El consumo semicrónico moderado de alcohol altera la foliculogénesis y la calidad núcleo-citoplasmática oocitaria, en el ratón / Moderate semi-chronic alcohol consumption alters folliculogenesis and oocyte nuclear-cytoplasmic quality in mice

Resumen El consumo crónico de alcohol es un problema de salud mundial que afecta particularmente a la población femenina. Sin embargo, los efectos de la ingesta semicrónica en cantidades moderadas a bajas en el ovario y el oocito son poco conocidos. En un modelo murino, se administró etanol al 10% en agua de bebida (hembras tratadas) o agua (hembras control) por 15 días, y luego de la superovulación o no (ovulación espontánea), se analizó el ciclo estral y la calidad ovárico-gamética. En las hembras tratadas, la frecuencia y duración del diestro aumentó, y las frecuencias de folículos y cuerpos lúteos disminuyeron vs hembras controles, valores que se restauraron luego de la superovulación. Sin embargo, en las hembras tratadas, la tasa de proliferación celular folicular y el desbalance de la expresión ovárica de VEGF (factor de crecimiento endotelial) persistieron luego de la superovulación. El número de ovocitos ovulados con metafase II anormal, fragmentados y activados partenogenéticamente fue mayor en las hembras tratadas respecto las controles. En conclusión, el consumo semicrónico moderado de alcohol produce anestro, ciclo estral irregular, foliculogénesis deficiente y anomalías núcleo-citoplasmáticas en los oocitos ovulados. Estas alteraciones podrían constituirse en un factor etiológico de pérdida gestacional temprana y desarrollo embrionario anormal luego del consumo de alcohol.

Abstract Chronic alcohol consumption is a global health problem that particularly affects the female population. However, the ef-fects of semi-chronic ethanol intake in low-moderate amounts on the ovary and oocyte are poorly understood. In a mouse model, 10% ethanol was administered in drinking water (treated females) or water (control females) for 15 days, and after superovulation or not (spontaneous ovulation), the estrous cycle and ovarian-gametic quality were analyzed. In treated females, the frequency and duration of the diestrus increased, and the frequencies of follicles and corpus luteum decreased vs control females, values that restored after superovulation. However, in treated females, the follicular cell proliferation rate and the imbalance in ovarian expression of VEGF (endothelial growth factor) persisted after superovulation. The number of ovulated oocytes with abnormal metaphase II, fragmented and parthenogenetically activated was higher in treated females than in control ones. In conclusion, moderate semi-chronic alcohol consumption produces anestrum, irregular estrous cycle, poor folliculogenesis, and nuclear-cytoplasmic abnormalities in ovulated oocytes. These alterations could constitute an etiological factor of early gestational loss and abnormal embryonic development after alcohol consumption.

The morphology of paca (Cuniculus paca) testis with high dose of letrozole an aromatase inhibitor / La morfología del testículo de paca (Cuniculus paca) con altas dosis de letrozol, un inhibidor de la aromatasa

SUMMARY: The study reported the influence of the high and acute dose of Letrozole on the testis morphology in paca (Cuniculus paca), an aromatase inhibitor that reduces the endogenous estrogen, the essential hormone for spermatogenesis. Morphological changes were observed in seminiferous epithelium with germ cells with apoptotic characteristics and presence of vacuoles and nuclei in pycnose.

RESUMEN: El objetivo de este estudio fue analizar la influencia de una dosis alta de Letrozol en la morfología de los testículos de la paca (Cuniculus paca), un inhibidor de la aromatasa que reduce el estrógeno endógeno, la hormona esencial para la espermatogénesis. Se observaron cambios morfológicos en el epitelio seminífero con células germinales con características apoptóticas y la presencia de vacuolas y núcleos en picnosis.

Protective effects of Tribulus terrestris hydroalcoholic extract against cisplatin-induced germ cell apoptosis in male mice / Efectos protectores del extracto hidroalcohólico Tribulus terrestris contra la apoptosis de células germinales inducida por cisplatino en ratones machos

SUMMARY: Toxic effects of anti-cancer and other drugs on the normal tissues could be reduced by the herbal plants and their fractions. This study investigated the protective effect of Tribulus terrestris (TT) on Cisplatin- induced cytotoxicity germ cell apoptosis in male mice. In this experimental study, thirty male Balb/c mice were divided randomly into 5 groups (n=6). A single dose of Cisplatin (5.5 mg/kg) and differ-ent concentrations of Tribulus terrestris were administrated for 14 consecutive days. Reverse transcription polymerase chain reaction (RT-PCR) of apoptosis-re-lated genes was performed with RNA extracted from testes of the mice. Statistical analysis was done using one-way ANOVA. In the Cisplatin group, there was a significant increase in mRNA expression of p53 (P=0.008), bax (P=0.004) and the ratio of bax/Bcl-2 (P=0.000), whereas there was an decrease in the expression of Bcl-2 (P=0.003), as compared to control group. In Cis+TT groups, the data showed that different concentrations of TT could improve the harmful effects caused by the Cisplatin. The best protective effects were achieved in Cis+TT (300 mg/kg). Tribulus terrestris protects testicular germ cell against Cisplatin induced apoptosis by affecting related genes regulation.

RESUMEN: Los efectos tóxicos en los tejidos normales, de los medicamentos contra el cáncer al igual que otras medicamentos podrían mejorar con el uso de plantas medicinales y hierbas. Este estudio investigó el efecto protector de Tribulus terrestris (TT) sobre la apoptosis de células germinales por citotoxicidad inducida por cisplatino en ratones machos. En este estudio se dividieron treinta ratones Balb/c macho aleatoriamente en 5 grupos (n = 6). Se administró una sola dosis de cisplatino (5,5 mg / kg) y diferentes concentraciones de Tribulus terrestris durante 14 días consecutivos. La reacción en cadena de la polimerasa de transcripción reversa de los genes relacionados con la apoptosis, se realizó con ARN extraído de los testículos de los ratones. El análisis estadístico se realizó usando ANOVA de una vía. En el grupo cisplatino, hubo un aumento significativo en la expresión de mRNA de p53 (P = 0,008), bax (P = 0,004) y la relación de bax / Bcl-2 (P = 0.000), mientras que hubo una disminución en la expresión de Bcl-2 (P = 0,003), en comparación con el grupo control. En los grupos Cis + TT, los datos mostraron que las diferentes concentraciones de TT podrían mejorar los efectos nocivos causados por el cisplatino. Los mejores efectos protectores se lograron en Cis + TT (300 mg / kg). Tribulus terrestris protege las células germinales testiculares contra la apoptosis inducida por cisplatino al afectar la regulación de los genes relacionados.

Effects of Ghrelin on germ cell apoptosis and proinflammatory cytokines production in Ischemia-reperfusion of the rat testis

Testicular torsion is a medical emergency that requires surgical intervention to reperfuse the affected testis. Ischemia reperfusion injury is usually associated with proinflammatory cytokine generation and apoptosis of germ cells in the testes. In this study we investigate the effect of ghrelin on the proinflammatory cytokines levels and germ cell apoptosis in testicular ischemia reperfusion. 45 male rats were selected for the study and randomly divided into 3 groups, each containing 15 rats. Animals in the testicular torsion and ghrelin treated groups were subjected to unilateral 720 counterclockwise testicular torsion for 1 hr and then reperfusion was allowed after detorsion for 4 hr, 1 and 7 days. The ghrelin-treated group received intraperitoneal injection of ghrelin 15min before detorsion. The expression levels of bcl-2-associated X protein and proliferating cell nuclear antigen in testicular tissue in the different groups were detected by immunohistochemical assay and tissue cytokines interleukin-1beta, tumor necroses factor-alpha and interleukin-6 were measured using enzyme-linked immunosorbent assay. After being treated by ghrelin, the population of immunoreactive cells against BAX in the spermatocytes on day 7 after reperfusion significantly decreased when compared to tortion/ detortion-saline animals [p=0.024]. In contrast, PCNA expression in the spermatocytes and spermatogonia were not significantly different between tortion/ detortion-ghrelin and tortion/ detortion-saline groups on both experimental days. Administration of ghrelin significantly attenuated the testicular tumor necroses factor-alpha and interleukin-6 levels compared with the untreated animals, but had no significant effect on the level of interleukin-1beta. Ghrelin offers remarkable anti-inflammatory and anti-apoptotic effects in testicular ischemia reperfusion injury

Contribution of environmental pollutants to male infertily: A working model of germ cell apoptosis induced by plasticizers

Bisphenol A [2,2-bis(4-hydroxyphenyl)propane] (BPA), 4-nonylphenol (NP) and di(2-ethylhexyl)phthalate (DEHP), and its metabolite mono-2-ethylhexyl phthalate (MEHP) are chemicals found in plastics, which act as endocrine disruptors (EDs) in animals, including human. EDs act like hormones in the endocrine system, and disrupt the physiologic function of endogenous hormones. Most people are exposed to different endocrine disruptors and concern has been raised about their true effect on reproductive organs. In the testis, they seem to preferentially attack developing testis during puberty rather than adult organs. However, the lack of information about the molecular mechanism, and the apparently controversial effect observed in different models has hampered the understanding of their effects on mammalian spermatogenesis. In this review, we critically discuss the available information regarding the effect of BPA, NP and DEHP/ MEHP upon mammalian spermatogenesis, a major target of EDs. Germ cell sloughing, disruption of the blood-testis-barrier and germ cell apoptosis are the most common effects reported in the available literature. We propose a model at the molecular level to explain the effects at the cellular level, mainly focused on germ cell apoptosis.

Chronic ethanol consumption in mice does not induce DNA damage in somatic or germ cells, evaluated by the bone marrow micronucleous assay and the dominant lethal mutation assay

Although alcohol is known to be a carcinogen for humans, ethanol-genotoxicity studies are incomplete. Ethanol seems not to be a bacterial mutagen, but the results are conflicting in rodent assays. We investigate the genotoxicity in the bone marrow micronucleus (MN) test and in the dominant lethal mutation (DLM) assay using two long-term ethanol exposure protocols. In the MN test, mice consumed three doses (5, 10 and 15% v/v) for 32 weeks. MN induction was compared to two control groups of 5- and 38-week-old mice (the ages of the treated mice when the treatment was initiated and when they were killed, respectively). For the three groups treated with ethanol there was no significant increase in MN induction as compared to the first control group, but observed MN frequencies were significantly lower than in the 38-week-old control group. This suggests a protective effect against genotoxic damage caused by aging, probably due to ethanol action as a hydroxyl radical scavenger. In the DLM assay, male mice drank ethanol at 15% or 30% (v/v) for 20 weeks. In both groups the number of dead implants was similar to the control, but there was a significant reduction in total implants, indicating a pre-implantation loss.

Histomorphometric study of effects of bicalutamide on spermatogenesis in male rats

To study the effects of Bicalutamide on spermatogenesis in male rats. Laboratory based randomized controlled trial. Anatomy Department, Armed Forces Postgraduate Medical Institute [AFPGMI], Rawalpindi in collaboration with National Veterinary Laboratories [NVL], Islamabad from April 2008 to May 2008. Forty adult male Sprague Dawley rats weighing 200-300 grams were randomly divided into two groups, Group A and Group B, each consisting of 20 animals each. Group A was taken as control group and was administered 5 cc of distilled water orally daily for 24 days while group B [Experimental group] was given 5 cc of distilled water daily containing bicalutamide 10 mg/ kg/ day for 24 days. All the animals were sacrificed on the next day after the last dose. The testes were removed and fixed in 10% formalin and then processed for paraffin embedding. Five micron thick sections were made. Haematoxylin, Eosin and PAS stains were used. Histomorphometric analysis was done and parameters, including the tubular diameter, height of seminiferous epithelium and germ cell count were noted. Statistically significant differences were found in tubular diameter, height of seminiferous epithelium and germ cell count in testes of experimental group when compared with the control group. The results showed that the mean tubular diameter, the height of the germinal epithelium of the seminiferous tubules and the number of germ cells were significantly reduced in by the experimental group showing that bicalutamide suppresses spermatogenesis in the Sprague - Dawley rats

effect of human gonadotropin treatment on recipient mouse germ cell proliferation following spermatogonial stem cell transplantation of neonatal donor mice

Spermatogonia are the male germ line stem cells whose life long expansion is needed for permanent production of spermatozoa. The present study was designed to examine the effect of hCG treatment on germ cell proliferation following stem cell transplantation in mice. Spermatogonial stem cells were isolated from neonatal mice testes and characterized by alkaline phosphatase, immunoreactivity and morphological analysis. hCG was injected into normal and cell transplanted mice. We then evaluated the testosterone levels and cell number in normal mice. After that, cyclin B1 gene expression was investigated in transplanted mice. Different doses of busulfan were injected to investigate the effects of chemotherapy on morphological criteria and preparation of recipient mice for transplantation. In this report we show proliferative potential of spermatogonial stem cells after cytotoxic treatment, transplantation efficiency by semi-quantitative RT-PCR, and hCG effect on stem cell regeneration in normal mice and following cell transplantation. The results indicate the spermatogonial stem cells can proliferate after transplantation, and the efficiency of their transplantation depends on hormonal treatment. Therefore, hormonal treatment after stem cell transplantation will be a powerful avenue for increasing the efficiency of transplantation and fertility restoration

[Buserelin inhibits apoptosis in male germ cells induced by busulfan in mouse testis]

The aim of this study was to investigate the effect of buserelin on apoptosis of male germ cells induced by busulfan in adult male mice. Male adult NMRI mice were divided into four group of eight each. Group 1 [control] administered PBS for 21 days subcutaneously, group 2 given 0.4 micro g buserelin for 21 days subcutaneously, group 3 given single dose of 30 mg/kg busulfan intraperitoneally and group 4 given both busulfan and buserelin for 21 days. The animals were sacrificed and their testes were dissected 35 days after the treatment. Evaluations were made by determining Johnson's score and apoptosis were assayed by terminal- deoxynucleotidyl- transferase-mediated dutp nick end labeling [TUNEL]. Statistical analyses were performed using ANOVA test. Recovery status and Johnson's score in group 4 were significantly higher than those of busulfan treated group 7.71 +/- 0.69 VS 4.46 +/- 0.56 [p< 0.001]. Apoptotic cells number cells were significantly more numerous in busulfan treated group than those of control 23.28 +/- 7.10 VS 3.54 +/- 1.02 [p<0.001]. While buserelin substantially reduced germ cell apoptosis in fourth group 10.50 +/- 2.91 in comparison with third group 23.28 +/- 7.10, [p<0.001]. Administration of buserelin after testicular damage by busulfan enhances the regeneration of spermatogenesis in mouse through inhibition of apoptosis in germ cells

Gametocyte Clearance in Uncomplicated and Severe Plasmodium falciparum Malaria after Artesunate-Mefloquine Treatment in Thailand

Artemisinin-based combination therapy (ACT) is currently promoted as a strategy for treating both uncomplicated and severe falciparum malaria, targeting asexual blood-stage Plasmodium falciparum parasites. However, the effect of ACT on sexual-stage parasites remains controversial. To determine the clearance of sexual-stage P. falciparum parasites from 342 uncomplicated, and 217 severe, adult malaria cases, we reviewed and followed peripheral blood sexualstage parasites for 4 wk after starting ACT. All patients presented with both asexual and sexual stage parasites on admission, and were treated with artesunate-mefloquine as the standard regimen. The results showed that all patients were asymptomatic and negative for asexual forms before discharge from hospital. The percentages of uncomplicated malaria patients positive for gametocytes on days 3, 7, 14, 21, and 28 were 41.5, 13.1, 3.8, 2.0, and 2.0%, while the percentages of gametocyte positive severe malaria patients on days 3, 7, 14, 21, and 28 were 33.6, 8.2, 2.7, 0.9, and 0.9%, respectively. Although all patients were negative for asexual parasites by day 7 after completion of the artesunate-mefloquine course, gametocytemia persisted in some patients. Thus, a gametocytocidal drug, e.g., primaquine, may be useful in combination with an artesunate-mefloquine regimen to clear gametocytes, so blocking transmission more effectively than artesunate alone, in malaria transmission areas.

Effect of phosphamidon on the testes of albino rats: a histological study.

The present study shows the qualitative and quantitative histological changes in testes of albino rats treated with two doses of phosphamidon 35 and 50 parts per million(ppm) for 1 month time period. Rats were treated by drinking water containing 35 ppm (low dose) and 50ppm (high dose) concentration of phosphamidon for 30 days. After 30 days, they were sacrificed, the testes were fixed in vivo and were taken out. The histological slides of these testes were prepared and were studied under light microscope. The decrease in the weight of testes and diameter of seminiferous tubules, increase in the interstitial space, the decrease in the numbers of germ cells and supporting cells, Cytoplasmic vacuolization of the germ cells, distortion of seminiferous tubules were the findings of present study. phosphamidon seems to be toxic on male reproductive system if exposed for prolong period. The awareness regarding the impact of phosphamidon should be given to farmer and they should be encouraged to practice biological means to control pests and herbs instead of these harmful chemical compounds.

[ morphologic and morphometric study of adult mouse testis following different doses of busulfan administration]

Anti-cancer drugs have adverse effects on spermatogenesis. Therefore, information on their role for the prevention of germinal epithelium destruction is necessary. The aim of this study was morphologic and morphometric evaluations of testes, measurement of volume and volume density of testes parameters, measurement of tubular diameters, germ and somatic cell counts following administration of different doses of busulfan in adult mice. In the present study, 42 male NMRI mice aged 6-8 weeks were used. The animals were divided into 5 groups. Case groups received a single dose of busulfan by intraperitoneal injection as 5, 10, 20 and 40 mg/kg in the first, second, third and forth groups respectively. The control group received only the solvent for busulfan. All the animals were killed 35 days after treatment and their testes were dissected out and processed for light microscope studies. Then morphometric studies were performed on testicular parameters. The data were analyzed using ANOVA and Tukey test and p values less than 0.05 were considered significant Busulfan administration in 5, 10, 20 and 40 mg/kg doses significantly reduced most morphometric parameters in testes with a maximum effect in the 40 mg/kg group. Volumes of testes, tubules and germinal epithelia were decreased significantly in the experiment groups [p<0.05] however, the volume of interstitial tissue increased [p<0.05]. Tubular diameters and thickness of epithelia were also decreased in the experiment groups. Number of germ cells was reduced, but number of sertoli cells was not affected. The number of leydig cells were not affected in 10 and 20 mg/kg busulfan treated groups, however in the 40 mg/kg treated group they were increased significantly [p<0.003]. In 5 mg/kg treated group there were no significant differences in morphologic and morphometric studies. Busulfan could reduce testicular parameters and disrupt spermatogenesis through affecting both germ and somatic cells in a dose dependent manner. Therefore, the side effects of busulfan on spermatogenesis should be considered during cancer therapies

Induction of seminiferous tubular atrophy by single dose of 5-fluorouracil (5-FU) in Wistar rats.

Antimetabolite, 5-fluorouracil (5-FU) is known to cause testicular damage by epithelial sloughing and cell killing. However, it is not known whether 5-FU induces tubular atrophy and the fate of exfoliated germ cells. Present study was conducted to evaluate these effects of 5-FU on rat testis. Animals were injected, single dose of 5-FU (10.50 & 100 mg/kg, i.p.) and sampled at 1, 3, 15 and 30 day following the treatment. The testes were perfusion fixed by Bouin's fluid. Five micron thick paraffin sections of testes and epididymis were stained with haematoxylin and eosin. Slides were examined for the incidence of abnormal tubules (per 200 tubules), tubular diameter (STD), epithelial height (SEH) and for the presence of germ cells in the epididymis. Data were analysed by Mann-Whitney 'U' test. The testes weight, STD, SEH were decreased (P < 0.05-0.01) in treated animals. The abnormal tubules were increased in a dose dependent manner with atrophic tubules seen on 30 d. The exfoliated germ cells have not blocked the post testicular ductal system and found in the epididymis in a dose dependent manner. The present study concludes that 5-FU causes tubular shrinkage and atrophy. Further, epididymis is involved in the phagocytosis of germ cells.

Effect of vitamin A excess on germ cell development in prepubertal rat testis.

Vitamin A in graded doses of 125, 250 and 375 U.S.P./kg body wt, po, for 10 days (d 21-30) drastically reduced the testicular weight by 25 to 62% and seminiferous tubular diameter by 14 to 35% in prepubertal rats in lowest and highest doses of the treatment. The treatment induced disproportionate enlargement of nuclei and cytoplasm of the germ cells; predominantly the preleptotene and pachytene spermatocytes. These abnormal germ cells, often with 2 or 3 nuclei displayed vacuolated cytoplasm surrounding pyknotic or granulated or dispersed chromatin granules within the nuclei in a dose proportionate manner. The round spermatids were the most sensitive cell types which completely disappeared in two higher doses of treatment. Vacuolation of Sertoli cell cytoplasm in about 25% of the tubules with associated increase in intertubular space was also observed in rats treated with the highest dose of the vitamin. Circulatory levels of FSH, LH and testosterone remained unaltered following the vitamin excess treatment. Therefore, it is suggested that excess vitamin A even for shorter duration like the present one is detrimental to developing cell types and prevents the progress of the spermatogenic process beyond the round spermatid stage.

Genotoxic effects of pirmor on the germ cells of Drosophila melanogaster

Pirimor, a carbamate pesticides, was evaluated for its genotoxic effects on male germ cells and sterility in Drosophila melangogaster, by the well-known sex linked recessive lethal test. 2 concentrations LC50 and LC5 [0.47% and 0.33%] were used following one route of administration adult feeding. After single exposure, the results showed that at high concentration Pirimor induced significant increase of mutation frequencies in the first 2 broods of spermatogenesis which decreased gradually in the other germ-cell stages. On the contrary, value of lethals at LC5 [0.33%] were insignificant in all broods of spermatogenesis. Steriles induced by Pirimor were higher in the first 2 broods than the others stages at both concentrations. Lethalities as well as sterility exhibited a concentration and stage-dependent effect

Inhalation toxicity of methylisocyanate: assessment of germ cell mutagenicity and reproductive effects in rats.

Adult male Wistar rats were exposed to methylisocyanate (MIC, 3.2 mg/l, single inhalation exposure for 8 min under static condition) or ethyl methanesulphonate (EMS, 150 mg/kg, single ip dose) for the assessment of germ cell mutagenicity and reproductive effects. Sequential matings of treated males with normal females on days 1-7, 8-14 and 15-21 post-exposure did not indicate any induction of dominant lethal mutation (increased frequency of preimplantation losses and early fetal deaths) by MIC but it was significantly induced by EMS as compared to respective controls. Males, necropsied after 21 days of exposure, showed no effect of MIC on epididymal sperm density and morphology. EMS also had no effect on sperm density but it significantly induced morphological abnormalities in sperm as compared to untreated controls. There was an acute and transitional reduction in reproductive performance (10-21%) of MIC-exposed males during days 1-14 post-exposure followed by recovery to the normal level during days 15-21 post-exposure. The progeny of MIC-exposed males was also normal in terms of litter size, litter weight, neonatal survival and body weight gain in litters up to 10 days post-partem. It is concluded with the evidence at hand that the observed failure of MIC to cause germ cell mutagenicity is related to its poor biodistribution to the target site(s) and a transient reduction in the reproductive performance of MIC-exposed males is a result of general stress and disconsolate copulation.