Immunohistochemical localization of substance P-like immunoreactivity in the hypothalamus of the lizard, Podarcis sicula, R

The distribution of substance P (SP) immunoreactivity was investigated in the hypothalamus (preoptic area included) of the lizard by single and double immunocytochemical procedures, SP-immunopositive cell bodies were seen in the paraventricular nucleus and periventricular hypothalamic gray (including the paraventricular organ) together with some more lateral elements. Extensive nerve fibers were seen in the white matter and surrounding the paraventricular and supraoptic neurons, and more caudally reaching the hypothalamic periventricular gray, suggesting a massive involvement of SP-like substance in the control of hypothalamic neuroendocrine areas.     

Estradiol receptor in the lizard liver (Podarcis s. sicula). Seasonal changes and estradiol and growth hormone dependence

This study shows that in the liver of the oviparous lizard, Podarcis s. sicula, the estrogen receptor (ER) level increases during the reproductive period (spring) when vitellogenesis occurs. This phenomenon interested both unfilled and filled ER present in the cytosolic and nuclear fractions. The increase in unfilled cytosolic and filled nuclear receptor was positively correlated to the plasma level of vitellogenin. The level of liver ER approximated that of mammalian liver ER and, therefore, it is higher than that reported for the liver of several nonmammalian species. At electrofocusing, liver ER distributes in two pH ranges (pH 6.5-7.5 and 8.0-8.8, respectively). The first form predominated in nuclei of reproductive females or of spayed estrogenized females and could represent the activated form of receptor. Ovariectomy was followed by a decrease in liver ER which can be induced in spayed females by estradiol administration. Pituitary growth hormone (GH) seemed to exert a synergic effect on estradiol liver estrogen receptor regulation. In lizards treated both with estradiol and GH, in fact, there was a significant increase in nuclear filled ER rather than an increase in the level of total nuclear ER.     

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.     

Exogenous vitellogenesis and micropinocytosis in the lizard, Podarcis sicula, treated with follicle-stimulating hormone

The regulation of oocyte growth and of exogenous vitellogenesis by micropinocytosis has been studied in lizard Podarcis sicula kept at 28 degrees during the winter stasis and stimulated by follicle-stimulating hormone (FSH). Under these experimental conditions, oocyte auxocytosis as well as vitellogenesis is stimulated, while the follicular hierarchy is preserved. At the ultrastructural level, the flow of exogenous yolk precursors toward the oocyte increases and the pathway taken by them through intercellular spaces and zona pellucida is the same as that taken by peroxidase (tracer). Yolk precursor endocytosis is found only in oocytes greater than 1500 microns in diameter and takes place through the formation of several coated pits and vesicles. It is suggested that membrane receptors necessary for micropinocytosis are available only in such oocytes. Last, a different permeability of the ovarian follicle to exogenous yolk precursors during the different stages of oocyte growth and endovarian control of vitellogenesis are suggested.     

Plasma sex steroid binding proteins (SSBP) in the male lizard, Podarcis s. sicula, during the reproductive cycle.

In male Podarcis s. sicula plasma, a sex steroid binding protein [SSBP(s)] binds testosterone (T) and estradiol-17 beta (E2) with moderate affinity (Kd = 0.23 +/- 0.08 x 10(-8) for 3H-E2, and 0.24 +/- 0.07 x 10(-8) for 3H-T) and high capacity. The SSBP binding affinity is unchanged throughout the sexual cycle, although its capacity is higher in nonreproductive males (winter and postreproductive period). This change may be related to changes in plasma T and E2 levels, and is likely to be involved in mechanisms whereby free steroid is delivered to target organs. SSBP, under isoelectrofocusing, is distributed between pH 5.5-6.5 and pH 7.1-7.5. The concentration of these two forms varies during the annual cycle.     

Immunohistochemical localization of somatostatin-like immunoreactivity in the hypothalamus of the lizard, Lacerta sicula

Somatostatin-like (SRIF) immunoreactivity was shown in the preoptic area and posterior hypothalamus of lizard (Lacerta sicula R.), using both indirect immunofluorescence and PAP-technique. Immunoreactive neurons are seen in the preoptic paraventricular nucleus, in the caudal hypothalamus, and close to the hypophysial recess where they form a massive group of cerebrospinal-fluid-contacting elements. SRIF-immunoreactive fibers are seen also in the outer zone of the median eminence. Employing double-staining methods (double peroxidase-antiperoxidase sequence, according to Vandesande and Dierickx ((1976) Cell Tissue Res. 175, 289–296) and PAP-immunofluorescence) the immunoreactivity for SRIF was separated from that for neurophysins.     

Sex steroids levels in the plasma and testis during the reproductive cycle of lizard Podarcis s. sicula Raf.

Progesterone (P), 17-OH-progesterone (17-OH-P), androstenedione (A), dehydroepiandrosterone (DHEA), testosterone (T), 5 alpha-dihydrotestosterone (5 alpha-DHT), and 17 beta-estradiol (E2) were measured by RIA in plasma and testes of 114 males of the oviparous lizard Podarcis s. sicula raf, a species that displays annual hibernating cycles. Hormones were determined each month from January until December, except for August. Testosterone peaked at 174.8 ng/ml of plasma after emergence (March), while 5 alpha-DHT and A peaked in April. Plasma DHEA increased during hibernation. During the refractory period there were progressive increases in P and E2 plasma levels. The testicular peak of T, in March, coincided with that observed in plasma. The striking increases in testicular T and A in early July occurred at a time when plasma androgen concentrations were low. 5 alpha-DHT increased in April when spermatogenesis with spermiation occurred and then decreased alongside a second peak of T. There is an apparent separation of plasma and testicular androgen concentrations during the reproductive cycle.     

Atrial natriuretic factor: localization in the adrenal gland of the lizard Podarcis sicula and effects on pituitary-adrenal axis activity

The occurrence of atrial natriuretic factor (ANF) immunoreactivity was investigated in the adrenal gland of the lizard Podarcis sicula by avidin-biotinylated peroxidase complex (ABC) immunocytochemical technique: ANF immunoreactivity was present in the chromaffin tissue, and was absent in the steroidogenic tissue. The role of ANF in the modulation of the pituitary-adrenal axis activity was investigated in vivo by intraperitoneal administration of ANF. The effects were evaluated by examination of the morphological and morphometrical features of the tissues, as well as the plasma levels of adrenocorticotropic hormone (ACTH), corticosterone, aldosterone, norepinephrine, and epinephrine. ANF (28 microg/100 g body wt) did not affect ACTH plasma levels, that remained almost unchanged; in contrast, corticosterone plasma levels increased from 6.45 +/- 0.070 ng/ml in carrier-injected lizards to 9.69 +/- 0.080 ng/ml 24 h after the injection; aldosterone levels decreased from 2.19 +/- 0.010 ng/ml in carrier-injected specimens to 0.58 +/- 0.003 ng/ml 24 h after the experimental treatment. In the chromaffin tissue, an increase in the number of epinephrine cells and a decrease in the number of norepinephrine cells were observed, decreasing the numeric norepinephrine/epinephrine cell ratio, from 1.4/1 of control specimens to 0.3/1 24 h after ANF administration. Moreover, norepinephrine plasma levels decreased from 998 +/- 4.600 pg/ml in carrier-injected specimens to 321 +/- 2.230 pg/ml 24 h after ANF administration; epinephrine plasma levels were elevated from 614 +/- 3.410 pg/ml in carrier-injected specimens to 1672 +/- 10.800 pg/ml 24 h after the experimental treatment. The presence of ANF in the adrenal gland suggests that, also in reptiles as in other vertebrates, this peptide, locally released from the chromaffin cells, may modulate the activity of the adrenal gland, probably in a paracrine manner. The effects of ANF on the adrenal gland suggest that this peptide may affect reptilian salt and fluid homeostasis.     

Reproduction in the Mexican leaf frog, Pachymedusa dacnicolor. V. Immunohistochemical localization of gonadotropin-releasing hormone in the brain.

The presence and distribution of immunoreactive gonadotropin-releasing hormone (GnRH) in brains of adult male and female Pachymedusa dacnicolor has been studied immunohistochemically using antisera against mammalian, chicken-II, and salmon GnRHs. The distribution map of the immunoreactive-GnRH elements in the brain of P. dacnicolor is extremely simple, being limited to the anterior preoptic area-infundibulum-median eminence circuit. No sex- or reproductive status-related difference in either the distribution pattern or intensity of immunoreaction was revealed in this study. This is also the first immunohistochemical evidence of the presence of different structural forms of GnRH in the brain of an amphibian.     

Circadian rhythms of plasma melatonin in the ruin lizard Podarcis sicula: Effects of pinealectomy

Plasma melatonin was measured in lizards (Podarcis sicula) at six different times of day under conditions of constant temperature and darkness. Intact animals showed a circadian rhythm of melatonin with a peak in the subjective night of 207 pg/ml (median) and a trough during the subjective day that was below the minimum detection level of the assay (50 pg/ml). Pinealectomy abolished the circadian rhythm of plasma melatonin; median levels were near or below the minimum detection level at all times sampled. The data suggest that the pineal is the only source of rhythmic blood-borne melatonin in Podarcis sicula, and are consistent with the hypothesis that changes in the free-running period of the locomotor rhythm induced by pinealectomy in this species are due to withdrawal of rhythmic melatonin from the blood.     

Ovarian gonadotropin-releasing hormone-like protein(s): demonstration and characterization

Both GnRH and prostaglandin F2 alpha inhibit LH-stimulated cAMP accumulation and progesterone secretion in isolated luteal cells. Moreover, since ovarian GnRH receptors have been demonstrated, this study was conducted to determine if GnRH-like substances were present in the luteinized rat ovary. Detection of GnRH-like activity was based on a sensitive and specific ovarian membrane GnRH radioreceptor assay and an immunoassay specific for GnRH. Ovaries were extracted with an aqueous medium containing formic acid, HCl, trifluoroacetic acid, and NaCl. Material present in the supernatant fraction which adsorbed to Waters C18 Sep-Paks was subsequently eluted with acetonitrile and lyophilized. The redissolved ovarian extract showed substantial radioreceptor activity, but very little immunoassayable activity. The GnRH-like activity of the ovary was sensitive to proteolytic enzyme digestion and to incubations at 50 C for as little as 5 min, had an apparent mol wt greater than 1,000 but less than 10,000, and was not soluble in ether. Extracts of plasma did not exhibit radioreceptor or immunoreactive activity, whereas hypothalamic extracts exhibited both radioreceptor and immunoreactive activities. Liver and kidney extracts showed less radioreceptor activity than ovarian extracts and very little immunoreactive activity. Two peaks of radioreceptor activity appeared when the ovarian extract was further fractionated by reverse phase HPLC. The two peaks of GnRH-like activity were clearly separated from GnRH or [D-Ala6, des-Gly10] GnRH ethylamide, an analog used in this study, when these were included in ovarian extracts. It is concluded that rat ovaries contain a GnRH-like protein(s) with membrane binding properties similar to those of GnRH but with other characteristics distinctly different from those of GnRH. The ovarian GnRH-like protein is immunologically different from GnRH, sensitive to elevated temperatures which do not effect GnRH, and chromatographically different from GnRH during reverse phase HPLC.     

Oocyte plasma membrane proteins and the appearance of vitellogenin binding protein during oocyte growth in the lizard Podarcis sicula

In the ovary of the lizard Podarcis sicula, the micropinocytotic uptake of the yolk exogenous precursor (i.e., vitellogenin; VTG) occurs only in the reproductive period and involves the plasma membrane of > or =2000-microm oocytes. This paper analyzes the intrinsic proteins extracted from the plasma membrane of growing oocytes to identify the vitellogenin binding protein during the different stages of the annual ovarian cycle of this species. Despite the well-known ultrastructural changes of the oocyte plasma membrane, SDS-PAGE failed to show marked variation in the total number of membrane proteins during the most significant stages of oocyte auxocytosis. Nevertheless, ligand blotting, using homologous VTG and anti-VTG, revealed that an congruent with115-kDa protein of the oocyte plasma membrane bound plasma vitellogenin only in the reproductive period (spring-summer) in both vitellogenic and nonvitellogenic oocytes. During the nonreproductive period, this molecule was never observed. However, it could be induced in the coldest months (winter) by hypophyseal gonadotropins.     

Plasma vitellogenin and 17 beta-estradiol levels during the annual reproductive cycle of Podarcis s. sicula Raf.

Plasma vitellogenin and 17 beta-estradiol concentration were determined during the annual reproductive cycle of the female lizard Podarcis s. sicula Raf. living around Naples. Plasma vitellogenin was purified from estrogenized males for characterization and to raise specific immune serum. Using ELISA, plasma vitellogenin titers were determined in relation to ovary weight; plasma 17 beta-estradiol was measured by RIA method. Native vitellogenin was present as two polypeptide bands: alpha and beta. The electrophoretic patterns, studied in normal male and estrogenized male and female, showed vitellogenin to be a protein present in female and in estrogenized male plasma but not in normal males. Lizard monomeric VTG, determined by SDS-PAGE, was about 200 kDa. Correlations between seasonal ovarian weight variations and plasma vitellogenin and 17 beta-estradiol suggest that ovarian development in Podarcis depends on plasma vitellogenin synthesis, which in turn relies on plasma estradiol levels. The two ovulatory waves observed in this study coincided with the two peak values of plasma vitellogenin and 17 beta-estradiol.     

Diverse molecular forms of gonadotropin-releasing hormone in an elasmobranch and a teleost fish

Immunoreactive and biologically active gonadotropin-releasing hormones (GnRHs) in dogfish (Poroderma africanum) and teleost (Coris julis) brain extracts were studied by high-performance liquid chromatography (HPLC), radioimmunoassay with region-specific antisera, and assessment of luteinizing hormone (LH)-releasing activity in a chicken dispersed pituitary cell bioassay. In dogfish brain extract, seven GnRH molecular forms with LH-releasing activity were demonstrated. Three of these forms coeluted with synthetic mammalian GnRH; His5,Trp7,Tyr8-GnRH; and Trp7,Leu8-GnRH on HPLC. The peaks coincident with His5,Trp7,Tyr8-GnRH and Trp7,Leu8-GnRH had immunological and biological properties identical to those of the synthetic peptides. However, the molecular form coeluting with mammalian GnRH had immunological and biological properties different from those of mammalian GnRH and is thus a novel molecular variant of GnRH. The four remaining forms are also novel GnRHs or structurally unrelated peptides with LH-releasing activity. Dogfish systemic blood contained immunoreactive GnRH. In teleost brain extract, three biologically active GnRH forms with LH-releasing activity were present. The major peak of GnRH immunoreactivity coeluted with Trp7,Leu8-GnRH, and a second immunoreactive form coeluted with His5,Trp7,Tyr8-GnRH. The third biologically active peak is a novel, early-eluting molecular variant of GnRH or a structurally unrelated peptide with LH-releasing activity.     

Shift from noradrenaline to adrenaline production in the adrenal gland of the lizard,Podarcis sicula,after stimulation with vasoactive intestinal peptide (VIP)

The aim of this study was to investigate the distribution and function of VIP in the adrenal gland of the lizard, Podarcis sicula. We have shown by immunohistochemistry that VIP fibers were localized exclusively around clusters of chromaffin cells in the dorsal ribbon of the lizard adrenal gland. Moreover, a strong positivity for this peptide was observed within ganglial cells and within most chromaffin cells of the gland. To investigate the effects of VIP on the adrenal gland, we have treated lizards with several doses of this peptide and we have shown that injections of exogenous VIP increased plasma levels of catecholamines and corticosteroids, but not of ACTH. This probably suggests a direct effect of VIP on the control of adrenal hormone secretion without the involvement of the hypothalamo-hypophyseal axis. Our results also establish that the increased levels of the hormones were modulated in a time- and dose-dependent manner. Therefore, our morphological studies showed a clear increased function of steroidogenic cells. In the medullary region, VIP administration induced not only a functional enhancement of adrenaline release from adrenergic cells, but also a shift of noradrenaline cells to adrenaline ones.     

Seasonal-dependent effect of temperature on the response of adenylate cyclase to FSH stimulation in the oviparous lizard, Podarcis sicula

The study of environmental factors affecting vertebrate reproduction has long interested both developmental and evolutionary biologists. Although photoperiod has been considered to be an important environmental parameter for vertebrates such as birds, temperature is probably a primary external factor responsible for reproductive cyclicity in reptiles. In spite of the progress made in the understanding of reptilian reproductive strategies and adaptations, much remains to be learned about the interplay between endocrine physiological factors, such as hormones, and environmental parameters. In this report, we have examined the effects of in vivo administered FSH on oocyte recruitment during the most significant periods of the reproductive cycle of the lizard, Podarcis sicula. The results show that when FSH is administered in proximity to the reproductive period, it stimulates oocyte growth and ovulation; when the hormone is administered at the beginning of the winter stasis it affects ovarian activity without inducing ovulation. Ovarian adenylate cyclase activity is moderately sensitive to in vitro FSH stimulation during the pre- and post-reproductive periods. The sensitivity to hormone stimulation increases significantly during the reproductive period and winter stasis. We have also tested the hypothesis that environmental temperature affects the responsiveness of ovarian adenylate cyclase to FSH stimulation. For such a purpose, we exposed animals to 28 degrees C or 4 degrees C in different periods of the ovarian cycle. The results show that, whenever the temperature applied mimics the thermal regime of the coming season, adenylate cyclase sensitivity to FSH shifts towards levels that anticipate the natural responsiveness.     

Immunohistochemical localization of adrenocorticotropin in the rat brain.

ACTH and some of its fragments have been shown to play a role in a variety of adaptative mechanisms. To clearly identify the nervous structures containing ACTH in the rat brain, an immunohistochemical localization of this peptide was conducted at both light and electron microscopic levels. Nervous fibers staining for ACTH were found to be largely distributed throughout regions of hypothalamus, thalamus, and midbrain. Positive fibers could also be occasionally observed in the spinal cord. Immunostained neuronal cell bodies were only detected in the arcuate nucleus. Essentially, the same results were obtained 2 and 8 weeks after hypophysectomy. In animals pretreated with colchicine, the intensity in the staining of cell bodies was markedly increased, making possible the detection of a larger number of cell bodies. At the electron microscopic level, it was demonstrated that ACTH is contained in dense core vesicles present in nervous fibers and endings. These results indicate that ACTH of nonpituitary origin is synthesized in the central nervous system and could probably be considered as a neurotransmitter of still undefined function.     

Immunohistochemical localization of thyrotropin-releasing hormone in the brain of carp, Cyprinus carpio

The localization of immunoreactive thyrotropin-releasing hormone (IR-TRH) in the forebrain and pituitary of carp was studied immunohistochemically using the peroxidase-antiperoxidase technique. In the hypothalamus. IR-TRH was present in the neuronal processes extending from the preoptic nucleus (NPO) to the nucleus recessus lateralis (NRL). Cell bodies appeared to be present in the inside of the medial NRL. Most of these neurons were fusiform and bipolar. Immunoreactive-beaded fibers streamed from the anterior part of the NRL toward the nucleus posterioris periventriculas and nucleus lateral tuberis pars posterioris. Vertical strands of the beaded fibers ran in the nucleus lateral tuberis pars anterioris. In the pituitary, the reaction product was found in the neural lobe, where intense immunoreactivity was evident along neural fibers entering the intermediate lobe. Staining could be detected only rarely in the anterior lobe. IR-TRH-beaded fibers were present in the olfactory stalk as well as in the caudal and inner parts of the olfactory bulb. In contrast to the high concentration of IR-TRH in the olfactory bulb, immunohistochemical data from this work indicated weak immunoreactivity in this region.