Avaliação de parâmetros morfométricos e morfofuncionais em testículos de ratos tratados com fluoxetina e suplementados com vitamina B12

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Histopathologycal findings in the ovaries and uterus of albino female rats promoted by co-administration of synthetic steroids and nicotine

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação da técnica de coloração AgNOR em testículos de ovinos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Transplante interespecífico de espermatogônias-tronco de Brycon orbignyanus em Astyanax altiparanae (Teleostei: Characidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Influência materna nas caracteristicas morfológicas de ovários e testículos de fetos bovinos da raça Nelore

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Correlação da circunferência escrotal, citologia testicular e parâmetros do sêmen do epidídimo de touros

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ciclo testicular e espermatogênese de Devario aequipinnatus (cypriniformes, cyprinidae) submetido a diferentes temperaturas

Pós-graduação em Ciência e Tecnologia Animal - FEIS

Glyphosate impairs male offspring reproductive development by disrupting gonadotropin expression

Sexual differentiation in the brain takes place from late gestation to the early postnatal days. This is dependent on the conversion of circulating testosterone into estradiol by the enzyme aromatase. The glyphosate was shown to alter aromatase activity and decrease serum testosterone concentrations. Thus, the aim of this study was to investigate the effect of gestational maternal glyphosate exposure (50 mg/kg, NOAEL for reproductive toxicity) on the reproductive development of male offspring. Sixty-day-old male rat offspring were evaluated for sexual behavior and partner preference; serum testosterone concentrations, estradiol, FSH and LH; the mRNA and protein content of LH and FSH; sperm production and the morphology of the seminiferous epithelium; and the weight of the testes, epididymis and seminal vesicles. The growth, the weight and age at puberty of the animals were also recorded to evaluate the effect of the treatment. The most important findings were increases in sexual partner preference scores and the latency time to the first mount; testosterone and estradiol serum concentrations; the mRNA expression and protein content in the pituitary gland and the serum concentration of LH; sperm production and reserves; and the height of the germinal epithelium of seminiferous tubules. We also observed an early onset of puberty but no effect on the body growth in these animals. These results suggest that maternal exposure to glyphosate disturbed the masculinization process and promoted behavioral changes and histological and endocrine problems in reproductive parameters. These changes associated with the hypersecretion of androgens increased gonadal activity and sperm production.

Spermatogenesis in goats with and without scrotum bipartition

The objective of the present research was to quantify the seminiferous epithelium cells, spermatogenesis efficiency and characterize the ultrastrucure of Sertoli cells in goats. Eighteen goats were used and divided into three groups: Group I - goats without bipartition of the scrotum; Group II - animals with bipartition of the scrotum in up to 50% of the testicular length; Group III - goats with bipartition of the scrotum in more than 50% of the testicular length. The goat testes in Group III had a greater number of primary spermatocytes (25.37 +/- 4.55 cells per cross sections), spermatids (112 +/- 15.12 cells per cross sections), and Sertoli cells (9.46 +/- 1.74 cells per cross sections) than the animals in Groups land (P <0.05). The spermatogenic mitotic, meiotic, and general efficiency were greater in animals in Group III (1.25 +/- 0.28; 5.12 +/- 1.63; 6.44 +/- 1.96) when compared to those in Groups I and II. Sheet-like processes originated from the Sertoli cell body as simple and smooth structures which involved almost all the surface of germ cells. Slender cord-like processes originated from Sertoli cells and also from the sheet-like processes. The relative frequency of the cycle stages showed differences among the groups of goats studied, and the highest frequency was in Stage 3 (20.68% for goats in Group I, 21.15% for those in Group II, and 16.89% for the animals in Group III). In conclusion, goats with bipartition of the scrotum have a greater number of germ and Sertoli cells per cross section of seminiferous tubule, that indicated a greater sperm production when compared to the other groups, and the ultrastructure of the Sertoli cell process did not present any relationship with bipartition of the scrotum. (C) 2012 Elsevier B.V. All rights reserved.

Glutamate-induced obesity leads to decreased sperm reserves and acceleration of transit time in the epididymis of adult male rats

Background: Given the established fact that obesity interferes with male reproductive functions, the present study aimed to evaluate sperm production in the testis and storage in the epididymis in a glutamate-induced model of obesity. Methods: Male rats were treated neonatally with monosodium glutamate (MSG) at doses of 4 mg/kg subcutaneously, or with saline solution (control group), on postnatal days 2, 4, 6, 8 and 10. On day 120, obesity was confirmed by the Lee index in all MSG-treated rats. After this, all animals from the two experimental groups were anesthetized and killed to evaluate body and reproductive organ weights, sperm parameters, plasma hormone levels (FSH, LH and testosterone), testicular and epididymal histo-morphometry and histopathology. Results: Significant reductions in absolute and relative weights of testis, epididymis, prostate and seminal vesicle were noted in MSG-treated animals. In these same animals plasma testosterone and follicle-stimulating hormone (FSH) concentrations were decreased, as well as sperm counts in the testis and epididymis and seminiferous epithelium height and tubular diameter. The sperm transit time was accelerated in obese rats. However, the number of Sertoli cells per seminiferous tubule and stereological findings on the epididymis were not markedly changed by obesity. Conclusions: Neonatal MSG-administered model of obesity lowers sperm production and leads to a reduction in sperm storage in the epididymis of adult male rats. The acceleration of sperm transit time can have implications for the sperm quality of these rats.

Repeated inhalation of crack-cocaine affects spermatogenesis in young and adult mice

To investigate the effects of repeated crack-cocaine inhalation on spermatogenesis of pubertal and mature Balb/c mice, ten young (Y-ex) and ten adult (A(ex)) Balb/c mice were exposed to the smoke from 5 g of crack with 57.7% of pure cocaine in an inhalation chamber, 5 days/week for 2 months. The young (Y-c) and adult (A(c)) control animals (n = 10) were kept in a specially built and controlled animal house facility. The morphologic analysis of both testes of all animals included the analysis of quantitative and qualitative histologic parameters to assess the effect of crack-cocaine on spermatogenesis and Leydig cells. Apoptosis was determined by immunolabeling with caspase-3 antibodies. Compared to the Y-c animals, Y-ex animals showed a significant reduction in the number of stage VII tubules per testis (p = 0.02), Sertoli cells (p < 0.001) and elongated spermatids (p = 0.001). Comparisons between the Y-ex and A(ex) groups identified a significant reduction in the number of Sertoli cells (p < 0.001) and round spermatids (p < 0.001) in the Y-ex group and a significant increase in apoptotic Leydig cells (p = 0.04) in the A(ex) group. The experimental results indicate that crack-cocaine smoke inhalation induced spermatogenesis disruption in chronically exposed mice, particularly in pubertal mice.

Absence of inactivating mutations and deletions in the DMRT1 and FGF9 genes in a large cohort of 46,XY patients with gonadal dysgenesis

Despite advances in our understanding of the mechanisms involved in sex determination and differentiation, the specific roles of many genes in these processes are not completely understood in humans. Both DMRT1 and FGF9 are among this group of genes. Dmrt1 controls germ cell differentiation, proliferation, migration and pluripotency and Sertoli cell proliferation and differentiation. Fgf9 has been considered a critical factor in early testicular development and germ cell survival in mice. We screened for the presence of DMRT1 and FGF9 mutations in 33 patients with 46,XY gonadal dysgenesis. No deletions in either DMRT1 or FGF9 were identified using the MLPA technique. Eight allelic variants of DMRT1 were identified, and in silico analysis suggested that the novel c.968-15insTTCTCTCT variant and the c.774G>C (rs146975077) variant could have potentially deleterious effects on the DMRT1 protein. Nine previously described FGF9 allelic variants and six different alleles of the 3' UTR microsatellite were identified. However, none of these DMRT1 or FGF9 variants was associated with increased 46,XY gonadal dysgenesis. In conclusion, our study suggests that neither DMRT1 nor FGF9 abnormalities are frequently involved in dysgenetic male gonad development in patients with non-syndromic 46,XY disorder of sex development. (C) 2012 Published by Elsevier Masson SAS.

A bradykinin-potentiating peptide (BPP-10c) from Bothrops jararaca induces changes in seminiferous tubules

Abstract: Background: The testis-specific isoform of angiotensin-converting enzyme (tACE) is exclusively expressed in germ cells during spermatogenesis. Although the exact role of tACE in male fertility is unknown, it clearly plays a critical function in spermatogenesis. The dipeptidase domain of tACE is identical to the C-terminal catalytic domain of somatic ACE (sACE). Bradykinin potentiating peptides (BPPs) from snake venoms are the first natural sACE inhibitors described and their structure–activity relationship studies were the basis for the development of antihypertensive drugs such as captopril. In recent years, it has been showed that a number of BPPs – including BPP-10c – are able to distinguish between the N- and C-active sites of sACE, what is not applicable to captopril. Considering the similarity between tACE and sACE (and since BPPs are able to distinguish between the two active sites of sACE), the effects of the BPP-10c and captopril on the structure and function of the seminiferous epithelium were characterized in the present study. BPP-10c and captopril were administered in male Swiss mice by intraperitoneal injection (4.7 μmol/kg for 15 days) and histological sections of testes were analyzed. Classification of seminiferous tubules and stage analysis were carried out for quantitative evaluation of germ cells of the seminiferous epithelium. The blood-testis barrier (BTB) permeability and distribution of claudin-1 in the seminiferous epithelium were analyzed by hypertonic fixative method and immunohistochemical analyses of testes, respectively. Results: The morphology of seminiferous tubules from animals treated with BPP-10c showed an intense disruption of the epithelium, presence of atypical multinucleated cells in the lumen and degenerated germ cells in the adluminal compartment. BPP-10c led to an increase in the number of round spermatids and total support capacity of Sertoli cell in stages I, V, VII/VIII of the seminiferous epithelium cycle, without affecting BTB permeability and the distribution of claudin-1 in the seminiferous epithelium. Interestingly, no morphological or morphometric alterations were observed in animals treated with captopril. Conclusions: The major finding of the present study was that BPP-10c, and not captopril, modifies spermatogenesis by causing hyperplasia of round spermatids in stages I, V, and VII/VIII of the spermatogenic cycle.

Localizzazione della topoisomerasi IB sui telomeri di S. cerevisiae e ruolo della sua attività catalitica sulle modificazioni istoniche

Il superavvolgimento del DNA nelle cellule, regolato dalle DNA Topoisomerasi, influenza molti processi biologici, quali la trascrizione, la replicazione, la ricombinazione ed il rimodellamento della cromatina. La DNA Topoisomerasi IB eucariotica, (Top1), è un enzima efficiente nella rimozione dei superavvolgimenti del DNA in vitro e la sua principale funzione cellulare è la rimozione dei superavvolgimenti positivi e negativi generati durante la trascrizione e la replicazione. Risultati recenti hanno fornito evidenze sperimentali del coinvolgimento di Top1 in meccanismi multipli di regolazione dell’espressione genica eucariotica, in particolare nella fase di inizio e maturazione dei trascritti. Tuttavia, le funzioni di Top1 non sono ancora state stabilite a livello globale. Pertanto, nella presente tesi di dottorato abbiamo risposto a questa domanda con l’analisi dei profili di trascrizione genica globale e con studi di immunoprecipitazione della cromatina (ChIP) in cellule di S. cerevisiae. Circa il 9% dei geni sono influenzati da Top1, e l’analisi dei profili di espressione mostra che Top1 wt aumenta l’utilizzo del glucosio e dei pathway per la produzione di energia, con specifica diminuzione della trascrizione dei geni telomerici e subtelomerici. Abbiamo inoltre dimostrato che Top1 wt, ma non il suo mutante inattivo, aumenta la velocità di crescita cellulare nelle cellule di lievito studiate. Le analisi di ChIP mostrano che, in confronto all’assenza dell’enzima, Top1 wt diminuisce l’acetilazione dell’istone H4, compresa quella specifica della lisina 16, nel telomero destro del cromosoma XIV mentre la mutazione che inattiva l’enzima aumenta in maniera marcata l’acetilazione dell’istone H4 e la di-metilazione della lisina 4 dell’istone H3. Top1 wt incrementa anche il reclutamento di Sir3 nelle regioni di confine della cromatina silenziata dello stesso telomero. Studi di immunoprecipitazione indicano che l’enzima interagisce direttamente con la struttura della cromatina telomerica poichè entrambe le proteine, quella wt e quella inattiva, sono localizzate sulle ripetizioni telomeriche dei cromosomi di lievito. Questi risultati dimostrano che Top1, una proteina non essenziale in lievito, ottimizza i livelli globali dei trascritti per una crescita più efficiente di cellule in fase esponenziale. Indagando il meccanismo che è alla base della specifica repressione dei geni telomerici, abbiamo dimostrato che Top1 favorisce delle modifiche posttraduzionali degli istoni che indicano una struttura della cromatina repressa nelle regioni telomeriche.

Le neoplasie del gatto. Ricerche istologiche e immunoistochimiche

Il contributo di ricerca di questo lavoro è consistito nel rivalutare da un punto di vista diagnostico, classificativo e statistico, una casistica di neoplasie del gatto archiviate in 23 anni (1984-2006). Nei primi 20 anni sono state diagnosticate 1696 neoplasie su un totale di 3682 campioni prevenuti nello stesso periodo, con una prevalenza delle patologie di tipo neoplastico del 46,06%. Nei tre anni successivi sono state individuate 382 neoplasie su un totale di 601 campioni archiviati, con una prevalenza del 61,02%. Le neoplasie avevano per l'81,8% un comportamento maligno, e questo dato risulta coerente con il dato più recente sulla malignità (85,6%) e con quello fornito da altri autori. L'età dei soggetti affetti era simile nei due studi, compresa fra pochi mesi e 22 anni, con il maggior numero di casi fra 9 e 13 anni. Le due casistiche presentano inoltre dati simili riguardo gli organi coinvolti anche se i tumori della mammella sono notevolmente diminuiti negli ultimi anni, con un aumento corrispondente di neoplasie cutanee. I fibrosarcomi, che hanno subito un drastico aumento tra il 1993 e il 1994 si sono mantenuti costanti in tutti gli anni successivi, dato ancora oggi quasi certamente correlabile alle pratiche vaccinali. I linfomi sono risultati piuttosto infrequenti in entrambe le casistiche, comparate con i dati della letteratura in cui vengono riportate percentuali nettamente maggiori. I tumori del cavo orale e dell'intestino hanno mantenuto circa le stesse percentuali di insorgenza nel corso degli anni. Tra i tumori presenti in questo studio, sono stati approfonditi i tumori a carico delle sierose (mesoteliomi) e del polmone attraverso metodiche immunoistochimiche e ultrastrutturali. Nei 23 anni sono stati diagnosticati 10 casi di mesotelioma, di cui 8 coinvolgenti la pleura e due il peritoneo. Su questi casi è stata applicata una metodica immunoistochimica con un pannello anticorpale utilizzato di routine in patologia umana per la diagnosi di mesotelioma e per la diagnosi differenziale con i carcinomi polmonari. Tutti i casi presentavano una doppia positività per vimentina e citocheratine ad ampio spettro; anticorpi specifici come CK5/6 e HBME-1 hanno presentato una buona specificità. Anche la microscopia elettronica si è rivelata di ausilio, mettendo in evidenza la presenza di tonofilamenti citoplasmatici e di microvilli, tipici delle cellule mesoteliali. Le neoplasie polmonari, analogamente a quelle umane hanno, sono notevolmente aumentate negli ultimi anni. Dei 24 casi raccolti tra il materiale di archivio, 14 sono stati diagnosticati negli ultimi 5 anni. Si è rilevata una significativa predisposizione nei gatti di razza Persiana, e le metastasi più frequenti sono state a carico dei linfonodi, del miocardio e dei cuscinetti plantari, analogamente a quanto segnalato in letteratura. L’istotipo più rappresentato è stato il carcinoma squamoso, seguito da adenocarcinomi papillari e bronchioloalveolari. Il pannello immunoistochimico applicato, finalizzato a individuare l’origine broncogena o ghiandolare della neoplasia, è risultato utile per la diagnosi delle forme squamose poco differenziate (CK 5/6 e CK 14). Infine è stato testato l’anticorpo di elezione nella diagnosi differenziale fra le neoplasie primitive e secondarie del polmone, il TTF-1, che però non ha cross-reagito con tessuti di gatto; la diagnosi di neoplasia primitiva polmonare si è dunque basata sull’assenza, anche nei dati anamnestici, di masse in altri distretti corporei.