Thesis (M. Tech.) -- Central University of Technology, Free State, 2007 / Four different South African indigenous (Naked Neck (NN), Ovambo (OVB), Venda (VD) and Potchefstroom Koekoek (PK) chicken breeds were used in this study. From each of the four breeds of chicken, 40 hens and 8 cocks were selected randomly. Two groups each of sixteen cocks were subsequently formed: high performing (HP) and low performing (LP) groups to determine the relationships between cock semen viability and the fertility of artificially inseminated South African indigenous layer breeds. Semen was collected following five minutes of sexual massage (5SM) and evaluated for semen volume (ml), sperm motility (%), live sperm (%) and total sperm (x109/ml). Semen from each cock was then used to inseminate five hens per breed, in each treatment. Each hen was inseminated twice a week throughout the duration of the trial. During the experimental period, each hen was inseminated with 0.05 ml diluted semen. The artificially inseminated hens were examined for average egg weight (g), fertility (%), hatchability of set eggs (%), live chicks (%), normal chicks (%) and chick weight (g). A total of 1600 eggs, i.e. 400 eggs from each breed were collected in three batches following artificial insemination from individually caged hens and were hatched to compare hatching parameters among breeds. The hatchability traits of hens of the four breeds (NN, OVB, PK and VD) were compared. Hatching egg weight had significant (P < 0.05) difference among the four breeds. The results of this study indicate that semen viability exemplified by ejaculate volume, sperm motility; live sperm and total sperm per ejaculate were significantly (P < 0.01) superior in the HP cocks compared to the LP cocks. Hens inseminated with semen from the HP cocks in each experimental group resulted in higher egg weight (g), fertility (%), hatchability of set eggs (%), live chicks (%), normal chicks (%) and chick weight (g). Significant positive relationships existed between semen volume and sperm motility (P < 0.05), semen volume and live sperm cells (P < 0.01), semen volume and total sperm (P < 0.01) in NN, OVB and VD, with negative correlations in PK. Some positive correlations were found between sperm motility and live spermatozoa (P < 0.01), sperm motility and total sperm (P < 0.01), live sperm and total sperm (P< 0.01) in NN, OVB, PK and VND. Fertility was the highest in the HP group. Fertility was also the highest in PK, intermediate and similar in OVB and NN and lowest in VD (P<0.05). Breed had a significant effect on hatchability of fertile eggs (P<0.05). Hatchability of total eggs set was highest in PK and NN, intermediate in OVB and lowest in VD (P<0.05). Breed had a significant effect on live, normal chicks and chick weight (P<0.05). Live chick was the highest in NN, whereas at day-old, normal chick and chick weight at hatching were the highest (23.50 ± 0.11) (P<0.05) in PK (98.14 ± 0.67 vs. 37.90 ± 0.28 g), intermediate and similar in NN (87.90 ± 0.63 vs. 23.50 ± 0.11) and OVB (87.75 ± 0.45 vs. 32.81 ± 0.49 g) and the lowest but with an acceptable value in VD (76.85 ± 0.46 vs. 26.90 ± 0.36 g). There were some correlations among different hatchability traits depending on breed. The correlations were more profound among PK. It was clear that chick weight as percent of egg weight was not just a function of egg weight, and that genotype also played an important role favouring the heavier breeds. The results obtained in this study on the relationships between cock semen viability and the fertility of artificially inseminated South African indigenous layer breeds elucidate that the use of high performing (HP) cocks following five minutes of sexual massage, prior to semen collection and artificial insemination of layers is a practical method for optimising sperm viability and subsequent fertility of hens. The results of this study suggest that the Potchefstroom Koekoek (PK) cocks and hens are superior to the Naked Necks (NN), Ovambo (OVB) and Venda (VD). The Ovambo and Naked Neck cocks ranked second in donating quality semen as well as in improving the fertility and hatchability traits of the indigenous chicken breeds. Thus selection of high performing cocks through five minutes sexual massage prior to semen collection and use is recommended for poultry AI breeding programmes.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy

Un nouveau facteur de transcription, appartenant à la famille des protéines à domaine bHLH, a récemment été isolé dans notre laboratoire. Initialement appelé « clone bc8 » puis HRT1, ce facteur présentait des similitudes avec les protéines Hairy and Enhancer of split qui interviennent notamment dans le phénomène d’inhibition latérale lors de la formation du tissu neural. Des études d’hybridation in situ réalisées chez l'embryon de xénope ont suggéré un rôle important de XHRT1, la protéine HRT1 de xénope, dans le développement neural. Nous avons recherché les partenaires protéiques de XHRT1 par la technique du double-hybride afin de mieux comprendre son mécanisme d’action moléculaire dans la neurogenèse. Tout d’abord nous avons construit les outils appropriés pour l’élaboration du travail, à savoir, les clones de levures exprimant les appâts spécifiques des domaines de la protéine étudiée et la création d’une banque d’ADNc du xénope au stade de la neurulation. Ensuite, trois criblages ont été réalisés. Dans le premier cas, nous avons recherché les partenaires des domaines bHLH et Orange (bHLH-O). Le domaine bHLH est en effet responsable de la dimérisation de ce type de protéine. Le domaine Orange qui suit le domaine bHLH, pourrait participer dans le choix du partenaire d’hétérodimérisation. Nous avons isolé deux facteurs de type bHLH-Orange apparentés à HRT1, XHairy1 et XHairy2b et confirmé leur interaction avec XHRT1. Les domaines impliqués dans ces interactions sont les bHLH-O pour les trois facteurs. Ce même criblage nous a permis d’isoler un nouvel ADNc qui code une protéine sans domaine apparent connu actuellement. Nous avons montré que cette protéine reconnaissait spécifiquement le domaine Orange de HRT1 mais pas celui des autres facteurs de type bHLH-O. Elle a été baptisée BOIP pour Bc8 Orange Interacting Protein. Le rôle physiologique de cette interaction n’a pu être démontré. Nous avons établi que la protéine BOIP pouvait aussi s’homodimériser. Nous avons aussi déterminé son profil d’expression chez le xénope et la souris. Son transcrit est hautement présent dans les testicules adultes. La protéine pourrait donc jouer un rôle important dans la spermatogenèse. Les deux autres criblages, utilisant les domaines situés dans la partie C-terminale de XHRT1, ont apporté des nouveaux partenaires potentiels, mais ces interactions n’ont pu être confirmées dans un système indépendant. Enfin, en étudiant plus en détail les interactions entre XHRT1 et XHairy1 ou XHairy2b, nous avons mis à jour une possible fonction de spécificité dans le choix du partenaire dans la région C-terminale de HRT1. La formation de ces dimères pourrait jouer un rôle dans la formation du tube neural mais également dans d’autres différenciations tissulaires.

SPAG16 is the murine orthologue of Chlamydomonas reinhardtii PF20, a protein known to be essential to the structure and function of the “9 + 2” axoneme. The “9 + 2” axoneme provides the cytoskeletal core of all eukaryotic motile cilia and flagella. In Chlamydomonas, the Pf20 gene encodes a single protein present in the central pair of the axoneme. Loss of Pf20 prevents central pair assembly and results in flagellar paralysis. The murine Spag16 gene encodes two proteins. While 71 kDa SPAG16L is found in all murine cells with motile cilia or flagella, 35 kDa SPAG16S transcript and protein are detected only in male germ cells, suggesting a unique role distinct from general axoneme formation. Transgenic mouse studies published previously by our lab have shown that abrogation of both SPAG16 isoforms causes arrest of spermatogenesis, and the mutant allele is not transmitted to offspring by chimeric males. Mice homozygous for a knock-out of SPAG16L alone are infertile, but show no abnormalities in spermatogenesis. The defects seen in chimeric Spag16 mutant mice, unaccounted for by loss of SPAG16L, indicate a possible role for SPAG16S in the specialized process of male germ cell development. Our results demonstrate that SPAG16S is predominantly found in specific regions within the nucleus of round spermatids. These nuclear sub-domains also contain SC35, a known marker of nuclear speckles enriched in pre-mRNA splicing factors. Putative interaction partners of SPAG16S are also shown to play critical roles in the peri-nuclear region during the round spermatid transition to the condensation and elongation stage of spermiogenesis, the final specialization point in sperm development. The distinct localization of SPAG16S at this critical juncture, its interaction with discretely localized proteins at a critical temporal junction in spermatogenesis, and its ability to modulate SPAG16L expression, suggest that SPAG16S plays an important role in the gene expression machinery of male germ cells, and represents an evolutionary distinction in axoneme gene function.

The PRDM9 (PR domain containing 9) protein is an epigenetic factor that trimethylates lysine 4 of histone H3 and thereby determines the future meiotic double-strand breaks - sites important for proper segregation of homologous chromosomes. Males of the Mus musculus domesticus (Mmd) origin with homozygous deletion in Prdm9 (Prdm9-/- ) are sterile with a complete arrest in meiotic prophase I, in contrast to the same mutant males of the M. m. musculus (Mmm) subspecies. The aim of this diploma thesis was to identify the genomic loci responsible for the phenotypic difference of these Prdm9-/- males. The major research tool was a population of 182 Mmm x Mmd Prdm9-/- males. The mapping method of quantitative trait loci (QTLs) was based on relating the genotypes of single-nucleotide and microsatellite polymorphisms to the observed phenotypes. At least two QTLs on Chr X were identified. The Mmm alleles of these QTLs reduced fertility of Prdm9-/- males. Both QTLs were confirmed and narrowed down using two types of subconsomic strains. It was not possible to confirm other QTLs, particularly on autosomes. This QTL mapping is the first step towards the identification of genes that modify the resulting phenotype of Prdm9-/- animals. This identification should help designing studies of human infertility that...

A number of chemicals may have a negative impact on the environment and wildlife. Endocrine disruptors (EDs), which can mimic estrogen, interfere with natural hormones in organism and can have a negative effect on the reproductive system. Such substances include zearalenone (ZEA) - mycotoxin, produced by the fungi Fusarium. Despite the non-steriodal structure of its molecule, ZEA and its derivates possess potent estrogenic activity. The influence of ZEA on reproductive parameters and changes in expression of selected genes were tested in the outbred line of mice. This study showed significant effects of ZEA on number of reproductive performances. This effect was observed at lower examined dose, to which are humans normally exposed, and at higher doses, both showed changes in the tested parameters.

Le gène Spatial est exprimé par des cellules hautement polarisées : les cellules épithéliales du thymus, neuronales du système nerveux central et germinales du testicule. La différenciation de ces cellules est accompagnée d'une polarisation de la distribution de Spatial, dans des structures microtubulaires hautement organisées telles que la manchette, le flagelle et la dendrite.Mon projet a porté sur l’identification de la fonction du gène Spatial au niveau du thymus et du cerveau murins. De plus, j’ai été impliquée dans la caractérisation du gène Spatial chez l’homme et l’évaluation de son impact sur la spermatogenèse et l’infertilité humaine.Nos résultats montrent qu’au niveau du thymus, Spatial est détecté tout au long de l’organogenèse thymique jusqu’aux stades adultes. Son profil d’expression correspond à un « promiscuous gene » impliqué dans l’acquisition de la tolérance des lymphocytes T. Au niveau du système nerveux central, Spatial présente une distribution somatodendritique dans les cultures de neurones hippocampiques et son expression est fortement détectée lors de la poussée dendritique. Nous avons montré que le transport de Spatial du corps cellulaire vers les dendrites est dépendant de la kinésine Kif17. De plus, Spatial semble être impliqué dans la formation des dendrites via la voie de signalisation stimulée par le Nerve Growth Factor.Chez l’homme, le gène H-Spatial est fortement exprimé au niveau du testicule et son expression est spécifique de la spermiogenèse, étape de différenciation des spermatides rondes en spermatozoïdes. Chez les patients infertiles asthéno- et/ou tératozoospermiques, présentant des anomalies de mobilité et de forme des spermatozoïdes, le niveau d’ARNm d’H-Spatial est fortement réduit. Ces résultats suggèrent qu’H-Spatial est un marqueur potentiel de l’infertilité masculine.J’ai également participé à la validation des dendrimères PAMAM (poly-amidoamine) comme vecteurs efficaces pour le transfert de siRNA et d’ADN in vitro sur différents lignées cellulaires et in vivo sur des thymus murins. Ce système pourrait être un moyen thérapeutique pour traiter des immunodéficiences liées aux lymphocytes T. / Spatial gene is expressed in highly polarized cells such as, thymic epithelial cells, testicular germ cells and neuronal cells of the central nervous system. The differentiation of these cells is accompanied by the polarized distribution of Spatial in highly organized microtubule structures such as the manchette, the flagellum and dendrites. This project aims to identify the function of Spatial gene in the thymus and in the brain. Moreover, we characterize Spatial gene in humans and evaluate its impact on spermatogenesis and human infertility.Our results showed that, in the thymus, Spatial is detected throughout the thymic development, until adulthood. Its expression profile corresponds to a ‘promiscuous gene’, implicated in the acquisition of T lymphocyte tolerance.At the level of the central nervous system, Spatial showed a somatodendritic distribution in hippocampal neuron cultures. Moreover, its expression was highly detected during dendritic growth. We have also shown that the transport of Spatial from the cell body to the dendrites is dependent on the kinesin Kif17. In addition, our results suggest that Spatial seems to be implemented in dendrite formation by the Nerve Growth Factor mediated signaling pathway.In the humans, H-Spatial is highly expressed in the testis and its expression is specific to spermiogenesis: the phase of differentiation of round spermatids to spermatozoids. In infertile astheno and/or teratozoospermic patients with sperm shape and mobility anomalies, H-Spatial levels were drastically reduced. These results propose H-Spatial as a potential marker for human male infertility.Finally, we have equally participated in the validation of PAMAM (poly-amidoamine) dendrimers as efficient vectors for the transfer of DNA and siRNA in vitro, in different cell lines; and in vivo, in murine thymi. This system could serve as a new therapeutic model for treating diseases linked to T lymphocytes.

Chez les vertébrés, le processus de la spermatogénèse est directement contrôlé par deux hormones gonadotropes hypophysaires, Fsh et Lh. Chez les salmonidés, les profils de sécrétion des 2 hormones diffèrent et présentent des variations significatives au cours du cycle de développement spermatogénétique, suggérant que Fsh et Lh interviennent à des étapes différentes du processus. A la différence des mammifères, chez les poissons les 2 gonadotropines exercent une forte activité stéroïdogène, et par ailleurs il a été rapporté par plusieurs auteurs que leurs récepteurs seraient moins sélectifs vis-à-vis des 2 ligands. Ainsi, le périmètre des actions respectives de Fsh et de Lh n'est pas défini chez les poissons. D'autre part, les mécanismes de l'action de Fsh qui ne seraient pas relayés par les stéroïdes sont très mal connus. Chez la truite, nous avons déterminé que chaque gonadotropine agit essentiellement par l'intermédiaire de son récepteur respectif. L'analyse des variations du transcriptome testiculaire après un traitement in vitro par les hormones de la reproduction (Fsh, Lh, androgènes) a permis 1- de révéler des actions distinctes de Fsh et de Lh sur l'expression des gènes, 2- de mettre en évidence deux mécanismes d'action de la Fsh, l'un dépendant et l'autre indépendant de la production de stéroïdes et 3- d'identifier plusieurs acteurs d'interaction cellulaire régulés par Fsh, et probablement impliqués dans les étapes précoces de prolifération ou de différenciation des cellules germinales, tels que l'hormone antimüllérienne, la midkine, l'insulin-like growth factor1b, la follistatine-like 3 et l'activine, 4-de proposer une implication de Fsh dans les évènements tardifs de maturation et d'excrétion du sperme. Au-delà des acquis concernant les régulations endocriniennes et moléculaires chez la truite, ces travaux constituent un apport de connaissances qui peut être étendu à d'autres téléostéens pour décrypter l'action propre à Fsh dans le déclenchement de la maturation pubertaire. Enfin, nous montrons qu'une vaccination contre les récepteurs des gonadotropines constitue une voie potentielle de contrôle des maturations précoces en élevage. / In vertebrates, spermatogenesis is under the direct control of two pituitary gonadotropic hormones named Fsh and Lh. In salmonids, the 2 hormones are differentially secreted in the plasma through the reproductive cycle, suggesting that Fsh and Lh are involved in the regulation of different steps of the process. Unlike in mammals, both fish gonadotropins exert a strong steroidogenic activity and, besides, some authors reported for their receptors a much lower selectivity towards the two ligands. Yet, their respective roles are not established in fish. Furthermore, the mechanisms of Fsh action that would not be mediated by steroids are poorly investigated. In trout, we showed that each gonadotropin mainly acts through its cognate receptor. The analysis of the variations of testicular transcriptome following an in vitro treatment with reproductive hormones (Fsh, Lh and androgens) permitted 1- to reveal that Fsh and Lh have distinct effects on gene expression, 2- to highlight two mechanisms of Fsh action, one dependent on the steroid production and the second one independent of that production, 3- to identify several Fsh regulated factors involved in cellular interactions and particularly in the control of germ cell proliferation / differentiation (anti mullerian hormone, midkine, insulin-like growth factor 1b, follistatin-like 3 and activin), 4- to propose an involvement of Fsh in late events of sperm maturation and release. In addition to knowledge on endocrine and molecular regulations in trout, this work provides a fund of knowledge useful in other teleosts to decipher the action of Fsh in triggering puberty onset. Finally, we showed that an immunization against the gonadotropin receptors is a potential method to delay sexual maturation in farmed fish.

Neste estudo foram investigados os efeitos da inalação crônica de cocaína crack na espermatogênese de camundongos púberes e maduros. Camundongos Balb/c machos de duas diferentes idades, jovem e adulta (n=20), foram expostos à fumaça de 5g de cocaína crack em uma câmara de inalação, 5 dias por semana, durante 2 meses. Animais controle (n=10) foram mantidos em biotério durante o período de experimentação. Análises morfométricas quantitativas de cortes histológicos de testículos foram feitas em microscopia óptica. A qualidade da espermatogênese foi avaliada durante a fase VII de maturação do epitélio seminífero, através da quantificação dos tipos celulares normais e degenerados presentes nos espaços intra e intertubular (células germinativas, células de Sertoli e células de Leydig). As diferenças foram consideradas significativas quando p<0,05. O número de túbulos seminíferos em fase VII por testículo mostrou significante redução (p=0,023) em animais jovens expostos. Houve redução significativa observada no número de células de Sertoli (p=0,000) e espermátides alongadas (p=0,005) de animais jovens expostos. A degeneração celular é aumentada em todos os grupos expostos, com maior severidade no grupo jovem (p=0,000). A inalação de fumaça de cocaína crack induz a alterações na espermatogênese, sendo sua toxicidade maior em animais jovens expostos durante a fase de maturação gonadal . Estes achados são de interesse na saúde pública e mais investigações devem ser feitas focando efeitos similares em homens. / In the present study, the effects of chronic inhalation of crack cocaine on the spermatogenesis of pubertal and mature mice were investigated. Balb/c mice of two different ages, young and adult (n=20), were exposed to the smoke of 5g of crack in an inhalation chamber for 5 days a week during 2 months. Correspondents control animals (n=10) were kept in animal house during experimentation. Morphological quantitative analyses of testis were made in optical microscope. The spermatogenesis was evaluated during phase VII of maturation of the seminiferous epithelium. The number of spermatogenesis cell types (germ cells and Sertoli cells), the germ cell degenerations and the intertubular Leydig cells population was scored. Differences were considered significant when p<0.05. The number of tubular phase VII per testis showed significant (p= 0,023) reduction in young exposed animals. Significant reductions (p=0,000) were observed in Sertoli cells and spermatids elongated (p=0,005) in young intoxicated animals. Apoptosis is also increased in all intoxicated groups being more severe in young groups (p=0,000). Inhalation of crack cocaine smoke induced spermatogenesis disruption of chronic exposed mice. Crack toxicity was more severe in pubertal mice when sexual gonad undergoes maturation. We think that our findings should be of public health concern and that further investigations focusing similar effects on human males are warranted.