The present work was done to investigate the cell localization of testicular aromatase activity and its regulation in immature pig testis using an in vitro model. Leydig cells and Sertoli cells were isolated from immature pig testes and cultured alone or together in the absence or presence of human chorionic gonadotropin (hCG) or porcine follicle-stimulating hormone (pFSH) for 2 days. At the end of incubation, the amounts of testosterone (T), estrone sulfate (E1S) and estradiol (E2) were measured. Then the cells were incubated for 4 h in the presence of saturating concentrations of Δ4-androstenedione (3 μM) and the amounts of E1S and E2 were measured again (aromatase activity). The ability of Sertoli cells to produce estrogens was very low and neither hCG nor pFSH had any significant effect. hCG stimulated, in a dose-dependent manner, the secretion of T and E1S by Leydig cells cultured alone as well as the aromatase activity of these cells. The main estrogen produced by Leydig cells was E1S. pFSH also stimulated the above parameters of Leydig cell function; this may have been due to the contamination of this hormone with luteinizing hormone (LH). Coculture of Leydig cells with Sertoli cells without gonadotropins had very small effects on T and E1S production and on aromatase activity. However, treatment of coculture with increasing concentrations of hCG had a dramatic effect on Leydig cell functions. For each hCG concentration, the amounts of T and E1S secreted, as well as the aromatase activity of the coculture, were 2- to 3-fold higher than those of Leydig cells cultured alone. This potentiating action of Sertoli cells on hCG effects on Leydig cells depends upon the Sertoli cell:Leydig cell ratio. Treatment of coculture with increasing concentrations of pFSH also enhanced T production, but to a lesser extent than hCG. However, at greater concentrations, FSH was as effective as hCG for E1S production and aromatase activity. Again, the potentiating action of Sertoli cells on pFSH stimulation of the aromatase activity depends upon the Sertoli cell:Leydig cell ratio in the coculture. Finally, when the coculture was treated with greatest effective concentrations of hCG (0.1 nM) and pFSH (30 and 300 ng1m1), the stimulation of the aromatase activity was significantly higher than that produced by each hormone alone; no such effect was observed on T production. The above results show the following: 1)1n immature pigs, in contrast to immature rats, the main site of aromatization is Leydig cells. 2 )This activity is regulated by hCG, and Sertoli cells potentiate this stimulatory effect. 3) FSH has a small stimulatory effect on the aromatase activity of Leydig cells cultured alone, but in the coculture, the effect of this hormone is equipment to that of hCG. Taken together, the above results suggest that the secretory products of Leydig cells treated with hCG may stimulate the secretion by Sertoli cells off actors that in turn stimulate Leydig cells, and that stimulation of Sertoli cells with FSH also enhances the secretion of factors that also stimulate Leydig cells.
Several mutations in the human phenylalanine hydroxylase (PAH) gene have been described and it may be interesting to tentatively correlate mutant genotypes and clinical phenotypes of phenylketonuria (PKU).Twelve mutations were searched for using classical techniques of molecular biology in a total of 126 patients. 3 phenotypes were arbitrarily defined: typical PKU, atypical PKU or Mediterranean form, and persistent mild hyperphenylalaninemia. The patients were classified according to the residual in vivo PAH activity.Mutations were found in 64 patients. One mutation was found in each of the 2 alleles in 20 children. Only one mutation could be identified in the other 44. Only 45 children could be assigned to a phenotype. Mutations leading to total loss of PAH activity were associated with typical PKU (in homozygotes or compound heterozygotes). Mutations leading to residual PAH activity were associated in homozygotes with atypical PKU; they were also associated in compounded heterozygotes with atypical PKU, irrespective of the fact that the other allele suppressed enzyme activity or was unknown. A mutation which changed the affinity of PAH for phenylalanine was associated with mild hyperphenylalaninemia.The clinical heterogeneity of PKU can be correlated with identified mutations of the human PAH gene. Molecular studies do not help to predict either the phenotype or the long-term outcome. However, description of the biochemical changes combined with identification of the mutation should lead to a better understanding of consequences of mutation for PAH activity.
