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.     

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.     

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.     

Neuropeptide Y modulates pituitary-adrenal axis activity in the lizard, Podarcis sicula

The role of neuropeptide Y (NPY) in the modulation of the pituitary-adrenal axis activity in a lizard, Podarcis sicula, was investigated by in vivo NPY administration. The effects were evaluated by examination of the morphological and morphometrical features of the tissues as well as the plasma levels of ACTH, corticosterone, aldosterone, norepinephrine, and epinephrine. Intraperitoneally administered NPY (27 nmol /100g body wt) raised ACTH plasma levels (from 5.23+/-0.06 pg/ml in carrier injected specimens to 6.83+/-0.01 pg/ml, 24 h after the injection). In the steroidogenic cells a strong decrease of lipid amount was found; corticosterone plasma level increased from 6.28+/-0.02 ng/ml in carrier injected lizards to 7.96+/-0.01 ng/ml 24 h after the injection); aldosterone levels were raised from 1.88+/-0.02 ng/ml in carrier injected specimens to 6.38+/-0.05 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 (NE/E) cell ratio, from 1.4/1 of control specimens to 0.5/1 24 h after NPY administration. Moreover, norepinephrine plasma level were elevated from 922+/-4.30 pg/ml in carrier injected specimens to 3075+/-11.30 pg/ml 24 h after NPY administration; epinephrine plasma level increased from 502+/-2.40 pg/ml in carrier injected specimens to 2759+/-8.70 pg/ml 24 h after the experimental treatment. Consistent with these findings, morphological observations showed many chromaffin cells weakly stained and with a reduced content of secretory granules. These results suggest that, in P. sicula, NPY may play a role in the modulation of the pituitary-adrenal axis activity. Previous studies localized NPY in the epinephrine cells of P. sicula adrenal gland; taken together, these results suggest that this peptide might participate in the regulation of adrenal gland activity, enhancing corticosteroid and catecholamine secretion in a paracrine/autocrine manner. The mechanism of action of NPY is discussed.     

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.     

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.     

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.     

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.     

A single chicken oocyte plasma membrane protein mediates uptake of very low density lipoprotein and vitellogenin.

Specific cell-surface receptors mediate the uptake of plasma proteins into growing oocytes of oviparous species, thereby forming yolk. Quantitatively the most important yolk precursors are the lipoproteins, very low density lipoprotein, and vitellogenin. We show that a single major chicken oocyte plasma membrane protein with an apparent molecular mass of 95 kDa as determined by SDS/PAGE under nonreducing conditions is the receptor for both of these ligands. Binding activities for the two ligands copurified on ligand affinity matrices and were inhibited by the same antibody preparations, and the ligands competed with each other for binding to the 95-kDa protein. In addition to these biochemical and immunological lines of evidence for the identity of the vitellogenin receptor with the very low density lipoprotein receptor, genetic proof was obtained. We have previously shown that the mutant nonlaying "restricted-ovulator" hen carries a defect in the gene responsible for functional expression of the oocyte 95-kDa protein. Here we demonstrate that this single gene defect in the restricted-ovulator hen has detrimental consequences for the binding not only of very low density lipoprotein but also of vitellogenin to the 95-kDa receptor normally present in oocytes. The intriguing bifunctionality of this chicken oocyte membrane protein possibly relates to its crucial role in receptor-mediated control of oocyte growth.     

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.     

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.     

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 demonstration of the presence and localization of diverse molecular forms of gonadotropin-releasing hormone in the lizard (Podarcis s. sicula) brain.

The immunohistochemical presence and the distribution pattern of four different molecular forms of gonadotropin-releasing hormone (GnRH) were investigated in the brain of both sexes of the lizard, Podarcis s. sicula. Animals used in this study were collected in November and April, representing two different periods of the reproductive cycle. The antisera used were those raised against synthetic mammalian GnRH, chicken GnRH-I and II, and salmon GnRH. Strong immunoreaction was obtained for salmon, chicken-I, and chicken-II GnRHs, whereas a very weak reaction was seen for the mammalian form of GnRH. The distribution of immunoreactive-GnRH perikarya and fibers did not vary with the sex, the reproductive condition of the animals, or the antiserum used. Also, the intensity of immunoreaction with any one antiserum was quite similar in both periods of the year and in all brains examined. The immunoreactive perikarya was seen as two distinct groups, one in the mesencephalon and the other in the infundibulum. Immunoreactive fiber endings were seen in the telencephalon, the optic tectum, the anterior preoptic area, the median eminence, the central grey matter, the rhombencephalon, and the cerebellum. No immunoreactive perikarya were seen in the telencephalon or the anterior preoptic area.     

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.     

Vitellogenesis in the lizard Lacerta vivipara jacquin. I. Purification and partial characterization of plasma vitellogenin

The polypeptide moiety of lizard plasma vitellogenin was reproducibly dissociated and separated by SDS-PAGE into two subunits: Vg alpha 2-2.2 X 10(5) Da and Vg beta 1-1.1 X 10(5) Da. In estrogenized females, active incorporation of inorganic 32P into both vitellogenin chains present in the plasma, and in the culture medium of the liver, was identified following autoradiography of SDS-polyacrylamide gels. Lacerta vivipara vitellogenin was isolated from pooled plasma collected from heavily estrogenized males by the two-step precipitation procedure of H. S. Wiley, L. Opresko, and R. A. Wallace, (1980, Anal. Biochem. 97, 145-152), followed by chromatography on DEAE-cellulose. This preparation of L. vivipara vitellogenin was of sufficient purity to generate in rabbits an immune serum which cross-reacted very slightly with plasma free of vitellogenin (rocket immunoelectrophoresis). Using the double immunodiffusion procedure it was shown that the anti-vitellogenin serum recognized identical antigenic determinants in plasma from a vitellogenic female or from estrogenized lizards, and in crude vitellus. The immunodetection of L. vivipara native vitellogenin consistently allowed two circulating forms to be distinguished. After autoradiography of a rocket immunoelectrophoresis plate it was demonstrated that both native forms had incorporated inorganic 32P into the polypeptide moiety and/or the phospholipid moiety.     

The insulin-like growth factor binding proteins in uncultured human cartilage: increases in insulin-like growth factor binding protein 3 during osteoarthritis

OBJECTIVE: To assess changes in the insulin-like growth factor binding proteins (IGFBPs) in uncultured cartilage during stages of osteoarthritis (OA), and to determine if OA cartilage is capable of autocrine secretion of IGFBPs. METHODS: Articular cartilage was dissected from fibrillated and nonfibrillated sites of 11 human femoral heads, and extracted in buffer containing 8M urea. IGFBPs were identified by immunoprecipitation and subsequent analysis by (125)I-IGF-2 Western ligand blotting (WLB), radioimmunoassay, or 2-site immunoradiometric assay (IRMA). IGFBPs were assessed in cartilage extracts by WLB. IGFBP-3 content was determined by IRMA and synthesis by metabolic labeling with (35)S-cysteine in organ cultures. RESULTS: Sample grouping into 3 distinct OA strata was supported by gross pathology of the femoral heads, histologic grading of cartilage slices, and biochemical analysis of the glycosaminoglycan and protein content of the extracts. Group I was normal/mild OA, group II was intermediate OA, and group III was severe OA. IGFBP-2 was present in all samples, IGFBP-4 in sporadic samples, and BP-3 in group II-III samples. By IRMA, group I had a mean +/- SD of 6.26 +/- 2.6 ng IGFBP-3/mg soluble protein (IGFBP-3) (n = 6), group II had a mean +/- SD 14 +/- 7.5 IGFBP-3 (n = 10), and group III had a mean +/- SD 17.03 +/- 8.94 IGFBP-3 (n = 6). Analysis of variance showed group differences (F[3,19] = 3.84, P = 0.04), and post hoc tests revealed that IGFBP-3 levels were higher for group III versus group I (P = 0.04). OA cartilage synthesized IGFBP-3. CONCLUSION: Increases in net cartilage content of IGFBP-3 occurred in intact OA cartilage, reaching statistically significant elevation in severe disease. There was autocrine IGFBP-3 production in OA cartilage.     

Minimal exclusion of plasma membrane proteins during retroviral envelope formation

The retrovirus forms its envelope by budding at the plasma membrane (PM). This process is primarily driven by its cytoplasmic core-precursor protein, Gag, as shown by the efficient formation of virus-like Gag particles in the absence of its envelope protein, Env. Most interestingly, several studies have demonstrated incorporation of various PM proteins into retrovirus, but the underlying mechanism of this phenomenon has remained elusive. We have purified Moloney murine leukemia virus Gag particles by sedimentation in an iodixanol gradient and donor PMs by flotation in a sucrose gradient and compared their protein compositions at equal lipid basis. We found that most PM proteins are present at similar density in both membranes. The inclusion of PM proteins was unaffected by incorporation of Env protein into the envelope of the Gag particles and whether these were produced at high or low level in the cells. These findings indicate that most PM proteins become incorporated into the retrovirus envelope without significant sorting. This feature of retrovirus assembly should be considered when studying retrovirus functions and developing retrovirus vectors.     

Metabolic regulation of the growth hormone independent insulin-like growth factor binding protein in human plasma

This study examines the regulation of circulating GH-independent insulin-like growth factor binding protein, BP-28. Commencing at 22.00 h, BP-28 in 5 normal adults rose 11-fold to peak values of 120 +/- 12 micrograms/l, remained elevated between 01.00 and 08.00 h, then fell rapidly following a meal. If meals were omitted, BP-28 remained at peak levels throughout the day. The fasting BP-28 level was higher in women (141 +/- 22 micrograms/l, N = 5) than men (59 +/- 14 micrograms/l, N = 7), and pregnancy caused a further 2-fold elevation. Oral glucose rapidly lowered BP-28 in diabetic and nondiabetic pregnant women, nonpregnant women, and men. In a heterogeneous group of 18 subjects, insulin (0.1 U/kg iv), with or without simultaneous administration of GnRH and TRH, elicited a 3- to 4-fold rise in BP-28, commencing 60 min after the nadir of plasma glucose, and independent of the response in GH, PRL, TSH, LH or cortisol. We conclude that BP-28 levels in adults are metabolically regulated, and postulate a role for this protein in the maintenance of glucose homeostasis.