Sandra Maria Miraglia

Functional and morphometric evaluation of offspring kidney after intrauterine undernutrition

Abstract  Pregnant rats were subjected to 50% food restriction during the first or the second half of pregnancy, or throughout pregnancy. The effects of intrauterine food restriction, on kidney function and morphometry were studied in newborn and adult (3 months) offspring. No differences in glomerular diameter were observed in newborn restricted rats compared with controls. The number of glomeruli was significantly lower both in newborn and 3-month-old restricted rats. However, glomerular diameter was increased in 3-month-old rats, which suggests that hypertrophic stimuli were present. The medulla/cortex ratio increased in adult rats submitted to food restriction during pregnancy, a finding that agrees with the preserved sodium and acid excretion, and the normal osmolar and free water clearance observed in these groups. These results show that the reduction in glomerular number is still present 3 months after birth in the progeny of mothers submitted to severe food restriction during pregnancy, suggesting impairment of glomerulogenesis even after birth. Intra utero undernutrition can be regarded as an experimental model of glomerular hypertrophy.

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. Anat Rec, 291:797–808, 2008.

Morphological alterations and intratubular lipid inclusions as indicative of spermatogenic damage in cimetidine-treated rats

Doses of cimetidine (50 mg/kg b/w) were administered to adult male Wistar rats over 52 consecutive days. Besides plasma testosterone levels, morphological and morphometric aspects of the seminiferous tubules as well as histochemical analysis of the lipid content by oil red O were emphasized. Abnormal tubules exhibiting disorganization of their cellular association, loss of germ cells, and multinucleated giant spermatids were usually found. Significant reductions of testis weight and tubular diameter at specific stages (VII-IX), as well as lack of contact between Sertoli cells and spermatids in tubules at stage IX, suggest a possible interference of cimetidine on the histoarchitecture of the seminiferous epithelium. The dense concentration of lipid inclusions in tubules at postspermiation stages indicates phagocytosis and degradation of germ cells. Since no change in serum testosterone levels was verified in cimetidine-treated rats, the authors could not exclude the possibility that besides an antiandrogenic effect, other biochemical factors necessary for normal spermatogenesis could be involved in the testicular alterations.

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

BackgroundDoxorubicin 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.MethodsPrepubertal 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 22dpp. The testes were collected at 40, 64 and 127dpp, 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.ResultsThe rats treated with doxorubicin showed reduction of transferrin labeling in the seminiferous epithelium at 40 and 64dpp, 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 discussionThese 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.

Apoptosis during the seasonal spermatogenic cycle of Rana catesbeiana

In the bullfrog Rana catesbeiana, testicular weight is constant throughout the year, but the volume densities of germinative and interstitial compartments undergo inverse changes from winter (non‐breeding) to summer (breeding). The occurrence of apoptosis in the seminiferous lobules of bullfrogs was investigated in these two periods using sections stained with haematoxylin and eosin (H&E), the TUNEL (terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling) method and transmission electron microscopy. TUNEL‐positive cells were observed in the seminiferous lobules, and ultrastructural morphological details confirmed the occurrence of cell death by apoptosis. In summer, the occurrence of several spermatogenic processes (in addition to spermiogenesis and spermiation), and then the overconsumption of Sertoli cell‐derived pro‐survival factors, could be responsible for the increased density of apoptotic cells. Alternatively, the low apoptotic frequency in winter could be related to the constant homeostasis in the germinative compartment given that most lobules are filled with primary spermatocytes. As volume densities of interstitial and germinative compartments undergo inverse seasonal variations through the year, the incidence of apoptosis (in summer) could play a part in controlling the spermatogenic process, maintaining the lobular size when interstitial tissue is maximally developed. In winter, the low apoptotic cell density leads to spermatogenic recrudescence and, thereby, the production of an adequate quantity of spermatozoa for the next breeding period. Thus, apoptosis may participate not only in the maintenance of spermatogenic homeostasis, but also in the cyclical control of the different spermatogenic processes according to seasonal changes of the testicular compartments as a whole.

Structural alterations in the seminiferous tubules of rats treated with immunosuppressor tacrolimus

BackgroundTacrolimus (FK-506) is an immunosuppressant that binds to a specific immunophilin, resulting in the suppression of the cellular immune response during transplant rejection. Except for some alterations in the spermatozoa, testicular morphological alterations have not been described in rats treated with tacrolimus. In the present study, we purpose to evaluate if the treatment with tacrolimus at long term of follow-up interferes in the integrity of the seminiferous tubules.MethodsRats aging 42-day-old received daily subcutaneous injections of 1 mg/kg/day of tacrolimus during 30 (T-30) and 60 (T-60) days; the rats from control groups (C-30 and C-60) received saline solution. The left testes were fixed in 4% formaldehyde and embedded in glycol methacrylate for morphological and morphometric analyses while right testes were fixed in Bouins liquid and embedded in paraffin for detection of cell death by the TUNEL method. The epithelial and total tubular areas as well as the stages of the seminiferous epithelium and the number of spermatocytes, spermatids and Sertoli cells (SC) per tubule were obtained.ResultsIn the treated groups, seminiferous tubules irregularly outlined showed disarranged cellular layers and loss of germ cells probably due to cell death, which was revealed by TUNEL method. In addition to germ cells, structural alterations in the SC and folding of the peritubular tissue were usually observed. The morphometric results revealed significant decrease in the number of SC, spermatocytes, spermatids and significant reduction in the epithelial and total tubular areas.ConclusionTacrolimus induces significant histopathological disorders in the seminiferous tubules, resulting in spermatogenic damage and reduction in the number of Sertoli cells. A careful evaluation of the peritubular components will be necessary to clarify if these alterations are related to the effect of FK-506 on the peritubular tissue.

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.

Vitamin B-12 Supplement Exerts a Beneficial Effect on the Seminiferous Epithelium of Cimetidine-Treated Rats

Treatment of gastric ulcer with cimetidine reduces acid secretion and interferes in the vitamin B12 absorption. Regarding the harmful effect of cimetidine on the seminiferous tubules, the aim of the present study was to verify if prolonged treatment with cimetidine causes vitamin B12 deficiency and whether the testicular damages are attenuated by vitamin B12 supplementation. Adult male rats received, for 50 days, cimetidine (CMTG), cimetidine and vitamin B12 (CMT/B12G), vitamin B12 (B12G) and saline solution (CG). Vitamin B12 and homocysteine plasma levels were evaluated and the testes were embedded in glycol methacrylate for the morphometric analyses of total, epithelial and luminal areas of the seminiferous tubules, number of Sertoli cells and frequencies of tubules according to stages and containing Sertoli and germ cells in the lumen. Terminal deoxynucleotidyl-transferase mediated dUTP nick end labeling (TUNEL) method and proliferating cell nuclear antigen (PCNA) immunohistochemistry were carried out. CMTG showed TUNEL-positive Sertoli cells and significant reductions in the epithelial and total tubular areas, number of Sertoli cells and frequency of tubules VII-VIII. In the CMT/B12G, the number of Sertoli cells and the epithelial and total tubular areas were similar to CG. The number of Sertoli cells (in B12G) and the frequency of tubules at stages VII-VIII (in B12G and CMT/B12G) increased significantly; PCNA-positive Sertoli cells were found in these groups. Although cimetidine was not able to induce vitamin B12 deficiency, this drug causes tubular atrophy due to Sertoli cell damage and loss of germ cells. However, vitamin B12 supplement is able to stimulate spermatogenesis and restore the number of Sertoli cells, softening the harmful effect of cimetidine on spermatogenesis.

Carbamazepine damage to rat spermatogenesis in different sexual developmental phases

Carbamazepine (CBZ) is a first-line antiepileptic drug (AED), although it is also utilized for treatment of psychiatric disorders and neuropathic pain. The utilization of CBZ has been associated with damage to male reproduction including hormonal alterations, sexual dysfunction and reduction of sperm quality. Wide and long-term use of CBZ has been a common schedule for children and adolescents, despite the fact it alters the testosterone level in adult rats and humans. In addition, hypothalamic-pituitary-gonadal (HPG) axis during pre-puberty and puberty is more susceptible to toxic agents than in adult phase. The objective of this work was to evaluate the side effects of CBZ on the spermatogenic process of rats from pre-puberty to puberty and sexual maturation. Damage on the seminiferous epithelium, testicular interstitial oedema, reductions of testosterone levels and an increase in oestradiol levels were observed in rats, which were CBZ-treated since the weaning. The results suggest that CBZ, when administered from pre-puberty, provokes specific side effects on rat testes, resulting in more severe damage in the adult phase.

Effects of prenatal and lactation nicotine exposure on rat testicular interstitial tissue

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.