Inhibition of complement-mediated cytotoxicity of antisera by fluid secreted by the seminal vesicle of the house mouse

The fluid from the seminal vesicles of the house mouse inhibited complement-mediated cytotoxicity of antisera against both sperm and lymphocytes. This inhibition was not reduced by heating or by absorption with sperm. Fractionation of the seminal vesicle fluid on Sephadex G-100 columns revealed three peaks of inhibitory activity, one of which appeared in the void volume of the columns. This inhibitory action of the seminal vesicle fluid may protect sperm from immunological attack in the female reproductive tract. It could explain the observation that immunization of female house mice with sperm does not prevent pregnancy. The relationship between this activity in the seminal vesicles of house mice, which is directed against the cytotoxicity of antisera, and the inhibition of cell-mediated responses to sperm reported for human and bull semen have not been investigated.     

Pre-B-cell colony-enhancing factor is a secreted cytokine-like protein from the human amniotic epithelium

OBJECTIVE: The purpose of this study was to determine whether pre-B-cell colony-enhancing factor is a secreted cytokine in the human amnion and to study its chemotaxic and antiapoptotic properties. STUDY DESIGN: Pre-B-cell colony-enhancing factor secretion was studied from amniotic epithelial-like WISH cells and primary amniotic epithelial cells that were seeded on squares of immobilon-P membrane and stimulated with lipopolysaccharide or tumor necrosis factor-alpha, respectively. The pre-B-cell colony-enhancing factor protein was detected both intracellularly and after secretion, as bound to the membrane, by immunostaining and densitometry. Medium and cell lysates that were obtained from WISH cells that were treated with lipopolysaccharide alone or together with a pre-B-cell colony-enhancing factor antisense oligonucleotide to block pre-B-cell colony-enhancing factor translation were also analyzed for secreted pre-B-cell colony-enhancing factor by Western blotting and densitometry. A chemotaxic effect of pre-B-cell colony-enhancing factor on human neutrophils was compared with the chemoattractants interleukin-8 and N-Formyl-Met-Leu-Phe methyl ester in a rapid fluorescence-based neutrophil migration assay. Apoptosis was induced in primary amniotic epithelial cells and fibroblasts by actinomycin D (1 microg/mL); the antiapoptotic effects of pre-B-cell colony-enhancing factor on early apoptosis were measured by the annexin V assay, and the late effects were determined by measurement of nuclear matrix protein in the media. RESULTS: Treatment of amnion cells that adhered to immobilon-P membrane to induce the secretion of pre-B-cell colony-enhancing factor showed significantly (P<.05) more pre-B-cell colony-enhancing factor protein surrounding the cells compared with the controls. Although the addition of lipopolysaccharide to cultured WISH cells caused the secretion of pre-B-cell colony-enhancing factor into the medium, co-treatment with an antisense oligonucleotide to pre-B-cell colony-enhancing factor obliterated it. Analysis of the cell lysates showed no significant change, which suggests that most of the pre-B-cell colony-enhancing factor protein had been secreted. No significant chemotaxic effects of pre-B-cell colony-enhancing factor were observed; however, pre-B-cell colony-enhancing factor treatment (100 ng/mL), together with actinomycin D, cancelled the early induction of apoptosis, although there was a dose-dependent and significant late antiapoptotic effect on primary amnion epithelial cells (P<.001) and fibroblasts (P<.01). CONCLUSION: Pre-B-cell colony-enhancing factor is a secreted protein from amniotic epithelial cells. Although it had no chemotaxic effects, it was antiapoptotic for both amniotic epithelial cells and fibroblasts and may protect these cells against apoptosis that is induced by chronic distension, labor, or infection.     

Immunosuppressant material in human seminal fluid. Inhibition of blast transformation and of NK activity by seminal fluid patients of a male infertility clinic

Human seminal plasma from normal or patients with abnormal parameters of the ejaculates contains an inhibitory material that expresses potent in vitro inhibitory activity on PHA-M-induced blast transformation and NK activity. Using the test of inhibition of NK activity, the semen samples from individuals with higher concentrations of fructose had higher inhibitory activity. The results described herein suggest that inhibitory activity for blast transformation may be present in the prostatic fluid while the NK inhibition aspects are correlated with the vesicle-marker (fructose). Inhibition of the immune responses by human seminal plasma of the effector functions indicates the interesting implication that soluble factors may indirectly protect against or promote human autoimmune infertility disease.     

Inhibition of the human sperm acrosome reaction by a high molecular weight factor from human seminal plasma

Human seminal plasma possesses a factor (acrosome reaction-inhibitory factor) that is precipitated by high speed centrifugation and that inhibits the ionophore- and dbc AMP-induced acrosome reaction of capacitated human spermatozoa but only if it is added toward the end of the capacitation period. Acrosome reaction-inhibitory factor can be partially purified by cation exchange chromatography and appears to differ from another factor that can be obtained by ultracentrifugation of human seminal plasma and that prevents the fertilization of mouse gametes.     

Inhibition of follicle-stimulating hormone- and adenosine-3',5'-cyclic monophosphate-induced progesterone production by calcium and protein kinase C in the rat ovary

In this study we examined the effects of A23187 (a calcium ionophore) and 12-O-tetradecanoyl phorbol-13-acetate, a known activator of protein kinase C, on progesterone production. Granulosa cells obtained from pregnant mare serum gonadotropin-primed rats were maintained in primary culture. Treatment with follicle-stimulating hormone (0.5 microgram/ml), 8-bromo-adenosine-3',5'-cyclic monophosphate (2 mmol/L), or cholera toxin (0.1 microgram/ml) for 5 hours or 24 hours markedly stimulated progesterone production. The concomitant presence of A23187 attenuated the elevated levels of progesterone induced by follicle-stimulating hormone, 8-bromo-adenosine-3',5'-cyclic monophosphate, or cholera toxin, with or without the presence of a phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (0.2 mmol/L). Likewise, treatment of the cells with 12-O-tetradecanoyl phorbol-13-acetate suppressed follicle-stimulating hormone-induced progesterone production, whether or not 1-methyl-3-isobutylxanthine was present in the cultures. The effect of 12-O-tetradecanoyl phorbol-13-acetate was not mimicked by phorbol-13-monoacetate or 4 alpha-phorbol-12, 13-didecanoate. These results indicate that both A23187 and 12-O-tetradecanoyl phorbol-13-acetate inhibit follicle-stimulating hormone-induced progesterone production, in part at a step or steps beyond adenosine-3',5'-cyclic monophosphate generation and degradation. They further support a role of calcium and protein kinase C in the intraovarian action of luteinizing hormone-releasing hormone.     

Inhibition of the primate ovarian cycle by a porcine follicular fluid protein(s)

Monkeys received twice daily intramuscular injections of 3 mg of purified porcine follicular fluid protein(s) for the first 14.5 days of the menstrual cycle. Two of five treated monkeys had anovulatory menstrual cycles. Three monkeys had cycles characterized by long follicular phases, low follicular and luteal phase serum estradiol concentrations, and subnormal luteal progesterone production. Serum gonadotropin concentrations were not affected by the follicular fluid protein(s). The data demonstrate in the nonhuman primate that porcine follicular fluid contains a protein(s) that acts at the ovarian level to inhibit gonadotropin action.     

Uptake and digestion of 125I-labelled bovine serum albumin by the rat visceral yolk sac cultured in vitro as a closed vesicle

A system for culturing the rat visceral yolk sac in vitro as a closed vesicle--the 'giant' yolk sac--has been employed to investigate the vectorial nature of the uptake and digestion of exogenous protein substrates. Uptake of 125I-labelled formaldehyde-denatured bovine serum albumin by such yolk sacs was found to be similar to that observed in yolk sacs removed directly from the mother at 17.5 days' gestation, provided that homologous serum was used as a culture medium. However, unlike the control yolk sacs, giant yolk sacs tended to accumulate substrate within the tissue with increasing culture time. The concentration of digestion products released to the inside of the closed vesicle was found to be greater than that released to the surrounding culture medium at time intervals up to five hours. Giant yolk sacs preloaded with 125I-labelled bovine serum albumin were found to release material to the culture medium or the inside of the vesicle almost entirely in the acid-soluble (digested) form. This system is a useful model for studying the polar nature of epithelia, particularly those involved in the uptake and transport of nutritional and/or informational macromolecules.     

Inhibition of epithelial ovarian cancer by Minnelide,a water-soluble pro-drug

Objective

Minnelide is a water-soluble pro-drug of triptolide, a natural product. The goal of this study was to evaluate the effectiveness of Minnelide on ovarian cancer growth in vitro and in vivo.

Methods

The effect of Minnelide on ovarian cancer cell proliferation was determined by real time electrical impedance measurements. Multiple mouse models with C200 and A2780 epithelial ovarian cancer cell lines were used to assess the efficacy of Minnelide in inhibiting ovarian cancer growth.

Results

Minnelide decreased cell viability of both platinum sensitive and resistant epithelial ovarian cancer cells in vitro. Minnelide with carboplatin showed additive effects in vitro. Minnelide monotherapy increased the survival of mice bearing established ovarian tumors. Minnelide, in combination with carboplatin and paclitaxel, improved overall survival of mice.

Conclusions

Minnelide is a promising pro-drug for the treatment of ovarian cancer, especially when combined with standard chemotherapy.     

Expression of cathepsin P mRNA, protein and activity in the rat choriocarcinoma cell line, Rcho-1, during giant cell transformation

Lysosomal proteases perform critical functions in protein turnover and are essential for normal growth and development. Cathepsin P is a member of a newly discovered family of lysosomal cysteine proteases uniquely expressed in rodent placenta (PECs), and is closely related to human cathepsin L. Using the rat choriocarcinoma cell line model, Rcho-1, mRNA for the PECs cathepsins P, M, Q, R, 1, 2 was found to increase in expression during differentiation into a trophoblast giant cell phenotype. By contrast, expression of cathepsin L was not regulated. A specific enzyme assay was developed to show that activity of cathepsin P mirrored mRNA expression during differentiation. Cathepsin P protein co-localizes with cathepsin B, indicating that the enzyme probably functions in the endosomal-lysosomal compartment. This study demonstrates that the PEC genes produce functional proteases that can perform specific placental roles that are probably performed by broader specificity proteases in human placenta.     

Inhibition of human spermatozoa-zona pellucida binding by a combinatorially derived peptide from a synthetic target

Intact zona-free human oocytes were screened using a combinatorial peptide library selection protocol. Pieczenik Peptide Sequence 1 (PPS1) HEHRKRG binds human spermatozoa. A complementary and unique binding sequence HNSSLSPLATPA (PPS2) was developed from the first PPS1 ligand that binds to the human zona pellucida or oolemma. Cytoplasm-free zonae from unfertilized eggs were obtained and used as an assay system to test the effects of exposure to these two ligands. Spermatozoa were inserted into evacuated zonae and their behaviour and binding activity were assessed at regular intervals. The behaviour of spermatozoa exposed to PPS1 and unlabelled spermatozoa injected into unexposed zonae was similar as far as binding was concerned (50 and 54% binding), but PPS1 exposed spermatozoa had higher motility and displacement, marked by their escape from the zona pellucida. Zonae exposed to PPS2 inhibited the interaction between injected spermatozoa and the inside of the zona when compared with controls (8.3 and 53.8% attached respectively, P < 0.001). The sperm-zona pellucida interaction described in this paper is applied as a functional assay for molecular interactions of sperm binding and can be used to assess function for potential surface markers on gametes. It is shown here that a unique binding ligand (PPS2) can be synthesized from another complimentary ligand (PPS1) without the need for a known intermediate substrate. PPS1 and PPS2 may have properties that can be used to target processes involved in conception and assisted reproduction. A movie sequence taken approximately 30 min after injection of spermatozoa into empty human zonae pellucidae shows behaviour of non-manipulated spermatozoa into zonae not exposed or exposed to ligand. This may be purchased for viewing on the Internet at www.rbmonline.com/Article/2159 (free to web subscribers).     

Isolation,in vitro maturation,and fertilization of germinal vesicle oocytes obtained from the intact murine ovary

The purpose of this investigation was to attempt to develop a process, utilizing a murine model, which would allow more efficient harvesting from the intact ovary and maturation in vitro of germinal vesicle (GV) oocytes. The recovery process yielded 25.5±4.5 ( ±SE) cumulusfree GV oocytes per animal. Treatment groups included culture medium (CM) supplemented with either estradiol (E₂), follicle stimulating hormone (FSH), human chorionic gonadotropin (hCG), or prolactin (PRL). Among the hormone-free controls 83.2±1.6% of oocytes underwent GV breakdown, whereas 25.3±2.6% developed to the first polar body stage (PB-1) following 18 hr of incubation (n=29 trials). Oocytes progressing to the PB-1 stage were inseminated in vitro. In vitro fertilization (IVF) of pooled in vitro matured (IVM) PB-1 oocytes (judged by two-cell formation) was 19.9%, which was significantly lower than in the group of in vivo matured oocytes (74.7%). E₂ significantly increased the percentage of GV breakdown (control, 76.8±2.5%; E₂ at 10 ng/ml, 92.9±2.5%,P<0.001; E₂ at 100 ng/ml, 93.7±2.1%,P<0.001; and E₂ at 1 g/ml, 86.7±3.3%,P<0.05) but not PB-1 formation. Neither FSH nor hCG significantly increased GV breakdown or PB-1 formation. Prolactin treatment resulted in an increased percentage of PB-1 formation (control, 25.3±2.5%; PRL at 2 g/ml, 35.0±2.9%; and PRL at 20 g/ml, 34.1±1.9%;P<0.01), fertilization (control, 15.3±5.1%; PRL, 33.6±8.5;P<0.01), and subsequent development (control, 3.5±2.3%; PRL, 18.8±5.6%;P<0.01). We conclude that recovery and IVM of GV oocytes is feasible, however, further work is necessary to define optimal conditions.     

Adenocarcinoma arising from the respiratory ciliated epithelium in a benign cystic teratoma of the ovary

Benign cystic teratoma of the ovary (BCTO) is the most common benign ovarian tumor, accounting for 15–20% of all ovarian tumors. It is usually diagnosed in the third and fourth decades of life. Malignant transformation is rare, occurring in approximately 1–2% of reported cases, with squamous cell carcinoma being the most common form. Adenocarcinoma arising from mature cystic teratoma is extremely rare. We present the patient with a BCTO, where a malignant transformation of respiratory ciliated epithelium resulted in well differentiated adenocarcinoma. Although respiratory epithelium is often found in BCTOs, adenocarcinoma arising from this cell type is uncommon. To our knowledge, this is the fourth reported case of adenocarcinoma arising from the respiratory epithelium of a BCTO.