Recombinant plasmids specific for bovine adrenocortical cytochromes P-450scc and P-45011 beta have been identified and characterized. Using these cDNA inserts as probes, it is found that tissue specificity of gene expression for these two proteins is as expected. Cytochrome P-450scc mRNA is found in adrenocortical and corpus luteum RNA while cytochrome P-45011 beta mRNA is found only in adrenocortical RNA. Neither mRNA was detected in heart, liver or kidney RNA. Cytochrome P-450scc mRNA is found to be 1.9 kb in length while cytochrome P-45011 beta mRNA is found to be 4 kb in length. Treatment of bovine adrenocortical cells with ACTH or other modulators results in increased levels of cytochrome P-450scc and cytochrome P-45011 beta mRNAs. Cytochrome P-450scc is found to be encoded by a limited number or even a single gene.

Adrenal Cortex↗

PubMedDate not supplied

Lipoproteins and the regulation of adrenal steroidogenesis.

ACTH has both short-term (acute) and long-term (chronic) effects to regulate steroid hormone biosynthesis in the adrenal cortex. The acute action of ACTH involves the mobilization of cholesterol and its binding to cytochrome P-450scc. The long-term action of ACTH involves the regulation of synthesis of the various enzymes involved in steroidogenesis. Evidence is presented that cholesterol may have a role to play in this regulatory process as it does in the short-term action of ACTH, consistent with the concept that substrates of specific forms of cytochrome P-450 are frequently able to regulate the synthesis of these specific forms.

Adrenal Cortex↗

PubMedDate not supplied

Genetic mutations resulting in loss of aromatase activity in humans and mice.

Aromatase enzyme is the product of the CYP19 gene. Human aromatase deficiency is a rare disorder and is usually caused by single base-pair changes resulting in amino acid substitution or premature stop codons. In most cases, the affected mother presents with virilization in the third trimester of pregnancy. Affected female newborns have pseudohemaphrodism with clitoromegaly and hypospadias. The cause of these presentations in pregnancy is the inability to convert fetal dehydroepiandrosterone to estrogen in the placenta and subsequent conversion to androgens in the periphery. Affected male newborns present with tall stature secondary to failed epiphyseal fusion. They also have delayed bone age, osteopenia, and undermineralization, which can be corrected with the addition of estrogen, highlighting estrogen's critical role in men as well as women. The aromatase knock-out male mouse (ArKO) has shortened femur length and bone undermineralization. Female ArKO mice at 10-12 weeks have multiple ovarian follicles arrested in the antral phase and stromal hyperplasia. By 1 year the ovaries become grossly dysmorphic with numerous cystic follicles and fibrous stroma. Male ArKO mice testes demonstrate arrest of spermatogenesis at the level of round spermatids and Leydig cell hyperplasia. ArKO mice also exhibit evidence of insulin resistance and visceral adiposity.

Animals↗

PubMedDate not supplied

Characterization of the fertility of male aromatase knockout mice.

Previous studies employing the male aromatase knockout (ArKO) mouse have indicated that local expression of estrogens appears to be important for the progression of spermatogenesis. In the absence of estrogen biosynthesis round spermatids are observed to undergo apoptosis and thus fail to differentiate into mature, elongated spermatids. This lesion appears to arise between the ages of 18 weeks and 1 year. To ultimately determine if the disruption to spermatogenesis arises earlier than 18 weeks, we performed an intensive study to examine the fertility of younger male ArKO mice. This involved an analysis of their mating capacity together with an extensive stereological analysis, determination of the in vitro potential of mature sperm, and sexual behavior. ArKO and wild-type (w/t) males at 7 weeks of age were placed with w/t females for 7 weeks. At age 14 weeks, the males were killed and the testes removed. ArKO mice were observed to sire significantly fewer litters than the w/t mice; 5 out of the 10 sired no litters at all. Stereological analysis performed on the removed testes found a significant decrease in round spermatid numbers between w/t and ArKO mice at this age; however, there were no differences in all other germ cells and Sertoli cell numbers. When mature spermatozoa were analyzed, sperm from 15-week-old ArKO mice had a significant reduction in motility. This was further reduced by 1 year of age with a decrease in concentration. A preliminary examination of sexual behavior found that ArKO mice did not attempt to mount the females, in contrast to the w/t mice, which mounted consistently during the time period. In conclusion, we observed that ArKO mice have reduced fertility at age 14 weeks. This may be due in part to a disruption in spermatogenesis because the phenotype does appear to arise earlier than 18 weeks, possibly leading to abnormalities in the mature spermatozoa. Or, in part, this may be attributable to an impairment in the development of copulatory behavior, which is consistent with the available evidence that points to a crucial role for estrogens in the neural development and initiation of male sexual behavior.

Animals↗

PubMedDate not supplied

Effects of conditioned medium from different cultured cell types on aromatase expression in adipose stromal cells.

OBJECTIVE: To determine whether serum-free (SF) conditioned media (CM) from several human breast cancer cell lines and primary stromal cell cultures contain factor(s) that mimic the marked stimulatory effects of serum on aromatase activity and aromatase P450 (P450arom) gene expression in adipose stromal cells in culture (ASC) in the presence of dexamethasone (DEX). METHODS: Adipose stromal cells, harvested from fresh adipose specimens, were grown to confluence, switched to SF media, and then incubated in the presence or absence of DEX with CM from T47-D breast cancer cells, pre-treated with or without 17 beta-estradiol (E2), and with CM from stromal cell cultures. Aromatase activity of the ASC was determined by the [3H] water release assay. Total RNA was isolated, and reverse transcription-polymerase chain reaction was performed to determine the expression of various 5'-termini. RESULTS: T47-D CM stimulated aromatase activity in a concentration-dependent manner, similar to that of serum, in ASC incubated with DEX. Estrogen potentiated this in a dose-dependent fashion. The ASC CM and endometrial stromal cell CM also markedly induced aromatase activity in ASC. Heat inactivation destroyed the stimulating ability of CM. The majority of P450arom 5'-termini expressed by ASC incubated with CM plus DEX contained the promoter I.4-specific sequence. CONCLUSIONS: Conditioned media from several breast cancer cell lines and primary stromal cell cultures can mimic the effects of serum in the presence of DEX to stimulate aromatase activity in ASC. These results suggest that undefined, heat-labile and proteinaceous factors are present in CM that stimulate P450arom expression in a fashion similar to that of serum.

Adipose Tissue↗