

Interleukin-1 beta inhibits in vitro pulsatile progesterone secretion and stimulates prostaglandin F2 alpha secretion by micro-retrodialyzed baboon corpus luteum.

Interleukin-1 beta (IL-1 beta) modulates steroidogenesis and prostaglandin (PG) secretion by dispersed luteal cells of some non-primate species. To determine if IL-1 beta affects progesterone (P) and PGF2 alpha secretion by the baboon corpus luteum (CL), we microretrodialyzed the intact CL for 48 h; 12 h media (baseline), 12 h with IL-1 beta (3 IU/h), 12 h media only (post- IL-1 beta) and 12 h with cAMP (5 nmol/h). Four CL from the midluteal phase (LH + 8 days) were studied. P was measured by a sensitive and specific radioimmunoassay and PGF2 alpha by an enzyme immunoassay in the 10-min fractions of retrodialysates collected. P secretions were analyzed for peaks by PC Pulsar (3.0) and total P retrieved/12 h for each experimental segment was calculated. P secretion was pulsatile. Pulses of P declined from 8.2 +/- 1.2/12 h (mean +/- SEM) before to 5.0 +/- 1.2 h after IL-1 beta treatment (P = 0.022), but increased to 10.2 +/- 4.3/12 h with cAMP. Interpulse interval increased significantly from 92 +/- 23 min (baseline) to 137 +/- 31 min (p = 0.025) after IL-1 beta treatment. Total P secreted decreased significantly from 2471 +/- 515 nmol/12 h (baseline) to 1480 +/- 167 nmoles/12 h during IL-1 beta and 788 +/- 85 nmoles/12 h after IL-1 beta (P = 0.015). P was immediately suppressed after starting IL-1 beta in 2 CL but declined only towards the end of treatment in the other 2 CL. PGF2 alpha secretion increased during IL-1 beta with a further increase after IL-1 beta, while P secretion was progressively inhibited. Therefore, IL-1 beta is luteolytic to the primate midluteal phase CL by inhibiting P while simultaneously stimulating PGF2 alpha secretion, demonstrating paracrine-autocrine interaction within the luteal tissue.

Oxytocin and its receptor in pregnancy and parturition: current concepts and clinical implications.

OBJECTIVE: To present our current understanding of oxytocin and its receptors during pregnancy and parturition and their potential clinical applications. DATA SOURCES: A MEDLINE search was conducted for pertinent articles from 1966 to October 1996 related to oxytocin and its receptor and their clinical implications during pregnancy and parturition. Review articles, book chapters, and published trials were also searched. METHODS OF STUDY SELECTION: Only references in English that were deemed relevant were used. When possible, human data and sometimes animal data pertinent to understanding the interaction of oxytocin and its receptors were selected. TABULATION, INTEGRATION, AND RESULTS: Oxytocin is synthesized in the hypothalamus and in many reproductive tissues during pregnancy, whereas the receptors are synthesized in reproductive tissues. The genes for oxytocin and its receptors are on chromosomes 20 and 3, respectively. Oxytocin and its receptors are regulated by sex steroids and by oxytocin itself. The paracrine and autocrine mechanisms regulating oxytocin and its receptor within the fetoplacental-uterine unit are central to the control of uterine contractions and parturition. Such current understanding provides the basis for appropriate oxytocin regimens to induce or augment labor, to inhibit preterm labor by blockade of oxytocin receptors, and to achieve cervical ripening. CONCLUSION: Advances in our knowledge of oxytocin and its receptor have provided rational and sound principles for current concepts about their role in parturition, the appropriate use of oxytocin to stimulate the pregnant uterus or ripen the cervix, and the use of oxytocin antagonist to inhibit uterine contractions and preterm labor.

Clinical, endocrine, and metabolic effects of two doses of gestrinone in treatment of pelvic endometriosis.

OBJECTIVE: Our purpose was to determine and compare the efficacy and hormonal and metabolic effects of 1.25 mg with 2.5 mg of gestrinone given twice a week in the treatment of mild and moderate pelvic endometriosis. STUDY DESIGN: A phase II, prospective, randomized, double-blind study involving 11 patients given gestrinone 1.25 mg (five patients) or 2.5 mg (six patients) orally twice a week for 24 weeks was performed. Revised American Fertility Society scores were determined by laparoscopy before and at the end of treatment. Serum hormone (free thyroxine, free testosterone, estradiol, progesterone, follicle-stimulating hormone, luteinizing hormone), sex hormone binding globulin, and lipid concentrations were measured before, throughout, and for 6 months after treatment. Quantitated computerized tomography of thoracic 12 through lumbar 4 vertebral bodies were determined before, at the end of, and 6 months after treatment. RESULTS: Gestrinone 2.5 mg significantly reduced the endometriosis implant score from 10.3 +/- 2.8 to 3.8 +/- 0.8 (p = 0.05). Both doses significantly reduced serum progesterone and sex hormone binding globulin levels. Estradiol, free testosterone, free thyroxine, follicle-stimulating hormone, and luteinizing hormone levels were not significantly affected. Spinal bone increased significantly by 7.1% with 2.5 mg but lost significantly by 7.1% with 1.25 mg gestrinone; these changes had not reversed completely 6 months after stopping treatment. CONCLUSIONS: In mild to moderate pelvic endometriosis 2.5 mg of gestrinone twice a week was more effective and had a more positive effect on bone mass than did 1.25 mg of gestrinone.

Immunohistological localization and expression of alpha-actin in the baboon (Papio anubis) corpus luteum.

We have recently shown the presence of E-cadherin and of alpha- and gamma-catenins in human and baboon corpora lutea. These are components of adherens junctions between cells. The cytoplasmic catenins link the cell membrane-associated cadherins to the actin-based cytoskeleton. This interaction is necessary for the functional activity of the E-cadherins. Our aim therefore was to determine the presence of alpha-actin in the baboon corpus luteum, to further establish whether the necessary components for E-cadherin activity are present in this tissue. An antibody specific for the smooth muscle isoform of actin, alpha-actin, was used for these studies. The results using immunohistochemistry show that (a) alpha-actin is present in steroidogenic cells of the active corpus luteum, theca externa of the corpus luteum, cells of the vasculature, and the tunica albuginea surrounding the ovary. The intensity of immunoreactivity for alpha-actin varied, with the cells of the vasculature reacting more intensely than the luteal cells. A difference in intensity of immunoreactivity was also observed among the luteal cells, with the inner granulosa cells showing stronger immunoreactivity than the peripheral theca lutein cells. There was no detectable immunoreactivity in the steroidogenic cells of the atretic corpus luteum. However, in both the active and atretic corpora lutea, alpha-actin-positive vascular cells were dispersed within the tissue. (b) Total alpha-actin (luteal and non-luteal), as determined by Western blot analyses, does not change during the luteal phase and subsequent corpus luteum demise (atretic corpora lutea). (c) hCG stimulated the expression of alpha-actin and progesterone secretion by the early luteal phase (LH surge + 1-5 days) and mid-luteal phase (LH surge + 6-10 days) cells in culture, but only progesterone in the late luteal phase (LH surge + 11-15 days). The data show that alpha-actin is present in luteal cells and that its expression is regulated by hCG, thus suggesting that E-cadherin may form functional adherens junctions in the corpus luteum.

Epidermal growth factor receptors in uteroplacental tissues in term pregnancy before and after the onset of labor.

Using saturation binding assays and Scatchard analyses, we determined the concentrations and binding affinities of epidermal growth factor (EGF) receptors in human myometrium (n = 13) and decidua (n = 10) before and during labor and in placenta (n = 15), chorion (n = 17), and amnion (n = 17) before labor, during labor, and after vaginal delivery. Each tissue was individually assayed. In myometrium and chorion, EGF receptors increased significantly from 5.6 +/- 0.8 and 13.5 +/- 1.7 fmol/mg protein (mean +/- SEM) before labor to 11.1 +/- 2.8 and 26.7 +/- 3.0 fmol/mg protein, respectively, after the onset of labor (P < 0.05). In amnion, EGF receptors increased from 12.8 +/- 2.7 fmol/mg protein before labor to 33.0 +/- 2.3 fmol/mg protein during labor, but decreased significantly (5.9 +/- 1.2 fmol/mg protein) with vaginal delivery (P < 0.05). Decidual and placental concentrations of EGF receptors did not change significantly with labor. The binding affinity of EGF receptors in all tissues studied did not change significantly with labor, as reflected by their respective association and dissociation constants. Up-regulation of EGF receptors in myometrium, chorion, and amnion with spontaneous labor may enhance stimulation of prostanoid production and stimulate uterine activity.

In vitro microdialysis of baboon corpus luteum: effects of oxytocin on total and pulsatile progesterone secretion.

Baboon corpora lutea (two each from the early, mid- and late luteal phases) were individually microretrodialyzed in vitro for 48 h, 12 h initial baseline, 12 h retrodialysis with OT (9 mU/h), 12 h without OT and 12 h with cAMP (5 mmol/h). Progesterone (P) was measured by a sensitive and specific radioimmunoassay in 10-min fractions of retrodialysates and analyzed for P peaks by PC-pulsar 3.0. Neither OT nor cAMP had any effect on the characteristics of P pulses. In early and late luteal phase CL, OT inhibited P secretion within 1 h of administration followed by increased P secretion late during OT perfusion. In midluteal phase, OT did not affect P secretion. In all CL, P secretion was sustained or further increased during the 12 h after stopping OT. cAMP also sustained baseline or stimulated P secretion. In contrast, OT either increased total P output/12 h (28 to 49% above baseline) with a further increase of 21% to 296% above baseline after stopping OT, or inhibited total P output by 4% to 13% percent with a further decline of 51% to 61% after stopping OT. Thus, while overall OT is luteotropic, its dual effect (initial inhibition followed by stimulation) suggests direct and indirect effects through paracrine-autocrine mechanisms.

Immunocytochemical localization and expression of E-cadherin and beta-catenin in the human corpus luteum.

We have previously shown that the protein connexin-43 which forms the connexons in gap junctions is present in the human corpus luteum. Abundant expression of connexin-43 is seen in the mid-luteal phase corpora lutea. Since the formation of gap junctions in a tissue requires the presence of adherens junctions formed by the cadherins, our aim in these studies was firstly to localize immunocytochemically E-cadherin and beta-catenin (a cytoplasmic protein associated with E-cadherin) in the human corpus luteum, and secondly to determine the concentrations of these proteins in the early, mid- and late luteal phase human corpora lutea. E-cadherin was localized to the periphery of luteal cells and was not detected in non-luteal tissue. beta-catenin was observed in the cytoplasm of the luteal cells. Abundant expression of E-cadherin was observed by Western analysis in the early luteal phase and the level of expression was significantly different from that observed in the mid- and late luteal phase corpora lutea. In contrast the concentrations of beta-catenin were higher in the mid-luteal phase compared to the early luteal phase. The differential expression of the cell adhesion molecule E-cadherin suggests that it may play a significant role in cell-to-cell communication in the corpus luteum, and in the cyclic development and demise of this tissue.

Immunocytochemical localization and expression of E-cadherin, beta-catenin, and plakoglobin in the baboon (Papio anubis) corpus luteum.

We have previously shown that gap junctions and gap junction-associated protein connexin 43 are present in the human and baboon corpus luteum. Abundant expression of connexin 43 is seen in the midluteal phase corpora lutea. Since the formation of gap junctions requires the presence of adherens junctions formed by the cadherins, our aim in these studies was 1) to immunocytochemically localize E-cadherin and the associated proteins, beta-catenin and gamma-catenin (plakoglobin), in the baboon corpus luteum; 2) to determine by Western analysis the levels of these proteins in early, mid-, and late luteal phase and atretic baboon corpora lutea; and 3) to examine whether or not cell-cell contact in cells in culture is disrupted by the addition of the antibody for E-cadherin. E-cadherin was localized to the peripheral cell membranes of luteal cells at all stages examined, except atretic corpora lutea, with the strongest immunoreactivity in the early luteal phase. Both beta-catenin and plakoglobin were localized in the cytoplasm of the luteal cells. Immunoreactivity for all three peptides was not observed in nonluteal tissue. By Western analysis, abundant expression of E-cadherin was observed in the early luteal phase, and the level of expression was significantly different from that observed in the mid- and late luteal phase corpora lutea. In contrast, the levels of beta-catenin and plakoglobin were higher in the midluteal phase compared to the early luteal phase. Addition of the E-cadherin antibody to early luteal phase cells in culture disrupted the cell-cell contacts between cells. Thus, cell adhesion involving E-cadherin may play a significant role in the cyclic development and demise of this tissue.

Localization and expression of zonula occludens-1 tight junction-associated protein in baboon (Papio anubis) corpora lutea.

The identification of the cell junction-forming proteins connexin-43, a gap junction protein and E-cadherin, which is a component of adherent junctions, in the corpus luteum of both humans and baboons suggests that cell-cell interactions and metabolic cooperation must occur in this tissue. Occluding junctions are a third type of junction which form a physical barrier between cells. Thus, our aims in this study were firstly to examine the presence of the tight junction-associated protein zonula occludens-1 (ZO-1) by immunohistochemistry, and secondly to determine the concentrations of this protein in the early-, mid- and late luteal phase baboon corpora lutea of the menstrual cycle by a Western analysis. ZO-1 was localized mainly at the periphery of the luteal cells, and the intensity of immunoreactivity varied through the luteal phase, with comparatively stronger immunoreactivity in the mid-luteal phase than the early and late luteal phases. Atretic corpora lutea were devoid of activity. By Western analysis, bands of immunoreactivity were observed at 225 kDa, further confirming the presence of the protein. Maximum activity, as determined by densitometry, was observed in the mid-luteal phase. These data infer the presence of tight junctions in the corpus luteum and suggest that expression of the ZO-1 protein forming these junctions may be hormonally regulated within this tissue.