Renato Massa

The transformation of testosterone into dihydrotestosterone by the brain and the anterior pituitary

Abstract Slices of rat pituitary gland, hypothalamus, amygdala, cerebral cortex and prostate have been incubated in vitro with labelled testosterone; the metabolites formed have been identified. Testosterone is convened into 17β-hydroxy-5α-androstan-3-one (androstanolone, dihydrotestosterone, DHT) by all tissues examined. The prostate is the structure which effects such a conversion to the greatest extent; the pituitary gland and the hypothalamus are also very active; the cerebral cortex and the amygdala are also able to transform testosterone into DHT, but the rate of conversion is not as great as that found in the tissues previously mentioned. Androstenedione, 5α-androstan-3, 17-dione and 5α-androstan-3α, 17β-diol are also formed by some of the tissues. Castration increases and treatment with exogenous testosterone decreases the transformation of testosterone into DHT at the pituitary and the hypothalamic level. Both at the hypothalamic and the pituitary level, the addition in vitro of progesterone, 11-deoxycorticosterone, 11-deoxycortisol and corticosterone reduces the transformation of testosterone into DHT; on the contrary, the addition of pituitary FSH increases the conversion of testosterone into its ‘active’ metabolite. The ability to transform testosterone into DHT is much higher in all structures examined (with the exception of the prostate) in prepuberal than in adult rats.

Photoperiodic control of testosterone metabolism, plasma gonadotrophins, cloacal gland growth, and reproductive behavior in the Japanese quail.

Abstract The changes in plasma gonadotrophins and testosterone, in cloacal gland area, and in reproductive behavior were observed in male Japanese quails after transfer to long days or testosterone implantation and were correlated with the testosterone metabolism in the hyperstriatum, hypothalamus, pituitary, and cloacal gland. Long days stimulate the growth of the cloacal gland and at the same time enhance its production of androstenedione from testosterone. This increased 17β-hydroxysteroid dehydrogenase activity is correlated at the individual level with cloacal gland area but not with plasma testosterone. Similarly the changes observed in some reproductive activities (aggressive behavior and struts) are correlated with individual differences in the brain metabolism of testosterone (hyperstriatal or hypothalamic production of androstenedione), but not with plasma testosterone. The plasma luteinizing hormone is also related to testosterone metabolism rather than to the circulating level of the hormone (negative correlation with the production of 5α-dihydrotestosterone in the pituitary). The testosterone metabolism in target organs thus appears of critical importance in the control of reproduction and seems largely responsible for the occurrence of individual differences. After exposure to 12 long days the mean testosterone metabolism in the pituitary is also strongly affected (increased 5β-reduction) though not in the same way as after 2 months of photostimulation. It is suggested that these metabolic changes at the pituitary level may play some role in the induction of the increased responsiveness to luteinizing hormone-releasing hormone which can be observed in quails after exposure to 7 long days.

Ambient temperature effects on photo induced gonadal cycles and hormonal secretion patterns in Great Tits from three different breeding latitudes.

The present study determines how populations of Great Tits (Parus major) breeding in southern, mid and northern European latitudes have adjusted their reproductive endocrinology to differences in the ambient temperature during the gonadal cycle. A study based on long-term breeding data, using the Colwell predictability model, showed that the start of the breeding season has a high predictability ( approximately 0.8-0.9) at all latitudes, and that the environmental information factor (I(e)) progressively decreased from mid Italy (I(e)>4) to northern Finland (I(e)<1). The results indicate that integration of supplementary information, such as ambient temperature, with photoperiodic initial predictive information (day length), becomes progressively more important in maintaining the predictability of the breeding season with decreasing latitude. This hypothesis was verified by exposing photosensitive Great Tits from northern Norway, southern Sweden and northern Italy to sub-maximal photo-stimulatory day lengths (13L:11D) under two different ambient temperature regimes (+4 degrees C and +20 degrees C). Changes in testicular size, plasma levels of LH and testosterone were measured. The main results were: (1) Initial testicular growth rate, as well as LH secretion, was affected by temperature in the Italian, but not in birds from the two Scandinavian populations. (2) Maximum testicular size, maximum LH and testosterone levels were maintained for a progressively shorter period of time with increasing latitude, regardless of whether the birds were kept on a low or a high ambient temperature. (3) In birds from all latitudes, the development of photorefractoriness, as indicated by testicular regression and a decrease in plasma levels of LH and testosterone, started much earlier (with the exception for LH Great Tits from northern Scandinavia) when kept on +20 degrees C than when kept on +4 degrees C. The prolonging effects of a low temperature was more pronounced in Mediterranean birds, than in birds from Scandinavia, and more pronounced in Great Tits from southern Scandinavia than in Great Tits from northern Scandinavia. Ecological implications of the results are discussed, as well as possible impact of global warming on the breeding success of European Great Tits from different breeding latitudes.

Conversion of testosterone into 5α-reduced metabolites in the anterior pituitary and in the brain of maturing rats

Abstract The anterior pituitary of normal female rats shows a considerable 5α-reductase activity. Such an activity is higher in the very first days of life and decreases from birth until the 28th day of age. The anterior pituitary of normal male rats also shows 5α-reductase activity, but this is smaller than that of the anterior pituitary of female animals. Significant differences between activities present in the two sexes can be observed from birth until the 21st day of age. A slight difference in the 5α-reductase activity of the hypothalami of maturing male and female animals has also been observed. As previously stated, the 5α-reductase activity of the anterior pituitary of female rats is much higher than that of male animals on day 14 of life. Castration performed on neonatal male rats brings about an increase in the 5α-reductase activity of their anterior pituitaries evaluated on day 14 of age. The levels of the enzyme become very similar to those found in the anterior pituitary of normal females of the same age. Conversely, the neonatal administration of testosterone propionate to female rats induces a decrease of the 5α-reductase activity of their pituitaries measured on day 14 of life. Such an activity becomes very similar to that of normal males of the same age. Analogous variations, although of a smaller magnitude, have been observed at hypothalamic level following neonatal castration in male animals or following neonatal administration of testosterone to female animals.

Seasonal changes in testosterone metabolism in the pituitary gland and central nervous system of the European starling (Sturnus vulgaris)

Abstract The metabolism of testosterone in the pituitary gland, the hypothalamus, and the hyperstriatum of the male European starling was studied in the breeding season (May) and at the beginning and the end of the photorefractory period (July and November). In the pituitary gland the percentage conversion of testosterone to androstenedione and to 5α-DHT did not show seasonal variation, while the conversion of testosterone to 5β-DHT and to 5β-3α-diol was increased two- to threefold when the birds became photorefractory (July and November). In the hypothalamus, the formation of the 5α-reduced metabolites did not show seasonal variation, while the formation of androstenedione was significantly greater in November than in July. In contrast, the formation of 5β-reduced metabolites in the hypothalamus was greater in May and July than in November. In the hyperstriatum, the formation of 5β-reduced metabolites was also greater in July and November than in May. These observations show that seasonal changes in the metabolism of testosterone in the pituitary gland and central nervous system of the starling are mainly characterized by changes in the formation of 5β-reduced metabolites and androstenedione.

TESTOSTERONE METABOLISM: A NECESSARY STEP FOR ACTIVITY?

The 5α-reductase activity and the 3α-hydroxysteroid-dehydrogenase activity of levator ani and skeletal muscles, submaxillary gland, pancreas, kidney, anterior pituitary and prostate collected from adult normal male rats have been analyzed using an “in vitro” approach. Labelled testosterone has been used as the substrate. It has been found that the prostate, the anterior pituitary, the submaxillary gland and the pancreas have a rather elevated 5α-reductase activity. The kidney has a limited capacity to form 5α-reduced metabolites of testosterone. The levator ani and skeletal muscles do not seem to possess any 5α-reductase activity. The activity of the 3α-hydroxysteroid-dehydrogenase was found to be totally unrelated to the 5α-reducing capacity of each structure.

Effect of progesterone on the sexual behavior of the male Japanese quail

The reduced metabolites of testosterone produced in the central nervous system of birds are known to be involved in the regulation of male sexual behavior. Since progesterone may compete with testosterone for 5 alpha- and 5 beta-reduction, it may also interfere with the sexual behavior of birds. In order to test this hypothesis, progesterone was administered to male quail either transferred from short days to long days or kept in short days and treated with testosterone. Sexual behavior and crowing were scored at intervals for 21 days and the size of the cloacal gland was measured at the same times. On Day 21, the birds were killed and their testes were weighed. The administration of a large dose (1 mg/day) of progesterone depressed the sexual behavior of the birds stimulated either by long days or by the administration of testosterone. It is suggested that progesterone may compete with testosterone for the active sites of 5 alpha- and 5 beta-reductase; alternatively, its effect may be due to an antiandrogenic activity.

The NADPH-diaphorase-containing system in the brain of the budgerigar (Melopsittacus undulatus)

Abstract.In the present investigation we studied the presence and distribution of histochemically detected neuronal NADPH-diaphorase (ND) in the brain of the budgerigar, Melopsittacus undulatus. Positive neurons are widely distributed throughout the central nervous system. ND-containing neurons are present in the telencephalon and the paleostriatal-parolfactory lobe complex. Positive cells were observed also in the neostriatum, including the main auditory area (field L), in several nuclei of the archistriatum and in the hyperstriatum (accessory, dorsal, and ventral). In the diencephalon, positive neurons were present both in the lateral hypothalamic and periventricular areas, and in a segregate area at the confluence of the anterior commissure and the lateral prosencephalic bundle. A group of positive perikarya was located lateral to the dorsal part of the IIIrd ventricle, and continued laterally into the thalamus. Weakly stained neurons were observed in the thalamic dorsomedial posterior nucleus. In the mesencephalon, ND-containing neurons were scattered in the reticular formation (pars lateralis and pars medialis) and in the optic tecta. A large population of positive neurons was observed in the substantia nigra, the ventral area of Tsai and the nucleus interpeduncularis. Positive neurons extended through the tegmental nuclei to the locus coeruleus. In the cerebellum, the granular neurons were weakly stained and the internal cerebellar nuclei were surrounded by a wide network of positive fibers. In the medulla the number of positive cells was highly reduced, but stained neurons were observed in the cochlear as well in the vestibular nuclei. The data here presented suggest that the distribution of ND-containing neurons in the brain of the budgerigar is different from those of the chicken and quail. The locations of positive neurons suggest also a possible involvement in sound perception and production pathways, and visual perception.

PHOTOPERIODIC CONTROL OF TESTOSTERONE METABOLISM IN THE CENTRAL AND PERIPHERAL STRUCTURES OF AVIAN SPECIES

Abstract In many avian species LH secretion is photoperiodically stimulated and is endogenously interrupted during the “photorefractory period”. In order to elucidate the possible role of 5α- and 5β-reduced metabolites of testosterone in these phenomena, the metabolism of testosterone has been studied in vitro in the pituitary gland, in the hypothalamus, in the hyperstriatum and in the cloacal gland of the Japanese quail. The pituitary gland of the quail is able to convert testosterone to 5α-reduced metabolites (5α-dihydrotestosterone, 5α-androstan-3α,17β-diol), to 5β-reduced metabolites (5β-dihydrotestosterone, 5β-androstan-3α,17β-diol) and to androstenedione. The conversion to the 5α-reduced metabolites is significantly higher in the glands of the quails maintained in “long days” and is hardly detectable in those of animals kept in “short days”; a further increase in the conversion of testosterone to 5α-reduced metabolites is observed in the pituitaries of castrated quails maintained in “long days”; in contrast the conversion of testosterone to 5β-reduced metabolites is not significantly affected by the photoperiod. Trace amounts of 5α-reduced metabolites are also found after the incubation of testosterone with the hypothalamus but not with the hyperstriatum. In these two nervous structures, and especially in the hyperstriatum, the 5β-reduced metabolites of testosterone are quantitatively predominant. The cloacal gland is able to convert testosterone in vitro both to androstenedione and to 5α- and 5β-reduced metabolites. The conversion to the 5α-reduced metabolites is significantly higher in the glands of birds kept in “long days” than in those of birds maintained in “short days”; the conversion to 5β-reduced metabolites is not significantly affected by the photoperiod. The administration of 5α- and 5β-reduced metabolites of testosterone to castrated male quails has shown that androgens having a 3-keto-Δ 4 , 3-keto-5α, or 3α-ol-5α configuration are able to lower the high castrate level of plasma LH and to promote the growth of the cloacal gland. The 5β-reduced metabolites do not have any activity either at central or at peripheral level. These results are compatible with the hypothesis that the 5α-reduced metabolites of testosterone play a role in the mechanisms controlling LH release and in maintaining androgen-dependent peripheral structures in birds as in mammals. Further studies are necessary for establishing the role of the 5β-reduction of testosterone.

Studies on the mode of action of androgens in the neuroendocrine tissues

Abstract Experiments were performed to study the factors which might influence the activity of the 5α-reductase-3α-hydroxysteroid dehydrogenase system in the hypothalamus and in the anterior pituitary of male and female rats and to investigate the modulatory effects of a chronic treatment of testosterone (T) and of its 5α-reduced metabolites on LH and FSH secretion. Estradiol benzoate (EB), in a dose of 50ng/rat/day either for 7 or 14 days, did not have any effect on the enzymatic activities of the anterior pituitary and of the hypothalamus of castrated animals of both sexes and on the hypothalamus of castrated male rats. On the other hand the same dose significantly enhanced the testosterone conversion in the hypothalamus of castrated female rats. Higher dose of EB (5 μg/rat/day) proved able to bring back to precastration levels the 5α-reductase activity of the anterior pituitary of animals of both sexes in the 7-day schedule of administration and to further reduce it in the 14-day schedule. The same dose, while ineffective on the activity of the enzymes of the hypothalamus of the female, was highly active in suppressing it in the males. The treatment with 800 μg/rat for 5 days of prolactin did not modify the rate of conversion of T in the anterior pituitary and in the hypothalamus of normal male rats and in the gland of castrated males. A significant suppression of the enzymatic activities of the hypothalamus of castrated male rats was observed. T, dihydrotestosterone (DHT), 5α-androstan-3α, 17β-diol (3α-diol) were given, in a dose of 2 mg/day for 6 days, to castrated male and female rats in order to assess their inhibiting effect on LH and FSH secretion. DHT and 3α-diol were shown to be better suppressors of LH than T itself in both sexes. With regard to FSH no steroid was able to affect FSH release in castrated female and male rats with the exception of DHT which showed some inhibiting effect in males. The data suggests that EB and prolactin, by modifying the rate of conversion of T into DHT and 3α-diol, may modulate the effects of T at anterior pituitary and at hypothalamic levels. In addition they indicate that T probably exerts its negative feedback effect on gonadotropin secretion following its conversion into DHT and 3α-diol.