Androgen and oestrogen modulation by D-aspartate in rat epididymis.

Testosterone (T) synthesised in Leydig cells enters the epididymis and may there be converted into dihydrotestosterone (DHT) by 5α-reductase (5α-red) or into 17ß-oestradiol (E2) by P450 aromatase (P450-aro). D-aspartate (D-Asp) is known to induce T synthesis in the testis. In this study, we investigated the effects of in vivo D-Asp administration in two major regions of the rat epididymis (Region I: initial segment, caput, corpus; Region II: cauda). The results suggest that exogenous D-Asp was taken up by both regions of rat epididymis. D-Asp administration induced a rapid increase in T, followed by a more gradual decrease in the T:DHT ratio in Region I. In Region II, T levels rapidly decreased and the T:DHT ratio was consistently lower relative to the control. Expression of 5α-red and androgen receptor genes showed a good correlation with DHT levels in both regions. D-Asp treatment also induced an increase of both E2 levels and oestradiol receptor-α (ERα) expression in Region I, whereas neither E2 levels nor ERα expression were affected in Region II. The early increase of P450-aro expression in Region I and late increase in Region II suggests a direct involvement of D-Asp modulation in P450-aro gene expression. Our results suggest that D-Asp modulates androgen and oestrogen levels and expression of androgen and oestrogen receptors in the rat epididymis by acting on the expression of 5α-red and P450-aro genes.

Hatching enzyme from the sea-squirt Ciona intestinalis: purification and properties.

We have purified a 34 kDa hatching enzyme from the water in which the embryos of the sea-squirt Ciona intestinalis hatch. This enzyme was obtained in homogeneous form as judged from SDS-PAGE and HPLC gel filtration. The enzyme possesses proteolytic activity and is able to digest the chorion of the egg of C. intestinalis. It is a metalloproteinase and contains one atom of Zn per molecule. The optimum pH is 8.5. The enzyme shows hydrolytic activity towards the -CO-NH- bonds, which are hydrolyzed by the members of the serine proteinase family. It has a trypsin-like activity in that it cuts the bond of Arg and Lys at P1 position of the scissile bond -P1-P1', but it differs from trypsin insofar as it hydrolyzes the peptide bond on either side of Arg and Lys. The purified enzyme is inhibited by the common metal-chelators and by the classical trypsin proteinase inhibitors. The apparent K(m) values at 37 degrees C and pH 8.5 toward tosyl-Gly-Pro-Arg-NHNap, tosyl-Gly-Pro-Lys-NHNap and Bz-Arg-Gly-Arg-NHNap were 0.125, 0.5 and 2.5 mM, respectively. The results obtained in this study suggest that the hatching enzyme from C. intestinalis exhibits both trypsin-like activity and metalloproteinase activity.

Endocrine roles of D-aspartic acid in the testis of lizard Podarcis s. sicula.

In the lizard Podarcis s. sicula, a substantial amount of D-aspartate (D-Asp) is endogenous to the testis and shows cyclic changes of activity connected with sex hormone profiles during the annual reproductive phases. Testicular D-Asp content shows a direct correlation with testosterone titres and a reverse correlation with 17beta-estradiol titres. In vivo experiments, consisting of i.p. injections of 2.0 micromol/g body weight of D-Asp or other amino acids, in lizards collected during the three main phases of the reproductive cycle (pre-reproductive, reproductive and post-reproductive period), revealed that the testis can specifically take up and accumulate D-Asp alone. Moreover, this amino acid influences the synthesis of testosterone and 17beta-estradiol in all phases of the cycle. This phenomenon is particularly evident during the pre- and post-reproductive period, when endogenous testosterone levels observed in both testis and plasma were the lowest and 17beta-estradiol concentrations were the highest. D-Asp rapidly induces a fall in 17beta-estradiol and a rise in testosterone at 3 h post-injection in the testis and at 6 h post-injection in the blood. In vitro experiments show that testicular tissue converted L-Asp into D-Asp through an aspartate racemase. D-Asp synthesis was measured in all phases of the cycle, but was significantly higher during the reproductive period with a peak at pH 6.0. The exogenous D-Asp also induces a significant increase in the mitotic activity of the testis at 3 h (P < 0.05) and at 6 h (P < 0.01). Induction of spermatogenesis by D-Asp is recognized by an intense immunoreactivity of the germinal epithelium (spermatogonia and spermatids) for proliferation cell nuclear antigen (PCNA). The effects of D-Asp on the testis appear to be specific since they were not seen in lizards injected with other D- or L-forms of amino acids with known excitatory effects on neurosecretion. Our results suggest a regulatory role for D-Asp in the steroido-genesis and spermatogenesis of the testis of the lizard Podarcis s. sicula.

The role of D-aspartic acid and N-methyl-D-aspartic acid in the regulation of prolactin release.

In this study, using an enzymatic HPLC method in combination with D-aspartate oxidase, we show that N-methyl-D-aspartate (NMDA) is present at nanomolar levels in rat nervous system and endocrine glands as a natural compound, and it is biosynthesized in vivo and in vitro. D-aspartate (D-Asp) is its natural precursor and also occurs as an endogenous compound. Among the endocrine glands, the highest quantities of D-Asp (78 +/- 12 nmol/g) and NMDA (8.4 +/- 1.2 nmol/g) occur in the adenohypophysis, whereas the hypothalamus represents the area of the nervous system where these amino acids are most abundant (55 +/- 9 and 5.6 +/- 1.1 nmol/g for D-Asp and NMDA, respectively). When D-Asp is administered to rats by ip injection, there is a significant uptake of D-Asp into the adenohypophysis and a significant increase in the concentration of NMDA in the adenohypophysis, hypothalamus and hippocampus, suggesting that D-Asp is an endogenous precursor for NMDA biosynthesis. Experiments conducted on tissue homogenates confirm that D-Asp is the precursor of the NMDA and that the enzyme catalyzing this reaction is a methyltransferase. S-adenosyl-L-methionine (SAM) is the methyl group donor. In vivo experiments consisting of ip injections of sodium D-aspartate show that this amino acid induced a significant serum PRL elevation and this effect is dose and time dependent. In vitro experiments conducted on isolated adenohypophysis or adenohypophysis coincubated with the hypothalamus, showed that the release of PRL is caused by a direct action of D-Asp on the pituitary gland and also mediated by the indirect action of NMDA on the hypothalamus. Then, the latter induces the release of a putative factor that in turn stimulates the adenohypophysis reinforcing the PRL release. In conclusion, our data suggest that D-Asp and NMDA are present endogenously in the rat and are involved in the modulation of PRL release.

Secretion of D-aspartic acid by the rat testis and its role in endocrinology of the testis and spermatogenesis.

The D-isomer of aspartic acid (D-Asp) has been found in rat testes. In the present study, samples of testicular venous blood plasma, rete testis fluid, interstitial extracellular fluid, luminal fluid from the seminiferous tubules, testicular parenchymal cells, epididymal spermatozoa and peripheral blood plasma were collected and analyzed for D-Asp by two methods, an enzymatic and a chromatographic HPLC method. The two methods gave very similar results for all samples. The highest concentrations of D-Asp (about 120 nmol/ml) were found in testicular venous blood plasma, with slightly lower concentrations in rete testis fluid (95 nmol/ml) and epididymal spermatozoa (80 nmol/g wet weight). Lower levels were found in testicular parenchymal cells (which would comprise mostly spermatids and spermatocytes), luminal fluid from the seminiferous tubules and interstitial extracellular fluid (26, 23 and 11 nmol/ml respectively). However, these values were all higher than those for peripheral blood plasma (6 nmol/ml). It would appear that D-Asp is being secreted by the testis mostly into the venous blood, passing thence into the rete testis fluid and being incorporated into the spermatozoa at the time or after they leave the testis. The distribution of D-Asp is thus quite different from that of testosterone, and its role and the reason for its high concentration in the male reproductive tract remain to be elucidated.

D-aspartic acid is implicated in the control of testosterone production by the vertebrate gonad. Studies on the female green frog, Rana esculenta.

In the present study we report the occurrence of D-aspartic acid (D-Asp) in the ovary of the green frog Rana esculenta and its putative involvement in testosterone production by the gonad. In the ovary, D-Asp concentrations undergo significant variations during the main phases of the sexual cycle. In spawning females (March), its concentration was low (2.5 +/- 1.1 nmol/g ovary) and during the post-reproductive period (June) it increased and reached its peak level (58.0 +/- 10.1 nmol/g) in October. In that month, vitellogenesis occurs in a new set of ovarian follicles and continues until the next spring. The concentrations of D-Asp in the ovary and of testosterone in the ovary and in the plasma were inversely correlated during the reproductive cycle: when endogenous D-Asp was low (March), testosterone was high (36.9 +/- 4.8 ng/g ovary; 23.1 +/- 2.76 ng/ml plasma) and, in contrast, when the D-Asp concentration was high (October), the testosterone concentration was low (0.86 +/- 0.21 ng/g ovary and 5.0 +/- 1.3 ng/ml plasma). In vivo experiments, consisting of injection of D-Asp (2.0 mumol/g body weight) into the dorsal lymphatic sac of adult female frogs, demonstrated that this amino acid accumulates significantly in the ovary. After 3 h, moreover, it caused a decrease in testosterone level in the plasma of about 80%. This inhibition was reversible: within 18 h after the amino acid injection, as the D-Asp concentration in the ovary decreased, the testosterone titre was restored in both ovary and plasma. In vitro experiments, conducted in isolated ovarian follicles, confirmed this phenomenon and identified these gonadal components as the putative D-Asp targets. Other amino acids (L-Asp, D-Glu, L-Glu, D-Ala and L-Ala) used instead of D-Asp were ineffective. These findings indicate that D-Asp is involved in the control of androgen secretion by the ovary in this amphibian species, revealing a more complex system for control of this androgen synthesis than was previously believed to exist.

Testicular endocrine activity is upregulated by D-aspartic acid in the green frog, Rana esculenta.

This study investigated the involvement of D-aspartic acid (D-Asp) in testicular steroidogenesis of the green frog Rana esculenta and its effect on stimulation of thumb pad morphology and glandular activity, a typical testosterone-dependent secondary sexual characteristic in this amphibian species. In the testis, D-Asp concentrations vary significantly during the reproductive cycle: they are low in pre- and post-reproductive periods, but reach peak levels in the reproductive period (140-236 nmol/g wet tissue). Moreover, the concentrations of D-Asp in the testis through the sexual cycle positively match the testosterone levels in the gonad and the plasma. The racemase activity evaluated during the cycle expresses its peak when D-Asp and testosterone levels are highest, that is, during the reproductive period, confirming the synthesis of D-Asp from L-Asp by an aspartate racemase. Short-term in vivo experiments consisting of a single injection of D-Asp (2.0 micro mol/g body weight) demonstrated that this amino acid accumulates significantly in the testis, and after 3 h its uptake is coupled with a testosterone increase in both testis and plasma. Moreover, within 18 h of amino acid administration, the D-Asp concentration in the testis decreased along with the testosterone titer to prestimulation levels. Other amino acids (L-Asp, D-Glu and L-Glu) used instead of D-Asp were ineffective, confirming that the significant increase in testicular testosterone was a specific feature of this amino acid. In long-term experiments, D-Asp had been administered chronically to frogs caught during the three phases of the reproductive cycle, inducing testosterone increase and 17beta-estradiol decrease in the gonad during the pre- and post-reproductive period, and vice versa during the reproductive period. The stimulatory effect of D-Asp on testosterone production by the testis is consistent with the stimulation of spermatogenesis and the maturation of thumb pads occurring in D-Asp-treated frogs. In these last animals, there was an increase of seminiferous ampoule area and a higher number of spermatids and sperm. Moreover, in spermatogonia I and II and in spermatocytes, a proliferating cell nuclear antigen (PCNA) intense immunopositivity was observed. In addition, the thumb pads of D-Asp-treated frogs compared with controls showed a significantly thicker epithelial lining, a wider area of their glands with taller secretion cells, and more numerous, PAS-positive-rich secretions. Finally, these results provide functional evidence for a biologic role of D-Asp in amphibian male steroidogenesis; therefore, this unusual amino acid could be considered a modulatory agent for reproductive processes.

Sex and reproductive status related brain content of mammalian and chicken-II GnRHs in Rana esculenta.

Mammalian and chicken-II forms of gonadotropin-releasing hormone (mGnRH and cGnRH-II, respectively) have been measured simultaneously in the brain, pituitary, and peripheral terminal nerves (nasal area tissue) of adult males and females of a representative amphibian, Rana esculenta, during the annual reproductive cycle. Whereas in the male, brain concentrations of both GnRH forms showed significant reproductive status-related fluctuations, in the female brain only cGnRH-II content showed significant changes. The highest GnRH levels were recorded just prior to breeding in both sexes. In the pituitary both GnRHs were present in all seasons. In the peripheral terminal nerves, instead, only mGnRH was detected in all seasons confirming our previous immunohistochemical data. In both sexes furthermore, the brain and pituitary mGnRH levels were consistently much higher than those of cGnRH-II and there were no sex-related differences in the brain and pituitary content of GnRHs. Seasonal changes in brain GnRH levels may correlate with plasma sex steroid levels reinforcing the postulate that sex steroids affect GnRH neuronal systems.

FMRFamide in the amphibian brain: a comprehensive survey.

Mapping of FMRFamidergic neural circuitry in the amphibian brain has been done by immunohistochemical methods. Comparative evidence suggests that there are similarities and differences in the overall pattern of distribution of FMRFamide-ir elements in the brain among the three amphibian orders and within each order. FMRFamide is expressed in neurons in some circumscribed areas of the brain. A part of these neurons is concentrated in classical neurosecretory areas of the hypothalamus in a bilaterally symmetrical fashion. Similar neurons occur occasionally in the midbrain, but are virtually absent from the hindbrain. Anurans are unique among amphibians to show FMRFamide neurons in the medial septum and diagonal band of Broca. A viviparous gymnophione is known to possess a small population of such neurons in the dorsal thalamus. Together, the FMRFamide neurons contribute to an extensive fiber network throughout the amphibian brain. Descriptive developmental studies suggest that the rostral forebrain-located FMRFamide neurons originate in the olfactory placode and then migrate into the brain along the route of the vomeronasal-olfactory-terminal nerve complex. Olfactory placodal ablation in an anuran and a urodele provide experimental support to this contention. Other FMRFamide neuronal cell groups, in the hypothalamus and dorsal thalamus, are supposed to arise from non-placodal precursors. The neuroanatomical distribution (projection of immunoreactive processes to areas of the fore-, mid-, and hindbrain as well as to cerebrospinal fluid, co-localization with other neuropeptides, and presence in the median eminence) has furnished morphological correlates of possible functions of FMRFamide in the amphibian CNS. While amphibian FMRFamide-like or structurally related peptides remain to be isolated and characterized, the sum of the distribution pattern of FMRFamide-like immunoreactivity suggests that it may act as a neurotransmitter or a neuromodulator, and also may have endocrine regulatory functions.

Immunoreactive mammalian and chicken-II GnRHs in Rana esculenta brain during development.

Two forms of gonadotropin-releasing hormone (mammalian, mGnRH and chicken-II, cGnRH-II) were measured by radioimmunoassay in the nasal area (containing peripheral terminal nerve), brain and pituitary of Rana esculenta during larval development, metamorphosis, and until prior to becoming reproductively active. Small amounts of both forms of GnRH were first detected in the brain extract of early tadpoles (stage 26-27, when hindlimbs begin to develop). Later, there was a gradual, but constant, stage-dependent increase in the brain content of GnRHs, with the most remarkable increase recorded at postclimax and in young frogs. In tadpoles, postclimax froglets, and young frogs, the brain concentration of mGnRH was higher than that of cGnRH-II, with a ratio of approximately 2:1 in favor of mGnRH. In juveniles, however, the brain extract contained more cGnRH-II than mGnRH. No GnRH immunoreactivity was detected in the nasal area until stage 31. In successive stages of development, however, only mGnRH was present in the nasal area, and this confirmed our previous immunohistochemical analysis which showed that the peripheral terminal nerve contains only mGnRH-immunoreactive neurons and fibers. Although both GnRH forms were detected in the anterior (telencephalon, diencephalon) and posterior (mesencephalon, rhombencephalon) brain halves from juveniles, mGnRH content predominated in the anterior half, whereas in the posterior half cGnRH-II was present in greater amounts. Pituitaries from male and female postclimax froglets and young frogs contained both forms of GnRH in a ratio of approximately 10:1 in favor of mGnRH. This finding may shed light on the question of which GnRH(s) regulate gonadotropin release from the pituitary. The developmental changes in GnRH-immunoreactive content of the brain and pituitary have been discussed in the light of functional maturation of the brain-pituitary-gonad axis.

Development and distribution of gonadotropin-releasing hormone neuronal systems in the frog (Rana esculenta) brain: immunohistochemical analysis.

The ontogenesis of the gonadotropin-releasing hormone (GnRH) neuronal systems was studied in the brain of the frog, Rana esculenta. Attention was also focussed on the differential distribution of molecular forms of GnRH during development. The first GnRH-immunoreactive neurons appear in the mesencephalon of posterior limb-stage tadpoles. These neurons are shown to contain only chicken [His5,Trp7,Tyr8]GnRH (cGnRH-II). Later in development, mammalian [Tyr5,Leu7,Arg8] GnRH (mGnRH)-like peptide-containing neurons appear simultaneously in the terminal nerve as well as in the anterior preoptic area of the telencephalon. Subsequently, only after metamorphosis, mGnRH-containing neurons appear in the medial septal area of the posterior telencephalon. It is here shown that neurons containing the two forms of GnRH are distributed in distinct brain areas during development and in the adult: mGnRH-immunoreactive neurons in the terminal nerve, olfactory bulb, mediobasal telencephalon, medial septal area, anterior preoptic area, ventrolateral thalamus and infundibulum, whereas cGnRH-II neurons are located in the mesencephalon. We hypothesize that the terminal nerve/forebrain-located GnRH neurons express immunohistochemically late in development and originate extracranially migrating centrally, along the terminal nerve, during development, whereas those located in the mesencephalon express earlier and may have an intracranial site of origin.

Regional decreases of free D-aspartate levels in Alzheimer's disease.

N-methyl-D-aspartate (NMDA) receptors have been shown to be involved in learning and memory processes. In Alzheimer's disease, there is a reduction of NMDA receptors. Since D-aspartate is an endogenous agonist for the NMDA receptor, we hypothesised that if there are reduced levels of this amino acid in the Alzheimer's brain, this could raise the reduction of NMDA receptor signal transduction system and contribute to the marked memory deficits seen in these patients. Therefore, using a chromatographic HPLC method, the regional distribution of free D-aspartate levels in post-mortem human brain samples from patients with Alzheimer's disease (AD) (n = 5) and age-matched controls (n = 5) were determined. We found that the levels of D-aspartate are significantly lower in Alzheimer's patients compared to controls (range: from -35 to -47%; P < 0.01). However, no differences were found in the cerebellum, a region spared from the neuropathological changes of AD. These data suggest that decreased levels of D-aspartate could contribute to a lower NMDA receptor function and consequently contribute to the memory deficits seen in AD.

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.

Presence of a human-like thyroid stimulating hormone (TSH) in Ciona intestinalis.

Using a monoclonal antibody against human Thyroid Stimulating Hormone (TSH), we have found that the invertebrate Ciona intestinalis (phylum Chordata, subphylum Tunicata) contains a previously unreported protein which is immunoreactive for anti-human TSH. The amount of this hormone in the blood, endostyle and ovary of C. intestinalis was found to be 0.01+/-0.003, 1.05+/-0.2 and 3.61+/-1.25 microIU/g of tissue, respectively, using the RIA method, and a value of 0.013+/-0.0043, 1.16+/-0.30 and 3.85+/-1.32 microIU/g using an immuno-chemiluminescent method. In addition to possessing immunological properties, this protein is able to induce the synthesis of cAMP in rat thyroid cell culture (FRTL-5) and in Chinese Hamster Ovary cells (CHO) transfected with the cDNA for human TSH receptor. This indicates that the putative ciona TSH has the capability to react specifically with receptors for mammalian TSH. Maximum concentrations of ciona TSH-like factor occur during periods of sexual maturity (between May to July), whereas very low concentrations were assayed during the rest of the year suggesting that this hormone may be involved in hormonal function related to sexual maturity. From a phylogenetic point of view, the above data supports the hypothesis for a common origin of a thyroid hormonal system between Tunicata and vertebrates.

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

Mammalian testis contains D-aspartic acid (D-Asp), which enhances testosterone production. D-Asp, on other hand, also stimulates 17beta-estradiol synthesis in the ovary of some lower vertebrates. We studied boar testis in order to determine if D-Asp intervenes in 17beta-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 17beta-estradiol. The enzyme's K(m) 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 17beta-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 17beta-estradiol that they produce (autocrine effect).

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 17beta-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.

Putative steroid-binding receptors and nonreceptor components and testicular activity in the lizard Podarcis sicula sicula.

Labelled testosterone- and oestradiol-binding molecules have been found in the cytosol and nuclei of lizard testes. DNA-cellulose affinity chromatography was used to separate putative sex-steroid-binding receptors (adhering molecules) and nonreceptor components (nonadhering molecules). A putative androgen receptor (Kd: 10(-10) mol l-1; 3-9 fmol g-1 tissue) was found mainly in the nuclei of testicular cells when actively undergoing spermatogenesis. This suggests that, as in higher vertebrates, testosterone is implicated in spermatogenetic step regulation (meiosis and spermiogenesis) in lizard testis. In the cytosol, testosterone-binding molecules (Kd: 10(-9) mol l-1; 384-784 fmol g-1 tissue) with several properties of androgen-binding proteins are present from autumn to spring. The behaviour of these molecules is consistent with the role assigned to androgen-binding proteins as androgen reservoir. A putative oestrogen receptor is present throughout the sexual cycle, except during the culmination phase (breeding). The putative oestrogen receptor may be involved in the regulation of the first spermatogenetic step (spermatogonia multiplication) and in the induction of post-reproductive refractoriness. This phase is present in temperate-zone lizards. These studies show that the evaluation of sex-steroid-binding molecules is useful in considering the relationships between sex hormones and spermatogenetic activity in the testes of lizards.

Sex steroid binding proteins in the plasma of the green frog, Rana esculenta: changes during the reproductive cycle and dependence on pituitary gland and gonads.

Sex steroid binding proteins (SSBP) have been reported in the plasma of female and male Rana esculenta. In both sexes SSBP bind [3H]estradiol and [3H]testosterone with medium-high affinity and high specificity. Using ion-exchange chromatography SSBP resolve into two peaks eluting at 0.27 M (peak I) and 0.36 M (peak II) NaCl. Both peaks bind [3H]estradiol and [3H]testosterone equally well. Isoelectrofocusing showed that peak I focused at pH 6.0 and 7.5, whereas peak II focused at pH 6.0. SSBP capacity for [3H]estradiol and [3H]testosterone changes throughout the reproductive cycle, showing low levels during the nonbreeding period and increasing levels during the breeding period. Hypophysectomy and/or gonadectomy result in small changes in SSBP capacity. Short-term steroid hormone treatment of gonadectomized animals does not modify SSBP capacity.

GnRH and substance P regulate prostaglandins and sex steroids from reptilian (Podarcis sicula sicula) ovarian follicles and corpora lutea.

The in vitro effects of salmon gonadotropin-releasing hormone (sGnRH), substance P (SP), and their antagonists on prostaglandin F2 alpha (PGF2 alpha), prostaglandin E2 (PGE2), progesterone, androgens, and estradiol-17 beta release by follicles and corpus luteum (CL) of the oviparous lizard, Podarcis sicula sicula, were studied. Follicles and CL were divided according to the different developmental stages; follicles: pre-vitellogenic, early-vitellogenic, mid-vitellogenic, and fully grown; CL: CL1 (unshelled eggs in the oviducts), CL2 (shelled eggs in the oviducts), CL3 (eggs laid 6 hr previously), and CL4 (eggs laid 48 hr previously). SGnRH increased PGF2 alpha and progesterone release by mid-vitellogenic and fully grown follicles; SP increased PGE2 and estradiol-17 beta release by pre-vitellogenic, mid-vitellogenic, and fully grown follicles. SGnRH and SP decreased PGE2 and progesterone and increased PGF2 alpha by CL1 and CL2. The antagonists of these two neuropeptides induced the opposite effects of those of sGnRH and SP. The present data indicate that sGnRH and SP play different roles in the regulation of prostaglandins and sex steroid production by ovarian follicles and CL of P. s. sicula.