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To test the hypothesis that insulin-like growth factor (IGF-I) is required for the in vivo development of testicular Leydig cell function, either recombinant human GH [(hGH) (1.5 micrograms/g BW) or recombinant IGF-I (1 microgram/g BW) was injected three times daily into immature Snell dwarf mice (dw/dw) and into phenotypically normal control (Dw/-) for 7 days. In dw/dw mice hGH enhanced significantly body, liver, kidney, and testicular weight. In addition, hGH increased testicular LH receptors and the acute steroidogenic response to human CG, but there was no significant effect on basal plasma testosterone or plasma LH levels. The effects of IGF-I in body and kidney weight were less pronounced than those produced by hGH, but its effects on testicular weight and LH receptors, as well as on the acute steroidogenic response to human CG, were similar to that observed after hGH treatment. In Dw/- mice hGH had no effect on either body or organ weight or on testicular function, despite the fact that it induced a significant increase in plasma IGF-I levels. These results indicated that IGF-I is able to induce the maturation of Leydig cell function and that the effects of hGH on the testis are probably mediated by IGF-I. They also suggest that the delayed puberty associated with GH deficiency or resistance is most likely related to an IGF-I deficiency.

Ghrelin receptor ligands based on trisubstituted 1,2,4-triazole structure were synthesized and evaluated for their in vitro binding and biological activity. In this study, we explored the replacement of the alpha-aminoisobutyryl moiety by aromatic or heteroaromatic groups. Compounds 5 and 34 acted as potent in vivo antagonists of hexarelin-stimulated food intake. These two compounds did not stimulate growth hormone secretion in rodents and did not antagonize growth hormone secretion induced by hexarelin.

The murine lambda gene locus is organized as follows: V lambda 2-V lambda x-J lambda 2C lambda 2-psi J lambda 4C lambda 4-V lambda 1-J lambda 3C lambda 3-J lambda 1C lambda 1 where all segments have the same transcriptional orientation. The combinatorial process of gene recombination should allow the generation of eight distinct immunoglobulin light chains. We have therefore investigated the probability of obtaining such chains among the mature lambda B cell repertoire. We analyze serum lambda immunoglobulins and lambda B cell clones induced by treatment with rabbit anti-lambda antibodies coupled to LPS. Confirming previous data obtained by others, our results indicate that the rearrangements of lambda segments take place within each V lambda-J lambda-C lambda cluster, thereby defining a unit of recombination. Our results also provide no evidence for the use of undescribed segments as has been recently suggested by the finding of the V lambda x segment.

The existence of a cortical androgen-stimulating hormone (CASH), distinct from ACTH, regulating the secretion of human adrenal androgens has long been postulated. Recently, it has been reported that an 18-amino acid peptide, corresponding to the first part of the joining peptide of proopiomelanocortin [POMC-(79-96)], was able to stimulate the secretion of dehydroepiandrosterone from cultured human adult adrenocortical cells, but had no effect on cortisol production. We have studied the acute and long term effects of ACTH (10(-11) and 10(-9) M), CASH-18 (10(-8) M), or both on cortisol and dehydroepiandrosterone sulfate by human adult adrenocortical cells. Although ACTH increased steroid secretion and enhanced the steroidogenic responsiveness to further ACTH stimulation, CASH-18 alone or together with ACTH (10(-11) or 10(-9) M) had no effect. In addition, we were unable to demonstrate any specific binding of [125I]CASH-18 to human adrenocortical cells, although [125I] ACTH-(1-39) binds specifically to the same cell preparation.

The first complete map of a mammalian immunoglobulin gene locus is presented. Mouse A genes were mapped by pulsed-field gel electrophoresis. The gene order is V2-Vx-C2-C4-Vl-C3-Cl. The distance between V2 or Vx and the C2-C4 cluster is 74 or 55 kilobases (kb), respectively, whereas that between Vl and C3-C1 is only 19 kb; V2 and C3-Cl are at least 190 kb apart. Thus, the distances between the A subloci are inversely proportional to their frequencies of rearrangement. The related gene A5 is not within the 500 kb of the A locus mapped here.Mouse A genes have been found in four separate clusters of DNA clones which so far were physically unlinked: a cluster of clones containing the JC3 and JC1 genes, one of JC2 and the pseudogene JC4, and separate groups of clones for Vl and V2 (2, 31). Interestingly, the lambda genes rearrange in restricted combinations, VA1 only with CX1 or CX3 and VX2 almost exclusively with CX2 but very rarely with CA1 or CX3 (7). The newly discovered VXx gene has so far been seen rearranged only with CX2 (6,28). Recently, the order and orientation of A genes, except for Vx, were determined by deletion mapping with unique probes (18). The genes were all found to be in the same transcriptional orientation, and the order was determined to be V2-C2-C4-Vl-C3-Cl (18). This organization explains why V1-C2 rearrangements were not found but gives no clues about why rearrangements of Vl are about 5 to 10 times more frequent than rearrangements of V2. Furthermore, the location of Vx in this scheme was unknown. Since the sequence of Vx is as related to VK as to VX1-VX2 (6), its evolutionary association with the A locus presumably occurred in a way different from that of V1-V2 and thus its location and orientation may be unusual.We have cloned, in phage and cosmids, approximately 140 kilobases (kb) of DNA making up the VA and CA genes and their flanking regions. However, it was impossible to link up the A genes by chromosomal walking because of the paucity of unique sequences in this locus (18). We therefore used pulsed-field gel electrophoresis (PFGE) (3,30,33) transferred into lysis buffer (0.5 M EDTA, pH 9.5, 1% N-lauryl sarcosine, 0.25 mg of proteinase K per ml). The agarose inserts were incubated for 48 h at 50°C, washed extensively for 48 h in 10 mM Tris (pH 7.2)-i mM EDTA (TE) at 4°C, and stored at 4°C in TE. DNA inserts were digested in the restriction enzyme buffer specified by the manufacturer (New England BioLabs, Inc.) containing 4 mM spermidine. Each 40-,lI insert was incubated in 40 ,ul of 2x reaction mixture with 60 U of enzyme at 37°C overnight; on the next morning, a further 40 U of enzyme, 4 ,umol of spermidine, and sufficient lOx buffer and bovine serum albumin to bring these reagents to lx were added. Incubation was continued for 4 to 6 h at 37°C, and the inserts were rinsed with 3 ml of electrophoresis buffer (0.25x TAE [16]) for 30 min on ice and electrophoresed as indicated in the figure legends.5-Azacytidine treatment. 5-Azacytidine treatment was for 32 h in culture (9, 12); cel...

Spermatogenesis is a complex cellular process regulated by gonadotrophins and local cell-cell interactions. Stem cell factor (SCF) is one of the paracrine factors, produced by the Sertoli cells, involved in the local regulation of spermatogenesis. Measurement of its testicular level is important for addressing its role in testis physiopathology. However, the relative cell composition of experimental and pathological testis samples may lead to misinterpretation in relating SCF mRNA levels to the amount of RNA extracted from the whole tissue sample. Taking into account the relative RNA content of Sertoli cell origin should provide more significant data. In the present study, three sets of experiments were intended for modifying the proportion of RNA of Sertoli cell origin in RNA extracted from whole testis tissue samples: during postnatal development; following methoxy-acetic acid (MAA) administration; and after injecting a long-acting gonadotrophin-releasing hormone agonist (GnRHa). In a first step, we demonstrated clusterin mRNA level stability in purified Sertoli cell preparations between 20 days and adulthood, and following MAA or GnRHa treatment. In a second step, we used a competitive RT-PCR assay to measure SCF and clusterin mRNA levels and expressed the amount of SCF mRNA relative to the amount of clusterin mRNA under the above experimental conditions. The SCF/clusterin mRNA level ratio was found to remain roughly stable from 20 days post-partum to adulthood; i.e. during the development of spermatogenesis. MAA administration led to an overall increase in the SCF/clusterin mRNA level ratio between 7 and 14 days after administration, consistent with the replenishment of the testis with pachytene spermatocytes and round spermatids. Conversely, after long-acting GnRHa injection, the SCF/clusterin mRNA level ratio decreased only slightly from day 21 onward. Hence, the present studies indicate that, under physiopathological conditions, the amount of clusterin mRNA is a good marker of the amount of RNA of Sertoli cell origin in testis samples at day 20 or later; different experimental alterations of spermatogenesis are associated with different patterns of SCF mRNA levels; the relationship between FSH and SCF in vivo is not as simple as that described in vitro.

An in-vitro method was developed to study Sertoli-Leydig cell interactions in man, using testes removed at the time kidneys were removed for transplantation from 6 young adult men (aged 17-45 years) after cerebral death. After collagenase digestion of testicular tissue, Leydig cells were purified on discontinuous Percoll gradients. Two fractions of Leydig cells, 'L2' and 'L3' which differed in their buoyant density (1.05 g < L2 < 1.06 g and 1.06 g < L3 < 1.08 g), were obtained. The Sertoli cell-enriched preparation was obtained from seminiferous tubular fragments after sequential treatment with glycine buffer to remove peritubular-myoid cells, a second collagenase digestion, mechanical fragmentation and washes to remove germ cells. Purified Leydig cells were then cultured either alone or together with Sertoli cells in culture dishes coated with collagen, fibronectin and laminin in a chemically defined medium without serum. The influence of co-culture on basal testosterone secretion was examined in 3 successive 48 h periods.(ABSTRACT TRUNCATED AT 250 WORDS)