Generation and characterization of monoclonal antibodies to the neural crest.

The generation of monoclonal antibodies (MAbs) specific for quail neural crest may provide valuable tools for studying the differentiation of embryonic precursor cells. Unfortunately, relatively few antibodies are available because of the difficulty in obtaining sufficient cells for in vivo immunization strategies. We have overcome this problem by using intrasplenic immunization with formaldehyde-fixed cells harvested from neural crest cultures. In addition, booster injections of cultured whole-embryo cells were administered intraperitoneally. Following two fusions, a total of 18 hybridomas were generated with antibody reactivity to the cytoplasm of neural crest cells. Furthermore, 32 were reactive against both somite (a noncrest mesodermal control) and crest cultures, whilst 15 were not reactive. Out of those hybridomas reactive with neural crest, six designated 160D, 164D, OE, 12E, 120E and 124E were further characterized. Interestingly MAb supernatants OE, 12E, 120E, and 124E exhibited reactivity against some but not all neural crest cells suggesting that they might recognise subpopulations. Immunoglobulin isotyping of supernatants revealed that 4 (160D, 164D, OE, and 120E) were IgM and 2 (12E and 124E) were IgG(2b). On assessing their reactivity against human tissue sections, all six hybridoma supernatants cross-reacted with neuroendocrine cells within appendix, colon and rectum. These MAbs could provide novel reagents for the understanding of neural crest development.

Endometriosis and polycystic ovary syndrome: enhanced stimulatory effect of peritoneal fluid on progesterone release from human granulosa-lutein cells.

OBJECTIVE: To compare the activity of peritoneal fluid (PF) from women with and without endometriosis or polycystic ovary syndrome (PCOS) on P release from cultured human granulosa-lutein cells. DESIGN: Case-control study. SETTING: Medical school and hospital. PATIENTS: Women with mild to moderate endometriosis or PCOS and controls undergoing laparoscopic sterilizations. MAIN OUTCOME MEASURES: Human granulosa-lutein cells, obtained from the IVF clinic, were cultured with increasing volumes of steroid-extracted PF samples with and without hCG. Progesterone concentrations in the media after 72 hours culture were measured by RIA. RESULTS: Peritoneal fluid stimulated P release in a dose-dependent manner and, at the highest dose, the response was enhanced significantly by PF from women with endometriosis and PCOS compared with control samples. The effects of PF plus hCG were not simply additive. There was a large potentiation of the response, which was greater in women with endometriosis and PCOS. CONCLUSIONS: Peritoneal fluid contains factors that stimulate P release and potentiate the response to hCG. The increased activity of PF associated with endometriosis and PCOS in part may have some significance in abnormal ovarian function associated with these syndromes.

The physicochemical, immunological and biological properties of rat pituitary and plasma LH.

A comparison was made between the properties of LH derived from female rat pituitary glands and plasma. Samples were collected from adult intact rats 5 h before or at the time of the pro-oestrous preovulatory LH surge; 27-day-old rats untreated or given 5 IU pregnant mares' serum gonadotrophin (PMSG) s.c. on day 25, which induced LH release 54 h later and adult ovariectomized rats untreated or primed with either 5 micrograms oestradiol benzoate s.c. or 5 micrograms oestradiol benzoate followed 48 h later by 0.5 mg progesterone s.c., which induced LH release 4-6 h later. All pituitary LH samples were totally bound to an anionic ion-exchange resin (DE52), while only a small proportion of the plasma LH was bound. Only 0-10% plasma LH obtained from intact, ovariectomized (with and without steroids) and untreated immature rats was bound, while a greater proportion of bound LH (36%) was noted in rats treated with PMSG. Gel filtration indicated only slight differences between pituitary and plasma LH, the former eluting marginally earlier than whole plasma and the unbound and bound plasma forms derived after separation by DE52 resin. Affinity chromatography (Concanavalin A and Glycine maximus) showed that LH from both sources possesses high mannose oligosaccharides and that plasma LH does not bear terminal N-acetyl galactosamine residues, although 20% of the pituitary form does. Plasma obtained from pro-oestrous rats had greater bioactivity than had pituitary LH in stimulating testosterone from Leydig cells and progesterone from granulosa cells in vitro, and inducing ovulation in immature rats in vivo. Leydig cell bioassays for LH in fractions obtained from ion-exchange separation indicate that steroidogenic activity of unbound plasma LH is greater than bound pituitary LH when they were collected at times of enhanced release. When release was inhibited (oestrogen-primed ovariectomized rats or immature rats), the steroidogenic activity of plasma and pituitary LH were similar and an acidic steroidogenic component was present in the plasma that was not recognized immunogenically as LH. In summary, pituitary LH undergoes a conversion on release into the plasma that involves a change in binding characteristics on an ion-exchange resin. In conditions when LH release is enhanced there is an increase in bioactivity of plasma LH owing to modification either by steroids or some other plasma factor(s) that perhaps influence the structure of LH directly or by steroids acting indirectly to alter GnRH release, which then modifies LH structure. These structural changes are minor and probably involve alterations in the glycosyl attachments.

Inhibitory action of peritoneal macrophages on progesterone secretion from co-cultured rat granulosa cells.

Resident ovarian macrophages are recognized as potential regulators of ovarian function, and the majority of evidence suggests that such regulation is mediated through cytokine secretions, particularly interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha). In this study we examine the effects of co-cultured macrophages, stimulation and inhibition of the immune system, and IL-1 beta and TNF alpha on gonadotropin-induced progesterone secretion from rat granulosa cells. Granulosa cells were isolated from ovarian follicles of proestrous rats and were cultured for a period of 48 h. Peritoneal macrophages, obtained from untreated rats or from animals pretreated with either thioglycollate (TG) or lipopolysaccharide (LPS), were plated at concentrations of 10(5), 5 x 10(3), or 2 x 10(3) cells/ml. After their adherence to the base of the culture wells, 1 ml of serum-free McCoy's medium containing 3 x 10(5) granulosa cells/ml were added to the macrophages. Gonadotropin-induced progesterone secretion was markedly inhibited in the co-cultures, and the degree of inhibition was dependent on both the number of co-cultured macrophages and whether or not the macrophages had been pre-activated by either TG or LPS. TG-activated macrophages were most potent in this respect. Such effects were not due to cytotoxic effects of the macrophages on granulosa cells as determined by a colorimetric assay for cellular growth and survival. In fact, macrophages increased the number of viable granulosa cells after 48-h culture. Granulosa cells obtained from animals pretreated with LPS showed a reduced ability to respond to ovine LH, although suppression of the immune system with cyclosporin had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of lipopolysaccharide and cyclosporin on the endocrine control of ovarian function.

The effects of stimulating the immune system with lipopolysaccharide (LPS) or suppressing the immune system with cyclosporin (CS) on reproductive functions in the female rat were investigated. Animals were either treated acutely with LPS (2 mg kg-1) or cyclosporin (20 mg kg-1) on dioestrus day 1 and 2 or treated chronically over a period of 6 days (on alternate days with LPS, daily with CS). Chronic LPS treatment induced a state of constant dioestrus and decreased circulating concentrations of progesterone and oestradiol. Chronic CS treatment induced some irregularity in the 4-day vaginal smear pattern in a minority of animals and, while it had no effect on circulating concentrations of progesterone, oestradiol concentrations were suppressed compared with those measured in pro-oestrous animals. LH responses to GnRH were reduced in both perifused pituitary fragments and cultured pituitary cells obtained from animals pretreated with either LPS or CS. In contrast, a low dose of LPS (20 micrograms kg-1) given over 6 days did not disrupt ovarian cycles and reduced, but did not abolish, the second phase primed LH response. Neither drug had a direct effect on the pituitary LH responses to GnRH, except that pituitary cells exposed to high doses of CS for periods greater than 48 h did show attenuated LH responses to GnRH. This finding was not paralleled with high doses of LPS. The differential count of ovarian follicles from histological studies showed that LPS treatment was associated with significantly fewer large preovulatory follicles, whereas animals treated with CS showed a similar distribution of follicular volumes compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)