RESUMO
Smoking can lead to several diseases and cause a reduction in fertility in men and women. Among the various components of cigarettes harmful during pregnancy, nicotine stands out. It can cause a reduction in placental blood flow, compromising the development of the baby with neurological, reproductive and endocrine consequences. Thus, we aimed to evaluate the effects of nicotine on the pituitary-gonadal axis of rats exposed during pregnancy and breastfeeding (1st generation - F1), and whether the possible damage observed would reach the 2nd generation (F2). Pregnant Wistar rats received 2 mg/kg/day of nicotine throughout the entire gestation and lactation. Part of the offspring was evaluated on the first neonatal day (F1) for macroscopic, histopathological and immunohistochemical analyses of brain and gonads. Another part of the offspring was kept until 90 days-old for mating and obtainment of progenies that had the same parameters evaluated at the end of pregnancy (F2). The occurrence of malformations was more frequent and diversified in nicotine-exposed F2. Brain alterations, including reduced size and changes in cell proliferation and death, were seen in both generations of nicotine-exposed rats. Male and female gonads of F1 exposed rats were also affected. The F2 rats showed reduced cellular proliferation and increased cell death on the pituitary and ovaries, besides increased anogenital distance in females. The number of mast cells was not enough altered to indicate an inflammatory process in brain and gonads. We conclude that prenatal exposure to nicotine causes transgenerational alterations in the structures of pituitary-gonadal axis in rats.
Assuntos
Exposição Materna , Efeitos Tardios da Exposição Pré-Natal , Ratos , Gravidez , Feminino , Animais , Masculino , Humanos , Exposição Materna/efeitos adversos , Nicotina/toxicidade , Ratos Wistar , Placenta , ReproduçãoRESUMO
Doxorubicin has been largely used in anticancer therapy in adults, adolescents, and children. The efficacy of l-carnitine as an antioxidant substance has been confirmed both in humans and rats. Carnitine, present in testis and epididymis, is involved in sperm maturation. It is also effective in infertility treatment. As a continuation of a previous study, we evaluated whether some spermatic qualitative parameters, DNA integrity, chromatin structure, and fertility status, could be ameliorated by the carnitine treatment in adult rats, which were subsequently exposed to doxorubicin at pre-puberty. Pre-pubertal male rats were distributed into four groups: Sham Control; Doxorubicin; l-carnitine; l-carnitine + Doxorubicin (l-carnitine injected 1 h before doxorubicin). At 100 days of age, all groups were reassigned into two sets: One set was submitted to the evaluation of sperm motility, acrosome integrity, mitochondrial activity, sperm chromatin structure analysis (SCSA), and evaluation of the oxidative stress. The other set of rats was destined to the evaluation of reproductive competence. The percentage of spermatozoa with intact acrosome integrity was higher in the Carnitine+Doxorubicin group when compared with the Doxorubicin group. However, sperm motility and mitochondrial activity were not improved by carnitine pre-treatment. Both values of malondialdehyde and nitrite (indirect measurement of nitric oxide) concentrations were statistically higher in the only doxorubicin-treated group when compared to the Carnitine + Doxorubicin group. Fertility index and implantation rate were lower in Doxorubicin group, when compared to Carnitine + Doxorubicin group. Moreover, the percentage of spermatozoa with damaged DNA was higher in the Doxorubicin-treated group when compared to the Carnitine+Doxorubicin group. l-carnitine, when administered before doxorubicin, partially preserved the acrosome integrity, an important feature related to sperm fertilization ability that positively correlated with the reproductive competence and sperm DNA integrity at adulthood. In conclusion, l-carnitine attenuated the long-term alterations caused by doxorubicin in the germ cells and improved male reproductive capacity in adulthood.
Assuntos
Acrossomo/efeitos dos fármacos , Antibióticos Antineoplásicos/toxicidade , Antioxidantes/farmacologia , Carnitina/farmacologia , Doxorrubicina/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Animais , Citoproteção , Masculino , Ratos , Ratos Wistar , Análise do Sêmen , Motilidade dos Espermatozoides/efeitos dos fármacosRESUMO
We previously observed that nicotine, administered to rats (Wistar) during pregnancy and lactation periods, provokes, in the progeny, late morphofunctional alterations in Leydig cell, body weight increase in adulthood (90 days post partum, dpp) as well as seminiferous epithelium injury. Aiming to investigate whether the spermatogenic damage previously observed in adult progenies from pregnant and lactating nicotine-exposed rat dams are maintained or whether it is worsened in older rats, we analyzed the morphological testicular alterations after up to two complete periods of spermatogenesis (53 days each), spermatic parameters, and sperm DNA fragmentation. Pregnant and lactating rats were nicotine-exposed (2 mg/kg/day) through an osmotic minipump implanted on the first day of pregnancy and replaced after birth. Absolute Control (no minipump) and Sham Control (minipump without nicotine) groups were established. The offspring were killed at 90, 143, and 196 dpp. Significant alterations in morphometric and stereological testicular parameters, such as concentration of sperm number, daily sperm production, and plasma and intratesticular levels of cholesterol and testosterone were not observed in nicotine-exposed rats. Testicular histopathological analysis showed small intraepithelial vacuolization and an accentuated germ cell desquamation in exposed rats. However, the offspring from nicotine-exposed dams exhibited higher frequency of morphologically abnormal spermatozoa and lower sperm motility in comparison with control groups. In addition, nicotine-exposed groups showed a significant reduction in sperm mitochondrial activity and an increased sperm DNA fragmentation (Comet assay). These results indicate a late reproductive damage in the male progeny caused by maternal nicotine exposure, related to the decrease in sperm quality.
Assuntos
Nicotina/toxicidade , Efeitos Tardios da Exposição Pré-Natal , Espermatozoides/efeitos dos fármacos , Testículo/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Colesterol/metabolismo , Feminino , Lactação , Masculino , Tamanho do Órgão/efeitos dos fármacos , Gravidez , Ratos Wistar , Reprodução/efeitos dos fármacos , Contagem de Espermatozoides , Motilidade dos Espermatozoides/efeitos dos fármacos , Testículo/patologia , Testosterona/metabolismoRESUMO
Nicotine is largely consumed in the world as a component of cigarettes. It can cross the placenta and reach the milk of smoking mothers. This drug induces apoptosis, affects sex hormone secretion, and leads to male infertility. To investigate the exposure to nicotine during the whole intrauterine and lactation phases in Sertoli cells, pregnant rats received nicotine (2 mg/kg per day) through osmotic minipumps. Male offsprings (30, 60, and 90 days old) had blood collected for hormonal analysis (FSH and LH) and their testes submitted for histophatological study, analysis of the frequency of the stages of seminiferous epithelium cycle, immunolabeling of apoptotic epithelial cells (TUNEL and Fas/FasL), analysis of the function and structure of Sertoli cells (respectively using transferrin and vimentin immunolabeling), and analysis of Sertoli-germ cell junctional molecule (ß-catenin immunolabeling). The exposure to nicotine increased the FSH and LH plasmatic levels in adult rats. Although nicotine had not changed the number of apoptotic cells, neither in Fas nor FasL expression, it provoked an intense sloughing of epithelial cells and also altered the frequency of some stages of the seminiferous epithelium cycle. Transferrin and ß-catenin expressions were not changed, but vimentin was significantly reduced in the early stages of the seminiferous cycle of the nicotine-exposed adult rats. Thus, we concluded that nicotine exposure during all gestational and lactation periods affects the structure of Sertoli cells by events causing intense germ cell sloughing observed in the tubular lumen and can compromise the fertility of the offspring.
Assuntos
Gonadotropinas/sangue , Lactação/efeitos dos fármacos , Nicotina/toxicidade , Efeitos Tardios da Exposição Pré-Natal/sangue , Efeitos Tardios da Exposição Pré-Natal/patologia , Células de Sertoli/efeitos dos fármacos , Animais , Feminino , Masculino , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Ratos , Células de Sertoli/patologia , Células de Sertoli/fisiologia , Espermatogênese/efeitos dos fármacos , Espermatogênese/fisiologia , Testículo/efeitos dos fármacos , Testículo/patologiaRESUMO
Doxorubicin, an anticancer drug, is widely included in chemotherapy protocols to combat childhood cancer. Carnitine, an important quaternary amine, is present in testis and epididymis and is involved in sperm maturation; it has been used in infertility treatment. In a previous study, our group observed that L-carnitine given before etoposide, another chemotherapeutic drug, reduces the spermatogenic damage and protects germ cells against apoptosis. This study aimed to evaluate the antiapoptotic and cytoprotective actions of L-carnitine in long- and mid-term basis, on the seminiferous epithelium of doxorubicin-treated pre-pubertal rats. Forty-eight 30-day-old male Wistar rats were distributed into four groups: sham-control; doxorubicin; carnitine; carnitine/doxorubicin (L-carnitine injected 1 h before doxorubicin). The rats were submitted to euthanasia at 64 and 100 days of age and their testes were collected for biometric, morphometric, and histopathological analyses. The numerical density of apoptotic germ cells was obtained (TUNEL method). In adult phase (100 days), the following spermatic parameters were analyzed: mature spermatid (19 step) count and sperm daily production per testis; sperm number and transit time through the epididymal caput/corpus and cauda; frequency of morphologically abnormal spermatozoa (from epididymal fluid), as well as sperm DNA integrity (Comet assay). The testicular and spermatic parameters at both ages were improved in rats treated with carnitine before doxorubicin. At 64 days, the TUNEL-positive germ cell frequency was lower in the carnitine/doxorubicin-treated rats comparatively to the doxorubicin-treated rats. At 100 days of age, the sperm DNA fragmentation was also lower in the previously carnitine-treated rats, as evidenced by the analysis of three parameters. Carnitine reduced the late testicular and spermatic damages caused by doxorubicin, probably providing a partial cytoprotection against the deleterious action of doxorubicin administration to pre-pubertal rats. However, further studies shall be undertaken to investigate the protective mechanisms involved in such germ cell preservation.
Assuntos
Carnitina/farmacologia , Doxorrubicina/toxicidade , Maturidade Sexual/efeitos dos fármacos , Testículo/efeitos dos fármacos , Animais , Peso Corporal , Ensaio Cometa , Marcação In Situ das Extremidades Cortadas , Masculino , Tamanho do Órgão , Ratos , Ratos WistarRESUMO
Nicotine is largely consumed as a component of cigarettes. It induces apoptosis, interferes with endocrine function by changing the sex hormones secretion and leads to male infertility. Testosterone is produced from cholesterol by Leydig cells (LC), with the participation of testicular macrophages (MO). Thus, to investigate whether nicotine administration to pregnant and lactating rats changes cholesterol and sexual hormone levels and LC and MO populations of offspring, female rats received nicotine (2 mg/kg/day) through osmotic minipumps from the first day of pregnancy up to the end of weaning. At 1, 30, 60 and 90 days post-partum (dpp) the plasma cholesterol and testosterone levels were obtained, as well as the biometric, histopathological and stereological testicular parameters. Nicotine reduced the body weight, cholesterol levels and lipid droplet number in foetal LC at 1 dpp. The number of apoptotic LC did not change in the offspring of nicotine group at any age studied. No alterations in the numerical densities of MO and LC occurred at 60 and 90 dpp. Hypertrophy of mature LC and increase in cholesterol and testosterone levels were noted at 90 dpp. In conclusion, nicotine when administered to rats throughout pregnancy and lactation induces morphofunctional alterations of foetal and mature LC and affects cholesterol and testosterone levels.
Assuntos
Células Intersticiais do Testículo/fisiologia , Macrófagos/fisiologia , Nicotina/farmacologia , Efeitos Tardios da Exposição Pré-Natal , Animais , Apoptose/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Colesterol/sangue , Feminino , Estimulantes Ganglionares/farmacologia , Lactação , Células Intersticiais do Testículo/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Masculino , Agonistas Nicotínicos/administração & dosagem , Agonistas Nicotínicos/farmacologia , Gravidez , Ratos , Testosterona/sangueRESUMO
Obesity has been considered a public health issue in many countries and is of increasing concern for authorities over the past 6 years. The Zucker rat is a good experimental model for obesity and diabetes studies due to its metabolic characteristics that are similar to those developed by humans. A total of 12 obese Zucker rats and their lean littermates were killed in pubertal and young adult phases for assessing organ weights (testis and epididymis), testicular histomorphometric and stereological analyses, daily sperm production, and transit time in the epididymis. Sperm integrity was also investigated in the adult animals using the Comet assay. Alterations in organ weights, seminiferous epithelium architecture, sperm production, and transit time were noticed in the pubertal fatty rats. The volume density of the lymphatic space was decreased in both the ages. Adult animals had a significant increase in the extent of damage found in sperm DNA. Our results show for the first time that leptin receptor deficiency compromises sperm production during puberty and that genetic obese Zucker rats have increased sperm DNA fragmentation.
Assuntos
Dano ao DNA/fisiologia , Obesidade/genética , Reprodução/fisiologia , Espermatozoides/patologia , Espermatozoides/fisiologia , Animais , Fragmentação do DNA , Masculino , Obesidade/patologia , Ratos , Ratos Zucker , Contagem de Espermatozoides/métodosRESUMO
The rat has been elected as the main animal model in several studies involving reproduction. However, there are scarce and conflicting data related to its estrous cycle. It comprises phases characterized by different cell types in vaginal smears (proestrus, estrus, metestrus and diestrus). Nevertheless, this classification and the time span of each phase are controversial and may induce misleading interpretations. In addition, there are no reports regarding the quantification of all cell types in each phase, including pre-acidophilic cells and leukocytes. The goal of this study was to revisit the literature about the rat estrous cycle and to perform a detailed quantitative and qualitative description of its phases and the transitional periods among them. Vaginal smears were obtained twice daily for 20 days from Wistar rats and stained using the Shorr method. Cells were classified as small (SBC) or large (LBC) basophilic cells, nucleated (NAC) or enucleated (EAC) acidophilic cells, pre-acidophilic cells (PAC) or leukocytes. Ten fields per smear were analyzed and cellular frequencies were determined to distinguish the phases. Enucleated acidophilic cells were observed in all phases. The number of PAC was high during proestrus, but none were found during estrus. Frequency of NAC was higher during the transitional period between metestrus II and diestrus than during other phases. Leukocytes were first observed during metestrus and showed very high frequency during diestrus. This study demonstrated that the quantitative analysis of cell populations in vaginal smears improves the identification of the estrous cycle phases and may contribute to a more precise detection of cyclical alterations.(AU)
Assuntos
Animais , Ratos , /metabolismo , Esfregaço Vaginal , Reprodução/fisiologia , Ratos/classificaçãoRESUMO
The rat has been elected as the main animal model in several studies involving reproduction. However, there are scarce and conflicting data related to its estrous cycle. It comprises phases characterized by different cell types in vaginal smears (proestrus, estrus, metestrus and diestrus). Nevertheless, this classification and the time span of each phase are controversial and may induce misleading interpretations. In addition, there are no reports regarding the quantification of all cell types in each phase, including pre-acidophilic cells and leukocytes. The goal of this study was to revisit the literature about the rat estrous cycle and to perform a detailed quantitative and qualitative description of its phases and the transitional periods among them. Vaginal smears were obtained twice daily for 20 days from Wistar rats and stained using the Shorr method. Cells were classified as small (SBC) or large (LBC) basophilic cells, nucleated (NAC) or enucleated (EAC) acidophilic cells, pre-acidophilic cells (PAC) or leukocytes. Ten fields per smear were analyzed and cellular frequencies were determined to distinguish the phases. Enucleated acidophilic cells were observed in all phases. The number of PAC was high during proestrus, but none were found during estrus. Frequency of NAC was higher during the transitional period between metestrus II and diestrus than during other phases. Leukocytes were first observed during metestrus and showed very high frequency during diestrus. This study demonstrated that the quantitative analysis of cell populations in vaginal smears improves the identification of the estrous cycle phases and may contribute to a more precise detection of cyclical alterations.
Assuntos
Animais , Ratos , Esfregaço Vaginal , Reprodução/fisiologia , Ratos/classificaçãoRESUMO
BACKGROUND: In a previous study, we found that amifostine provides some protection to the seminiferous epithelium of prepubertal doxorubicin-treated male rats but does not improve their fertility status as adults. Based on these results, a long-term study was undertaken to evaluate the DNA damage caused to spermatogonia and the consequences for embryo development. METHODS: Twenty-four male prepubertal rats (30-day-old) were divided into four equal groups and treated with: doxorubicin (D--5 mg/kg), amifostine (A--400 mg/kg), amifostine/doxorubicin (AD--amifostine 15 min before doxorubicin) and control (C--0.9% saline solution). Sixty-four days after the treatment, animals were euthanized and the testes and epididymides were excised. The testes were fixed in Bouin's solution and historesin-embedded for histopathological analysis. Spermatozoa from the cauda epididymides were collected for chromatin structure analyses (Comet Assay and SCSA™). Adult rats (100-day-old) were mated with fertile females for embryo analyses on 2.5, 4.5 and 20 days post-coitum (d.p.c.). RESULTS: The seminiferous epithelium histopathology of AD group was better preserved compared with the D group. On the other hand, rats from the D and AD groups presented an increased percentage of sperm DNA strand breaks, as assessed by the comet assay, as well as an increased level of sperm chromatin denaturation, as assessed by the SCSA™ assay. In amifostine-treated groups (A and AD) there was a significant increase in the number of arrested embryos, as observed by the number of oocytes/zygotes on 2.5 d.p.c., when compared with control and doxorubicin groups; however, this number was increased when the AD group was compared with the A group. CONCLUSIONS: These results raise a concern about the effects of the association of these two drugs on the germ cell genome. Amifostine-doxorubicin-exposed rat spermatogonia produced long-term damage on sperm DNA, compromised conceptus development and reduced pregnancy outcome.