High-fat diet decreases H3K27ac in mice adipose-derived stromal cells.

OBJECTIVE: The study goal was to analyze the effects of a high-fat diet (HFD) on the histone 3 lysine 27 (H3K27) posttranscriptional modifications and the expression of histone-modifying enzymes in adipose-derived stromal cells (ASCs) from white adipose tissue (WAT). METHODS: Male C57BL/6J mice received control or HFD for 12 weeks. The ASCs were isolated from subcutaneous and visceral (epididymal) WAT, cultivated, and evaluated for expression of H3K27 trimethylation (H3K27me3) and H3K27 acetylation (H3K27ac) by Western blot. The transcription of histone-modifying enzymes was analyzed by real-time polymerase chain reaction. RESULTS: When compared with control, HFD ASCs showed a decrease in H3K27ac enrichment in subcutaneous and visceral WAT and ATP-citrate lyase expression in subcutaneous WAT. Curiously, the expression of CREB-binding protein was increased in visceral ASCs from HFD-fed mice. CONCLUSIONS: These results show that an HFD significantly reduces acetylation of H3K27 in ASCs and the expression of ATP-citrate lyase in subcutaneous ASCs, suggesting that, in this fat depot, the H3K27ac reduction could be partly due to lower acetyl-coenzyme A availability. H3K27ac is an epigenetic mark responsible for increasing the transcription rate and its reduction can have an important impact on ASC proliferation and differentiation potential.

Carnitine Diminishes Etoposide Toxic Action on Spermatogonial Self-renewal and Sperm Production in Adult Rats Treated in the Prepubertal Phase.

The aim of this study was to investigate carnitine action against negative effects of etoposide on stem/progenitor spermatogonia and on sperm production. Carnitine (250 mg/kg body weight/day) and etoposide (5 mg/kg body weight/day) were administered from 25-days postpartum to 32-days postpartum. Testes were collected at 32-days postpartum, 64-days postpartum, and 127-days postpartum, and submitted to the immuno-labeling of UTF1, SOX2, and PLZF proteins to identify undifferentiated spermatogonia populations. At 127-days postpartum, sperm were collected for analysis. Carnitine+etoposide group showed a higher numerical density of spermatogonia labeled for all studied proteins at 64-days postpartum (critical age) compared to the etoposide group. Moreover, there was an improvement of spermatic parameters and sperm DNA integrity in rats of the carnitine+etoposide group in comparison with rats of the etoposide group. The results suggest that carnitine improves the self-renewal of undifferentiated spermatogonia and promotes a partial protection on them, alleviating the etoposide harmful late effects and leading to an enhancement of the sperm parameters in adulthood.

Expression of genome defence protein members in proliferating and quiescent rat male germ cells and the Nuage dynamics.

During epigenetic reprogramming germ cells activate alternative mechanisms to maintain the repression retrotransposons. This mechanism involves the recruitment of genome defence proteins such as MAEL, PIWIL4 and TDRD9, which associate with piRNAs and promote Line-1 silencing. MAEL, PIWIL4 and TDRD9 form the piP-bodies, which organization and dynamics vary according to the stage of germ cell epigenetic reprogramming. Although these data have been well documented in mice, it is not known how this mechanism operates in the rat. Thus, the aim of this study was to describe the distribution and interaction of MAEL, PIWIL4, TDRD9 and DAZL during rat germ cell development and check whether specific localization of these proteins is related to the distribution of Line-1 aggregates. Rat embryo gonads at 15 days post-conception (dpc), 16dpc and 19dpc were submitted to MAEL, PIWIL4, TDRD9 and DAZL immunolabelling. The gonads of 19dpc embryos were submitted to the double-labelling of MAEL/DAZL, TDRD9/MAEL and PIWIL4/MAEL. The 19dpc gonads were submitted to co-immunoprecipitation assays and fluorescent in situ hybridization for Line-1 detection. MAEL and TDRD9 showed very similar localization at all ages, whereas DAZL and PIWIL4 showed specific distribution, with PIWIL4 showing shuttling from the nucleus to the cytoplasm by the end epigenetic reprogramming. In quiescent 19dpc gonocytes all proteins colocalized in a nuage adjacent to the nucleus. DAZL interacts with PIWIL4 and MAEL, suggesting that DAZL acts with these proteins to repress Line-1. TDRD9, however, does not interact with DAZL or MAEL despite their colocalization. Line-1 aggregates were detected predominantly in the nuclear periphery, although did not show homogeneous distribution as observed for the nuage. In conclusion, the nuage in quiescent rat gonocytes show a very distinguished organization that might be related to the organization of Line-1 clusters and describe the association of DAZL with proteins responsible for Line-1 repression.

Search for appropriate reference genes for quantitative reverse transcription PCR studies in somite, prosencephalon and heart of early mouse embryo.

qRT-PCR requires reliable internal control genes stably expressed in different samples and experimental conditions. The stability of reference genes is rarely tested experimentally, especially in developing tissues given the singularity of these samples. Here we evaluated the suitability of a set of reference genes (Actb, Gapdh, Tbp, Pgk1 and Sdha) using samples from early mouse embryo tissues that are widely used in research (somites, prosencephalon and heart) at different developmental stages. The comparative ΔCq method and five software packages (NormFinder, geNorm, BestKeeper, DataAssist and RefFinder) were used to rank the most stable genes while GenEx and GeNorm programs determined the optimal total number of reference genes for a reliable normalization. The ranking of most reliable reference genes was different for each tissue evaluated: (1) in somite from embryos with 16-18 somite pairs stage, the combination of Pgk1 and Actb provided the best normalization and Actb also presented high stability levels at an earlier developmental stage; (2) Gapdh is the most stable gene in prosencephalon in the two developmental stages tested; and (3) in heart samples, Sdha, Gapdh and Actb were the best combination for qPCR normalization. The analysis of these three tissues simultaneously indicated the combination of Gapdh, Actb and Tbp as the most reliable internal control. This study highlights the importance of appropriate reference genes according to the cell type and/or tissue of interest. The data here described can be applied in future research using mouse embryos as a model for mammalian development.

Identification of a Critical Window for Ganciclovir-Induced Disruption of Testicular Development in Rats.

Ganciclovir (GCV) has been implicated in the development of testicular alterations. Exposure on gestational day (GD) 10 in rats induced permanent effects, including focal reduction or absence of germ cells (Sertoli cell-only tubules). Because the timing of exposure can be critical for testicular effects, we exposed rat dams to 300 mg/kg GCV (3 100 mg/kg subcutaneous injections) on GD10, 14 and 19, when germ cells have high rates of migration, proliferation and are mitotically quiescent, respectively. Males exposed to GCV in utero on GD10 and 14 were evaluated for androgenization markers, serum and fecal androgens, and testicular histomorphometry at adulthood. Double-labeling immunofluorescence for DAZL and Ki67 were used to assess gonocytes number and the proliferative activity of germ and somatic cells in fetal testes on GD15 and 20, ie, 24 h after GCV exposure. Adult rats exposed on GD14 showed delayed puberty onset, despite normal androgen levels. Also, there was a 50% reduction in testicular weight and about 30% of seminiferous tubules lacking germ cells. Effects on GD10 animals were less pronounced. In the fetal testis, the number of gonocytes was reduced by 50% in rats exposed on GD14, but normal in GD19 fetuses. GCV also reduced Sertoli cell proliferation immunolabeling in GD19 fetuses and Sertoli cell number in adults. In conclusion, GCV toxicity on germ cells seems to be linked to their proliferation rate and GD14 is a critical window in rats, when GCV exposure causes an acute massive loss of germ cells that persists until adulthood.

Inactivation of AMMECR1 is associated with growth, bone, and heart alterations.

We report five individuals with loss-of-function of the X-linked AMMECR1: a girl with a balanced X-autosome translocation and inactivation of the normal X-chromosome; two boys with maternally inherited and de novo nonsense variants; and two half-brothers with maternally inherited microdeletion variants. They present with short stature, cardiac and skeletal abnormalities, and hearing loss. Variants of unknown significance in AMMECR1 in four male patients from two families with partially overlapping phenotypes were previously reported. AMMECR1 is coexpressed with genes implicated in cell cycle regulation, five of which were previously associated with growth and bone alterations. Our knockdown of the zebrafish orthologous gene resulted in phenotypes reminiscent of patients' features. The increased transcript and encoded protein levels of AMMECR1L, an AMMECR1 paralog, in the t(X;9) patient's cells indicate a possible partial compensatory mechanism. AMMECR1 and AMMECR1L proteins dimerize and localize to the nucleus as suggested by their nucleic acid-binding RAGNYA folds. Our results suggest that AMMECR1 is potentially involved in cell cycle control and linked to a new syndrome with growth, bone, heart, and kidney alterations with or without elliptocytosis.

An Initial Investigation of an Alternative Model to Study rat Primordial Germ Cell Epigenetic Reprogramming.

BACKGROUND: Primordial germ cells (PGC) are the precursors of the gametes. During pre-natal development, PGC undergo an epigenetic reprogramming when bulk DNA demethylation occurs and is followed by sex-specific de novo methylation. The de novo methylation and the maintenance of the methylation patterns depend on DNA methyltransferases (DNMTs). PGC reprogramming has been widely studied in mice but not in rats. We have previously shown that the rat might be an interesting model to study germ cell development. In face of the difficulties of getting enough PGC for molecular studies, the aim of this study was to propose an alternative method to study rat PGC DNA methylation. Rat embryos were collected at 14, 15 and 19 days post-coitus (dpc) for the analysis of 5mC, 5hmC, DNMT1, DNMT3a and DNMT3b expression or at 16dpc for treatment 5-Aza-CdR, a DNMT inhibitor, in vitro. METHODS: Once collected, the gonads were placed in 24-well plates previously containing 45µm pore membrane and medium with or without 5-Aza-CdR. The culture was kept for five days and medium was changed daily. The gonads were either fixed or submitted to RNA extraction. RESULTS: 5mC and DNMTs labelling suggests that PGC are undergoing epigenetic reprogramming around 14/15dpc. The in vitro treatment of rat embryonic gonads with 1 µM of 5-Aza-CdR lead to a loss of 5mC labelling and to the activation of Pax6 expression in PGC, but not in somatic cells, suggesting that 5-Aza-CdR promoted a PGC-specific global DNA hypomethylation. CONCLUSIONS: This study suggests that the protocol used here can be a potential method to study the wide DNA demethylation that takes place during PGC reprogramming.

Carbamazepine-exposure during gestation and lactation affects pubertal onset and spermatic parameters in male pubertal offspring.

Carbamazepine (CBZ) is an anti-epileptic drug that acts on Leydig cells, affecting steroidogenesis and causes fetal malformation. The aim of this study was to investigate the effects of CBZ on male sexual maturation and other male parameters. Rat dams were treated with CBZ during pregnancy and breastfeeding. The anogenital distance (AGD) and the anogenital index (AGI) were obtained. Testicular descent and preputial separation were also evaluated. The offspring was euthanized at PND 41 and 63. The accessory glands were weighed and the testes were collected for histopathological, morphometric and sterological analyses. The numerical density of Leydig cells and hormone dosage were obtained. CBZ caused an increase of AGI and a delay of testicular descent and of preputial separation. CBZ also caused a decrease of testosterone level and of sperm count and an increase of abnormal sperm. These results indicate that CBZ delays puberty onset and affects steroidogenesis and sperm quality.

Late morfofunctional alterations of the Sertoli cell caused by doxorubicin administered to prepubertal rats.

BACKGROUND: Doxorubicin is a potent chemotherapeutic drug used against a variety of cancers. It acts through interaction with polymerases and topoisomerase II and free radical production. Doxorubicin activity is not specific to cancer cells and can also damage healthy cells, especially those undergoing rapid proliferation, such as spermatogonia. In previous studies our group showed that etoposide, another topoisomarese II poison, causes irreversible damage to Sertoli cells. Thus, the aim of this study was to address the effects of doxorubicin on Sertoli cell morphology and function and on the seminiferous epithelium cycle when administered to prepubertal rats. METHODS: Prepubertal rats received the dose of 5 mg/Kg of doxorubicin, which was fractioned in two doses: 3 mg/Kg at 15dpp and 2 mg/Kg at 22 dpp. The testes were collected at 40, 64 and 127 dpp, fixed in Bouin's liquid and submitted to transferrin immunolabeling for Sertoli cell function analysis. Sertoli cell morphology and the frequency of the stages of the seminiferous epithelium cycle were analyzed in PAS + H-stained sections. RESULTS: The rats treated with doxorubicin showed reduction of transferrin labeling in the seminiferous epithelium at 40 and 64 dpp, suggesting that Sertoli cell function is altered in these rats. All doxorubicin-treated rats showed sloughing and morphological alterations of Sertoli cells. The frequency of the stages of the seminiferous epithelium cycle was also affected in all doxorubicin-treated rats. CONCLUSIONS AND DISCUSSION: These data show that doxorubicin administration during prepuberty causes functional and morphological late damage to Sertoli cells; such damage is secondary to the germ cell primary injury and contributed to enhance the spermatogenic harm caused by this drug. However, additional studies are required to clarify if there is also a direct effect of doxorubicin on Sertoli cells producing a primary damage on these cells.

Gonocyte development in rats: proliferation, distribution and death revisited.

Spermatogonial stem cells are responsible for the constant production of spermatozoa. These cells differentiate from the gonocytes, but little is known about these cells and their differentiation into spermatogonia. This study analyzed rat gonocyte proliferation, death and distribution as well as their differentiation into spermatogonia. Rat testes were collected at 19 dpc and at 1, 3, 5, 8, 11 and 15 dpp and submitted to apoptosis investigation through morphological analysis and TUNEL, p53 and cleaved caspase 3 labeling. Ki67 and MVH labeling was used to check gonocyte proliferation and quantification, respectively. OCT4 and DBA labeling were used to check gonocyte differentiation. Seminiferous cord length and gonocyte numerical density were measured to check gonocyte distribution along the seminiferous cords. Although a reduction of gonocyte number per testicular section has been observed from 1 to 5 dpp, the total number of these cells did not change. Apoptotic gonocytes were not detected at these ages, suggesting that the reduction in gonocyte number per testicular section was due to their redistribution along the seminiferous cords, which showed continuous growth from 19 dpc to 5 dpp. The first proliferating germ cells were observed at 8 dpp, coinciding with OCT4 upregulation and with the emergence of the first spermatogonia. In conclusion, this study suggests that (a) gonocytes do not die in the first week after birth, but are rather redistributed along the seminiferous cords just before their differentiation into spermatogonia; (b) mitosis resumption and the emergence of the first spermatogonia are coincident with OCT4 upregulation.

Detection of four germ cell markers in rats during testis morphogenesis: differences and similarities with mice.

Germ cells are the only cells capable of transmitting genetic information from generation to generation. Germ cell development has been widely studied in different species. Among mammals, the mouse is the model used in the majority of studies on germ cell differentiation, sex determination and genetics. In the present study, we suggest that the rat is also a very important model for the investigation of the mechanisms of germ cell development. To study rat germ cell development and compare it with that of mouse, the germ cell markers germ cell nuclear antigen 1 (GCNA1), OCT4, mouse vasa homologue (MVH) and specific surface embryonic antigen 1 (SSEA1) were immunolabeled at different phases of embryonic and postnatal development. SSEA1 and GCNA1 were not detected in rat primordial germ cells and fetal gonocytes. GCNA1 was detected postnatally and was present only in leptotene, zygotene and early pachytene spermatocytes. On the other hand, in mice, these markers were detected in germ cells as soon as 11.5 days postcoitum (dpc). MVH was detected in migrating rat primordial germ cells as well as in those that have already colonized the gonads, whereas in mice, MVH is detected only in germ cells that have reached the gonads. In rats, OCT4-positive germ cells were detected from 13 to 17 dpc, but not at 19 dpc or in postnatal testes. This is in contrast with mice that show OCT4 labeling in both embryonic and adult testes. These data suggest that primordial germ cell development in rats and mice shows considerable differences and that the rat may also be an important model to study the embryonic development of germ cells.

Biologia epididimária: maturação espermática e expressão gênica / Biología Epididimaria: maduración espermática y expresión génica / Epididymal biology: spermatic maturation and gene expression

O epidídimo dos mamíferos é um órgão formado por um ducto único e enovelado que liga os dúctulos eferentes ao ducto deferente, sendo anatomicamente dividido em segmento inicial, cabeça, corpo e cauda. A classificação baseia-se no estudo da altura do epitélio, do diâmetro tubular e da variação na frequência dos diferentes tipos celulares. A passagem dos espermatozoides, que são produzidos nos testículos, ao longo do ducto epididimário determina a aquisição da capacidade fertilizante e da motilidade progressiva, processo denominado maturação espermática. Durante a passagem através do epidídimo, o fluido ao qual os espermatozóides são expostos sofre alterações substanciais na composição, incluindo mudanças da osmolaridade, da proporção iônica, das reservas energéticas e de tipos protéicos. A expressão gênica programada e diferencial ao longo do epidídimo, responsável pela formação de um microambiente especializado, é importante para o sucesso do processo de maturação espermática. Embora vários estudos tenham identificado inúmeros genes com expressão seletiva e específica no epidídimo, os quais são importantes para o entendimento da expressão gênica complexa deste órgão, muito ainda permanece a ser determinado como, por exemplo, os tipos protéicos codificados por estes genes, bem como a identificação dos sítios de ação destas proteínas.

El epidídimo de los mamíferos es un órgano formado por un único ducto anovelado que conecta los dúctulos eferentes al ducto deferente, siendo dividido anatómicamente en segmento inicial, cabeza, cuerpo y cola. La clasificación se basa en el estudio de la altura del epitelio, del diámetro tubular y de la variación en la frecuencia de los diversos tipos celulares. El pasaje de espermatozoides (producidos en los testículos en toda la superficie del ducto epididimario), determina la adquisición de la capacidad de fertilización y dela motilidad progresiva, proceso denominado maduración espermática. Durante el pasaje a través del epidídimo, el líquido a lo que los espermatozoides se exponen sufre alteraciones substanciales en su composición, incluyendo cambios de la osmolaridad, del cociente iónico, de las reservas de energía y de los tipos proteínicos. La expresión génica programada y de distinción y diferencial a través del epidídimo, responsable de la formación de un microambiente especializado, siendo importante para el éxito del proceso de maduración espermática. Aunque algunos estudios hayan identificado genes innumerables con expresión selectiva y específica en el epidídimo, genes que son importante como para entender la expresión génica compleja de este órgano, todavía siguen muchos factores a ser determinados como, por ejemplo, los tipos de proteínas codificados por estos genes, así como la identificación de las áreas de acción de estas proteínas.

The mammalian epididymis consists of a single coiled duct that connects the efferent ducts to the different duct. Anatomically, four segments are distinguished in this organ: the caput, the corpus and the cauda epididymidis. This classification is based in the epithelium height, in the diameter of the duct and in the cell types present in each segment. During their transit through the epididymis, the sperm, produced in the testis, acquire progressive motility and become able to fertilize the oocyte, a process called sperm maturation. During this transit, the epididymis fluid changes its osmolarity, energetic reserves and proteins, in a way to promote sperm maturation. The programmed and differential gene expression along the epididymis provides the appropriate environment to sperm maturation. Although different studies have identified many epididymis-enriched and specific genes that are important to understand the transcriptome of this organ, many aspects of epididymis gene expression is still to be shown, such as what proteins are produced by the genes described so far, what are the functions of these proteins and where they act.

Carnitine reduces testicular damage in rats treated with etoposide in the prepubertal phase.

Etoposide is a chemotherapeutic agent that induces cell death by blocking topoisomerase II catalytic function. Although etoposide is effective in the treatment of cancer, it also causes the death of normal proliferating cells, including male germ cells. Administration of etoposide during the prepubertal phase causes diturbances in several testicular morphometric parameters and in Sertoli cells. Cytoprotection of the seminiferous epithelium is the only means of preserving potential male reproduction in prepubertal cancer patients. Carnitine, an amino acid naturally present in normal cells, is a promising cryoprotectant as it is concentrated in the epididymis and promotes sperm maturation. We have therefore investigated whether carnitine protects rat testes against etoposide and, thus, improves fertility in adulthood. Our results suggest that carnitine partially protects the testis against damage caused by etoposide, although the mechanism by which it happens remains unknown.

Amifostine protective effect on cisplatin-treated rat testis.

Cisplatin is a potent drug used in clinical oncology but causes spermatogenesis damage. Amifostine is a drug used against toxicity caused by ionizing irradiation and chemotherapeutic drugs. Since cisplatin provokes fertility and induces germ cell apoptosis and necrosis, we proposed to evaluate the amifostine cytoprotective action on testes of cisplatin-treated rats. Thirty-day-old prepubertal Wistar rats received a single cisplatin dose of 5 mg/kg and were killed after 3, 6, and 12 hr. The hematoxylin-eosin stained testicular sections were submitted to histological, morphometric, and stereological analysis. The terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) method was used to label apoptotic cells. TUNEL-positive and TUNEL-negative germ cells with abnormal nuclear morphology (ANM) were scored. Significant alterations of greater part of the parameters occurred in the cisplatin-treated group (CE) compared to the group that received amifostine before the cisplatin-treatment (ACE); however, testicular weight and volume did not vary between these groups. Tubular diameter was reduced in CE in comparison to ACE rats, while interstitial tissue and lymphatic space volume and volume density were significantly higher in CE rats; interstitial testicular edema probably occurred in cisplatin-treated rats. CE rats showed important histological alterations, which were more accentuated than in ACE rats. The numerical densities of apoptotic germ cells and TUNEL-negative cells with ANM were lower in ACE than in CE rats. In conclusion, the amifostine previously administered to prepubertal rats reduced the testicular damage caused by cisplatin. We conclude that amifostine partially protected the rat seminiferous epithelium against cisplatin toxicity.

Sertoli cell morphological alterations in albino rats treated with etoposide during prepubertal phase.

Sertoli cells are very important to spermatogenesis homeostasis because they control germ cell proliferation, differentiation, and death. Damages to Sertoli cells cause germ cell death and affect fertility. Etoposide is a potent chemotherapeutic drug largely used against a variety of cancers. However, this drug also kills normal cells, especially those undergoing rapid proliferation. In the testis, etoposide acts predominantly on intermediate and type B spermatogonia. Etoposide was shown to permanently alter Sertoli cell function when administered to prepubertal rats. Based on this, we decided to investigate whether etoposide can affect Sertoli cell morphology. For this, 25-day-old rats were treated with etoposide during 8 consecutive days and killed at 32, 45, 64, 127, and 180 days old. Testes were fixed in Bouin's liquid or in a mixture of 2.5% glutaraldehyde and 2% formaldehyde for analysis under light and electron microscopes, respectively. Sertoli cells showed morphological alterations such as the presence of chromatin clumps close to the nuclear membrane, nucleus displacement, and cytoplasmic vacuolization. Some Sertoli cells also showed nuclear and cytoplasmic degenerative characteristics, suggesting that etoposide causes severe damages to Sertoli cell.

Sertoli cell function in albino rats treated with etoposide during prepubertal phase.

Sertoli cell plays a key role in spermatogenesis. Many studies refer that this cell is not harmed by the majority of anticancer treatments known to cause damage to the testis. However, in the previous study we observed that etoposide, an efficient chemotherapeutic drug, provokes an increase in numerical density of the Sertoli cells. This phenomenon suggests that this cell was harmed by etoposide. Thus, we decided to investigate a possible direct action of etoposide on Sertoli cells analyzing the function of this cell and relating it with the integrity and damage of the seminiferous epithelium. Prepubertal albino rats received 5 mg/kg of etoposide for eight consecutive days and were sacrificed in different ages. The control groups received 0.9% saline solution. The testes were fixed in Bouin's liquid for transferrin immunolabeling and testicular labeled tissue volume density measurement. Except for the younger rats, all the etoposide-treated rats showed diminution of transferrin immunolabeling in the seminiferous epithelium, and consequently, of total labeled testicular tissue volume density. We concluded that the diminution of transferrin labeling in the seminiferous epithelium was not associated with germ cell absence such as commonly reported. The results suggest etoposide impairs Sertoli cell function.

Apoptosis and testicular alterations in albino rats treated with etoposide during the prepubertal phase.

Etoposide is a podophyllotoxin semiderivative that is used in a variety of chemotherapy treatments, including therapy for children tumors. This drug promotes the formation of a ternary DNA-topoisomerase II-etoposide complex that triggers apoptosis. The purpose of this work was to analyze the occurrence of apoptosis in the seminiferous epithelium of prepubertal, pubertal, and adult rats treated with 10, 20, and 40 mg/Kg of etoposide during the prepubertal phase, as well as the role of apoptosis in etoposide-induced testicular damage. The rat testes were fixed in Bouin's liquid, and the apoptotic cells were quantified by means of the hematoxylin and eosin (H&E) technique (all groups) and the terminal dUTP nick end labeling (TUNEL) method (prepubertal groups only). The results obtained from both the H&E and TUNEL methods showed an increased frequency of apoptosis in the seminiferous epithelium of treated animals, except for the subgroup that received the 10-mg/Kg dose and was sacrificed 12 hr after the treatment and for the etoposide-treated pubertal group, that did not show cells suggesting apoptosis during H&E analysis. The labeled cells were mainly primary spermatocytes and differentiated spermatogonia. The prepubertal rats showed an etoposide-dose-dependent diminution of differentiated spermatogonia. Etoposide treatment during the prepubertal phase increases the frequency of apoptosis in the seminiferous epithelium, and causes serious harm to male fertility. 2004.