Quantificação de células dos túbulos seminíferos e rendimento da espermatogênese em cutias (Dasyprocta aguti) criadas em cativeiros

O presente estudo teve como objetivo avaliar o rendimento da espermatogênese de cutias criadas em cativeiro, por intermédio das razões encontradas entre tipos celulares do epitélio seminífero. Os resultados apontaram que o rendimento da espermatogênese da cutia dos nove aos quatorze meses de idade não chegou a um ponto de estabilização. O coeficiente de eficiência de mitoses espermatogoniais não aumentou com a idade. O rendimento meiótico, o rendimento geral da espermatogênese e o índice de células de Sertoli mostraram variações numéricas em função da idade, entretanto, não detectadas estatisticamente.

Caracterização morfológica e frequência dos estádios do ciclo do epitélio seminífero em preás (Galea spixii Wagler, 1831) criados em cativeiro

Estudos baseados nas características testiculares estão altamente relacionados com a eficiência reprodutiva de varias espécies. Assim, o projeto desenvolvido teve como objetivo identificar as células do epitélio seminífero, caracterizar histologicamente suas associações, que formam os estádios, e determinar a frequência destes. Os fragmentos de testículos, com 30, 45, 60, 75, 90, 105, 120, 150 dias foram coletados no Centro de Multiplicação da Universidade Federal Rural do Semi-Árido (UFERSA), Mossoró/ RN. Passando pelos processos de fixação, lavagens em soluções de concentrações crescentes de álcoois (70-100%), desidratação em xilol, inclusão em Histosec®, preparação das lâminas histológicas, colorações em Hematoxilina e Eosina (HE) e suas fotomicrografias para a caracterização dos núcleos celulares do epitélio germinativo e a definição dos oitos estágios do ciclo do epitélio seminífero (CES) baseados no Método da Morfologia Tubular. Das faixas etárias analisadas todos os animais de 90-150 dias de idade apresentaram todos os estádios do CES. Os estádios I e III foram os que apresentaram maior e menor freqüência, respectivamente. Os animais caracterizados como pré-púberes (30 dias), púberes (45-90 dias de idade) e pós-púberes (105150 dias de idade) apresentaram os estádios I, VIII e IV com uma maior freqüência, respectivamente.

KINESIN MOTOR PROTEINS ARE ESSENTIAL FOR MALE GAMETOPHYTE DEVELOPMENT IN MARSILEA VESTITA

The male gametophyte of the semi-aquatic fern, Marsilea vestita, produces multiciliated spermatozoids in a rapid developmental sequence that is controlled post-transcriptionally when dry microspores are placed in water. Development can be divided into two phases, mitosis and differentiation. During the mitotic phase, a series of nine successive division cycles produce 7 sterile cells and 32 spermatids in 4.5-5 hours. During the next 5-6 hours, each spermatid differentiates into a corkscrew-shaped motile spermatozoid with ~140 cilia. This document focuses on the role of motor proteins in the regulation of male gametophyte development and during ciliogenesis. In order to study the mechanisms that regulate spermatogenesis, RNAseq was used to generate a reference transcriptome that allowed us to assess the abundance of transcripts at different stages of development. Over 120 kinesin-like sequences were identified in the transcriptome that represent 56 unique kinesin transcripts. Members of the kinesin-2, -4, -5, -7, -8, -9, -12, -13, and -14 families, in addition to several plant specific and ‘orphan’ kinesins are present. Most (91%) of these kinesin transcripts change in abundance throughout gametophyte development, with 52% of kinesin mRNAs enriched during the mitotic phase and 39% enriched during differentiation. Functional analyses show that the temporal regulation of kinesin transcripts during gametogenesis directly correlates with kinesin protein function. Specifically, Marsilea makes one kinesin-2 (MvKinesin-2) and two kinesin-9 (MvKinesin-9A and MvKinesin-9B) transcripts, which are present during spermatid differentiation and ciliogenesis. Silencing experiments showed that MvKinesin-2 and MvKinesin-9A are required for ciliogenesis and motility in the Marsilea male gametophyte; however, these kinesins display atypical roles during these processes. In contrast, spermatozoids produced after the silencing of MvKinesin-9B exhibit normal morphology. MvKinesin-2 is necessary for cytokinesis as well as for regulating ciliary length and MvKinesin-9A is needed for the correct orientation of basal bodies, events not typically associated with these proteins. In addition, Marsilea makes motile, ciliated gametophytes without the help of IFT dynein, outer arm dynein, or the BBsome. These results are the first to investigate the kinesin-linked mechanisms that regulate ciliogenesis in a land plant.

Germ Cell Transplantation in Felids: A Potential Approach to Preserving Endangered Species

With the exception of the domestic cat, all members of the family Felidae are considered either endangered or threatened. Although not yet used for this purpose, spermatogonial stem cell (SSC) transplantation has a high potential to preserve the genetic stock of endangered species. However, this technique has not previously been established in felids. Therefore, we developed the necessary procedures to perform syngeneic and xenogeneic SSC transplants (eg, germ cell [GC] depletion in the recipient domestic cats, enrichment and labeling of donor cell suspension, and the transplantation method) in order to investigate the feasibility of the domestic cat as a recipient for the preservation and propagation of male germ plasm from wild felids. In comparison with busulfan treatment, local x-ray fractionated radiation was a more effective approach to depleting endogenous spermatogenesis. The results of both syngeneic and xenogeneic transplants revealed that SSCs were able to successfully colonize and differentiate in the recipient testis, generating elongated spermatids several weeks posttransplantation. Specifically, ocelot spermatozoa were observed in the cat epididymis 13 weeks following transplantation. As donor GCs from domestic cats and ocelots were able to develop and form mature GCs in the recipient environment seminiferous tubules, these findings indicate that the domestic cat is a suitable recipient for SSC transplantation. Moreover, as modern cats descended from a medium-size cat that existed approximately 10 to 11 million years ago, these results strongly suggest that the domestic cat could be potentially used as a recipient for generating and propagating the genome of wild felids.

The frequency of follicle stimulating hormone receptor gene polymorphisms in Iranian infertile men with azoospermia

Background: Azoospermia is the medical condition of a man not having any measurable level of sperm in his semen. Follicle stimulating hormone (FSH) is a member of the glycoprotein hormone family that plays an important role in human reproduction because of its essential role in normal spermatogenesis. Various Single Nucleotide Polymorphisms (SNPs) have been reported within FSH receptor (FSHR) gene that may affect the receptor function. Objective: The present study aimed to investigate the correlation between two FSHR SNPs at positions A919G, A2039G, and susceptibility to azoospermia in a group of Iranian azoospermic men. The association between FSH levels within the sera and A919G and A2039G alleles and genotypes were also investigated. Materials and Methods: This case control study was performed on 212 men with azoospermia (126 non-obstructive and 86 obstructive) and 200 healthy Iranian men. Two FSHR gene SNPs were genotyped using PCR-RFLP method. The relationship between FSH levels within the sera and A919G and A2039G alleles and genotypes were also investigated. Results: Statistical analysis indicated that at A919G position, AA genotype and A allele were more frequent in obstructive azoospermia cases compared to non- obstructive or normal men (p=0.001). Regarding A2039G polymorphisms, no significant difference was observed between both azoospermia groups and the controls. The mean level of serum FSH was higher in the non-obstructive men compared to the obstructive patients (23.8 versus 13.8, respectively, p= 0.04). Conclusion: The results of the present study indicated that the genetic polymorphisms in the FSHR gene might increase the susceptibility to azoospermia in Iranian men.

Cartographie des cassures bicaténaires du remodelage chromatinien du spermatide et développement des outils techniques associés.

Résumé : La phase haploïde de la spermatogenèse (spermiogenèse) est caractérisée par une modification importante de la structure de la chromatine et un changement de la topologie de l’ADN du spermatide. Les mécanismes par lesquels ce changement se produit ainsi que les protéines impliquées ne sont pas encore complètement élucidés. Mes travaux ont permis d’établir la présence de cassures bicaténaires transitoires pendant ce remodelage par l’essai des comètes et l’électrophorèse en champ pulsé. En procédant à des immunofluorescences sur coupes de tissus et en utilisant un extrait nucléaire hautement actif, la présence de topoisomérases ainsi que de marqueurs de systèmes de réparation a été confirmée. Les protéines de réparation identifiées font partie de systèmes sujets à l’erreur, donc cette refonte structurale de la chromatine pourrait être génétiquement instable et expliquer le biais paternel observé pour les mutations de novo dans de récentes études impliquant des criblages à haut débit. Une technique permettant l’immunocapture spécifique des cassures bicaténaires a été développée et appliquée sur des spermatides murins représentant différentes étapes de différenciation. Les résultats de séquençage à haut débit ont montré que les cassures bicaténaires (hotspots) de la spermiogenèse se produisent en majorité dans l’ADN intergénique, notamment dans les séquences LINE1, l’ADN satellite et les répétions simples. Les hotspots contiennent aussi des motifs de liaisons des protéines des familles FOX et PRDM, dont les fonctions sont entre autres de lier et remodeler localement la chromatine condensée. Aussi, le motif de liaison de la protéine BRCA1 se trouve enrichi dans les hotspots de cassures bicaténaires. Celle-ci agit entre autres dans la réparation de l’ADN par jonction terminale non-homologue (NHEJ) et dans la réparation des adduits ADN-topoisomérase. De façon remarquable, le motif de reconnaissance de la protéine SPO11, impliquée dans la formation des cassures méiotiques, a été enrichi dans les hotspots, ce qui suggère que la machinerie méiotique serait aussi utilisée pendant la spermiogenèse pour la formation des cassures. Enfin, bien que les hotspots se localisent plutôt dans les séquences intergéniques, les gènes ciblés sont impliqués dans le développement du cerveau et des neurones. Ces résultats sont en accord avec l’origine majoritairement paternelle observée des mutations de novo associées aux troubles du spectre de l’autisme et de la schizophrénie et leur augmentation avec l’âge du père. Puisque les processus du remodelage de la chromatine des spermatides sont conservés dans l’évolution, ces résultats suggèrent que le remodelage de la chromatine de la spermiogenèse représente un mécanisme additionnel contribuant à la formation de mutations de novo, expliquant le biais paternel observé pour certains types de mutations.