Evidence for a protein-like factor from "rete testis" fluid that suppresses luteinizing hormone-releasing hormone (LHRH) pulses (LHRH statin): a new hormonal activity?

Signals that modulate LH-releasing hormone (LHRH) pulse frequency are fundamental mechanisms for regulating important reproductive processes. Gonadal steroids are presently considered to account for the entire gonadal feedback mechanism that modulates LHRH secretion. However, we have previously suggested that a testicular protein(s) present in charcoal-treated rete testis fluid (ctRTF) can suppress LH pulsatility in the ram. The present experiments were aimed at determining whether the disappearance of LH pulses induced by ctRTF administration implicate a hypothalamic or a pituitary site of action. Thus, we have examined the effects of ctRTF peripheral administration on 1) the LH response to LHRH, 2) LHRH portal blood levels, and 3) LHRH content in hypothalamic tissue. Finally, the effects of ctRTF administered into the third ventricle on plasma LH levels were assessed. The present results show that a testicular protein(s) is able to suppress LHRH pulse frequency without affecting amplitude and without any effect on the LH response to LHRH (LHRH Statin). The observation that an active dose administered by the intracerebroventricular route is 0.0005 the active dose needed by the peripheral route reinforces this evidence. These data lead to the new concept that the testicular signals that govern LHRH pulse frequency may be not only steroids, but also proteins.

A linkage disequilibrium map of the MHC region based on the analysis of 14 loci haplotypes in 50 French families.

A sample of 100 individuals from 50 French families of known pedigrees were typed for 14 loci of the HLA region (DPB1, DQB1, DQA1, DRB1, DRB3, 4, 5, C4B, C4A, Bf, C2, TNFa, TNFb, B, Cw, A). Linkage disequilibrium in each pair of loci was investigated by an exact test using a Markov chain algorithm. The results indicate no disequilibrium between DPB1 and the other loci, whereas the other class II genes are all significantly linked to each other. Linkage disequilibrium is also detected between some pairs of class I and class II-class I loci despite the long physical distance separating the loci (e.g. A-B, Cw-DRB1). On the other hand, some contiguous loci of the class III region are found to be in equilibrium with each other. Several hypotheses including selection, but also unequal allelic diversity at different MHC loci are discussed to explain this complex pattern of linkage disequilibrium.

A novel HLA-B*39 allele (HLA-B*3916) due to a rare mutation causing cryptic splice site activation.

A novel HLA-B*39 variant, found in an African patient with sickle cell anemia undergoing bone marrow transplantation is described. Initially suspected by inconsistent serological typing (B-blank, Bw6), then recognized by PCR-SSP, and finally characterized by nucleotide sequencing, this novel allele is designated HLA-B*3916. It differs from HLA-B*3910 by a point mutation (G to C) at position 17 of exon 3 causing glutamine to histidine change at codon 96 of alpha(2) domain, a conserved position among HLA class I alleles. cDNA sequence analysis further revealed the presence of both normally and abnormally spliced mRNA species in established cell lines. The abnormal species correspond to partial truncation of exon 3 presumably due to the nucleotide change in exon 3, which constitutes a new consensus acceptor splice site within this exon. We postulate that the observed blank is essentially the consequence of qualitative change in a critical region of this novel antigen as abnormal mRNA species are relatively less abundant than normal species. Because the residue 96 of the HLA class I heavy chain is directly involved in interaction with alpha(2)m, another interesting possibility is that an aminoacid change in this position would perturb such interaction and consequently could affect the serological specificity of B*3916, or its expression or both.

Blockade of the final phase of ovulation has no effect on the post-ovulatory surge of serum FSH in the rabbit.

Mating induces a surge of both LH and FSH in the blood of female rabbits, followed 10-12 h later by a surge of FSH only, which begins at the time of ovulation. We have studied the effect of suppression of ovulation on the post-ovulatory surge of FSH. In the first experiment, follicular fluid and oocytes were withdrawn from the largest follicles 8 h after coitus. In the second experiment, ovulation was inhibited by injecting the rabbits with 25 mg indomethacin/kg body weight 7.5 h after mating. Levels of serum FSH and LH were measured for 24-48 h after mating. Control rabbits ovulated normally in both experiments. The treatments did not significantly affect the levels of serum FSH in either experiment, although the second surge of FSH was slightly higher after fluid had been aspirated from the preovulatory follicles. These observations show that the post-ovulatory surge of serum FSH is not dependent upon the completion of ovulation and that it is programmed before 7.5-8 h post coitum.

Anastomosis of the ovarian vein to the hepatic portal vein in sheep induces ovarian hyperstimulation associated with increased LH pulsatility, but only in the absence of the contralateral ovary.

In this study, two experiments were performed, the first of which examined the ovarian response in ewes that were subject to unilateral ovariectomy (ULO) at different intervals (0-14 days) after surgical anastomosis (AN) of the ovarian vein to the mesenteric vein (n=7 ewes), or sham operation (SO; n=4 ewes). Hypertrophy and development of multiple follicular and luteal structures on AN ovaries were observed after ULO, while SO ovaries remained of normal size and appearance after ULO. The second experiment involving 11 ewes (five AN; six SO) aimed to clarify the mechanism by which AN following ULO-induced ovarian hypertrophy and increased follicle development. The results confirmed that there were more large (>5 mm) follicles on AN compared with SO ovaries; however, their rate of atresia was similar. Oestradiol and progesterone concentrations in follicular fluid of class 1 follicles (5-9 mm) were higher in AN ovaries than those in control follicles of the same size collected in the late follicular phase of an induced oestrous cycle. In AN ewes, intrafollicular progesterone concentrations increased while follicular aromatase activity and intrafollicular oestradiol, inhibin A, follistatin and activin A concentrations all decreased as follicle size increased. Oestradiol and progesterone concentrations were substantially higher in ovarian venous blood than in hepatic venous blood, both in AN and SO ewes, whereas inhibin A levels were not significantly modified by passage through the liver in either group. Mean plasma LH concentration, and LH pulse frequency and amplitude increased markedly after AN but were not affected by SO. Plasma FSH showed only a small transient increase after AN, presumably due to the maintenance of inhibin feedback. Injection of prostaglandin F(2)(alpha) 4 days later did not further modify LH or FSH secretion in either group. Full ovariectomy (FO) 9-14 days after AN or SO increased LH secretion markedly in SO ewes but to a lesser degree in AN ewes; FO induced a large and rapid increase in FSH levels in both groups. In conclusion, AN of the ovary to the liver via the mesenteric vein provides a useful model for studying the feedback between the ovary and the hypothalamo-pituitary system and the mechanisms controlling follicle development. The present results indicate that the pattern of LH secretion is an important factor controlling the terminal phase of follicle development in the ewe.

Influence of metabolic and genetic factors on tumour necrosis factor-alpha and lymphotoxin-alpha production in insulin-dependent diabetes mellitus.

The potential role of tumour necrosis factors (TNFs) in autoimmunity and insulin-dependent diabetes mellitus (IDDM) led us to determine in vitro TNF-alpha and lymphotoxin-alpha (LT-alpha, TNF-beta) production in IDDM patients according to TNF polymorphism. LT-alpha production of peripheral blood mononuclear cells (PBMC) was lower in diabetic subjects (m = 0.30 +/- 0.2 ng.10(-6) cells) than controls (m = 0.68 +/- 0.3 ng.10(-6) cells, p < 0.05), and early age-at-onset was correlated with low LT-alpha production (rs = 0.8, p = 0.0006). TNF-alpha production was the same in patients and controls, but patients with HbA1c > or = 8% had a higher TNF-alpha production (m = 3.05 +/- 1.2 ng.10(-6) cells) than those with HbA1c < 8% (m = 1.31 +/- 0.33 ng.10(-6) cells, p < 0.05). A study of the microsatellite TNFa region close to the LTA gene showed that the presence of the TNFa1 allele in HLA-(DR3) subjects was associated with increased risk of IDDM. TNFa1-positive subjects (both patients and controls) also had lower LT-alpha production than other subjects. These results indicate that low LT-alpha production is an additional risk factor for IDDM and that poor glycaemic control in patients is associated with enhanced PBMC TNF-alpha production which causes an imbalance between TNF-alpha and LT-alpha production in IDDM patient.

Plasma levels of LH, FSH, prolactin, oestradiol-17beta and progesterone during natural and induced oestrus in the dairy goat.

The patterns of LH, FSH, prolactin and oestradiol-17beta, before and during natural oestrus, and of progesterone during the following cycle were studied in four French Alpine dairy goats and compared with those obtained after synchronization of oestrus in the same animals. The highest concentration of oestradiol-17beta was measured at the beginning of oestrus and was followed 3 hours later by simultaneous rises of LH, FSH and prolactin. A second FSH peak was observed 48h after the first one. On D(3) (D(0) = day of oestrus) progesterone concentration was over 1 ng/ml. The luteal phase lasted 15 days. Peak concentrations of oestradiol-17beta and progesterone were higher in animals when oestrus was induced. This was attributed to their higher ovulation rate. The second FSH peak was lower, and the interval between oestradiol-17beta peak and gonadotrophin surge longer, than at natural oestrus.

[HLA markers and complotypes: risk factors in systemic lupus erythematosus?]. / Marqueurs HLA et complotypes: facteurs de risque dans le lupus érythémateux systémique?

Sixteen simplex and two multiplex families of subjects presenting with systemic lupus erythematosus (SLE) were studied and compared with 108 controls. The frequency of A1, B8 and DR3 alleles reported in the literature was not confirmed. Only the DR3 (chi 2 = 5.45, p less than 0.02, RR = 2.53) and CW4 (chi 2 = 6.72, p less than 0.01) alleles, not yet described by other authors, were noted as being associated with SLE. The DR3 C21 BFS C4 AQ0B1 B8 A1 haplotype was not found with statistically significant frequency (chi 2 = 2.55, p = NS). The distribution of haplotypes within the multiplex families confirmed the absence of link between the major histocompatibility complex and systemic lupus erythematosus. Finally, the association of a null allele of complement was confirmed (chi 2 = 5.08, p less than 0.05), but only the C4 BQ0 allele was associated with SLE (chi 2 = 12.27, p less than 0.001, RR = 3.78) instead of the C4 AQ0 allele reported in the literature as being associated with SLE. Some of the CMH alleles are thought to constitute a risk factor of SLE. The presence of silent alleles of complement (C2 Q0, C4 AQ0 and C4 BQ0) seems to play a decisive role in the occurrence and expression of this disease.

A comparison of ovulatory gonadotropic surge in two rabbit strains: no evidence for a relationship between LH or FSH surge and factors of prolificacy.

Plasma levels of LH and FSH before and after ovulation were measured in two rabbit strains (New Zealand A 1077 and Californian A 1066) having a different number of ovulations and rate of embryonic loss. Maximal concentrations and total secreted amounts before ovulation were slightly higher in New Zealand, but the difference was not significant. No relationship between the number of ovulations and the increase in plasma LH or FSH level was found in either strain. In most cases, there was no relationship between gonadotropic surge and early embryonic loss.

Highly specific antisera against native ovine follitropin (oFSH) obtained from crude antisera by affinity chromatography on solid-phase undissociable lutropin (LH) column.

Antibodies recognizing determinants of the alpha-subunit, which is common to all glycoprotein hormones, were eliminated from antisera against native oFSH by affinity chromatography. Since the free alpha-subunit is immunologically different from the alpha-subunit in the intact hormone, we did not use an alpha-subunit affinity column. Instead, the antisera were applied to an LH affinity column. However, because intact LH dissociates in the conditions used to elute the purified antibodies, we prepared an LH derivative with covalently-linked subunits and coupled it to gel matrix. By this method oFSH antisera were freed from their non-specific antibodies. The cross-reaction of ovine lutropin in the oFSH radioimmunoassay (RIA) was lowered from 2% to less than 0.1%. Moreover, as the columns can be used repeatedly over long periods with no apparent loss of efficiency, large volumes of antiserum can be treated in this manner.

Relaxin-like peptide in ascidians. II. Bioassay and immunolocalization with anti-porcine relaxin in three species.

A substance related to vertebrate relaxin, previously identified by radioimmunoassay and Northern hybridization in the ascidian Herdmania momus, was also purified and tested in bioassay in another species, Ciona intestinalis. In addition, immunocytochemistry with anti-porcine relaxin was performed, at the light and electron microscopic levels, on sections of ovary from three different species, living in various environmental conditions. A positive immunoreaction was located specifically in follicle cells surrounding mature oocytes. The role of this relaxin-like substance in ascidian reproduction is unknown.

[Distortion of the maternal segregation of the silent alleles of complement factor 4 in normal and diabetic families]. / Distorsion de la ségrégation maternelle des allèles silencieux du facteur 4 du complément dans des familles normales et des familles diabétiques.

54 normal Caucasian families and 169 families in whom at least one child had type I diabetes (IDDM) were genotyped for HLA-A, B, C, DR and for the complement factors Bf and C4. The paternal and maternal transmission of the different alleles and of haplotypes and complotypes in linkage desequilibrium have been analysed. No distortion of the paternal transmission has been observed in the offspring of the two series of families. On the contrary, a distortion of the maternal segregation of the silent alleles at the complement factor C4A and B locus was found: mothers transmitted C4AQ0 more often than expected to their male offspring (p less than 0.04 in normal families, p less than 0.001 in IDDM families) while they transmitted C4BQ0 in excess to their female offspring (p less than 0.01 and p less than 0.03 in normal and IDDM families, respectively).

[Risk factors in insulin-dependent diabetes]. / Facteurs de risque du diabète insulino-dépendant.

The relative risks (RR) of the immunogenetic markers of insulin-dependent diabetes mellitus (IDDM) have been calculated in a population of 235 IDDM patients compared with a control population. The highest relative risk was that of subjects heterozygous DR3/DR4 (RR = 47, P less than 0.001) which was still more increased in those who carry this combination associated with the RFLP cluster DQR4 (RR = 72). Further, a sample of 51 secondary affected siblings of IDD index cases has been compared with 265 non affected siblings (one child of each family, excluding index cases). The highest risks have been found in addition to DR3/DR4, for DR3 alone and particularly for the combination C4BQ0, DR3 (RR = 9, p less than 0.001) suggesting a role for this peculiar association in the familial penetrance. In the group of siblings HLA-identical with the index case, only two factors showed some capacity of discriminating between affected and non affected siblings: HLA-DR3 and age (less than or equal to 10 years old at onset of IDDM in the index case) (p less than 0.01). In the group of haploidentical siblings, the combination DR3/DR4 and the associations C4BQ0, DR3 and BfF1, DR3 significantly increased the susceptibility for higher familial occurrence of the disease. If confirmed by additional family series, this scale of risk factors could be helpful in predicting risk of IDDM to siblings of diabetic children.

[Familial studies of systemic lupus erythematosus. HLA markers and complotypes]. / Etudes familiales du lupus érythémateux systémique. Marqueurs HLA et complotypes.

Particular susceptibility to systemic lupus erythematosis (SLE) could be due to a certain alleles of class I, II or III of the major histocompatibility complex (MHC). The existence of total hereditary deficiencies of factor 2 or 4 of the complement in this syndrome suggests the presence of silent alleles which could conceivably play a determining role in the appearance of SLE. In this study, the HLA haplotypes and complotypes (C2, C4, Bf) were determined in 20 individuals suffering from SLE, and compared with 108 healthy, genotyped individuals. The results obtained showed a significant increase in the frequency of C4 BQ0 in patients compared with that found in controls (chi 2 = 12.27, p less than 0.001, Relative Risk = 3.78), and confirm the HLA association, DR3/SLE (chi 2 = 5.45, p less than 0.02, RR = 2.53).

Evidence that the corpus luteum of pregnancy contributes to the control of tonic secretion of LH in the ewe.

Concentrations of LH and FSH were measured in blood samples collected from the jugular vein at 20-min intervals for 7 h (09:00-16:00 h) on Days 60, 80, 100 and 120 of pregnancy in 5 intact ewes and 5 from which the CL had been excised on Day 70. In the 5 intact ewes, plasma LH concentrations remained low and unchanged between Days 60 and 120. During this period, pulsatile release of LH occurred irregularly and infrequently. Removal of the CL resulted in an increase in the basal values of LH and in the frequency and amplitude of LH pulses. Concentrations of FSH were relatively constant in all stages of pregnancy examined and were similar in both groups of ewes. These results show that (1) LH concentrations are low during the second half of pregnancy; and (2) LH, but not FSH, increases after CL excision, presumably by removing some luteal factor inhibitor of LH secretion.

Evidence for a gonadal nonsteroidal factor that specifically inhibits release of luteinizing hormone in ewes.

Bilaterally ovariectomized ewes were used to investigate the effect of systemic administration (i.v.) of charcoal-treated aqueous luteal extracts from ovine corpora lutea on plasma concentrations of pituitary gonadotrophins. Jugular blood samples were taken every 15 min at least 5 h before (control period) and 5 h after (treatment period) injection. In Expt 1, the administration of luteal extract from corpora lutea of days 70-76 of pregnancy, but not of the extract prepared from muscular tissue, resulted in a significant decrease of mean concentrations of luteinizing hormone (LH) (P < 0.02) and frequency of LH pulses (P < 0.01). Plasma follicle-stimulating hormone (FSH) concentrations were not affected by injections of either extract. These findings provide the first demonstration of the presence of a nonsteroidal factor in the corpus luteum of midpregnancy that selectively suppresses the secretion of LH. In Expt 2, mean concentrations of LH and FSH and frequency of LH pulses were unaffected by injections of luteal extracts from ovine corpora lutea of days 10-12 of the oestrous cycle or day 15 of pregnancy. These data suggest that some factor(s), probably from the fetoplacental endocrine unit, is required to ensure the production of a significant quantity of the luteal LH-inhibiting factor after day 15 of pregnancy. In Expt 3, treatment of luteal extract from corpora lutea of day 70 of pregnancy with proteolytic enzymes destroyed the LH-inhibiting activity, suggesting the proteic nature of the luteal LH-inhibiting factor. In Expt 4, plasma concentrations of LH were not affected by injection of charcoal-treated extract prepared from fetal cotyledonary tissue of days 110-120 of pregnancy suggesting that the LH-inhibiting factor exclusively originates from the corpus luteum during pregnancy. These experiments provide the first direct evidence for the existence of a potent nonsteroidal factor of luteal origin that specifically inhibits pulsatile secretion of LH, without influencing FSH release in female animals. We propose the term LH-release-inhibiting factor (LH-RIF) to describe this activity.