Genetics of Vesicoureteral Reflux.

Vesicoureteral reflux (VUR) is the retrograde passage of urine from the bladder to the upper urinary tract. It is the most common congenital urological anomaly affecting 1-2% of children and 30-40% of patients with urinary tract infections. VUR is a major risk factor for pyelonephritic scarring and chronic renal failure in children. It is the result of a shortened intravesical ureter with an enlarged or malpositioned ureteric orifice. An ectopic embryonal ureteric budding development is implicated in the pathogenesis of VUR, which is a complex genetic developmental disorder. Many genes are involved in the ureteric budding formation and subsequently in the urinary tract and kidney development. Previous studies demonstrate an heterogeneous genetic pattern of VUR. In fact no single major locus or gene for primary VUR has been identified. It is likely that different forms of VUR with different genetic determinantes are present. Moreover genetic studies of syndromes with associated VUR have revealed several possible candidate genes involved in the pathogenesis of VUR and related urinary tract malformations. Mutations in genes essential for urinary tract morphogenesis are linked to numerous congenital syndromes, and in most of those VUR is a feature. The Authors provide an overview of the developmental processes leading to the VUR. The different genes and signaling pathways controlling the embryonal urinary tract development are analyzed. A better understanding of VUR genetic bases could improve the management of this condition in children.

Effects of dietary Lactobacillus acidophilus and Bacillus subtilis on laying performance, egg quality, blood biochemistry and immune response of organic laying hens.

The objective of this study was to evaluate the effects of two different probiotic micro-organisms on the performance, egg quality and blood parameters of organically reared hens. A total of 900 16-week-old Hy-Line layer hybrids were randomly assigned to three groups of 300 birds each. The control (CTR) group was fed a corn-soya bean cake-based diet; the L group was fed the same diet supplemented with 0.1% Lactobacillus acidophilus, while the B group was fed the same diet supplemented with 0.05% Bacillus subtilis. Data were recorded at the beginning (weeks 5 and 6: T1) and at the end (weeks 19 and 20: T2) of the experiment, and no differences in hen performance were recorded between dietary groups or sampling times. All of the investigated clinical chemistry parameters, except GGT, were affected by diet (p < 0.05), with the best results recorded for the probiotic-treated groups. The immune-response values showed higher blood bactericidal activity in the B and L groups at T2 (p < 0.05) and a lower lysozime concentration in the B group at T1. Higher antibody production against Newcastle disease virus was observed in the L group compared to the CTR (p = 0.013). No differences in oxidative status were recorded, and no effects of diet on egg quality were observed. Among the physical egg characteristics, only the Roche scale colour was affected by diet (p < 0.05): the egg yolk was paler in the L group. The age of the hen was the most relevant factor affecting physical egg characteristics. The chemical parameters of the egg were almost unaffected by supplementation with probiotics except for the lipid content, which decreased with the L diet (p < 0.05). Both probiotic inclusions had beneficial effects on hen metabolism and welfare, and L. acidophilus induced the best immune response.

Circulating cytokines present in the serum of peripheral arterial disease patients induce endothelial dysfunction.

Peripheral arterial disease (PAD) is a chronic condition caused by atherosclerosis and is a severe complication of type 2 diabetes (T2D). We hypothesised that chronic condition of arterial disease engenders inflammation and endothelial damage in response to circulating cytokines released in the blood stream of PAD patients. We explored the levels of circulating cytokines in PAD patients with and without diabetes by multiplex cytokine array compared with non-PAD controls. Serum from PAD patients with or without diabetes showed high levels of VEGF, IFN-gamma, TNF-alpha, MCP-1, and EGF. VEGF levels correlated with TNF-alpha and IFN-gamma, significantly. Endothelial cells (ECs) were exposed to the different altered cytokines to evaluate changes in cell growth, migration and tubule-like formation, displaying impairment on proliferation, migration and tubule formation. Our findings demonstrate that a set of cytokines is significantly increased in the serum of PAD patients. These cytokines act to induce endothelial dysfunction synergistically. VEGF strongly correlated with TNF-alpha and IFN-gamma, opening new therapeutic perspectives.

Posterior urethral polyp causing haematuria in children.

Posterior urethral hemangioma (PUH) is a rare congenital lesion, included in group of polypoid or papillary lesion of the prostatic urethra. This lesion is responsible for a variety of symptoms in children that may be associated or isolated, sometimes its finding is occasional. The diagnosis is usually made by ultrasonography and cystourethrogram, but the gold standard is represented by the urethrocystoscopy with double possibility: diagnostic and therapeutic. The Authors report a case of 1-year-old boy with persisting haematuria, in whom a previews cystoscopy didn't find any cause of haematuria. An accurate urethrocystoscopy let to make diagnosis of prostatic urethral polyp, a transurethral resection was performed and pathological assessment confirmed the diagnosis of PUH.

Diagnosis of Hirschsprung's Disease: an age-related approach in children below or above one year.

AIM: The aim of this study was to evaluate the effectiveness of a differential diagnostic approach to Hirshchsprung's Disease (HD) on the basis of age. METHOD: Data on 185 consecutive children with suspected HD were subjected to an age-related diagnostic approach. The patients were divided into two groups according to age (A < 1 year; B > 1 year). Children in Group A had rectal suction biopsy (RSB) and contrast enema (CE), and in Group B anorectal manometry (ARM) was performed. Patients with a normal recto-anal inhibitory reflex (RAIR) underwent bowel disimpaction and medical treatment. Only selected cases in Group B underwent RSB and CE. RESULTS: In Group A (18 patients) CE showed a colonic transitional zone in three patients, whereas RSB led to the diagnosis of HD in nine. In Group B (167 patients) ARM was not possible in seven patients and it was normal in 140 (normal anal sphincter pressure: 83; hypertonia of the internal anal sphincter: 57). The RAIR was negative in 20 patients. RSB performed in 31 children in Group B confirmed HD in three patients. CONCLUSIONS: For patients with a neonatal onset of constipation RSB is the best diagnostic technique. Chronic constipation is rarely due to HD and ARM is a useful non-invasive screening tool.

Expression and localization of the deubiquitinating enzyme mUBPy in wobbler mouse testis during spermiogenesis.

Mouse ubiquitin-specific processing protease (mUBPy) is a deubiquitinating enzyme highly expressed in both brain and testis. In testis, it interacts with the DnaJ protein, MSJ-1; both mUBPy and MSJ-1 are located on the cytoplasmic surface of the developing acrosome and in the centrosomal region during spemiogenesis. Present data show the first appearance in testis of mUbpy mRNA and protein at 10 days post-partum (d.p.p.). In addition, to investigate on a possible role of mUBPy in sperm formation, we took advantage of mutant wr/wr (wobbler) mice characterized by male infertility, which is likely due to the lack of a real, functional acrosome. RT-PCR and Northern blot analyses show that mUbpy is up-regulated in adult wobbler testis. Furthermore, in wild-type testis mUBPy protein is primarily detected by Western blot in the soluble (cytosolic/nuclear) fraction during the first round of spermatogenesis and in the adult. By contrast, mUBPy is primarily detected in membranous/insoluble protein fraction when wobbler phenotype is clearly shown (30 d.p.p.) and in adult wobbler testis. By immunohistochemistry, whereas in wild-type animals mUBPy marks the profile of the acrosomic vesicle in differentiating spermatids, in wobbler mice only a detergent pre-treatment procedure allows to detect mUBPy immunoreactivity, which results in diffuse spotted granules inside the cytoplasm and around the nuclear shape. In conclusion, in wobbler testis expression of mUbpy is up-regulated, while a differential sorting of the protein characterizes wobbler spermatids where acrosome formation is impaired.

Meckel's diverticulum perforation in a newborn positive to Sars-Cov-2.

The health emergency linked to the Sars-Cov-2 infection represented an absolutely new problem for all health professionals. In particular, the information regarding the spread of the virus in the pediatric field and its manifestations are still incomplete. In this paper we present a case of neonatal infection which, as far as we know, represents one of the few published cases and which occurred in a patient who came to our attention for acute abdomen from intestinal perforation. The perforation was caused by Meckel's diverticulum, an event considered infrequent in the first year of life and almost exceptional in the neonatal period. This case required particular management, putting pediatric surgeons in front of new and difficult to solve problems. New onset clinical events, such as this one described, represent an opportunity for sharing useful data for the creation of universal protocols for the management of patients with problems that are becoming common and of which little is known.

Role of anorectal manometry in children with severe constipation.

OBJECTIVE: Constipation is one of the most frequent disorders of the digestive tract in children and it can be an important problem in paediatric and surgical practice. Most of the time, the cause is psychological or because of a slowing of colonic transit, but it can be a sign of organic gastrointestinal outlet obstruction. Some patients with chronic constipation are resistant to a medical approach and they present with a severe form of constipation that needs recurrent hospital admission. Anorectal manometry (ARM) is a noninvasive procedure and it helps to explain the mechanisms of defecation disorders. The aim of the present study was to evaluate the role of ARM in children with severe constipation. METHOD: From October 2003 to October 2006, in the Paediatric Surgery Unit, 85 children - aged more than 1 year - with severe constipation were seen. The mean age was 5 years (range, 1-13). At presentation, every child had abdominal and rectal examination in order to identify abdominal distension or faecal masses. Bowel preparation with enemas was performed before ARM in patient with a rectal faecaloma. Myoelectric activity of the internal anal sphincter and resting anal tone was recorded; recto-anal inhibitory reflex (RAIR) was tested to exclude Hirschsprung's disease (HD). Anal tone was considered normal until 50 cm H(2)O. When the RAIR was absent, the patient underwent rectal suction biopsies (RSB) for histology and acetylcholinesterase histochemistry. In cases of normal or high anal tone with the RAIR present, the child had bowel cleaning, medical treatment, 2- and 6-month follow-up. Children with ineffective treatment at follow-up underwent RSB. In case of HD, a laparoscopic-assisted endorectal pull-through (ERPT) according to Georgeson's technique was performed. RESULTS: Seventy per cent of the patients had bowel preparation before ARM. In four patients the ARM was impossible to assess because of crying. In 28 patients, the anal tone result was higher than 50 cm H(2)O and local treatment with anaesthetic agents was used for 8 weeks. Seventeen patients underwent RSB: 11 patients with RAIR absent/unclear, 4 noncooperative children and 2 patients with ineffective medical treatment at follow-up. HD was diagnosed in 2 patients and laparoscopic-assisted ERPT was performed. The remaining patients had good results at 6-month follow-up. CONCLUSION: ARM is a noninvasive diagnostic tool to study the mechanism of defecation in children with constipation in order to prescribe the appropriate treatment. This procedure can be used in every child - aged more than 1 year - with severe constipation and assessment of the RAIR can select the cases for RSB.

Features of microvessel density (MVD) and angiogenesis inhibitors in therapeutic approach of hepatocellular carcinoma (HCC).

OBJECTIVE: The curative hepatocellular carcinoma (HCC) therapy was traditionally based on surgical or loco-regional ablation approach. However, HCC is a solid tumor characterized by a highest level of vascularization; therefore, angiogenesis inhibitor could play a pivotal role in the pharmacological therapeutic approach. Despite the low number of approved drugs, a wide range of multi-kinase and MET inhibitor is currently being evaluated in phase II and III study. In this review, we described all the drugs that have shown efficacy in recently and ongoing trials. Moreover, the immunotherapy represents a recent challenge in the HCC treatment. The strategy based on the production of multi-epitope, multi-HLA peptide vaccine naturally processed and presented on primary tumor tissues of HCC patients. A further upgrade of cancer vaccine could be represented by the combination of metronomic chemotherapy and checkpoint inhibitors.

Characterization of Follicular Atresia Responsive to BPA in Zebrafish by Morphometric Analysis of Follicular Stage Progression.

Bisphenol A is an industrial chemical compound, pervasively polluting the environment and diet, classified as an endocrine disruptor because of its interference effects on the endocrine system. In zebrafish, BPA exposure induces follicular atresia. To acquire knowledge on this atretic effect, using a qualitative and quantitative histomorphological approach, we studied zebrafish ovarian follicular stage development in response to low BPA concentrations. Results show that BPA interferes with follicular progression by affecting the previtellogenic and vitellogenic phases. In particular, BPA exposure (i) increases follicular recruitment by acting on primary stage follicles, (ii) forces the follicular transition from stage III to stage IV producing enlarged stage IV follicles, and (iii) induces atresia by producing atretic follicles that are peculiarly enlarged (i.e., big atretic follicles). We suggest that BPA induces atresia by the primary effect on recruitment of stage I follicles. This forces follicular progression and produces stage IV follicles that are peculiarly enlarged that undertake the atretic development.

Transcription factor expression, RNA synthesis and NADPH-diaphorase across the rat brain and exposure to spatial novelty.

The molecular hypothesis of learning and memory processes is based on changes in synaptic weights in neural networks. Aim of this study was to map neural traces of exposure to a spatial novelty were mapped by (i) the transcription factors (TFs) c-fos, c-jun and jun-B using Northern blot and immunocytochemistry (ICC), (ii) RNA synthesis by (3)H-uridine autoradiography and RNA level, (iii) NADPH-diaphorase (NADPH-d) expression by histochemistry. Thus, adult male albino rats were exposed to a Làt-maze and sacrificed at different times. Non-exposed rats served as controls. The latter showed a low constitutive expression of TF, RNA synthesis and NADPH-d across the brain. Northern blots showed a three-fold increase in TFs in exposed versus non-exposed rats in the cerebral cortex. ICC showed in exposed rats several TFs positive cells in the granular and pyramidal layers of the hippocampus and later in all layers of the somatosensory cortex, in the granular layer of the cerebellar cortex. The TF-positivity was stronger in rats exposed for the first time, and was time and NMDA-dependent. Autoradiography for RNA synthesis showed positive cells in the ependyma, hippocampus and cerebellum 6h after testing, and in the somatosensory cortex 24h later. In addition, exposure to novelty induced NADPH-d in the dorsal hippocampus, the caudate-putamen, all the layers of the somatosensory cortex. and the cerebellum. The positivity was absent immediately after exposure, appeared within 2h and disappeared 24h later. A strong neuronal discharge by the convulsant pentylenetetrazol, strongly induced TFs but not din not affect NADPH-d 2h later. Thus, data suggest that the processing of spatial and emotional components of experience activates neural networks across different organization levels of the CNS.

Surgical management of Necrotizing Enterocolitis in an Incredibly Low Birth Weight infant and review of the Literature.

Survival of preterm infants have dramatically improved over the last decades. Nonetheless, infants born preterm remain vulnerable to many complications, including necrotizing enterocolitis (NEC). The severity of the disease and the mortality rate are directly correlated with decreasing gestational age and birth weight. Despite surgical treatment mortality rate remains very high in extremely premature infants, especially in newborns at the lowest limit of viability. Survival of infants of birth weight (BW) below 750 g has been increasingly reported in recent years, however the overall mortality in extremely low "BW" infants (ELBW) requiring surgery for NEC has not decreased over the past years. We describe our experience with a male preterm infant who survived after an ileostomy procedure for Bell stage II NEC, with improving neuromotor skills at 2 years follow up. Although standard indication to surgery is Bell stage III, in our case the choice of minimal laparotomy, exploration of the bowel and ileostomy at Bell stage II was safe and effective. Our experience suggest that surgery has not a negative impact on survival and ileostomy could prevent further damage of the bowel in NEC. We hypothesize that indication to surgery at an earlier stage may prevent further progression of the disease without a significantly negative impact on survival. Further studies are needed to confirm the appropriateness of this approach in ELBW infants.

Alcohol sclerotherapy: a new method for Bartholin gland cyst treatment.

AIM: Bartholin gland cysts are one of the most common gynecologic problems. Around 2% of women suffer from these pathologies. Bartholin gland cyst are generally asymptomatic, but sometimes extremely painful to restrict physical activity. The treatment choice is related to the patient's age, the size of the cyst or abscess and relapses, but different approaches are possible. The aim of this study is to investigate the efficicacy and safety of the alcohol sclerotherapy versus the only aspiration to cure symptomatic cysts or abscesses of the Bartholin's gland. METHODS: Between January 2002 and June 2004, 18 patients suffering from Bartholin symptomatic unilateral cysts or with abscess are selected. These patients have been divided into 2 groups and they have been treated with alcohol sclerotherapy or aspiration. The simple aspiration removes only the cyst fluid. The alcohol sclerotherapy allows to destroy the epithelial covering of the cyst by a coagulative necrosis and then a fibrosis which covers the cavity and prevents the reformation of liquid. RESULTS: Treatment has been satisfactory for all the patients, and treatment time has been shorter with alcohol sclerotherapy. None of the patients, in both groups, presented sexual dysfunctions or dyspareunia. CONCLUSIONS: Alcohol sclerotherapy might be an ideal and safe procedure in the treatment of the Bartholin's gland or abscesses with a low percentage of relapses.

Concomitant activation of MEK-1 and Rac-1 increases the proliferative potential of thyroid epithelial cells, without affecting their differentiation.

Activating point mutations in the Ras oncogene occur in a large number of human tumors, especially of epithelial origin. In thyroid follicular cells, ectopic expression of oncogenic H-Ras results in growth factor-independent proliferation, loss of differentiation and tumor formation in nude mice. In fibroblasts concomitant activation of the MAP kinase cascade and the small GTPase Rac-1 leads to full malignant transformation. We have tested the effects of these key downstream mediators of Ras in thyroid epithelial cells, by stably expressing either a constitutively active form of MEK-1 (MEK(deltaN3/S218E/S222D)), a constitutively active form of Rac-1 (Val12-Rac), or both. While the activation of one molecule or the other results in a weak phenotype, concomitant activation of both MEK-1 and Rac-1 in thyroid cells leads to growth factor-independent proliferation, morphological transformation and anchorage-independent growth. However, in contrast to Ras-transformed thyroid cells, the ones expressing the constitutively active forms of MEK-1 and Rac-1 maintain their differentiate phenotype and fail to form tumors when injected into nude mice. Thus, in thyroid epithelial cells, concomitant activation of MEK-1 and Rac-1 can reproduce only a subset of the Ras-induced effects and is not sufficient to cause full malignant transformation. Significantly, Ras-mediated increased proliferation and loss of differentiation can be dissociated in these cells.

Immediate early genes induced by H-Ras in thyroid cells.

Expression of oncogenic v-H-Ras in the thyroid cell line FRTL-5 (FRTL-5(Ras)) results in uncontrolled proliferation, loss of thyroid-specific gene expression and tumorigenicity. Concomitant expression of constitutively activated MEK and Rac, two major H-Ras downstream effectors, in FRTL-5 (FRTL-5(MEK/Rac)) recapitulates H-Ras effects on proliferation and morphology. In contrast to FRTL-5(Ras), however, FRTL-5(MEK/Rac) cells remain differentiated and are not tumorigenic. To find H-Ras induced genes potentially responsible for tumorigenicity and loss of differentiation, we have used subtractive suppression hybridization (SSH), a PCR-based cDNA subtraction technique, between de-differentiated and tumorigenic FRTL-5(Ras) cells and differentiated and non-tumorigenic FRTL-5(MEK/Rac) cells. We examined 800 of the cDNA clones obtained after subtraction and verified their levels of expression in the two cell lines by reverse northern, identifying 337 H-Ras induced genes. By sequence analysis, we clustered 57 different genes. Among these, 39 were known genes (involved in diverse signal transduction processes regulating mitogenic activity, cell survival, cytoskeletal reorganization, stress response and invasion) while the remaining 18 clones were novel genes. Among the 57 H-Ras specific clones, we identified those genes whose expression is induced early by H-Ras. We suggest that these immediate-early genes may play a crucial role in H-Ras-mediated transformation in thyroid epithelial cells.

Structure of msj-1 gene: a comparative analysis.

Msj-1 gene encodes a DnaJ protein highly expressed in spermatids and spermatozoa of both rodents and amphibians. We isolated and characterized the msj-1 gene in mice. A bioinformatic approach was then used to predict the putative promoter region, chromosomal localization, and its presence in the human genome. The analysis of msj-1 genomic sequence revealed that msj-1 is an intronless gene. Interestingly, two regions (A and B, separated by 10,682 bp) on human chromosome 2 having respectively 78% and 77% nucleotide identity with the murine msj-1 coding region were identified. This suggests the existence of an msj-1-like gene also in humans.

Functional antagonism between the estrogen receptor and Fos in the regulation of c-fos protooncogene transcription.

Estrogen hormones induce transient transcriptional activation of c-fos during the early phases of mitogenic stimulation of target cells. This is mediated by a functional estrogen response element (ERE) that in the human c-fos gene is localized 1kb up-stream of the transcription start site. This is the first known example of transient transcriptional activation induced by a steroid hormone acting via its nuclear receptor. Starting with the hypothesis that the product of c-fos (Fos) interferes with estrogen receptor (ER) activity on this gene promoter, generating in this way a feedback inhibition mechanism responsible for the rapid transcriptional down-regulation detected in vivo, we tested the effects of Fos overexpression on ER-mediated activation of the c-fos promoter in transfected HeLa cells. Transient transfection of an ER expression vector is followed by hormone-dependent trans-activation of reporter genes comprising the c-fos ERE linked to its own promoter. Coexpression of Fos in the cell induces a significant reduction in the activity of ER on the reporter genes. Fos antagonism is effective on both transcription activation functions of the receptor molecule and is independent of the nature of the target promoter. Furthermore, under the same experimental conditions, the estrogen-receptor complex antagonizes activation of an AP-1-responsive test gene by Fos. ER mutants deprived of the DNA-binding domain are efficient inhibitors of Fos activity, indicating that reciprocal antagonism is likely to be mediated by the formation of inactive complexes between the two factors. These results reveal the existence of a functional interference between the ER and Fos for regulation of c-fos protooncogene transcription. It is the first case in which the product of an estrogen-induced growth-related gene is shown to exert a negative feedback control on ER regulation of its own promoter.

In vivo functional analysis of the mouse estrogen receptor gene promoter: a transgenic mouse model to study tissue-specific and developmental regulation of estrogen receptor gene transcription.

Understanding the molecular and morphological basis of estrogen responsiveness in the various tissues and organs that make up an adult organism and its onset during ontogenesis requires identification of the genetic controls that determine timed expression of the estrogen receptor (ER) gene in multiple cell types. With this goal in mind, we describe here the results of the functional analysis of the mouse (m) ER gene promoter, carried out in vivo in transgenic mice. The mER gene promoter was cloned and spliced to the coding sequence of the bacterial lacZ gene (fused to the nuclear localization signal of SV40 large T: nls-beta-GAL) and then stably reintegrated into the genome of mice. Analysis of beta-GAL mRNA and protein expression in multiple organs of both female and male transgenic animals was then performed. Results show that the transgenic mER promoter, much like the endogenous one, is active in several organs and tissues of adult female and male mice. The first 0.4 kilobases of 5'-flanking DNA (up to -364) are sufficient to direct widespread expression of the transgene in mouse organs. This indicates that genetic elements functional in various cell types are included in this segment. Furthermore, the first exon and intron of the mER gene are necessary to achieve sexually dimorphic expression of the transgene in neurons located at specific sites within the central nervous system. These mER promoter transgenic mice will be useful in mapping estrogen- responsive cell types under different physiological and pathological conditions in vivo, in defining ontogenesis of estrogen action in the mouse, and in studying the mechanisms that regulate ER gene transcription.

A novel autoregulatory loop between the Gcn2-Atf4 pathway and (L)-Proline [corrected] metabolism controls stem cell identity.

Increasing evidence indicates that metabolism is implicated in the control of stem cell identity. Here, we demonstrate that embryonic stem cell (ESC) behaviour relies on a feedback loop that involves the non-essential amino acid L-Proline (L-Pro) in the modulation of the Gcn2-Eif2α-Atf4 amino acid starvation response (AAR) pathway that in turn regulates L-Pro biosynthesis. This regulatory loop generates a highly specific intrinsic shortage of L-Pro that restricts proliferation of tightly packed domed-like ESC colonies and safeguards ESC identity. Indeed, alleviation of this nutrient stress condition by exogenously provided L-Pro induces proliferation and modifies the ESC phenotypic and molecular identity towards that of mesenchymal-like, invasive pluripotent stem cells. Either pharmacological inhibition of the prolyl-tRNA synthetase by halofuginone or forced expression of Atf4 antagonises the effects of exogenous L-Pro. Our data provide unprecedented evidence that L-Pro metabolism and the nutrient stress response are functionally integrated to maintain ESC identity.

c-fos activity in Rana esculenta testis: seasonal and estradiol-induced changes.

Estradiol-17beta (E2) is suspected to exert a role in the regulation of testicular activity. Using a nonmammalian vertebrate model (the frog, Rana esculenta), we have investigated whether c-fos activity is detectable in the testis during the annual sexual cycle and whether E2 exerts a regulatory role on spermatogenesis through fos activity. FOS protein is available in testicular nuclear extracts (about 60 kDa) and, surprisingly, also in cytosolic extracts (about 60, 80, and 100 kDa). Estradiol induces primary spermatogonia (ISPG) proliferation [this effect is counteracted by antiestrogens (Tamoxifen and ICI 182-780)] and FOS appearance in testicular cytosolic extracts as well as c-fos transcription. Also, this effect is counteracted by ICI 182-780. Interestingly, the number of FOS immunopositive nuclei of ISPG strongly increases after E2 treatment, whereas a great increase of immunopositivity in the cytoplasm of ISPG is observed with the contemporaneous treatment with antiestrogens. In conclusion, our results demonstrate that E2 induces ISPG multiplication in the frog, R. esculenta, and, for the first time in a vertebrate species, that it triggers c-fos activity in the testis. Moreover, E2 may be involved in mechanisms related to FOS transport in the nucleus of ISPG to induce the mitotic activity.