The Role of microRNAs in the Gonocyte Theory as Target of Malignancy: Looking for Potential Diagnostic Biomarkers.

Some pediatric patients with cryptorchidism preserve cells with gonocyte characteristics beyond their differentiation period, which could support the theory of the gonocyte as a target for malignancy in the development of testicular neoplasia. One of the key molecules in gonocyte malignancy is represented by microRNAs (miRNAs). The goal of this review is to give an overview of miRNAs, a class of small non-coding RNAs that participate in the regulation of gene expression. We also aim to review the crucial role of several miRNAs that have been further described in the regulation of gonocyte differentiation to spermatogonia, which, when transformed, could give rise to germ cell neoplasia in situ, a precursor lesion to testicular germ cell tumors. Finally, the potential use of miRNAs as diagnostic and prognostic biomarkers in testicular neoplasia is addressed, due to their specificity and sensitivity compared to conventional markers, as well as their applications in therapeutics.

Neonatal exposure to monosodium glutamate induces morphological alterations in suprachiasmatic nucleus of adult rat.

Neonatal exposure to monosodium glutamate (MSG) induces circadian disorders in several physiological and behavioural processes regulated by the suprachiasmatic nucleus (SCN). The objective of this study was to evaluate the effects of neonatal exposure to MSG on locomotor activity, and on morphology, cellular density and expression of proteins, as evaluated by optical density (OD), of vasopressin (VP)-, vasoactive intestinal polypeptide (VIP)- and glial fibrillary acidic protein (GFAP)-immunoreactive cells in the SCN. Male Wistar rats were used: the MSG group was subcutaneously treated from 3 to 10 days of age with 3.5 mg/g/day. Locomotor activity was evaluated at 90 days of age using 'open-field' test, and the brains were processed for immunohistochemical studies. MSG exposure induced a significant decrease in locomotor activity. VP- and VIP-immunoreactive neuronal densities showed a significant decrease, while the somatic OD showed an increase. Major axes and somatic area were significantly increased in VIP neurons. The cellular and optical densities of GFAP-immunoreactive sections of SCN were significantly increased. These results demonstrated that newborn exposure to MSG induced morphological alterations in SCN cells, an alteration that could be the basis for behavioural disorders observed in the animals.

Disruption of gonocyte development following neonatal exposure to di (2-ethylhexyl) phthalate.

Pre- and/or post-natal administrations of di(2-ethylhexyl) phthalate (DEHP) in experimental animals cause alterations in the spermatogenesis. However, the mechanism by which DEHP affects fertility is unknown and could be through alterations in the survival and differentiation of the gonocytes. The aim of the present study was to evaluate the effect of a single administration of DEHP in newborn mice on gonocytic proliferation, differentiation and survival and its long-term effects on seminiferous epithelium and sperm quality. BALB/c mice distributed into Control and DEHP groups were used. Each animal in the DEHP group was given a single dose of 500 mg/Kg at birth. The animals were analyzed at 1, 2, 4, 6, 8, 10 and 70 days postpartum (dpp). Testicular tissues were processed for morphological analysis to determine the different types of gonocytes, differentiation index, seminiferous epithelial alterations, and immunoreactivity to Stra8, Pcna and Vimentin proteins. Long-term evaluation of the seminiferous epithelium and sperm quality were carried out at 70 dpp. The DEHP animal group presented gonocytic degeneration with delayed differentiation, causing a reduction in the population of spermatogonia (Stra8 +) in the cellular proliferation (Pcna+) and disorganization of Vimentin filaments. These events had long-term repercussions on the quality of the seminiferous epithelium and semen. Our study demonstrates that at birth, there is a period that the testes are extremely sensitive to DEHP exposure, which leads to gonocytic degeneration and delay in their differentiation. This situation can have long-term repercussions or permanent effects on the quality of the seminiferous epithelium and sperm parameters.

Molecular Characterization of Patients with Cryptorchidism: Preliminary Search for an Expression Profile Related to That of Testicular Germ-Cell Tumors.

Cryptorchidism (CO) is a risk factor for the development of testicular germ-cell tumors (TGCT). This is supported by reports showing the persistence of gonocytes in CO patients. These cells are proposed to be related to the development of germ-cell neoplasia in situ (GCNIS), which is considered the precursor stage/lesion of TGCT. Therefore, it is proposed that some patients with CO could express some molecular markers related to TGCT. In this study, we analyzed testicular tissue samples from CO, TGCT, and controls. We determined the expression of POU5F1, PLAP, and KIT by immunohistochemistry and that of the hsa-miR-371-373 cluster, hsa-miR-367, and LATS2, PTEN, and IGFR1 genes by RT-qPCR. We then carried out a bioinformatic analysis to identify other possible candidate genes as tumor biomarkers. We found that 16.7% (2/12) of the CO patients presented increased expression of POU5F1, KIT, PLAP, hsa-miR-371-373, and hsa-miR-367 and decreased expression of LATS2 and IGF1R. Finally, the genes ARID4B, GALNT3, and KPNA6 were identified as other possible candidate tumor biomarkers. This is the first report describing the expression of the hsa-miR-371-373 cluster, hsa-miR-367, LATS2, and IGF1R in the testicular tissues of two CO patients with cells immune-positive to POU5F1, PLAP, and KIT, which is similar to what is observed in TGCT.

Cryptorchidism: The dog as a study model.

Cryptorchidism (CO) or undescended testicle is an abnormality of male gonadal development that can generate long-term repercussions in men, such as infertility and germ cell neoplasia in situ (GCNIS). The origin of these alterations in humans is not completely clear, due to the absence of an animal model with similar testicular development as in humans with CO. This work intends to describe the testicular histological development of dogs with congenital CO, and determine whether the species could adequately serve as a study model for this pathology in humans. The study was carried out with 36 dogs, equally distributed in two groups: healthy control (CTRL) and CO groups. The contralateral testis to the undescended one in CO group of the animals was considered and analyzed. Each group was subdivided in three stages of development: (1) peripubertal stage (6-8 months), (2) young adult (9-48 months) and (3) senile (49-130 months). Histological development, the presence of cells with gonocyte morphology, cell proliferation, testicular lipoperoxidation and hormonal concentrations of testosterone, estradiol, FSH and LH were evaluated and described. In the cryptorchid testes, the first histological alterations appeared from the first stage of development and were maintained until the senile stage. A pronounced testicular lipoperoxidation occurred only in the second stage of development. The histological alterations due to CO were markedly evident in the young adult stage. Testosterone concentrations witnessed a decrease starting from in the second stage and kept on until the last stage. The contralateral testes of the CO animals showed alterations that positioned them between the control and CO testes. Testicular development of dogs with CO is similar to that of humans. The results of the study suggest that this species could serve as a suitable model for the study of CO in humans.

Alterations induced by chronic lead exposure on the cells of circadian pacemaker of developing rats.

Lead (Pb) exposure alters the temporal organization of several physiological and behavioural processes in which the suprachiasmatic nucleus (SCN) of the hypothalamus plays a fundamental role. In this study, we evaluated the effects of chronic early Pb exposure (CePbe) on the morphology, cellular density and relative optical density (OD) in the cells of the SCN of male rats. Female Wistar rats were exposed during gestation and lactation to a Pb solution containing 320 ppm of Pb acetate through drinking water. After weaning, the pups were maintained with the same drinking water until sacrificed at 90 days of age. Pb levels in the blood, hypothalamus, hippocampus and prefrontal cortex were significantly increased in the experimental group. Chronic early Pb exposure induced a significant increase in the minor and major axes and somatic area of vasoactive intestinal polypeptide (VIP)- and vasopressin (VP)-immunoreactive neurons. The density of VIP-, VP- and glial fibrillary acidic protein (GFAP)-immunoreactive cells showed a significant decrease in the experimental group. OD analysis showed a significant increase in VIP neurons of the experimental group. The results showed that CePbe induced alterations in the cells of the SCN, as evidenced by modifications in soma morphology, cellular density and OD in circadian pacemaker cells. These findings provide a morphological and cellular basis for deficits in circadian rhythms documented in Pb-exposed animals.

Protective effect of α-tocopherol on damage to rat testes by experimental cryptorchidism.

It is thought that the degeneration of germ cells associated with an increase in the temperature due to cryptorchidism involves oxidative stress. α-Tocopherol is a powerful antioxidant that prevents oxidation of polyunsaturated fats found in membranes and stabilizes peroxyl radicals. For this reason we were interested in determining the role of α-Tocopherol using experimental cryptorchidism, followed by orchidopexia in neonatal rats. Eighty-four, 10-day-postpartum (dpp) male rats (Wistar strain) were used and divided into 7 groups: healthy control, sham with α-Tocopherol treated with 30 or 100 mg/kg doses, sham vehicle, cryptorchidism treated with α-Tocopherol at 30 or 100 mg/kg doses and cryptorchidism vehicle. Cryptorchidism was surgically induced at 10 dpp. At 25 dpp the animals were treated with α-Tocopherol and the vitamin vehicle. Lipoperoxidation and testicular morphology was determined in half of the animals at 40 dpp (short term). The remaining animals underwent orchidopexia and fertility was determined at 90 dpp. Testicular morphology was determined at 120 dpp (long term) in these animals. A significant reduction of lipoperoxidation was observed in the cryptorchid group treated with α-Tocopherol compared to the untreated cryptorchid group, in addition to short-term histological alterations. At long term, we observed an increase in the area and maturation of the seminiferous epithelium, a decrease in apoptosis and histological alterations and an increase in fertility from α-Tocopherol treatment. α-Tocopherol treatment decreased lipoperoxidation, possibly stabilizing free radicals produced during cryptorchidism, reducing morphological testicular alterations and favoring fertility.

Disorders in the function of Sertoli cells and arrest in the differentiation of gonocytes in patients with cryptochidism and microlithiasis.

PURPOSE: This study aimed to correlate the presence of microlithiasis (ML) in cryptorchidism (CO) patients with the functionality of Sertoli cells and the arrest of gonocyte differentiation. METHODS: Testicular biopsies were obtained from 21 inguinal CO pediatric patients and were classified in two groups as follows: patients with ML and those without ML. In both groups, the number of Sertoli cells/seminiferous cords and their functionality were determined, considering the concentrations of inhibin B. In addition, the area and the histological alterations of seminiferous epithelium were evaluated. The arrest of gonocyte differentiation was determined by immunoreactivity to SALL4, AP2É£, PLAP and POU5F1. RESULTS: We found a statistical correlation between the presence of ML with the alterations in the functionality of Sertoli cells without reflecting in the differentiation of the gonocytes. CONCLUSION: The study of this population suggests that the association between CO and ML shows a malfunction of the Sertoli cells without necessarily causing arrest in the differentiation of gonocytes in these patients.

Chronic lead exposure alters photic entrainment of locomotor activity rhythm and neuronal photoactivation in the suprachiasmatic nucleus of the adult rat.

Chronic lead (Pb) exposure affects the circadian physiological processes regulated by suprachiasmatic nucleus (SCN), which is synchronized (entrainment) by light. Disorders in the entrainment capacity of an organism alter its performance to interact with the environment, thus affecting its health status. The objectives of the present study were to evaluate whether chronic early Pb exposure affects the entrainment of the circadian rhythm of locomotor activity by light and to explore the possible mechanisms involved. Adult male Wistar rats, control and chronically exposed to Pb (320 ppm) in drinking water from gestation to adult age, were used. Assessment of the metal level showed a significant increase of Pb in the blood, hypothalamus and prefrontal cortex of the experimental rats. Continuous registrations of locomotor activity (12 h:12 h light-dark cycle) depicted that Pb induces important delay of this activity when the light was turned off. The Pb exposed animals entrained faster with a photoperiod delay of 6 h, (lights on at 13:00 h), and maintained the significant delay in the onset of activity at lights out. In continuous darkness, the animals were exposed to a light pulse at circadian time 23. This resulted in a significant decrease of photo-stimulated neurons (immunoreactivity to c-Fos) in the SCN of the metal-exposed animals. These results show that chronic early Pb exposure alters the photic entrainment of the rhythm of locomotor activity, which is evidenced by a significant decrease in both the number of photo-stimulated neurons and neuronal population (Nissl stain) of the SCN.

Fetal and Postnatal Nicotine Exposure Modifies Maturation of Gonocytes to Spermatogonia in Mice.

Studies in laboratory animals have shown that male offspring from dams, exposed to nicotine during pregnancy and postnatal periods, show alterations in fertility, although the origin of this is still uncertain. In this study, we examined in a mouse model if the process of gonocyte maturation to spermatogonia was affected in male offspring from dams with nicotine administration during pregnancy and postnatal periods. BALB/C mice, with and without nicotine administrations in pregnancy and postnatal periods, were studied. The animals were euthanized at 3, 7, 10, 16, and 35 days postpartum (dpp). Testicular tissue samples were processed for histological, ultrastructural, and immunohistochemical studies; and testicular lipoperoxidation was determined. It was observed that in the nicotine-exposed animals, there was increased apoptosis and a reduction in the number of gonocytes that matured to spermatogonia. This gonocyte-spermatogonia maturation reduction was associated with a greater immunoreactivity to nicotinic acetylcholine receptors in the germ cells. Lipoperoxidation was similar in both groups until 16 dpp, with significant reduction at 35 dpp. Our findings suggest that nicotine intake during pregnancy and postnatal periods can affect the process of maturation of gonocytes to spermatogonia and the pool of available spermatogonia for spermatogenesis.

Cryptorchidism and Testicular Tumor: Comprehensive Analysis of Common Clinical Features and Search of SNVs in the KIT and AR Genes.

Allelic variants in genes implicated in the development of testicular germ cell tumor (TGCT) could be present in patients with cryptorchidism (CO). Currently; the mechanisms explaining this relationship are still unknown. In this study the common clinical features in patients with CO and TGCT and 6 variants of KIT and AR genes associated to TGCT were analyzed. Population analyzed included 328 individuals: 91 patients with CO; 79 with TGCT, 13 of them with previous CO diagnosis, and 158 healthy males. Of the 13 patients with TGCT and history of CO, one patient (7.7%) presented the heterozygous form of the variant rs121913507 and two patients (15.4%) presented homozygote genotype for the variant rs121913506 in KIT gene. Interestingly, the heterozygous form for the variant rs121913506 of KIT gene was identifying in all of 13 patients. The rs201934623, rs774171864, and rs12014709 variants of the AR gene did not show any clinical association. Our results strongly support that genetic component in CO could be conditioning for the development of TGCT. Notably, KIT gene variants might be determinants in the pathological association between TGCT and CO.

Progesterone treatment in rats after severe global cerebral ischemia promotes hippocampal dentate gyrus neurogenesis and functional recovery.

OBJECTIVE: Rats treated with progesterone (P4) after ischemia show an adequate functional performance despite a significant loss of hippocampal pyramidal neurons, suggesting that P4 could favour a permissive microenvironment for cerebral plasticity mechanisms. The possibility of P4 treatment promoting the survival of newly generated hippocampal neurons, in relation to the performance of ischemic rats in a spatial learning task, was assessed in this study. METHODS: Adult male rats were subjected to a severe global cerebral ischemia episode (30 min) and treated with P4 or its vehicle at 15 min, 2, 6, 24, 48 and 72 h of reperfusion. From day 4 to 8 post-ischemia 5-bromo-2-deoxyuridine (BrdU) was administered to label proliferating cells. Twenty-one days post-ischemia, the rats were exposed to the Morris water maze to assess behavioral parameters of spatial learning and memory. Subsequently, the brain was perfusion-fixed and immunofluorescence procedures were performed to quantify the number of new mature neurons (BrdU+/NeuN+) in the dentate gyrus (DG) of the hippocampus. RESULTS: Rats subjected to severe global cerebral ischemia and treated with P4 had a significantly better performance in spatial learning-memory tests, than those treated with vehicle, and a significantly higher number of new mature neurons (BrdU+/NeuN+) in the DG. CONCLUSION: These findings show that post-ischemia P4 treatment, following an episode of severe global cerebral ischemia, promotes the survival of newly generated hippocampal neurons in the DG, which may be one of the mechanisms of cerebral plasticity induced by the hormone, that underlie a successful functional performance in learning and memory tests.

Epigallocatechin-3-gallate protects the testis from damage generated by experimental cryptorchidism in rabbits.

Cryptorchidism (CO) is a risk factor for infertility in men. It is associated with an increase in oxidative stress which alters the differentiation of the gonocytes to spermatogonia. Epigallocatechin-3-gallate (EGCG) is an antioxidant that acts as a free radical scavenger and activates the antioxidant enzymes. The aim of this work was to investigate if EGCG plays a role in the protection of the testicle from alterations generated by CO and its possible mechanism. Male rabbits 7 days old were divided into four groups and distributed as follows: 1) control (C) treated with EGCG vehicle (V) (C/V); 2) C with administration of EGCG from 65 to 120 days postpartum (dpp) (C/EGCG); 3) CO induced by administration of 17ß-estradiol plus EGCG vehicle (CO/V) and 4) CO plus EGCG administration (CO/EGCG). The animals were euthanized at 120 dpp and their testes were processed to evaluate lipid peroxidation, activities of superoxide dismutase (SOD) and catalase (CAT) enzymes as well as serum testosterone (T) concentrations. In addition, the rates of apoptosis, cell proliferation and histological alterations were determined. The CO/EGCG group showed a significant reduction in lipid peroxidation, a significant increase in the anti-oxidant enzyme activities and concentrations of T. Also, there was a significant decrease in the histological alterations, absence of gonocytes and active spermatogenesis when compared with CO/V group. These results show that EGCG reduces lipid peroxidation and increases the activity of the endogenous anti-oxidant system which protects the testes from alterations produced by oxidative stress generated during experimental CO.

Neonatal treatment with clomipramine modifies the expression of estrogen receptors in brain areas of male adult rats.

The participation of estrogens in depression has been well recognized. To exert its effects, estradiol binds mainly to estrogen receptors ESR1 and ESR2 (α and ß, respectively), expressed in brain regions including the hippocampus, limbic regions and hypothalamic nuclei. In rodents, modified estrogen receptors expression in brain areas have been implicated in different signs similar to those observed in depressive patients. Neonatal clomipramine (CMI) treatment is a pharmacological manipulation that generates behavioral and neurochemical changes that persist throughout adulthood and resemble human depression. The aim of this study was to analyze whether CMI neonatal treatment modifies the expression of nuclear ESR1 and ESR2 in the hippocampus, amygdala basolateral (BLA), amygdala medial (MeA), hypothalamic medial preoptic area (mPOA) and raphe nucleus in male rats. Our results indicate that CMI treatment significantly induced an mRNA increase of ESR1 in the hypothalamus, additionally produce a reduction in the mRNA ESR2 expression in raphe accompanied of an increase in hypothalamus and amygdala. CMI treated rats show more immunorreactive cells to ESR1 (ESR1-ir) in mPOA, BLA, MeA, together with a reduction of these cells in the hippocampal CA1 region. Moreover, an increase in the number of immunorreactive cells to ESR2 (ESR2-ir), in BLA and MeA, was observed in CMI treated rats. Additionally, the hippocampal CA2 region and raphe nucleus showed a decrease in these cells. Also, neonatal CMI treatment induced a decrease in the number of cells of the pyramidal layer in CA1. Overall, the results suggest that neonatal CMI treatment in rats (during brain development) induces changes in estrogen receptors in different brain areas involved with the regulation of depressive-like behaviors.

Influence of SNPs in Genes that Modulate Lung Disease Severity in a Group of Mexican Patients with Cystic Fibrosis.

BACKGROUND: The variation in cystic fibrosis (CF) lung disease not always is explained by the CFTR genotype, so it has become apparent that modifier genes must play a considerable role in the phenotypic heterogeneity of CF, so we investigated the association of allelic variants in modifier genes that modulate the severity of lung function in a group of Mexican patients diagnosed with CF. METHODS: We included 140 CF patients classified according to lung phenotype and analyzed 17 single nucleotide polymorphisms (SNPs) by TaqMan® allelic discrimination. RESULTS: We demonstrated that patients with GG or GC genotype of the allelic variant rs11003125 (MBL2-550) of the MBL2 gene exhibit most of the lung manifestations at an earlier age; and the rs1042713 allelic variant of ADRB2 gene, showed statistical difference only with the age of first spirometry. When we used the dominant model, the MBL2 allele rs11003125 (MBL2-550; p = 0.022, Odds Ratio (OR) 2.87, 95% CI 1.14-7.27) was significantly associated with CF patients as risk factor, and the ADRB2 allele rs1042713 (p.Arg16Gly; p = 0.005, Odds Ratio (OR) 0.37, 95% CI 0.19-0.75) was significantly associated with CF patients as protect factor. CONCLUSIONS: Our findings suggest that the MBL2 and ADRB2 genes exerts an important genetic influence on the lung disease in our patients. Taking into account our results, we insist on not leaving aside this type of studies, since having techniques such as GWAS or WES will be able to advance in achieving a better quality of life for CF patients with severe lung disease.

Epigenetic and risk factors of testicular germ cell tumors: a brief review.

Testicular germ cell cancer (TGCT) is the most common malignancy among young adult males, which has become important due to its increased incidence and mortality in the population worldwide. The etiology is multifactorial. Recent studies have shown some associations between the development of isolated TGCT and certain risk factors, such as exposure to endocrine disruptors, cryptorchidism, and family history of cancer, in order to identify the key pieces in carcinogenesis. Some of the most important findings in recent years is the association of different genes, such as c-KIT/KITLG, expression of the miR-371-373 cluster and protein expression as c-KIT and POU5F1 in the development of this neoplasia, and the identification of new molecular markers as TGFBR3 gene, identifying aberrant methylation patterns in promoter regions of several genes, expression of miR-1297 which regulates PTEN and protein expression as DMTR1. In the future, a multidisciplinary research strategy could provide valuable new insights into the etiology of TGCTs, which support clinical diagnosis of TGCT in the next years to increase survival in this kind of patients.

The effect of estrogen on testicular gonocyte maturation.

During the first days of postnatal life in rats the male germ cells (gonocytes) proliferate and move towards the seminiferous tubule basal lamina maturing into spermatogonia. This process is necessary for spermatogenesis and can be affected by estrogen (E); therefore, it is important to determine whether the damaging mechanism induced by E administration during the postnatal period impairs gonocyte maturation. One-day-old rat pups were given 1 microg 17-beta-estradiol daily and studied at 3, 5, 8, 10 and 16 days of age, corresponding to the critical gonocyte differentiation period in the rat. Testicles were isolated and the number of gonocytes in contact with the basal lamina of the seminiferous tubule was estimated, as well as the proliferation rate and apoptosis of the gonocytes. We observed that the administration of E changed the migration of gonocytes towards the basal lamina, decreased cell proliferation and increased apoptosis, resulting in a decrease in spermatogonia and spermatocytes. The migration of gonocytes and subsequent proliferation is required for survival of this germ cell type. The lack of maturation and the death of gonocytes could be one of the causes of infertility following exogenous E treatment.

Development of germ cell neoplasia in situ in chinchilla rabbits.

The present study was designed to describe the development of germ cell neoplasia in situ in Chinchilla rabbit by administration of estradiol. The study was performed in rabbits distributed into two groups: control and 17 ß-estradiol. The determination of histological alterations and POU5F1 and c-kit proteins employed as biomarkers for the diagnosis of this neoplasia was carried out. Testicular descent and complete spermatogenesis were observed in the control group. The protein biomarkers were negative. However, in the rabbits treated with estradiol, the testes remained undescended with the gonocytes undifferentiated to spermatogonia. There were histological lesions owing to germ cell neoplasia in situ and positive to POU5F1 and c-kit proteins. These findings indicate that the chinchilla rabbit is an ideal model to study this neoplasia in which the histological characteristics and biomarkers of the disease could be clearly observed. Using this model we suggested that the persisting gonocytes could be responsible for the development of germ cell neoplasia in situ.

Analysis of POU5F1, c-Kit, PLAP, AP2γ and SALL4 in gonocytes of patients with cryptorchidism.

Cryptorchidism is a risk factor for the development of testicular germ cell tumors (TGCTs). The most common type of TGCT in cryptorchidism is seminoma. The intratubular germ cell neoplasia unclassified (ITGCNU) is a histological pattern preceding the development of seminomas and non-seminomas. It was suggested that in patients with cryptorchidism, the gonocytes remained undifferentiated with pluripotent abilities expressing proteins like POU domain class 5 transcription factor 1 (POU5F1), tyrosine kinase receptor c-Kit, placental-like alkaline phosphatase (PLAP), the transcription factor AP2γ and sal-like protein 4 (SALL4) that confer to the gonocytes this ability and therefore make them susceptible to develop ITGCNU. The aim of the present study was to determine if the gonocytes of patients with cryptorchidism express POU5F1, c-Kit, PLAP, AP2γ and SALL4 proteins after their differentiation period. Based on this, we evaluated samples of testicular tissue from newborns to 16-year old subjects with or without cryptorchidism in search of POU5F1, c-Kit, PLAP, AP2γ and SALL4 using immunocytochemical method, the results of which were validated by RT-PCR. The results showed that control subjects witnessed a down-regulation in the expression of these five proteins in the first year of life, which eventually disappeared. On the other hand, it was determined that 21.6% (8/37) of the patients with cryptorchidism continued to express, at least, one of the proteins analyzed in this study after the second year of life. And only 5.4% (2/37) of the patients were positive to the five markers. These data sustain the proposed hypothesis that in cryptorchid patients, ITGCNU arises from gonocytes that fail in their differentiation process to spermatogonia with conservation of the proteins (POU5F1, c-Kit, PLAP, AP2γ and SALL4) that maintain pluripotency and undifferentiated characteristics and which are responsible for making the gonocytes susceptible to malignancy. However, we cannot guarantee that these patients present neoplastic transformation.