PATZ1 gene has a critical role in the spermatogenesis and testicular tumours.

PATZ1 is a recently discovered zinc finger protein that, due to the presence of the POZ domain, acts as a transcriptional repressor affecting the basal activity of different promoters. To gain insights into its biological role, we generated mice lacking the PATZ1 gene. Male PATZ1(-/-) mice were unfertile, suggesting a crucial role of this gene in spermatogenesis. Consistently, most of adult testes from these mice showed only few spermatocytes, associated with increased apoptosis, and complete absence of spermatids and spermatozoa, with the subsequent loss of tubular structure. The analysis of PATZ1 expression, by northern blot, western blot and immunohistochemistry, revealed its presence in Sertoli cells and, among the germ cells, exclusively in the spermatogonia. Since PATZ1 has been indicated as a potential tumour suppressor gene, we also looked at its expression in tumours deriving from testicular germ cells (TGCTs). Although expression of PATZ1 protein was increased in these tumours, it was delocalized in the cytoplasm, suggesting an impaired function. These results indicate that PATZ1 plays a crucial role in normal male gametogenesis and that its up-regulation and mis-localization could be associated to the development of TGCTs.

Sequence analysis and androgen regulation of MHG07 (Male harderian gland) mRNA in male hamster harderian gland.

The hamster Harderian gland (HG), a compound tubuloalveolar gland located in the orbital cavity, displays sex dimorphism. The present study focuses on the sequence analysis of a cDNA clone named MHG07 and on the regulation of its expression by steroid hormones. MHG07 mRNA (5.0 kb) is expressed in male HG only. The MHG07 cDNA (1.74 kb) shows an ORF of 94 amino acids and has no significant homologies with other polypeptides/genes. Castration leads to the disappearance of MHG07 mRNA after 4 days, whereas treatment with testosterone impairs the effect of castration. No MHG07 mRNA has been found in either rat or murine HGs. Androgen (A) administration to female hamsters induces the appearance of MHG07 mRNA. In primary culture of male hamster HG, androgens increase the MHG07 expression and this effect is blocked by both flutamide and cycloheximide. Dose-response experiments show that, at low A concentration (10(-12) M), the MHG07 was higher than that of the control (2-fold). This effect reaches its zenith at 10(-8) M (10-fold). This picture is paralleled by androgen receptor mRNA expression. It is argued that the expression of MHG07 is under androgenic control.

Morphology of the Harderian gland of the Gecko, Tarentola mauritanica.

The Harderian gland of the gecko, Tarentola mauritanica, was studied at the histological, histochemical, and ultrastructural levels. It is a nonlobate compound acinar gland surrounded by a thin capsule of connective tissue. Numerous connective tissue-type mast cells, ultrastructurally similar to those described in other higher vertebrates, were identified in the interstitial tissue between the acini. Pyramidal or columnar-shaped secretory glandular cells were observed in the acini. In the glandular cells, two types of structures could be distinguished on the basis of their high or low electron density. Lipid droplets were found in the cytoplasm of the Harderian gland of both sexes. Histochemical tests showed that the Harderian gland of the gecko is a seromucous gland. The secretion is essentially merocrine, although an apocrine type of secretion is sometimes observed.

Atrial natriuretic peptide, bradykinin, and angiotensin II-like immunoreactivity in the harderian gland of the terrapin Pseudemys scripta: response to osmotic stress.

The Harderian gland of the terrapin Pseudemys scripta has four types of acinar cells. Type IV cells are very similar to the salt secreting cells of the salt secretory glands of various marine vertebrates. The presence and localization of the Ile5-Angiotensin II, Atrial Natriuretic Peptide, and Bradykinin has been investigated by immunohistochemical methods. Immunoreactivity is confined to the type IV cells. Changes in the environmental salinity resulted in different patterns in the immunoreactivity especially after incubation with Ab-Angiotensin II and Ab-Atrial Natriuretic Peptide. Immunoreactive Angiotensin II cells are more numerous in animals maintained in distilled water, when reabsorption of sodium is needed. In contrast, immunoreactive Angiotensin II cells are very few in animals maintained in seawater. On the contrary, the number of immunoreactive cells for Atrial Natriuretic Peptide is high in seawater maintained animals, and weaker in animals in distilled water. The type IV cell may be considered a candidate for ion regulation in the terrapin Harderian gland.

Cell biology of the harderian gland.

The harderian gland is an orbital gland of the majority of land vertebrates. It is the only orbital gland in anuran amphibians since the lacrimal gland develops later during phylogenesis in some reptilian species. Perhaps because it is not found in man, little interest was paid to this gland until about four decades ago. In recent years, however, the scientific community has shown new interest in analyzing the ontogenetic and morphofunctional aspects of the harderian gland, particularly in rodents, which are the preferred experimental model for physiologists and pathologists. One of the main characteristics of the gland is the extreme variety not only in its morphology, but also in its biochemical properties. This most likely reflects the versatility of functions related to different adaptations of the species considered. The complexity of the harderian gland is further shown in its control by many exogenous and endogenous factors, which vary from species to species. The information gained so far points to the following functions for the gland: (1) lubrication of the eye and nictitating membrane, (2) a site of immune response, particularly in birds, (3) a source of pheromones, (4) a source of saliva in some chelonians, (5) osmoregulation in some reptiles, (6) photoreception in rodents, (7) thermoregulation in some rodents, and (8) a source of growth factors.

Protein extraction and western blotting from methacarn-fixed tissue.

Polyacrylamide gel electrophoresis (PAGE) of proteins with subsequent western blotting has become a routine technique for the analysis of proteins from both cultured cells and fresh whole tissue. We have developed a method to extract proteins from methacarn-fixed tissue which renders them suitable for SDS-PAGE and western blotting. With a panel of antibodies to specific intermediate filaments, transforming growth factor-alpha (TGF-alpha), and albumin, immunohistochemistry was performed in parallel with western blotting on sections cut from methacarn-fixed samples of normal rat liver and liver from rats treated under a regime which induces oval cell proliferation. Immunohistochemistry enabled the determination of changes in tissue distribution and abundance of the target proteins, which was mirrored by the corresponding western blot data. This technique can be especially effective when used in conjunction with immunohistochemistry. Tissue samples are easy to prepare, avoiding the precautions which need to be taken when handling fresh tissue (Abstract: J Pathol 1994; 173S: No. 41).

Seasonal variations in the daily rhythm of melatonin and NAT activity in the Harderian gland, retina, pineal gland, and serum of the green frog, Rana esculenta.

Day-night variations of melatonin content and N-acetyltransferase (NAT) activity were studied in the Harderian gland (HG), retina, pineal gland, and serum of the green frog Rana esculenta. Throughout the year the retinal melatonin content was correlated with retinal NAT activity and was always higher than those in the pineal gland and HG. On the other hand, in these structures diurnal fluctuations in NAT activity were observed. There were clear seasonal differences in the magnitude of the nocturnal increase of retinal melatonin levels as well as in the nocturnal pattern of retinal NAT activity. In summer day-night variations of melatonin and NAT are absent. The prevailing photoperiod seems to affect melatonin and NAT circadian rhythms in R. esculenta.

Osmoregulation at the Zoological Station of Naples at the end of the 19th century.

The Zoological Station of Naples was founded in 1872 by Anton Dohrn as a research institute for zoology and comparative anatomy. Although the original fields of interest were the morphology of vertebrates and comparative embryology, a department of physiology was added to the station in 1888. Osmoregulation in marine organisms has been extensively studied, notably by Bottazzi, who investigated chemical composition, electrical conductivity, surface tension, osmotic pressure and extracellular viscosity in circulating fluids in man and lower animals. Bottazzi classified aquatic animals into 2 groups, a distinction that is accepted today. More recent workers at the station include Bern, who made important contributions to the study of the essential role played by prolactin in regulation of hydromineral metabolism in euryhaline teleost fish in a freshwater environment.

Regional and seasonal variations of RNA synthesis in the brain of the green frog, Rana esculenta.

Changes of RNA synthesis were demonstrated in neurons and ependymal cells of the green frog Rana esculenta during the annual cycle using the Mallory's trichrome stain as histochemical marker and autoradiography. Since the higher affinity of the nuclei for aniline blue is consistent with the increase of RNA content, the increase of RNA synthesis was expressed as percentage of the blue stained nuclei (% BSN). Neuronal transcription starts slowly in March or April, reaches a maximum in July and declines from September to November or December, depending on the brain region. In the ependymal cells, RNA synthesis starts in March and lasts until October. Neuronal transcriptional activity is found mostly in the glomerular layer of the olfactory bulb, in the striatum, nucleus accumbens septi, lateral and medial septal nuclei of the telencephalon, in the habenulae and various nuclei of the diencephalon, in the tectum opticum (particularly in the stratum griseum centrale), in the molecular layer of the cerebellum and in various nuclei of the rhombencephalon. The transcriptional activity of the ependymal cells is quite uniform in the lateral ventricles and the fourth ventricle, while it shows regional symmetric distribution in the third ventricle. Seasonal differences in transcriptional activity appear to be independent of seasonal thermic and photoperiodic fluctuations. In fact, temperature and photoperiod manipulations do not modify significantly the number of active nuclei. It is likely that the increase of RNA synthesis in nerve and ependymal cells corresponds to the resumption of neurotransmitter biosynthesis after hibernation. The simple Mallory's trichrome stain provides a reliable method for revealing increased transcriptional activity in histological sections.

Effect of castration and testosterone therapy on harderian gland protein patterns of the golden hamster (Mesocricetus auratus).

1. Sodium dodecyl sulphate 7-12% gradient polyacrylamide gel electrophoresis of male and female hamster Harderian gland whole homogenate shows a clear-cut sexual dimorphism, which consists of the presence of two male-specific glycoproteins (168 and 116 kDa) and two specific female proteins (210 and 190 kDa). 2. In the male, castration causes a significant decrease in the concentration of the two glycoprotein fractions. 3. Replacement therapy with testosterone propionate (T) restores the intact male pattern.

Resumption of testicular activity in Gobius paganellus after administration of ethane 1,2-dimethane sulfonate (EDS).

1. The effect of a single injection of ethane-1,2-dimethane sulfonate (EDS) was studied in the teleost fish, Gobius paganellus in two different periods of the year. 2. During June EDS did not induce any change, while during December the drug was highly effective in promoting testicular activity. 3. Nucleus/cytoplasm ratio of interstitial cells strongly decreased concomitantly with the detection of high testicular androgen levels. 4. The germinal compartment was well developed showing the appearance of all spermatogenic stages and the cavity of lobular compartments filled of spermatozoa. 5. Our data are the first evidence of a stimulatory activity of EDS on testes of a vertebrate species.

Testosterone induction of poly(A)(+)-RNA synthesis and [35S]methionine incorporation into proteins of Rana esculenta Harderian gland.

The role of androgens in the cyclic secretory activity of the Rana esculenta Harderian gland (HG) was studied. Total RNA showed a dramatic increase in October and May when the nuclear androgen receptors peak. During the resumption of the secretory activity a gradual increase of poly(A)(+)-RNA was detected; during the enhancement phase (May) a peak of the poly(A)(+)-RNA fraction was found. In in vitro experiments testosterone increased the incorporation of [3H]uridine into the poly(A)(+)-RNA fraction and also that of [35S]methionine into a newly synthesized protein fraction (100 kDa). The latter effect is prevented by the exposure of the cells to the antiandrogen, cyproterone acetate (CPA). These findings reveal that, besides hamsters, the HG is a target for androgens in the frog.

Ultrastructural investigation of the corpora atretica of the electric ray, Torpedo marmorata.

Follicular atresia was studied in the ovary of the electric ray, Torpedo marmorata, by light and electron microscopy. The course of atresia may be divided into four stages. The first two comprise the dissolution of the oocyte and its phagocytosis by the small cells of the granulosa epithelium. The third stage consists of the transformation of the granulosa epithelium into an active glandular structure and is accompanied by the development of a smooth endoplasmic reticulum. The fourth stage is marked by sclerosis and pigmentary degeneration of the atretic follicle. Together these observations suggest an endocrine steroidogenic role for the corpora atretica (preovulatory corpora lutea) in T. marmorata.

Plasma and follicular tissue steroid levels in the elasmobranch fish, Torpedo marmorata.

Steroid concentrations in plasma and follicular tissues (theca plus granulosa layers) were determined by radioimmunoassay in the aplacental viviparous ray, Torpedo marmorata, during various stages of the reproductive cycle. Steroids in the uterine fluid of pregnant animals and in preovulatory atretic follicles were also measured. In the follicular tissue of cyclic animals, levels of progesterone were always lower than those of estradiol-17 beta and androgens (testosterone plus 5 alpha-dihydrotestosterone). Estradiol-17 beta and androgen levels increased as the animals approached the ultimate maturational stage before ovulation. Androgens were not detectable in plasma, while estradiol-17 beta increased dramatically before ovulation. In pregnant animals, only small ovarian follicles (less than 5 mm in diameter) were observed, and these had hormone concentrations that were similar to those of the small follicles of cyclic animals. Progesterone was the only steroid detected in the uterine fluid of pregnant animals. In completely sclerotic atretic follicles of pregnant animals, steroids were not detected. Progesterone was the main hormone in atretic follicles undergoing yolk resorption. This suggests that the latter may contribute to the elevated plasma progesterone concentrations of pregnant animals.

Mallory stain may indicate differential rates of RNA synthesis: I. A seasonal cycle in the harderian gland of the green frog (Rana esculenta).

When Mallory's trichrome stain is used, acinar nuclei of the Harderian gland of Rana esculenta display different affinities for the dye. Some of the orangiophilic nuclei show affinity for aniline blue (blue nuclei). In the Harderian gland of Rana esculenta their number and the intensity of staining with aniline blue may vary during the year. The affinity for aniline blue disappears following digestion of paraffin sections with RNAase, but not with DNAase or trypsin. Furthermore, in vitro incubation with [5, 6-3H]-Uridine shows a selective incorporation by the majority of blue nuclei. Therefore, the affinity for aniline blue is likely due to increased RNA synthesis. The increment of nuclear RNA shown by these methods is supported by the quantitative determination of total RNAs during the resumption (October) and enhancement (May) of secretory activity, when the percentage of blue nuclei of the acinar cells is at its highest levels of the year. The affinity of RNA-rich nuclei for aniline blue, while others are strictly orangiophil, is discussed on the basis of molecular structure of the dyes used in the staining mixture. Mallory's trichrome stain appears to be an useful tool for detecting changes in cell nuclear status.

Androgen receptor in the Harderian gland of Rana esculenta.

An androgen receptor has been identified in the cytosolic and nuclear extracts of the Harderian gland of the frog, Rana esculenta. A single class of high-affinity binding sites was found: Kd = 1.9 +/- 1.3 (S.D.) nmol/l (n = 26) for the cytosolic extract and Kd = 0.9 +/- 0.8 nmol/l (n = 15) for the nuclear extract. The presence of binding activity in both nuclear and cytosolic extracts and the low rate of ligand-receptor dissociation are characteristics that distinguish this receptor from a steroid-binding protein. The Kd did not show any sex difference and did not exhibit any secretory activity-related change. Binding in both cytosolic and nuclear extracts was specific for androgens (testosterone = 5 alpha-dihydrotestosterone); oestradiol-17 beta showed a 30% cross-reaction; moreover, specific binding of [3H]oestradiol-17 beta was not detectable. The binding capacity of the Harderian gland increased progressively in both fractions from October to December, reaching a peak in May, and decreased suddenly during July to August. The lack of any morphological sex-related difference in the Harderian gland of the green frog might be accounted for by the high amount of circulating androgens as well as a similar concentration of androgen receptor in both sexes.

Regulation of the testicular activity in the marine teleost fish, Gobius paganellus.

Seasonal variations of intratesticular steroid hormones (androgens and estradiol-17 beta) and spermatogenic activity have been studied in the marine teleost fish, Gobius paganellus. In addition, in vivo and in vitro experiments have been carried out in order to investigate the control of androgen production by the testis. While estradiol was never detected, androgens were at low values in autumn and reached maximal levels in spring concomitantly with the highest testis weight and the highest efficiency of the spermatogenic wave. In vitro incubations were carried out using ovine luteinizing hormone (oLH) (400, 4000, and 40,000 micrograms/liter; 20 degrees for 6 and 24 hr). The effective dose 40,000 micrograms/liter was used to induce androgen stimulation in both autumn and spring testes. The responsiveness to oLH was enhanced in spring testis. Estradiol and a gonadotropin-releasing hormone analog GnRHA (HOE766) were ineffective in modulating androgen production either alone (1-1000 nmol/liter) or in concert with oLH during short-term incubations. In intact animals, GnRHA elicited, 3 hr after the injection (10 micrograms), a three-fold increase of intratesticular androgen content. In conclusion, we show that the annual androgen profile in G. paganellus parallels the spermatogenic activity and that the androgen production is not affected in these experimental conditions by putative intratesticular factors (e.g., estradiol-17 beta and GnRH-like substances) which, conversely, are effective in inducing androgen changes in several vertebrate species.

Indirect evidence for a physiological role exerted by a "testicular gonadotropin-releasing hormone" in the frog, Rana esculenta.

The possible physiological role of a putative testicular gonadotropin-releasing hormone (GnRH)-like material was studied in the frog, Rana esculenta. We have investigated (a) changes of mitotic index (MI) of primary spermatogonia (SPG) in GnRH agonist (GnRH-Ag)-treated testes in vitro; (b) changes of androgen concentrations in testes of intact frogs treated with a GnRH antagonist (GnRH-Ant); (c) variations of mitotic index of primary SPG in intact GnRH-Ant-injected animals and in testes incubated with GnRH-Ant; (d) changes of MI in hypophysectomized (PDX) animals treated with hypophysis (PD) homogenate, and hCG alone or in combination with GnRH-Ant; and (e) changes of androgen concentrations in plasma PDX frogs treated with hCG and hCG plus GnRH-Ant. Our results indicate that while GnRH-Ag induced accumulation of primary SPG mitosis, GnRH-Ant inhibited androgen production and natural occurring mitosis accumulation. Therefore, GnRH-Ant may counteract an endogenous peptide working into the testis.

Characterization of gonadotropin-releasing hormone (GnRH) binding sites in the pituitary and testis of the frog, Rana esculenta.

Frog, Rana esculenta, pituitary and testis gonadotropin-releasing hormone (GnRH) receptors were characterized by using 125I-chicken IIGnRH (cIIGnRH) as radiolabeled ligand. At 4 C equilibrium binding of 125I-cIIGnRH to pituitary homogenates was achieved after 90 min of incubation; binding of 125I-cIIGnRH to testis membrane fractions reached its maximum at 60 min of incubation. Binding of the radioligand was a function of tissue concentration, with a positive correlation over the range 0.5-2 tissue equivalents per tube. One pituitary and one testis per tube were used as standard experimental condition. Incubation of the pituitary homogenate with increasing concentrations of 125I-cIIGnRH indicated saturable binding at radioligand concentrations of 1 nM and above while for the testis membrane preparation saturation was achieved using 5 nM 125I-cIIGnRH. The binding of 125I-cIIGnRH was found to be reversible after addition of the cold analog and the displacement curves could be resolved into one linear component for both tissues. Scatchard analysis suggested the presence of one class of binding sites for both pituitary and testis (Pituitary: Kd = 1.25 +/- 0.14 nM and Bmax = 8.55 +/- 2.72 fmol/mg protein; testis: Kd = 2.23 +/- 0.89 nM and Bmax = 26.48 +/- 7.39 fmol/mg protein). Buserelin displaced the labeled 125I-cIIGnRH with a lower IC50 as compared with cIIGnRH cold standard, while Arg-vasopressin (AVP) was completely ineffective, confirming the specificity of binding.