Loredana Assisi

Involvement of D-Asp in P450 aromatase activity and estrogen receptors in boar testis.

Summary.Mammalian testis contains D-aspartic acid (D-Asp), which enhances testosterone production. D-Asp, on other hand, also stimulates 17β-estradiol synthesis in the ovary of some lower vertebrates. We studied boar testis in order to determine if D-Asp intervenes in 17β-estradiol synthesis in the testis of those mammals which produce significant amounts of estrogens as well as testosterone. The boar testis contains D-Asp (40 ± 3.6 nmol/g tissue) which, according to immunohistological techniques, is localized mainly in Leydig cells, and, to a lesser extent, in sustentacular (Sertoli), peritubular and some germ cells. The enzyme P450aromatase is present in Leydig cells and few germ cells. In vitro experiments showed that the addition of D-Asp to testicular tissue extracts induced a significant increase of aromatase activity, as evaluated by testosterone conversion into 17β-estradiol. The enzyme’s Km was not affected by D-Asp (about 25 nM in both control and D-Asp added tests). On the basis of these results we suggest that, as in the ovary, D-Asp is involved in the local control of aromatase activity of boar testis and, therefore, it intervenes in the 17β-estradiol production. In the testis, the D-Asp targets are presumably the Leydig cells, which having also a nuclear estrogen receptor are, in turn, one of the putative targets of the 17β-estradiol that they produce (autocrine effect).

Opposing effects of D-aspartic acid and nitric oxide on tuning of testosterone production in mallard testis during the reproductive cycle

BackgroundD-Aspartic acid (D-Asp) and nitric oxide (NO) play an important role in tuning testosterone production in the gonads of male vertebrates. In particular, D-Asp promotes either the synthesis or the release of testosterone, whereas NO inhibits it. In this study, we have investigated for the first time in birds the putative effects of D-Asp and NO on testicular testosterone production in relation to two phases of the reproductive cycle of the adult captive wild-strain mallard (Anas platyrhynchos) drake. It is a typical seasonal breeder and its cycle consists of a short reproductive period (RP) in the spring (April-May) and a non reproductive period (NRP) in the summer (July), a time when the gonads are quiescent. The presence and the localization of D-Asp and NO in the testis and the trends of D-Asp, NO and testosterone levels were assessed during the main phases of the birds reproductive cycle. Furthermore, in vitro experiments revealed the direct effect of exogenously administered D-Asp and NO on testosterone steroidogenesis.MethodsBy using immunohistochemical (IHC) techniques, we studied the presence and the distributional pattern of D-Asp and NO in the testes of RP and NRP drakes. D-Asp levels were evaluated by an enzymatic method, whereas NO content, via nitrite, was assessed using biochemical measurements. Finally, immunoenzymatic techniques determined testicular testosterone levels.ResultsIHC analyses revealed the presence of D-Asp and NO in Leydig cells. The distributional pattern of both molecules was in some way correlated to the steroidogenic pathway, which is involved in autocrine testosterone production. Indeed, whereas NO was present only during the NRP, D-Asp was almost exclusively present during the RP. Consistently, the high testosterone testicular content occurring during RP was coupled to a high D-Asp level and a low NO content in the gonad. By contrast, in sexually inactive drakes (NRP), the low testosterone content in the gonad was coupled to a low D-Asp content and to a relatively high NO level. Consequently, to determine the exogenous effects of the two amino acids on testosterone synthesis, we carried out in vitro experiments using testis sections deriving from both the RP and NRP. When testis slices were incubated for 60 or 120 min with D-Asp, testosterone was enhanced, whereas in the presence of L-Arg, a precursor of NO, it was inhibited.ConclusionOur results provide new insights into the involvement of D-Asp and NO in testicular testosterone production in the adult captive wild-strain mallard drake. The localization of these two molecules in the Leydig cells in different periods of the reproductive cycle demonstrates that they play a potential role in regulating local testosterone production.

NADPH-diaphorase and NOS enzymatic activities in some neurons of reptilian gut and their relationships with two neuropeptides.

 The distribution of neurons containing the enzymes NADPH-diaphorase (NADPH-d) and nitric oxide synthase (NOS) has been studied in the gastrointestinal tract of lizard (Podarcis s. sicula) and snake (Thamnophis sirtalis). The techniques employed were the NADPH-d/nitroblue tetrazolium histochemical method, and the indirect immunofluorescence applied to cryostat sections and to whole-mount preparations. The colocalization of NADPH-d with NOS, with vasoactive intestinal polypeptide (VIP) and with galanin (Gal) was also studied, and a Western blot analysis using an antibody directed against mammalian Gal was performed on lizard stomach extracts. NADPH-d positive nerve cell bodies and fibres were found in the myenteric and submucous plexuses throughout the gastrointestinal tract of both reptiles. These nerve structures were also present in the other intramural nerve plexuses, although in smaller quantities. Both in lizard and snake, the stomach revealed a positive nerve population that was more dense than elsewhere in the gut. The population of the NADPH-d-positive neurons observed in the lizard was larger than that observed in the snake. The distribution of both populations was similar to those that have been described in the gut of several mammalian and non-mammalian vertebrates. Both in lizard and snake, a one-to-one correspondence was noted between NOS- and NADPH-d-containing nerve cell bodies, and the nitrergic neurons containing Gal appeared to be more numerous than those containing VIP. Western blot analysis recognised a single band with a molecular weight (3.4 kDa) very similar to that of porcine Gal. It is hypothesised that at least some of the nitrergic neurons of the lizard and snake gut are inhibitory motor neurons innervating the circular smooth musculature. In addition, the colocalization of NOS and VIP in neurons enhances their inhibitory action. The role of the neurons containing both NOS and Gal remains unknown.

Presence, distribution and steroidogenic effect of the peptides orexin A and receptor 1 for orexins in the testis of the South American camelid Alpaca (Vicugna pacos)

The orexins A (oxA) and B are peptides discovered in the rat hypothalamus and successively found in some peripheral organs of the mammalian body. They binds two protein G-coupled receptors defined receptor 1 (ox1r) and 2 for orexins, the first of which is highly specific for oxA while the second binds both the peptides with equal affinity. This work aimed to detect the presence of oxA and ox1r in the testis of the South American camelid alpaca (Vicugna pacos) and investigate the role played by them on Leydig cell steroidogenesis. The species alpaca acquired, in the last years, increasing zootechnical interest for the quality of the wool produced and its breeding spread from the country of origin to USA, Australia and Europe. Immunohistochemistry allowed us to detect oxA in Leydig and Sertoli cells, spermatogonia, resting spermatocytes, round and oval spermatids. Ox1r-immunoreactivity was found in Leydig cells and round, oval and elongated spermatids. The expression of the two peptides in tissue extracts was established by using Western blotting technique. Such results demonstrated that in the alpaca testis exists in a cellular complex able to produce and/or internalize oxA. Finally, the effect of oxA on steroidogenesis was investigated by means of in vitro cultured thin testis slices which were added with oxA or/and Müllerian Inhibiting Substance (MIS), a steroidolitic agent basally produced by the Sertoli cell. OxA evoked increase of testosterone production while MIS a decrease. The consecutive addition of oxA and MIS, or vice versa, highlighted an antagonistic interplay between the two substances which has been thought to be the main molecular event at the basis of the oxA-stimulated steroidogenesis mechanism.

Aromatase and testosterone receptor in the liver of the female green frog, Rana esculenta

In the green frog, Rana esculenta, a peculiar feature of female reproductive endocrinology is an high level of circulating testosterone. Although several hypotheses have been set out to explain this phenomenon, the testosterone specific roles in female anuran have not been yet fully explored. This study results propose a testosterone implication in liver vitellogenin synthesis control, since in ovariectomized frogs the hormone induces an increase of circulating vitellogenin. The testosterone action could depend on its local conversion to 17beta-estradiol by aromatase which is present in frog liver tissue. Liver aromatase activity ranges from 7.5 to 26 fmoles E2 formed/mg protein/h and results higher as long as liver is engaged in vitellogenin synthesis. Aromatase activity seems depend on testosterone since it decreases after ovariectomy and is restored by testosterone injection in ovariectomized frogs. In green frog liver, testosterone binding molecules are present both in cytosol and nuclei. These molecule binding properties (Kd and Bmax in nM range; t 1/2 = 85 min; specificity) are in line with those of testosterone receptor of other lower vertebrate target tissue. In liver nuclei, testosterone receptor level undergoes modification throughout the sexual cycle which almost coincides with that of plasma testosterone level and liver aromatase activity. This could indicate that the testosterone induction of liver aromatase in frogs is via the testosterone receptor, as reported for aromatase of mammalian brain tissues.

Relationships between liver testosterone receptor isoforms and aromatase activity in female green frog, Rana esculenta

Testosterone receptors (AR) are present in the liver of the female green frog, Rana esculenta, which resolve into two fractions (A and B) by ion-exchange chromatography. Fraction A is primarily located in the nuclei, fraction B predominates in the cytosols, and both fractions show a high affinity and specificity for testosterone. Liver AR fraction levels vary dramatically during the frog sexual cycle. Fraction A levels are high only when the liver is engaged in vitellogenin production and the plasma testosterone levels are high: they are maximal when aromatase activity is most intense. Fraction B levels are high when the liver is not producing vitellogenin and the plasma testosterone levels are minimal. In addition, in vivo experiments carried out on ovariectomized females treated with testosterone show that testosterone induces both fraction A and liver aromatase activity. This induction may be a step in the process that allows the liver to obtain estrogen from plasma testosterone which induces vitellogenin synthesis.

Endogenous testicular D-aspartic acid regulates gonadal aromatase activity in boar

D-aspartic acid (D-Asp), aromatase enzyme activity and the putative D-Asp involvement on aromatase induction have been studied in the testis of mature boars. The peroxidase-antiperoxidase and the indirect immunofluorescence methods, applied to cryostat and paraffin sections, were used to evaluate D-Asp and aromatase distributions. D-Asp level was dosed by an enzymatic method performed on boar testis extracts. Biochemical aromatase activity was determined by in vitro experiments carried out on testis extracts. D-Asp immunoreactivity was found in Leydig cells, and, to a lesser extent, in germ cells. Analogously, aromatase immunoreactivity was present in Leydig cells, but absent from seminiferous tubule elements. In vitro experiments showed that the addition of D-Asp to testicular tissue acetone powder induced a significant increase of aromatase activity, as assessed by testosterone conversion to 17β-estradiol. Enzyme Km was not affected by D-Asp (about 25 nM in control and D-Asp added tests). These findings suggest that D-Asp could be involved in the local regulation of aromatase in boar Leydig cells and intervenes in this organ’s production of estrogens.

Expression of orexin B and its receptor 2 in rat testis.

The peptides orexin A (OxA) and orexin B (OxB) deriving from a common precursor molecule, prepro-orexin, by proteolytic cleavage, bind the two G-coupled OX1 and OX2 receptors. While OX1 selectively binds OxA, OX2 shows similar affinity for both orexins. Firstly discovered in the hypothalamus, orexins and their receptors have been found in other brain regions as well as in peripheral tissues of mammals, thus resulting involved in the regulation of a broad variety of physiological functions. While the functional localization of OxA and OX1 in the mammalian genital tract has been already described, the expression of OxB and OX2 and their potential role in the reproductive functions remain to be explored. Here, we investigated the presence of OxB and OX2 in the rat testis by immunohistochemical and biochemical analyses. The results definitely demonstrated the localization of OxB and OX2 in pachytene and second spermatocytes as well as in spermatids at all stages of the cycle of the seminiferous epithelium. The expression of both OX2 mRNA and protein in the rat testis was also established by RT-PCR and Western blotting, respectively. The analysis of the molecular mechanism of action of OxB in the rat testis showed that OxB, in contrast with OxA, is unable to promote steroidogenesis. These results translate into the regulation of diverse biological actions by OxA and OxB in the male gonad.

The relationships of nicotinamide adenine dinucleotide phosphate-d to nitric oxide synthase, vasoactive intestinal polypeptide, galanin and pituitary adenylate activating polypeptide in pigeon gut neurons.

The distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-d neurons and their relationship with nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP), pituitary adenylate activating polypeptide (PACAP) and galanin (Gal) were examined in the gastrointestinal (GI) tract of the pigeon Columbia livia. NADPH-d-histochemistry, indirect immunofluorescence and confocal analysis were applied to cryosections. Western blot analysis was also applied on pigeon gut. NADPH-d neurons were found throughout the pigeon GI tract and they were evident in the myenteric, circular muscle and submucous plexuses. Positive varicose nerve fibres were also distributed within the longitudinal muscle layers and in the lamina propria of the mucosa. The stomach was the segment richest in positivities. The copresence VIP/Gal/NOS as well as PACAP/VIP were revealed in some NADPH-d-neurons. We suppose that the nitrergic nerve population of the pigeon GI tract belong to the muscle motility regulation as an inhibitory descending nerve pathway. Moreover the presence of VIP, Gal and PACAP in some NADPH-d-containing neurons enhances the inhibitory actions of these neurotransmitters whereas PACAP and Gal role is actually unknown.

Galanin in the lizard oviduct: Its distribution and relationships with estrogen, VIP and oviposition

The distribution of neurons containing galanin immunoreactivity (Gal/IR) has been detected in the oviduct of the lizard Podarcis s. sicula during the main phases of its sexual cycle and after 17beta-estradiol treatment. Indirect immunofluorescence technique was applied both to cryostatic sections and whole mount preparations, and Western blot analysis, with an antibody directed against mammalian galanin (Gal), was performed with lizard oviduct extracts. Colocalization of Gal with vasoactive intestinal polypeptide (VIP) was also studied as well as Gal effects on egg deposition. In the quiescent oviduct of non-reproductive females, scanty Gal/IR fibres were found in the uterine-vaginal segment. During the reproductive period a gradual increase of positive nerve fibres and cell bodies were found distally in the lizard oviduct and the vagina revealed a reactive nerve population denser than elsewhere. Gal-IR nerve structures were present either in the musculature or mucosa and in the intermuscular layer they were organized in a nerve network. In the oviduct of non-reproductive females, 17beta-estradiol administration induced a significant increase of neurons containing Gal/IR. This hormone could be involved in the egg laying by means of galanin action and this hypothesis is supported by the induction of premature oviposition in pre-ovulatory females after Gal administration. Western blot analysis validates this peptide as true Gal, recognising one protein band with a molecular weight (3.2 kDa), similar to that of porcine Gal. Double labelling studies showed the co-presence of Gal and VIP in some neurons.