Estrogen formation in the early rabbit embryo.

Androgen formation (3beta-hydroxysteroid dehydrogenase activity) was detectable in the rabbit blastocyst on day 5 of gestation (before implantation); estrogen formation was first detectable on day 7. The capacity to form estrogen on the day of implantation suggests that estrogen formation in the blastocyst may play a role in the implantation process.

Female feathering in sebright cocks is due to conversion of testosterone to estradiol in skin.

Sebright cocks develop a female feathering pattern but revert to normal male feathering after castration. Administration of testosterone to castrated cocks causes male comb development and reappearance of female feathering. Dihydrotes-tosterone treatment supports development of a male comb but does not induce female feathering. Since testosterone but not dihydrotestosterone is converted to estradiol in the skin of the Sebright, the female feathering appears to be the result of increased conversion of testosterone to estradiol.

Cell-adhesive motif in region II of malarial circumsporozoite protein.

The segment of the malarial circumsporozoite (CS) protein designated Region II is highly conserved among different malarial species. A similar sequence is also present in several other proteins, including thrombospondin, properdin, and a blood-stage antigen of Plasmodium falciparum. By means of peptides synthesized from sequences of the Plasmodium vivax CS protein in the vicinity of Region II, it was found that two overlapping 18- to 20-amino acid peptides promoted the adhesion of a variety of human hematopoietic cell lines. The amino acid sequence valine-threonine-cysteineglycine (VTCG), contained within this common motif, was shown to be the critical sequence for the observed cell-adhesive properties.

pathogenesis of the henny feathering trait in the Sebright bantam chicken. Increased conversion of androgen to estrogen in skin.

In female chickens of all breeds development of female feathering pattern is mediated by estrogens, whereas normal males and castrated chickens of both sexes develop male feathering. Male chickens carrying the henny feathering trait (such as the Sebright bantam and golden Campine) develop a female feathering pattern but otherwise virilize normally. To examine the possibility that the henny feathering trait is the result of increased conversion of androgen to estrogen in skin, estrogen formation from [1,2,6,7-3H]testosterone was measured in tissue slices from control breeds and chickens with the henny feathering trait. Rates of estrogen formation were undetectable or low in all control tissues other than ovary, whereas rates were high in skin and skin appendages and detectable in many tissues from Sebright and Campine birds. The increased rate of estrogen formation in skin was demonstrable in Sebright chicks and in all areas of skin biopsied in the mature bird. Furthermore, plasma levels of 17 beta-estradiol were higher in Sebright and Campine than in control male cocks. Thus, increased formation of estrogen from androgen in the peripheral tissues probably explains the henny feathering trait.

Changes in amount and intracellular distribution of androgen receptor in human foreskin as a function of age.

To provide insight into the factors that control growth of the penis we measured the amount and intracellular distribution of specific high affinity androgen receptor in foreskins obtained at circumcision from 49 males varying in age from newborn to 59 yr. Total (cytosolic plus nuclear extract) androgen receptor decreased from approximately 40 fmol/g tissue weight in newborn foreskins to approximately 25 fmol/g by 1 yr of age. The amount of receptor rose in childhood to approximately 180 fmol/g in the late teenage years and fell thereafter to approximately 20-40 fmol/g in men older than 40 yr. The amount of receptor in the nuclear fraction increased at the time of puberty and subsequently decreased in parallel with the decline in total receptor level. These changes in androgen-receptor amount are similar when expressed per milligram DNA or per milligram protein.

Classification of human leukemia by membrane antigen analysis with xenoantisera.

Rabbit and monkey antisera after appropriate absorption were rendered specific for normal or leukemic lymphoid- and myeloid-associated antigens. Antisera defining a common peripheral blood T-cell antigen, a thymus leukemia antigen, HLA-DR or Ia-like antigen, common acute lymphoblastic leukemia antigen (CALLA), and a myeloid-monocyte (M) antigen were used in a microcytotoxicity assay to classify leukemic cells from 30 patients in a double blind study. The antisera to the M antigen reacted with adherent peripheral blood cells and polymorphonuclear leukocytes and failed to react with nonadherent mononuclear cells and enriched T-cells and chronic lymphocytic leukemia cells. The M antisera also reacted with U937, a monocytic-type cell line, and with HL60, a promyelocytic-type cell line, but failed to react with T and B lymphoblastoid cell lines. The specificities of the other antisera have been described in previous reports. Cells from three of the patients could not be phenotyped by microcytotoxicity testing. Cells from 25 patients had a consensus morphological or histochemical diagnosis of either acute lymphoblastic leukemia or acute nonlymphocytic leukemia. The serological classification of these patients using the five types of antisera listed above were consistent with the consensus diagnosis. In addition, the lymphoid cancers were further subclassified as to T-, B-, or thymus antigen types. There was no consensus lymphoid versus myeloid diagnosis on cells from two patient. The serological classification in both cases favored a diagnosis of myeloid rather than lymphoid leukemia.

Developmental pattern of 5 alpha-reductase activity in the rat gubernaculum.

5 alpha-Reductase and aromatase activities were measured in fetal and newborn rat gubernaculum and other tissues by radiometric assays using [1 beta-3H]testosterone as substrate. Aromatase activity was measured by the stereospecific release of tritium into the incubation medium, and 5 alpha-reductase was assessed by measuring the amount of 5 alpha-reduced steroids produced during the same incubations. Aromatase activity was low (less than 0.2 pmol/h.mg protein) in gubernaculum at all ages studied, whereas the activity in postnatal ovary ranged from 3.9-20 pmol/h.mg protein. 5 alpha-Reductase activity was relatively high in day 18 fetal gubernaculum (approximately 115 pmol/h.mg protein), but 2 days later in development (fetal day 20), 5 alpha-reductase activity had declined to approximately 21 pmol/h.mg protein. 5 alpha-Reductase activity was very low (less than 10 pmol/h.mg protein) in the postnatal gubernaculum. Histological examination of day 18 fetal gubernaculum indicated that it is composed of dense, poorly organized, mesenchymal tissue. By day 20 of gestational development, primitive muscle cells are recognizable in the periphery of the gubernaculum, and by day 3 of postnatal development the gubernaculum is composed almost entirely of muscle. These findings suggest that 5 alpha-reductase activity may be located in the mesenchymal cells and may be important in early differentiation of the gubernaculum in the male. The administration of a 5 alpha-reductase inhibitor to pregnant rats from days 14-22 of gestation inhibited the normal growth rate of the gubernaculum, as assessed by measuring the protein content of the gubernaculum in control and treated rats, but did not have any profound effect on the histological development of the gubernaculum.

Androgen metabolism in the prostate of the finasteride-treated, adult rat: a possible explanation for the differential action of testosterone and 5 alpha-dihydrotestosterone during development of the male urogenital tract.

Previous work has clearly demonstrated that inhibition of 5 alpha-dihydrotestosterone (DHT) formation in vivo is not as effective as total androgen ablation (castration) in causing involution of the prostate. It is likely that this is due to the fact that testosterone is partially effective in maintaining androgen action. To provide insight into this observation, the androgenic metabolites of testosterone, androstenedione, and 5 alpha-DHT, were measured in prostate tissue and in blood of 5 alpha-reductase inhibitor (finasteride)-treated adult male rats. Finasteride treatment caused a significant decrease in prostatic DHT levels and a profound increase in prostatic testosterone and androstenedione levels. Similarly, circulating DHT levels were decreased in finasteride-treated rats (0.02 ng/ml compared with 0.05 ng/ml seen in control rats); and circulating androstenedione and testosterone levels were significantly elevated in finasteride-treated animals compared with controls. The in vitro effects of finasteride were assessed on the metabolism of [3H]testosterone in a tissue-slice assays. In the prostate, the inhibition of 5 alpha-reductase activity resulted not only in the decreased formation of 5 alpha-reduced metabolites (primarily DHT and 5 alpha-androstanedione), but also an increase in the 17-oxo metabolite androstenedione. In contrast, the tissues derived from the embryonic wolffian duct (seminal vesicle and epididymis) formed relatively low amounts of 17-keto steroids. Because DHT is a high affinity ligand for the androgen receptor and androstenedione shows very little, if any, affinity for the receptor, these studies suggest that 5 alpha-reduction of testosterone may be a mechanism to amplify androgen action in urogenital tissues such as the prostate by preventing catabolism of testosterone to the inactive androgen, androstenedione, at the site of hormone action.

5 alpha-dihydrotestosterone formation is necessary for embryogenesis of the rat prostate.

To determine the role of 5 alpha-dihydrotestosterone (DHT; 17 beta-hydroxy-5 alpha-androstan-3-one) in formation of the embryonic prostate, we quantitated prostatic development in urogenital tracts of control male newborn and offspring of rats treated with a specific 5 alpha-reductase inhibitor (L652,931; Merck, Sharp, and Dohme) during prostate morphogenesis. Treatment with the 5 alpha-reductase inhibitor (50 mg/kg.day) from days 14-22 of gestation impaired development of the prostate and virilization of the external genitalia in male offspring compared to those in control animals. However, virilization of the internal genitalia (seminal vesicles, epididymis) was unaffected. Simultaneous administration of DHT (50 mg/kg.day) with the 5 alpha-reductase inhibitor restored prostate development and anogenital distances of males to normal and virilized the external genitalia of females. We conclude that DHT is the active androgen responsible for prostatic development in the rat.

Androgen receptors are similar in fetal and adult rabbits.

In an effort to explain the separate roles of testosterone and dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) in virilizing the male fetus, we compared the binding of these androgens to cytosolic receptors from urogenital tract tissues of fetal and adult male rabbits. As measured by a direct binding assay, fetal and adult androgen receptors are similar in respect to specificity, affinity, and amount of binding. Apparent dissociation constants for dihydrotestosterone binding averaged 1.1 nM for fetal receptor and 0.8 nM for adult androgen receptors. Average apparent dissociation constants for testosterone binding were 4- to 24-fold higher than those for dihydrotestosterone in fetal and adult tissues. Nonradioactive dihydrotestosterone and testosterone competed for [3H]dihydrotestosterone binding to the androgen receptor in both adult prostate and fetal urogenital sinus in a manner consistent with their affinity for binding, whereas estradiol, progesterone, and cortisol were weak competitors for [3H]dihydrotestosterone. On sucrose density gradients, both testosterone and dihydrotestosterone were bound to a protein with a sedimentation coefficient of approximately 8S. Although androgen receptors were detectable in urogenital tubercle and urogenital sinus of both male and female fetuses on days 18 and 29 of gestation, we were unable to characterize androgen binding in fetal Wolffian ducts. The nature of the androgen receptor in this tissue remains unresolved. These findings are consistent with the hypothesis that dihydrotestosterone formation acts to amplify the androgenic signal in both the fetus and adult, but is not absolutely required for virilization.

Changes in aromatase activity in the rat brain during embryonic, neonatal, and infantile development.

We assessed the activity of the aromatase enzyme complex in slices of brain from rats by measuring the release of 3H2O from [1 beta-3H]testosterone. In hypothalami from 12-day-old rats, the rate of aromatase activity was linear with time and amount of tissue. The reaction was saturated at a substrate concentration of 0.1 microM, and the apparent Km of the reaction was 27 nM. The production of 3H2O was inhibited by 4-hydroxyandrostenedione, with an apparent Ki of 20 nM. Aromatase activity was first detected in the diencephalon of 16-day-old fetuses and reached maximum rates in hypothalamic tissue between days 18 and 20 of gestation. The highest rate of activity per mg protein (approximately 4.8 pmol h-1 mg protein-1) was observed in the preoptic area (POA) on the 20th day of embryonic development. However, when expressed as a rate per tissue fragment, aromatase activity was as high in the medial basal hypothalamus as in the POA. After day 20 of gestation aromatase activity rapidly decreased in the POA and medial basal hypothalamus of both males and females. The lowest levels were observed between postnatal days 16 and 20. Aromatase activity was not detectable in cerebral cortex and cerebellum at any age studied. Since serum testosterone was higher in males than females during the first 4 days of postnatal life, and since aromatase activity is elevated in the hypothalamus at this time, our results support the current concept that local formation of estrogen mediates testosterone-induced masculinization of the brain during the neonatal period. However, our results also indicate that failure of the rat brain to undergo complete sexual differentiation before birth cannot be due to an inability of the fetal hypothalamus to aromatize androgens, since aromatase activity was higher in the hypothalamus than in any other fetal tissue.

Developmental pattern of increased aromatase activity in the Sebright bantam chicken.

Feathers in the male Sebright bantam chicken are feminized as the result of a mutation that causes an increase in the capacity for estrogen synthesis (aromatase activity) in skin. To determine when during development the increased aromatase activity is expressed, we measured the enzyme in tissue slices of Sebright bantam and control bantam chickens of days 3, 12, and 18 of embryogenesis, on day 1 after hatching, and in mature birds. An assay was used that measures the release of 3H2O from [1 beta-3H]testosterone during the aromatization process. Aromatase activity was expressed in the 3-day-old Sebright embryos, but was not detectable in control embryos of the same age. In older embryos and birds, aromatase activity was assayed in multiple tissues. Enzyme activity was detectable in all tissues examined from day 12 and day 18 Sebright embryos. In control embryos, aromatase was detectable in ovarian but not in extraglandular tissues. The enzyme activity in most tissues of the Sebright decreased within 1 day after hatching and continued to decrease thereafter so that in the adult Sebright, aromatase activity was high only in skin (and skin appendages) and ovary. We conclude that the normal allele of this gene acts to limit the rate of estrogen formation in extraglandular tissues.

The effect of a 5 alpha-reductase inhibitor on androgen physiology in the immature male rat.

To provide insight into the role of 5 alpha-dihydrotestosterone (DHT) in postnatal androgen physiology, we administered the 5 alpha-reductase inhibitor finasteride to male rats from birth through the onset of puberty. In 4-week-old control rats serum testosterone levels averaged 0.21 ng/ml, and DHT levels averaged 0.64 ng/ml. By 7 weeks of age, testosterone levels increased more than 7-fold to 1.57 ng/ml, while the circulating DHT level declined to 0.26 ng/ml. In both the 4- and 7-week-old inhibitor-treated animals, circulating DHT levels were 25-50% of control values, and circulating testosterone levels were higher than control values. In 7-week-old inhibitor-treated rats, the weights of prostate, penis, seminal vesicles, and epididymal tissues were only 30-50% those of the controls. However, DHT formation is apparently not critical for postnatal development of the preputial glands or the androgen-dependent perineal muscles, since the weights of these tissues were not affected by treatment with inhibitor. Treatment with the 5 alpha-reductase inhibitor had no apparent effect on testicular histology or daily sperm production despite the fact that testicular DHT content was lower (70%) and testosterone content was higher (250%) than those in controls. We conclude that DHT formation is important for the normal postnatal growth of the prostate, seminal vesicles, epididymis, and penis and may be important for normal feedback control of testosterone production in rats, but that its formation is not critical for the onset of spermatogenesis or the development of the preputial glands or the androgen-dependent perineal muscles.

Estrogen formation by the ovary of the rabbit embryo.

The conversion of [1,2,6,7-3H]- testosterone to radioactive estradiol was assessed in tissue slices of 18 different tissues from rabbit embryos that varied in age from 16 to 29 days gestation. Significant rates of estradiol synthesis were demonstrated only in ovaries [4.2 +/- 0.7 (mean +/- SEM) pmol/h/mg) protein], placenta (0.7 +/- 0.2 pmol/h/mg protein) and brain (0.3 +/- 0.1 pmol/h/mg protein). Estradiol formation was undetectable in day 16 gonads of both sexes and in tests at all ages examined, but by day 18 it was demonstrable in ovaries and rose rapidly to reach a level of 6 pmol/h/mg protein by day 19. The time of appearance of the enzymatic capacity to convert testosterone to estradiol in the ovary is similar to the onset of the enzymatic capacity to form testosterone by the fetal testis, suggesting that the acquisition of the enzymatic activies that allow specific endocrine function by these two tissues may be regulated by the same or similar factors during embryonic development.

The effect of a 5 alpha-reductase inhibitor on androgen-mediated growth of the dog prostate.

The administration of testosterone cypionate (0.4 mg/kg BW . day) to castrated male dogs caused a doubling of prostate weight within 4 weeks and an increase in the content of testosterone and dihydrotestosterone in the prostate. When the 5 alpha-reductase inhibitor 17-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (3 mg/kg BW . day) was administered simultaneously with testosterone cypionate, prostatic testosterone content increased from 0.5 +/- 0.2 to 4.1 +/- 1.3 ng/mg DNA, the increase in prostatic dihydrotestosterone content was prevented, and prostatic size decreased to half the starting weight. These results suggest that dihydrotestosterone formation plays a role in prostatic growth.

Aromatase activity in the developing rabbit brain.

The formation of 17beta-[3H]estradiol from [1,2,6,7-3H]testosterone was assessed in placenta and central nervous system tissues from rabbit embryos that varied in age from 13-28 days of gestation. In the fetal brain, significant rates of aromatase activity were limited exclusively to the forebrain, and the highest rates of activity (approximately 0.5 pmol/h/mg protein) were found in the diencephalon both male and female embryos between days 19 and 25 of gestation. These rates of aromatase activity are second only to the fetal ovary when expressed per mg protein; moreover, forebrain is the only tissue in the male embryo capable of synthesizing significant amounts of estrogens in vitro. When projected to the whole organ, the capacity of the diencephalon for aromatization exceeds the capacity of the fetal ovary approximately 9-fold. Placental aromatase activity was high (2.1 pmol/h/mg protein) on day 13 but fell to a level approximately 20-fold lower by day 19 of gestation. These findings indicate the potential importance of the forebrain as a source of estrogens during embryogenesis.

The synthesis and metabolism of gonadal steroids in pouch young of the opossum, Didelphis virginiana.

We have measured steroid hormone biosynthesis from pregnenolone in ovaries and testes, aromatization of testosterone in gonads and peripheral tissues, and 5 alpha-reduction of testosterone in peripheral tissues of developing opossum pouch young. Sex of the newborn opossums is discernible grossly with development of the pouch in females and the scrotum in males approximately 10 days after birth. Differentiated endocrine function of ovaries and testes was demonstrable as soon as development of the pouch or scrotum was apparent. The testes synthesized testosterone, and ovaries aromatized androgens to estrogens as assessed by the conversion of [1 beta-3H] testosterone to 3H2O. This endocrine differentiation of the gonads occurs days or weeks before differentiation of the male and female urogenital tracts. As in other species, 5 alpha-reduction of [1 beta-3H] testosterone was high in urogenital sinus and urogenital tubercle. However, 5 alpha-reductase activity was highest in mesonephros and structures derived from the mesonephros. In wolffian and mullerian ducts of pouch young less than 10 days old, 5 alpha-reductase activity was greater than 50 pmol/h . mg protein and decreased by 19 days of age to approximately 3 pmol/h . mg protein, a pattern different than in eutherian mammals in which testosterone itself appears to mediate virilization of the wolffian ducts. These studies provide the framework for studies of the endocrine control of phenotypic sexual differentiation in the opossum.

Regulation of immunoreactive androgen receptor in the adrenal gland of the adult rat.

The androgen receptor (AR) was measured by an immunoblot assay in adult tissues of both male and female rats. Relatively high levels of AR were detected in tissues of the male urogenital tract and in the adrenal glands and gonads of both sexes. Another group of tissues, including the male levator ani/bulbocavernosus muscles, preputial gland, scrotal skin, and vagina, had low, but detectable, levels of AR. In a third group of tissues, including the uterus, kidney, spleen, liver, gut, heart, lung, pituitary, and hypothalamus, AR was undetectable. In some androgen target tissues, such as the penis, androgens cause an apparent disappearance of AR from the tissue, and in other tissues, such as the ventral prostate, androgen therapy increases the amount of detectable AR. We compared the effect of androgen on AR levels in the adrenal gland and ventral prostate, tissues that differ markedly in their trophic responses to androgen. Castration appeared to have no effect on the amount of detectable AR in the adrenal gland, whereas it caused a profound decrease in AR levels in the ventral prostate. By contrast, 7 days after hypophysectomy, AR levels declined in both the adrenal gland and the ventral prostate. The effects of hypophysectomy plus castration were similar to those of hypophysectomy alone. Administration of ACTH to hypophysectomized rats for 7 days did not reverse the effects of hypophysectomy on adrenal AR, nor did treatment with levothyroxine, dexamethasone, rat GH, or rat PRL. Treatment of hypophysectomized rats with 5alpha-dihydrotestosterone for 7 days caused a dramatic increase in the amount of detectable AR in both the ventral prostate and the adrenal gland, but had a trophic effect only in the ventral prostate. These findings suggest that the amount of immunoreactive AR detected in both the adrenal gland and the ventral prostate is enhanced by androgens: testicular androgens in the case of the ventral prostate and adrenal androgen in the case of the adrenal glands.

Conversion of androgen to estrogen by the human fetal ovary.

The conversion of radiolabeled androgen to estrone and 17 beta-estradiol was assessed in tissues of human embryos that varied from phenotypically indifferent stages (1-3 cm crownrump length) to midgestation (15. 1-20 cm crownrump length). Significant rates of estrogen synthesis were demonstrated only in ovaries, liver, and brain. Estrogen synthesis was undetectable in gonads from 1-3 cm fetuses, but by the 3.1-5-cm stage it had reached an average rate of 1.9 pmol . h-1 . mg protein -1 in ovaries and remained at this level of activity through the latest stages examined. Estrogen formation was undetectable in testes at all stages examined, but the time of appearance of the capacity to form estrogens in the fetal ovary is similar to the onset of the capacity of the fetal testis to synthesize testosterone. The capacity of the fetal ovary to form estrogen develops before histological differentiation of the tissue.

5 alpha-reduced androgens in the human fetal testis.

The androgen content was measured in testes from 34 male and in ovaries from 30 female embryos that varied in age from less than 12 to approximately 20 weeks. The 5 alpha-reduced androgens dihydrotestosterone and 3 alpha-androstanediol were found in testes at a level of about a 30th of that of testosterone at all ages examined, whereas very little or no testosterone, androstenedione, or either of the 5 alpha-reduced androgens were detected in the ovaries. Whether dihydrotestosterone plays a role in the development of the testes is unknown.