Dissertations / Theses on the topic 'Spermatogenesis'

Over the past decade many publications attest to the substantial increase in public and scientific concern regarding the potential of industrial and environmental chemicals to interfere with male reproductive function. Spermatogenesis, (the process of sperm formation) depends on a series of complex biological interactions which can be interrupted at various stages by chemical insult. Recently a number of these chemicals have been used experimentally to directly disrupt testicular function to provide an insight into the processes involved in normal spermatogenesis. Based on these investigations the primary objective of the work in this thesis was to study the time course of development of testicular lesions, their stage specificity, and subsequent recovery of normal spermatogenesis using morphological and functional characteristics of function in vivo and in vitro. The chemicals used in these studies included meta dinitrobenzene (mDNB), nitrobenzene (NB), the isomers of mononitrotoluene (mNT) and 2-methoxyacetic acid (MAA). Within 24h of a single oral administration of mDNB or NB to rats there was a progressive decrease in testicular weight and increase in serum follicle stimulating hormone (FSH) levels coincident with widespread germ cell degeneration, as determined by histological examination of the testis. The degree of disruption induced by mDNB and its reversibility were time - but not stage - dependent with a decline in both sperm number and motility in parallel with germ cell degeneration. NB however had a more specific effect causing the initial degeneration of leptotene spermatocytes and subsequently more extensive germ cell loss involving round spermatids at a number of stages. During the recovery of spermatogenesis a considerable change was observed in the frequency of occurrence of spermatogenic stages in comparison to controls. These changes remained significantly different up to at least 70 days post-treatment. The isomers of mNT induced small but statistically significant dose-dependent decreases in testicular weight. Spermatogenesis in these animals remained qualitatively normal, so detailed histological studies were not undertaken. Further investigation into the effects of the testicular toxicants listed above was performed in vitro using Sertoli cell monocultures and Sertoli cell-germ cell co-cultures. mDNB, NB and the isomers of mNT produced dose-dependent increases in germ cell exfoliation (from co-cultures). mDNB and NB also produced histological changes in culture including Sertoli cell vacuolation and phagocytosis of germ cells, and induced dose-dependent increases in the secretion of lactate and pyruvate, two established indices of Sertoli cell function in vitro. Sertoli cells secrete over 100 different proteins, only a handful of which have been characterised and a role in spermatogeneis proposed. One of these proteins is inhibin. The potenial of inhibin secretion as a further index of Sertoli cell function (or dysfunction) in monocultures and co-cultures has been investigated. mDNB, NB, 2-mNT and 3-mNT all induced dose-dependent alterations in the secretion of inhibin in vitro. Historically, FSH has been postulated to control inhibin secretion in a classical negative feedback manner. Recent evidence obtained in vivo and in co-culture suggests that its secretion may be controlled by specific type(s) of germ cells. Seminiferous tubule cultures represent a potentially more useful culture system to investigate this further. They present a unique opportunity to study Sertoli cell-germ cell interactions under more physiological conditions since the integrity of the seminiferous epithelium is maintained in vitro. Previous investigators have demonstrated that MAA specifically destroys pachytene spematocytes in vivoand this specificity of action was utilised to study which germ cel-Sertoli cell interactions control the secretion of inhibin. At selected times after MAA treatment when specific complements of germ cells were absent or grossly depleted, seminiferous tubules were isolated and cultured to measure inhibin secretion under basal or stimulated conditions. The results demonstrate that elongate spematids are the primary germ cell type which exerts major control over inhibin secretion under both basal and FSH-stimulated conditions. In conclusion, these studies demonstrate: Firstly that these compounds can be used as a means to investigate normal spermatogenesis. Secondly, effects of these compounds on spermatogenesis can be demonstrated both in vivo and in vitro. Finally, that the seminiferous tubule culture technique represents a more physiological approach with which to investigate in vivo - in vitro comparative toxicity with respect to chemically induced disruption of spermatogenesis.

The aims of the studies in this thesis were, firstly, to investigate the feasibility of detecting proteins derived from the seminiferous epithelium in testicular interstitial fluid, spermatic vein blood, testicular vein blood and also in peripheral blood. Secondly, to identify proteins might be potential markers of adverse effects on spermatogenesis. An antiserum was raised against seminiferous tubule secreted proteins and was used in conjunction with Western blot analysis to detect testicular proteins in biological fluids and also to screen a testis cDNA library to identify novel secretory products. Western blot analysis was only able to detect proteins secreted by seminiferous tubules in testicular interstitial fluid, but not in spermatic, testicular or peripheral venous plasma from adult rats. The antiserum specifically detected three proteins in testicular IF from control animals. The disruption of spermatogenesis by short-term testicular heat treatment 24 hours earlier (43°C for 30min) increased the abundance of these proteins and induced the appearance of several other less abundant proteins, all with molecular masses below 25 kDa. Two of the proteins present in IF were identified as germ cell secretory products, phosphatidylethanolamine binding protein and an androgen regulated protein (ARP-2). These results suggest that germ cell, as well as Sertoli cell, secretory products can gain access to the interstitium under both normal physiological conditions and more easily after testicular damage. The antiserum was unable to detect these proteins in peripheral blood even after heat treatment; this may be due to the insensitivity of the techniques used. However, this approach has identified two candidate proteins for the development of specific immunoassays, which will then enable more definitive assessment of whether proteins secreted by the seminiferous epithelium can be measured in blood.

Patterns of methylation at the 5-position of cytosine are postulated to be involved in several mammalian processes such as the regulation of gene expression, genomic imprinting and X chromosome inactivation. Disruptions in methylation patterns affect embryonic development and are involved in carcinogenesis, indicating the importance of regulating these patterns. The establishment of methylation patterns is believed to be initiated during gametogenesis and continue during early embryonic development. DNA (cytosine-5)-methyltransferase (EC2.1.1.37) (DNA methyltransferase) is the only known mammalian enzyme that catalyzes the formation of 5-methyl cytosine. Here, DNA methyltransferase is proposed to play a key role in establishing and maintaining methylation patterns during male gametogenesis and is predicted to be regulated during spermatogenesis. The regulation of DNA methyltransferase mRNA during testicular development in mouse and rat was first established as preliminary evidence for the regulation of DNA methyltransferase during spermatogenesis. Highly enriched populations of spermatogenic cell types were then isolated from mouse testes and examined for DNA methyltransferase mRNA and protein expression. Expression of DNA methyltransferase protein was proportional to the levels of a 5.2 kb mRNA. Substantial levels of protein and 5.2 kb mRNA were observed in mitotic types A and B spermatogonia, preleptotene spermatocytes, meiotic leptotene/zygotene spermatocytes and postmeiotic round spermatids but barely detectable in pachytene spermatocytes in which a large decrease in 5.2 kb mRNA levels and the appearance of a 6.2 kb transcript was observed. The subcellular localization of DNA methyltransferase in germ cells was also regulated. These findings reveal DNA methyltransferase to be highly regulated during spermatogenesis and support a role for the enzyme in events associated with germ line methylation changes such as developmental gene regulation, genomic imprintin

　　Vad1.3 is an evolutionarily-conserved, testis-specific gene identified from a retinol-treated Vitamin A-deficiency (VAD) rat model. VAD1.3 is expressed throughout spermiogenesis at the acrosome of spermatids and epididymal spermatozoa, suggesting a role in acrosome biogenesis or acrosome reaction. The present study aimed to explore the functional role of VAD1.3 in spermatogenesis and sperm functions by the cellular and gene-knockout approaches. 　　Double immunofluorescent microscopy confirmed the co-localization of VAD1.3 and syntaxin 1 in mouse spermatids and spermatozoa. Deletion analysis of the Vad1.3 gene in transfected mouse spermatocyte GC2-spd and human cervical cancer HeLa cells revealed a polarized peri-nuclear/Golgi expression pattern for the N-terminal GFP-VAD fusion proteins which contain a bipartite nucleus localization (BNL) motif, but a nuclear expression pattern for the C-terminal GFP-VAD. The N-terminal sequences of VAD1.3 mediated its interaction with syntaxin 1, as demonstrated by both co-localization and co-immunoprecipitation studies. The full-length GFP-VAD co-localized with the Golgi markers and was redistributed into the endoplasmic reticulum after brefeldin A treatment, suggesting that VAD1.3 was recruited through the ER-Golgi-acrosome pathway. 　　Vad1.3+/- mice was previously generated by the conventional knockout approach. The heterozygous mice had normal spermatogenesis during postnatal days and adulthood (6-8 weeks). At the age of 8-19 months, 6 out of 17 heterozygous mice but no wild-type exhibited a decrease in the epididymal sperm count and testicular weight (p < 0.05). Histological analyses unveiled disarrangement of the seminiferous epithelium and sloughing of germ cells, predominantly spermatids, which was mediated partially by apoptosis as a higher percentage of TUNEL-positive cells were detected in these heterozygous mice (p < 0.05). This phenotype was associated with a decrease in the mRNA (p < 0.05) and protein levels of VAD1.3 in the testis. 　　Crossing of the Vad1.3+/- mice produced wild-type and heterozygous offspring in a ratio of 1:3, but no Vad1.3-/- mice were found. There was no significant difference between the heterozygous intercrosses and the wild-type intercrosses in the number of oocytes ovulated, the developmental rate of embryos from zygotes to blastocysts, the number of implantation site, resorption site or the offspring could result from defective fertilization between Vad1.3 null gametes rather than developmental lethality. The role of VAD1.3 in fertilization was supported by the inhibitory effects of the anti-VAD1.3 antibody on in vitro fertilization and progesterone-induced acrosome reaction. Immuno-staining revealed that VAD1.3 was present in the acrosome-intact spermatozoa but not in acrosome-reacted spermatozoa, indicating a role of VAD1.3 in ZP-binding or acrosome reaction rather than sperm-egg fusion. In oocytes VAD1.3 was distributed in the cytoplasm near the cortex. litter size. Only a few Vad1.3-/- embryos were found at the zygotic (3.7%) and 2-cell (3%) stages in the heterozygous intercrosses. These findings suggested that the absence of the Vad1.3-/- 　　In sum, VAD1.3 may play important roles in fertilization and spermatogenesis in mice. The BNL motif of VAD1.3 directs its Golgi expression and the N-terminal sequence of the protein mediates its interaction with syntaxin 1. The use of tissue-specific knockout approach may help to answer the functional role of VAD1.3 in future.
published_or_final_version
Obstetrics and Gynaecology
Doctoral
Doctor of Philosophy

Studying the spermatogenesis of horse is beneficial for the horse industry by identifying the causes of chromosomal abnormalities, which cause embryonic loss, congenital abnormalities and infertility. Little is known about the spermatogenesis in horse. This is the first report that investigates the horse spermatogenesis in detail, particularly metaphase I (MI) and prophase I (PI) stages of the first meiotic division. Meiotic recombination is considered to be the major outcome of meiosis. It is essential for proper chromosome segregation and formation of normal haploid gametes. Analysis of recombination frequency and distribution are crucial for genomic and association studies. Any alteration of the recombination frequency and positioning can cause non-disjunction and generation of aneuploidy. The frequency and distribution of chiasmata were estimated at MI chromosomes from fourteen fertile stallions. The average frequency of autosomal chiasmata was 49.45 ± 2.07, corresponding to a genetic length of 2,472.5 cM. All autosomal bivalents had at least one chiasma. The majority of chromosomes have one or two chiasmata, which are mostly distally localized. The frequency and the distribution as well as the genetic length of chiasmata were also estimated for the first time in eight different individual autosomes. Immunofluorescent localization was used to characterize the early stages of the first meiotic division as well as to examine the frequency and the distribution of DNA mismatch repair protein MutL Homologous Protein 1 (MLH1) foci on synaptonemal complexes (SCs) from sex fertile stallions. The mean frequency of autosomal recombination foci was 50.11±2.35. All autosomal bivalents had at least one recombination focus. In general, foci were located near the distal ends with some foci interstitially distributed. The distribution of MLH1 foci indicated positive interference; however, foci were very close to one another in rare instances. The average SCs relative length was highly correlated with the average number of MLH1 foci. MLH1 have been proposed to mark crossover sites at PI since the frequency and distribuation of MLH1 foci closely correspond to the frequency and distribution of chiasmata on MI chromosomes. iii | P a g e Spermatozoa viability, which include spermatozoa head and tail membrane integrity, acrosomal integrity and mitochondrial function assessment are the main sperm analysis parameters considered in this thesis to evaluate the stallion fertility using epididymal collected semen samples. The mean percentage of spermatozoa with viable heads and tails, using Chicago sky blue stain, was 81.26 ± 5.06. FITC-Pisum sativum agglutinin (FITC-PSA) and MitoTracer green were used successfully to assess the spermatozoal acrosomal status as well as the mitochondrial function, respectively. The mean percentage of spermatozoa with integrated acrosome was 93.85 ± 1.9, while for functional mitochondria was 95.63 ± 1.63. In conclusion, this finding is the cornerstone to understanding the genetic basis of normal horse spermatogenesis. Simultaneous assessment of different functional sperm parameters as well as investigating the synapsis and recombination frequency and distribution, at PI or MI, would assist with predictions of stallion fertility prior to breeding. In addition, this study will enable investigators to use linkage analysis in identifying and localising different genetic loci associated with specific traits.

Mestrado em Biomedicina Molecular
The Amyloid precursor protein (APP) and Tau protein are related to histopathological hallmarks of Alzheimer’s disease, a progressive and complex neurodegenerative disease. APP is a type 1 integral transmembrane glycoprotein. There are two predominant proteolytic processing pathways of APP, the nonamyloidogenic pathway, and the amyloidogenic pathway. Tau is one of the microtubule-associated proteins, and its binding affinity for microtubules is regulated by phosphorylation of serine and threonine residues. APP and Tau protein expression has also been reported in the testis. However, their function and posttranslational modifications in the testis have not been established. Thus, we analyzed the expression of these two proteins and their phosphorylation patterns during spermatogenesis using wild-type mice and rats as models. Through immunohistochemistry we revealed that APP, Tau protein, Serine/Threonine Protein Phosphatase (PP) 1α and PP1γ are expressed throughout spermatogenesis. APP and Tau are phosphorylated during meiosis. In contrast to total-APP localization, phosphorylated APP at Thr668 was specially localized in spermatocyte nuclei. These results suggest that phosphorylation of APP and Tau protein contribute to spermatogenesis, especially in meiosis. However, further research is required to validate our results and unravel the specific function of APP and Tau protein during spermatogenesis and meiosis.
A proteína precursora de amilóide (PPA) e a proteína Tau estão relacionadas com os marcos histológicos da doença de Alzheimer, uma doença neurodegenerativa progressiva e complexa. A PPA é uma glicoproteína integral transmembranar que tem duas predominantes vias de processamento proteolítico, a via não-amiloidogénica e a via amiloidogénica. A proteína Tau encontra-se associada aos microtúbulos, sendo a afinidade dessa ligação regulada pela fosforilação de resíduos de serina e treonina. A expressão da PPA e da proteína Tau também já foi reportada no testículo. No entanto, ainda não foram estabelecidas as suas funções e modificações pós-traducionais neste tecido. Assim, analisamos a expressão destas duas proteínas e o seu padrão de fosforilação durante a espermatogénese usando como modelos murganhos e ratos wild-type. Através de imuno-histoquímica revelamos que a PPA, a proteína Tau, a proteína serina/treonina fosfatase (PP) 1α e a PP1γ são expressas em toda a espermatogénese. Fosforilação da PPA e da proteína Tau foi detetada durante a meiose. Em contraste com a localização da PPA-total, a PPA fosforilada na treonina 668 está especialmente localizada no núcleo dos espermatócitos. Estes resultados sugerem que a fosforilação da PPA e da proteína Tau contribui para a espermatogénese, especialmente na meiose. No entanto, são necessários estudos futuros para validar os nossos resultados e desvendar a função específica da PPA e da proteína Tau durante a espermatogénese e na meiose.

Temperature-related sequence 4 (Trs4) is a transcript identified from the testis of a cryptorchid rat model. TRS4 is firstly detected in round spermatids on postnatal day 21 mice, and is localized on the acrosome and tails of elongating spermatids and mature spermatozoa, suggesting a role in spermatogenesis. The present study aimed to investigate the regulatory and functional roles of Trs4 in spermatogenesis. To investigate the hormonal regulation of Trs4 in vitro, mouse germ cell were co-cultured with Sertoli (TM4) cell-line for 24 hours with 1μM or 10nM testosterone and the expression of Trs4 was quantitated. It was found that testosterone treatment at physiological level (10nM) significantly down-regulated Trs4 mRNA expression. However, TRS4 protein level did not change significantly. To investigate the functional roles of Trs4 and its interacting proteins (Rshl2, Gstmu1 and Ddc8) in spermatogenesis, the scrotal heat-treated rat model was used. The expression of Trs4, Rshl2, Gstmu1 and Ddc8 transcript were investigated on day 1, 9, 25, 37, 46, 56 and 79 post-heat treatment. Trs4, Rshl2 and Ddc8 transcripts were down-regulated on day 9 after heat-treatment, and recovered from day 37 onward. Immunohistochemical staining of TRS4 and RSHL-2 proteins showed they specifically localized in the cytoplasm of elongating spermatids in rat seminiferous tubules, suggesting that they might interact in regulating the development of elongating spermatids. Ten sessions of electroacupuncture (EA) was applied to the rats on day 9-36 to study the effect of EA in spermatogenesis. It was found that EA did not have any effect on the expression of Trs4, Rshl-2 and Ddc8 during recovery from hyperthermia when compared to the heat-treated only group. However, qPCR analysis on Gstmu1 mRNA expression showed a significant decrease in expression on day 46, and consistent with our TUNEL assay indicating a decrease in apoptosis in the EA group. Therefore, it is possible that Trs4 interacts with Gstmu1 and acts as anti-apoptotic factor for cell survival. To understand functional roles of Trs4 in mouse spermatogenesis, five Trs4 chimeric mice were generated in our laboratory and germline transmissible 〖Trs4〗^(flox/+) mice were identified. Sycp1-Cre mice were used to breed with Trs4 floxed mice to generate Trs4 floxed mice expressing Cre recombinase in spermatocytes (〖Trs4〗^(flox/+);〖Sycp-Cre〗^(+/0)) and aiming to generate Trs4 heterozygous KO mouse. However, after screening 74 offspring, no Trs4 heterozygous KO mouse was identified in this study and was likely to be caused by the low efficiency of recombination in Sycp1-Cre mice. Then, Zp3-Cre mice that expressing Cre-recombinase in oocyte were employed. Crossing of 〖Trs4〗^(flox/+):Zp3-〖Cre〗^(+/0) mice and Trs4 floxed mice could not produce heterozygous or homozygous Trs4 KO mouse, but suggested an aberrant recombination process happened in vivo. PCR assay confirmed DNA rearrangement occurred in regions flanking the exon 4 to the second LoxP site in Trs4 floxed mice. Furthermore, the absence of 〖Trs4〗^(flox/flox) homozygous transgene suggested a possibility of insertional mutagenesis in the transgene. Future study is needed to confirm and characterize the insertion site of the floxed allele in order to generate Trs4 knockout mice for fertility study.
published_or_final_version
Obstetrics and Gynaecology
Master
Master of Philosophy

Development can be seen as the consequence of alternative gene expression in different cell types. The focus of this study is the hierarchy of gene expression that controls the process of spermatogenesis, at the RNA level. RBM is an RNA-binding protein (encoded by the Y chromosome) thought to be important for the production of sperm. Its primary structure is reminiscent of SR protein splicing factors and it colocalises with them in the nucleus of germ line cells in the testes. If RBM were involved in splicing it should interact with components of the splicing machinery, so a yeast two-hybrid screen was employed and several candidate RNA-binding proteins were retrieved. These included two testes-specific novel proteins with ubiquitous known homologues. Overall the prey fell into three functional categories: signal transducers and RNA processors (STAR proteins), general heterogeneous ribonucleoproteins (hnRNPs) and SR protein splicing factors. This suggests a global theory for RBM function, that it relays signals from the cytoplasm to effect key changes in gene expression at the level of splicing. Evidence was obtained that these proteins colocalise in vivo and bind in vitro in a phosphorylation-dependent manner that could be the key to its essential regulation in nature.

La spermatogenèse est le processus par lequel les cellules germinales se différencient pour former les spermatozoides. Elle se déroule à l’intérieur des tubes séminifères. Pendant la période embryonnaire, les précurseurs des cellules germinales adultes constituent un pool de cellules appelées cellules germinales primordiales (Primordial Germ Cells, PGCs), qui vont migrer pour aller coloniser les gonades (Durcova-Hills and Capel, 2008; Surani et al., 2008). Au cours de leur migration, les PGCs vont subir une reprogrammation épigénétique de l’ensemble de leur génome, qui leur sera nécessaire pour passer de l’état somatique à l’état de totipotence (Ohinata et al., 2005). Durant cette reprogrammation, l’ADN est massivement démethylé, entrainant l’activation de plusieurs gènes qui sont importants pour le développement des PGCs, mais également l’activation des éléments transposables (ETs) (Hajkova et al., 2008; Sasaki and Matsui, 2008; Surani and Hajkova, 2010). Ces derniers constituent environ 50% du génome des mammifères. Ils sont subdivisés en deux classes et sont connus par leur capacité à être mobilisés dans le génome (Zamudio and Bourc'his, 2010). Cette mobilisation se fait au hasard et constitue ainsi un risque considérable de mutations, qui peuvent provoquer des tumeurs, des pathologies de développement et une infertilité (Zamudio and Bourc'his, 2010). Pour cela, leur expression doit être contrôlée pour maintenir l’intégrité du génome de la lignée germinale. Pour toutes ces raisons, les PGCs ainsi que les cellules germinales en cours de méiose ont développé des stratégies de défenses pour contrôler la mobilisation et l’expression des ETs basées entre autre sur la voie des piwi-interacting RNA (piRNA) (Chuma and Pillai, 2009; Pillai and Chuma, 2012b). Dans le laboratoire du Pr. Stéphane Viville, mes travaux de thèse se sont concentrés sur l’étude d’un gène nommé Tex19 pour Testis Expressed gene chez la souris. Nous avons démontré que ce gène est spécifique des mammifères et est dupliqué chez le rat et la souris en deux paralogues nommés Tex19.1 et Tex19.2. Deux domaines hautement conservés ont été identifiés par alignement multiple des protéines TEX19 et nommés MCP et VPTEL. Ces domaines ne présentent aucune homologie avec des domaines déjà caractérisés, prévenant ainsi toute prédiction de leurs fonctions (Kuntz et al., 2008). L’étude du profil d’expression de Tex19.1 et Tex19.2 a montré que ces deux gènes sont exprimés dans l’ectoderme et les PGCs. Ils sont aussi co-exprimés dans le testicule de l’âge embryonnaire à l’âge adulte. Néanmoins, seul Tex19.1 est exprimé dans les ovaires et le précurseur du placenta appelé cône ectoplacentaire (Celebi et al., 2012). Le knockout (KO) de Tex19.1 provoque une infertilité masculine chez la souris avec un arrêt de la spermatogenèse au stade pachytène, accompagnée d’une surexpression d’un rétrotransposon, MMERVK10C (Ollinger et al., 2008). Récemment, il a été démontré que Tex19.1 joue aussi un rôle dans le développement du placenta (Reichmann et al., 2013). Au cours de mes trois années de thèse, nous avons approfondie l’étude du KO de Tex19.1dans le testicule, les cellules embryonnaires souches (Embryonic Stem Cells, ESCs) et le placenta (Tarabay et al., 2013). Nous avons également étudié le phénotype observé suite au double KO de Tex19.1 et Tex19.2. [...]
We recently characterized two new mammalian specific genes, Tex19.1 and its paralog Tex19.2. Both genes are expressed in pachytene spermatocytes in adult testes. In addition, Tex19.1 is expressed in pluripotent cells (ES, EG, iPS and PGC cells), the inner cell mass of the blastocysts and the placenta. In order to decipher Tex19 functions, we generate three types of knockout (KO): i) KO of Tex19.1 ii) KO of Tex19.2 iii) double KO (DKO) of both genes. All Tex19.1-/- KO animals are growth-retarded and half of them die just after birth. This phenotype is probably linked to placenta defects. Surviving adults Tex19.1-/- KO males display a variable spermatogenesis phenotype, associated with an up-regulation of one endogenous retrovirus, MMERVK10C. Tex19.2 KO mice exhibit a subtle phenotype. Few seminiferous epitheliums are degenerated while the rest appear normal. DKO show a fully penetrant phenotype similar to the most severe Tex19.1-/- phenotype. DKO males exhibit small testes. Despite the presence of spermatogonia and spermatocytes, spermatogenesis is blocked at the pachytene stage. By RNA deep-sequencing on 10 days old DKO and WT testes, prior to histological phenotype, 114 genes are significantly up-regulated and 320 genes significantly down-regulated in the DKO compared to the WT. Gene ontology analyses show that among of these genes, two essential pathways are altered: meiosis and the piRNA pathway. Consistent with that, GST-pulldown and immunoprecipitation experiments demonstrate that MIWI, MILI, MAEL and MVH are partners of TEX19. Considering PIWI proteins function in the silencing of transposable elements through the piRNA pathway, we checked if TEX19 paralogs bind piRNA. By immunoprecipitation using WT and KO testes, we show that both TEX19.1 and TEX19.2 bind small RNA of 30 nucleotides through their VPTEL domain. This study highlights the pivot role of Tex19 paralogs in three essential functions of mammalian life cycle, i.e. pluripotency, placenta-supported in utero growth and fertility. The functional similarities of both paralogs, through the expression control of one endogenous retrovirus and the binding of piRNAs, lead us to propose that Tex19 paralogs are new members of the piRNA pathway

Mestrado em Biomedicina Molecular
The lamina-associated polypeptide 1 (LAP1) is a type II transmembrane protein of the inner nuclear membrane encoded by the human gene TOR1AIP1. LAP1 is involved in maintaining the nuclear envelope structure and appears be involved in the positioning of lamins and chromatin. In the nuclear envelope, LAP1 is suggested to exist as a complex with A-type and B-type lamins, torsins and emerin. The presence of such complexes suggests that LAP1 may cooperate functionally with these proteins in tissues where they play a critical role. Therefore, the identification of LAP1 binding partners and the signalling pathways where LAP1 participates, is crucial for a better understanding of LAP1 functions. The work described in this thesis addresses novel human LAP1 associated proteins found through bioinformatic tools. Public databases allowed for the discovery of the LAP1 interactome, which was manually curated, identifying several functionally relevant proteins. Subsequently, the integration of multiple bioinformatic tools established novel functions to LAP1 such as DNA damage response and telomere association. In conjunction, bioinformatic results also reinforced the association of LAP1 with mitosis, and the already identified role of LAP1 in nuclear morphology. Interestingly, this association of LAP1 with the regulation of the nuclear envelope structure and mitosis progression, shares functional elements with spermatogenesis. Therefore, this work additionally described the localization of LAP1 and some of its interactors throughout the spermatogenic cycle, in mouse and human testis. The results established that the activity of LAP1 during the mouse spermatogenic cycle is most evident from stage VIII until the end of spermiogenesis, which is characteristic of manchette development. Concomitantly, some LAP1 interactors studied in this work share a similar localization, namely, PP1γ2, Lamin B1 and Lamin A/C. The results obtained from the study of LAP1 throughout different periods of the male reproductive system attributed potential new biological functions to LAP1. Thereby, this work can be the foundation of future studies regarding LAP1 and the regulation of multiple cellular processes and disease conditions.
A proteína 1 associada com a lâmina (LAP1), codificada pelo gene humano TOR1AIP1, encontra-se localizada na membrana interna do núcleo. Funcionalmente a LAP1 está associada à manutenção da estrutura do envelope nuclear e ao posicionamento da lâmina e da cromatina. No envelope nuclear a LAP1 forma complexos com várias proteínas, nomeadamente lâminas tipo A e B, torsinas e emerina. A atividade destes complexos sugere a existência de uma função cooperativa entre a LAP1 e as proteínas nomeadas anteriormente, que será essencial, particularmente nos tecidos em que a sua expressão é substancial. Assim, a identificação de novos interactores da LAP1 e das suas respetivas vias de sinalização, será crucial para identificar e compreender as funções biológicas desta proteína. Ao longo deste trabalho foram utilizadas ferramentas bioinformáticas que possibilitaram a descoberta de novas proteínas associadas com a LAP1. Em primeiro lugar, o interactoma da LAP1 foi estabelecido através do acesso a bases de dados públicas, procedendo-se posteriormente à análise manual de cada uma destas interações. De seguida, a integração de múltiplas ferramentas bioinformáticas possibilitou a determinação de novas funções associadas à LAP1, designadamente a resposta a danos no DNA e a interação com os telómeros. Simultaneamente, os resultados bioinformáticos reforçaram a participação da LAP1 no processo de mitose e a intervenção desta proteína na manutenção da morfologia nuclear. Em particular, a função da LAP1 na regulação da mitose e da estrutura do envelope nuclear parece ter semelhanças com processo de espermatogénese. Assim, ao longo deste trabalho foi também estudada a distribuição da LAP1 ao longo deste processo de diferenciação, assim como de alguns dos seus conhecidos interactores, no testículo de ratinho e de humano. Em ratinho, foi possível determinar que a atividade da LAP1 durante a espermatogénese é mais significativa desde a fase VIII até ao fim da espermiogénese, sendo característica do desenvolvimento da manchete. Concomitantemente, alguns dos interactores avaliados possuem uma localização análoga à da LAP1, nomeadamente, as lâminas B1 e A/C, e a proteína serina/treonina fosfatase 1γ2 (PP1γ2). Os resultados obtidos após o estudo da LAP1 ao longo do sistema reprodutor masculino possibilitaram a atribuição de novas e potenciais funções biológicas à LAP1. Assim, este trabalho poderá servir de ponto de partida para estudo futuros da LAP1 e da sua função na regulação de múltiplos processos celulares e doenças.

Expression of the Tudor domain containing protein 6 (TDRD6), which is restricted to the male germ line, starts at day 16 of spermatogenesis, i.e. in pachytene spermatocytes. TDRD6 is a 250 kDa protein, which we recently found to be cleaved at the C-terminal end during germ cell development, resulting in a 230 kDa product. Neither is the process of cleavage itself nor are the functions of the two different forms known. The 230 kDa isoform is the most prominent form in round spermatids, where it localizes to the chromatoid body (CB), i.e. a single filamentous, perinuclear granule. One characteristic component of the CB is the RNA helicase MVH. CBs contain components of the microRNA (miRNA) pathway, including Piwi-interacting RNAs (piRNAs), as well as MIWI, MIWI2, and MILI, the mouse homologs of the Piwi proteins, which bind piRNAs and also act in transposon regulation. We showed that TDRD6 interacts with MIWI and MILI in vitro, and a direct interaction with MVH was shown before. To reveal the function of TDRD6, we generated Tdrd6-/- mice, which lack the protein. These mice are generally healthy but the males are sterile, due to the absence of mature spermatozoa. The most striking intracellular phenotype of Tdrd6-/- mice is the highly aberrant architecture of chromatoid bodies in round spermatids. Tdrd6-/- CBs appear as diffuse, disrupted, and less condensed structures. Their interior is largely missing, and only a “ghost”-like structure remains, expected to be significantly impaired in function. Other CB components like MVH, MIWI and MILI are expressed in Tdrd6-/- testes, but they cannot localize to the disrupted CBs. This suggests a role for TDRD6 in assembling the chromatoid body complex by recruiting other proteins. The CB is important for storage and translational regulation of mRNA, through interaction with miRNAs. In Tdrd6-deficient testes 10 % of all known murine miRNAs are differently expressed, whereas most of the mature miRNAs are up-regulated, indicating less turnover, and thus, accumulation of mature miRNAs. Since some precursor miRNAs are up-regulated as well, we assume, that TDRD6 affects miRNA transcription most likely by indirectly influencing transcriptional regulation of miRNA genes. In Tdrd6-/- mice an overall abnormal mRNA gene expression pattern was observed by microarray analyses. Of all mis-regulated genes 36 % are located to the centromer-proximal region of Chr 8, and 11 % are located to the distal end of Chr 1. This mis-regulation might be due to a common transcriptional regulation. The orthologous regions on the human chromosomes show altered chromosomal structures in many different carcinomas. If TDRD6 plays a role in carcinogenesis has to be investigated.

La línia germinal conté el passat i el futur d’una espècie, en la qual la informació genètica parental es recombina mitjançant la meiosi i és transmesa a la descendència. Així, entendre com el genoma s’organitza i es regula en l’espai nuclear durant la formació de cèl·lules germinals és essencial per comprendre les bases de la fertilitat i el seu impacte en la diversitat genètica. En cèl·lules somàtiques, el genoma s’organitza en territoris cromosòmics que estan formats per compartiments de cromatina plegats en dominis d’associació topològica (TADs) i bucles d’ADN. No obstant això, es coneix poc sobre l’organització de l’genoma en la línia germinal i com les reorganitzacions cromosòmiques poden modular-la. En aquest context, aquesta tesi té com a objectius: (i) entendre l’organització tridimensional de l’genoma durant l’espermatogènesi de ratolí i la seva relació amb la funció gènica i la localització de proteïnes aïllants (CTCF i cohesines), (ii) investigar les implicacions de les fusions Robertsonianes (Rb) en el plegament del genoma i la recombinació, i (iii) caracteritzar la variabilitat de PRDM9 en poblacions naturals de ratolins Rb, incloent el sistema Rb de Madeira i el de Barcelona (BRbS). Per això, hem combinat anàlisis citològics amb tecnologies de seqüenciació massiva i desenvolupat un protocol de citometria per obtenir poblacions cel·lulars germinals enriquides, incloent espermatogonis, espermatòcits primaris, espermàtides rodones i espermatozoides. Els nostres resultats revelen que l’estructura d’ordre superior del genoma és extremadament dinàmica durant l’espermatogènesi, on els espermatogonis presenten compartiments i TADs que desapareixen durant la meiosi i es restableixen posteriorment en cèl·lules post-meiòtiques. A més, hi ha una correlació entre la transcripció i els compartiments A, amb gens actius específics de tipus cel·lular relacionats amb la progressió de l’espermatogènesi, la fecundació i el desenvolupament embrionari. Addicionalment, hem trobat una correlació entre la localització de cohesines i transcripció activa tant en cèl·lules meiòtiques com post-meiòtiques, suggerint que les cohesines regulen la transcripció. Les fusions Rb reorganitzen la localització espacial dels cromosomes i en espermatòcits primaris, augmenten les interaccions heteròlogues, promovent nous entorns de regulació. En espermàtides rodones, les fusions afegeixen restriccions mecàniques que redueixen les interaccions inter-cromosòmiques. A més, les fusions Rb afecten tant a el nombre com a la distribució cromosòmica dels punts de recombinació, especialment en els metacéntrics fusionats en homozigosi, mentre que la presència de metacéntrics heterozigots asinapsats indueix una heterocromatinizació de la vesícula sexual. La reducció de la recombinació també es detecta en les anàlisis de desequilibri de lligament basats en SNPs, detectant alta divergència genètica en poblacions Rb comparades amb estàndard. Addicionalment, hem caracteritzat una gran variabilitat de PRDM9, sent especialment alta al sistema Rb insular de Madeira en comparació amb el sistema continental BRbS. Aquestes diferències es poden atribuir a la combinació de diferents factors: (i) la història evolutiva de cada sistema Rb, (ii) la prevalença de fusions Rb afectant la diversitat genètica, i en menor grau, (iii) restriccions funcionals meiòtiques (per exemple, asimetria en els hotspot de recombinació). En conjunt, aquesta tesi mostra que la cromatina pateix una remodelació profunda durant l’espermatogènesi específica de tipus cel·lular, en la qual l’activitat transcripcional es correlaciona amb l’estat de la cromatina i la localització de les cohesines. Addicionalment, les fusions Rb alteren l’organització del genoma en la línia germinal, afectant a la recombinació meiòtica i la diversitat genètica.
La línea germinal contiene el pasado y el futuro de una especie, en la que la información genética parental se recombina mediante la meiosis y es transmitida a la descendencia. Así, entender como el genoma se organiza y se regula en el espacio nuclear durante la formación de células germinales es esencial para comprender las bases de la fertilidad y su impacto en la diversidad genética. En células somáticas, el genoma se organiza en territorios cromosómicos que están formados por compartimentos de cromatina plegados en dominios de asociación topológica (TADs) y bucles de ADN. Sin embargo, se conoce poco acerca de la organización del genoma en la línea germinal y como las reorganizaciones cromosómicas pueden modularla. En este contexto, esta tesis tiene como objetivos: (i) entender la organización tridimensional del genoma durante la espermatogénesis de ratón y su relación con la función génica y la localización de proteínas aislantes (CTCF y cohesinas), (ii) investigar las implicaciones de las fusiones Robertsonianas (Rb) en el plegamiento del genoma y la recombinación, y (iii) caracterizar la variabilidad de PRDM9 en poblaciones naturales de ratones Rb, incluyendo el sistema Rb de Madeira y el de Barcelona (BRbS). Por ello, hemos combinado análisis citológicos con tecnologías de secuenciación masiva y desarrollado un protocolo de citometría para obtener poblaciones celulares germinales enriquecidas, incluyendo espermatogonias, espermatocitos primarios, espermátidas redondas y espermatozoides. Nuestros resultados revelan que la estructura de orden superior del genoma es extremadamente dinámica durante la espermatogénesis, donde las espermatogonias presentan compartimentos y TADs que desaparecen durante la meiosis y se reestablecen posteriormente en células post-meióticas. Además, hay una correlación entre la transcripción y los compartimentos A, con genes activos específicos de tipo celular relacionados con la progresión de la espermatogénesis, la fecundación y el desarrollo embrionario. Adicionalmente, hemos hallado una correlación entre la localización de cohesinas y transcripción activa tanto en células meióticas como post-meióticas, sugiriendo que las cohesinas regulan la transcripción. Las fusiones Rb reorganizan la localización espacial de los cromosomas y en espermatocitos primarios, aumentan las interacciones heterólogas, promoviendo nuevos entornos de regulación. En espermátidas redondas, las fusiones añaden restricciones mecánicas que reducen las interacciones inter-cromosómicas. Además, las fusiones Rb afectan tanto al número como a la distribución cromosómica de los puntos de recombinación, especialmente en los metacéntricos fusionados en homocigosis, mientras que la presencia de metacéntricos heterocigotos asinapsados induce una heterocromatinización de la vesícula sexual. La reducción de la recombinación también se detecta en los análisis de desequilibrio de ligamiento basados en SNPs, detectando alta divergencia genética en poblaciones Rb comparadas con estándar. Adicionalmente, hemos caracterizado una gran variabilidad de PRDM9, siendo especialmente alta en el sistema Rb insular de Madeira en comparación con el sistema continental BRbS. Tales diferencias se pueden atribuir a la combinación de distintos factores: (i) la historia evolutiva de cada sistema Rb, (ii) la prevalencia de fusiones Rb afectando a la diversidad genética, y en menor grado, (iii) restricciones funcionales meióticas (por ejemplo, asimetría en los hotspot de recombinación). En conjunto, esta tesis muestra que la cromatina sufre una remodelación profunda durante la espermatogénesis específica del tipo celular, en la que la actividad transcripcional se correlaciona con el estado de la cromatina y la localización de las cohesinas. Adicionalmente, las fusiones Rb alteran la organización del genoma en la línea germinal, afectando a la recombinación meiótica y a la diversidad genética.
The germline holds the past and the future of a species, as parental genetic information is recombined through meiosis and transmitted to the offspring. Thus, understanding how the genome is organized and regulated in the nuclear space during the formation of germ cells is essential to comprehend the bases of fertility and its impact on genetic diversity. In the last twenty years, many studies have shown that in somatic cells, the genome is organized in chromosome territories which are formed by chromatin compartments folded into topological associated domains (TADs) and DNA loops. However, little is known about how the genome is organized in the germline and how chromosomal reorganizations modulate genome architecture. In this context, this thesis aims to: (i) understand the three-dimensional organization of the genome during mouse spermatogenesis and its interplay with gene function and occupancy of insulator proteins (CTCF and cohesins), (ii) investigate the implications of Robertsonian (Rb) fusions in genome folding and meiotic recombination, and (iii) characterize the variability of PRDM9 in natural house mouse populations with Rb fusions: the Madeira Rb system and the Barcelona Rb system (BRbS). We combined cytological analysis with next generation sequencing technologies, and we developed an efficient cell sorting protocol to obtain enriched germ cell fractions including spermatogonia, primary spermatocytes at early and late prophase I, round spermatids and sperm. Our results revealed that the higher-order structure of the genome is extremely dynamic during spermatogenesis, where spermatogonia presents somatic-like compartments and TADs, that disappear during meiosis to be re-established later on in post-meiotic cells. Moreover, transcription correlates with A compartments throughout spermatogenesis, with cell-specific active genes involved in spermatogenesis progression, fertilization and embryonic development. In addition, we found a correlation between cohesin occupancy and active transcription in both meiotic and post-meiotic cells, suggesting a transcription-regulating role of meiotic cohesins. Although germ cells with Rb fusions presented the main features of genome architecture, Rb fusions reorganize the spatial chromosome occupancy. In primary spermatocytes, Rb fusions increase heterologous interactions, promoting the formation of novel regulatory environments. In round spermatids, Rb fusions reduce inter-chromosomal interactions as a result of mechanistic constrains. The cytological data shows that the increase in heterologous interactions is concomitant with the presence of asynapsed heterozygous metacentrics, which induce the full heterochromatinization of the sex body. Furthermore, Rb fusions affect the number and chromosomal distribution of crossovers in primary spermatocytes, especially in the case of fused metacentrics in homozygosis. The reduction in recombination was also observed in the analysis of linkage disequilibrium based on SNP genotyping, which translated into high levels of genetic divergence in Rb populations when compared to standard mice. In addition, our characterization of PRDM9 variability detected an unprecedented variability in natural house mouse populations, being especially high in the insular Madeira Rb system when compared to the continental BRbS. Such differences could be attributed to the combination of different factors: (i) the evolutionary history of each Rb system, (ii) the prevalence of Rb fusions affecting genetic diversity, and to a lesser extent (iii) meiotic functional constrains (i.e., recombination hotspot asymmetry). Taken together, this thesis shows that chromatin undergoes profound remodeling during spermatogenesis in a cell-specific way, where transcriptional activity correlates with the chromatin state and cohesin occupancy. In addition, Rb fusions alter genome organization in the germline, having an impact on meiotic recombination and genetic diversity.

In studies carried out previously, transcripts that are expressed in the testis were isolated from a human adult testis cDNA library. The characterisation of these cDNA clones was carried out by a number of methods. In order to determine the sizes of the transcripts and the tissues in which they are expressed, the clones were analysed by Northern blotting using human multiple tissue blots. It was seen that out of the 38 clones analysed, four were exclusively expressed in the testis and a further 17 were preferentially expressed in the testis, suggesting that they may have a role in testis function. Two clones produced multiple transcripts in the testis. This may be the result of post-transcriptional processing of the mRNA molecules in the testis. Southern analysis of the testis-specific and testis-elevated clones demonstrated the conservation of the human cDNA clones with other mammalian species. There was greater conservation between human and pig DNA compared with human and rodent DNA. This allowed the human clones to be used as probes for mRNA in situ hybridization studies using sections of pig testis. The expression patterns of the transcripts was determined at the cellular level by mRNA in situ hybridization. It was seen that all of the clones that produced a signal were expressed in the primary spermatocytes, when RNA synthesis is maximal. There was also germ cell-specific patterns of expression, indicating that the gene products are required at different stages of the spermatogenic cycle. The spermatogenic cells from pig testis were separated into germ cell enriched populations by centrifugal elutriation, and RNA was extracted from each cell fraction and used to prepare cDNA. Amplification of the cDNA with gene-specific primers showed in which germ cells the gene is expressed. The sensitive technique of RT-PCR provides information on low-level transcripts that do not produce a signal in mRNA in situ hybridization studies.

Spermatogenesis takes place in the seminiferous epithelium and it is a tightly regulated process that produces spermatozoa from spermatogonia. During spermatogenesis, germ cells have to traverse the seminiferous epithelium, from basal to adluminal compartment and finally reach the luminal edge of the seminiferous tubules at spermiation. During the transit of germ cells, they have to get across the blood-testis barrier (BTB), which is formed by adjacent Sertoli cells. Thus, although BTB is considered as one of the tightest blood-tissue barrier, the BTB undergoes cyclic restructuring to “open” transiently for the translocation of germ cells. However, the integrity of the BTB has to remain intact as the BTB is essential for the developing germ cells behind the barrier. For example, the BTB serves as an immunological barrier to “seal” developing germ cells from the systemic circulation. Since how the BTB restructuring is regulated remains elusive, the study herein aims to provide some information regarding to this events. The importance of the BTB to spermatogenesis was demonstrated by treating rats with 50 (lowdose) or 250 mg/kg b.w (high-dose) of adjudin. Although the BTB of rats was perturbed in both groups at week 6 post treatment, as shown by an in vivo BTB functional assay, the BTB of the low-dose group was found to have “resealed” at week 20 whereas the BTB of the high-dose group remained disrupted. Besides, despite almost all germ cells were depleted in both group of rats upon week 2 post treatment, spermatogonia were still present in the testis of rats no matter high- or low-dose of adjudin was used. However, spermatogenesis only recovered in low-dose treated group, which have an intact BTB. This suggests that after spermatogenesis is disrupted, its regeneration of spermatogenesis needs more than the existence of spermatogonia in which an intact BTB is required. After demonstrating the necessity of the BTB for spermatogenesis, the next question I addressed was how its restructuring was modulated. The involvement of mammalian target of rapamycin (mTOR) in manipulating the BTB was investigated. mTOR is able to form two distinct signaling complexes namely mTOR complex 1 (mTORC1) or mTORC2 by assembling with raptor or rictor, respectively. rpS6, which is a downstream molecule of mTORC1 was activated specifically during BTB restructuring and knockdown of rpS6 in cultured Sertoli cells was able to promote the TJ-barrier by inducing deposition of F-actin and BTB proteins at the cell-cell interface, suggesting the role of phosphorylated rpS6 is to “open” the BTB for the transit of spermatocytes. Moreover, the knockdown of rictor led to perturbation of TJ-barrier formed by cultured Sertoli cells via a PKC-α depending actin reorganization, causing internalization of BTB proteins. This indicates mTORC2 is necessary for the maintenance of the BTB and hence the two mTOR complexes work antagonistically to regulate the BTB in which mTORC1 is activated to promote the BTB restructuring while the expression of mTORC2 is essential to sustain the BTB integrity.
published_or_final_version
Biological Sciences
Doctoral
Doctor of Philosophy

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2006.
Includes bibliographical references (leaves 106-114).
Mouse Cdyl was originally identified based on homology to the human gene CDY (Lahn and Page, 1999), which is found in four copies on the human Y chromosome (Kuroda-Kawaguchi et al., 2001). Because CDY is expressed specifically in the testis (Lahn and Page, 1999) and is found in Y chromosomal regions that are deleted in some infertile men (Kuroda-Kawaguchi et al., 2001), CDY is thought to have an important role in male fertility and spermatogenesis. However, human studies have not yet been able to directly implicate CDY in male infertility. Even though mouse Cdyl is not located on the Y chromosome, it is the closest known mouse homologue of human CDY and is expressed highly in the testes (Lahn and Page, 1999), which suggests that mouse Cdyl provides a suitable model for the study of human CDY function. However, unlike human CDY, mouse Cdyl is expressed in tissues other than the testis (Lahn and Page, 1999). We have generated mice deficient for Cdyl to study its role in spermatogenesis and characterized their phenotype on a pure BALB/c background. Nearly 2/3 of Cdyl knockout mice die shortly after birth, but those that survive to adulthood appear healthy except for spermatogenic defects.
(cont.) Mice lacking Cdyl produce spermatozoa with misshapen heads and exhibit substantial germ cell death. The loss of germ cells is evident in some knockout mice as early as 3.5 weeks of age, affects spermatogonia, spermatocytes, and spermatids, and is so severe that at 5 months of age the seminiferous tubules of Cdyl knockout mice appear nearly empty. These results demonstrate that Cdyl plays a crucial role in spermatogenesis and suggest that the homologous human gene CDY does as well. However, our results do not support a previously suggested hypothesis that Cdyl participates in the global acetylation of histone H4 in spermatid nuclei (Lahn et al., 2002), as hyperacetylated histone H4 was detected in both wildtype and knockout spermatids. To directly study the role of the human gene CDYin spermatogenesis, we have attempted to rescue the Cdyl-/- spermatogenic phenotype by constructing a transgenic mouse that expresses human CDY and breeding the CDY transgene onto a Cdyl -/- background. Preliminary data from these crosses suggests that human CDY can not rescue the spermatogenic phenotype observed in Cdyl-/- mice, as Cdyl -/- TgCDY mice still exhibited germ cell loss.
(cont.) However, we currently possess TgCDY mice only on a C57BL/6 background, and in the course of these rescue experiments we observed that the spermatogenic phenotype of Cdyl-l- mice is not as severe on a C57BL/6 background, on a mixed C57BL/6x129 background, or on a mixed C57BL/6xBALB/c background as it is on a pure BALB/c background. We believe that future studies, performed on a pure BALB/c background, will be able to better address whether human CDYcan rescue the Cdyl knockout mouse spermatogenic phenotype.
by Jesse Potash.
Ph.D.

For cells to function in a complex organism, they must differentiate and acquire their specific morphology. To study how cells achieve this I have chosen the spermatozoon of the nematode Caenorhabditis elegans. Genes involved in virtually every step of sperm development have been identified by mutations in C. elegans and these mutations dissect the process of differentiation. During spermiogenesis spherical non-motile spermatids differentiate into bipolar amoeboid spermatozoa. Chapters II and III describe the characterization of two genes that affect this differentiation. For both genes, spe-27 and spe-12, mutant hermaphrodites are sterile whereas mutant males are fertile, and spermatids only activate in response to a subset of in vivo and in vitro activators. I present a detailed genetic, phenotypic and molecular analysis of spe-27 mutants. Spe-27 maps to chromosome IV between two previously cloned genes, so I could isolate and sequence a genomic clone containing spe-27 by transformation rescue with clones from the physical map. The protein sequence predicted from the genomic and cDNA sequences shows no significant similarity with currently known proteins. Three mutant alleles were found to be two splicing defects and a missense mutation. Differential northern analysis and in situ hybridizations indicate that spe-27 is expressed in spermatocytes. I propose a model for spermatid activation in which the spe-27 gene product is a member of a signaling pathway necessary for hermaphrodite sperm activation, and present but redundant in the males. I hypothesize that the spe-12 gene product is another member of the signaling pathway necessary for hermaphrodite spermiogenesis. I isolated six new spe-12 alleles. Chapter IV describes collaborative research to characterize the spe-6 gene, which is involved in the localization of sperm-specific proteins to specialized organelles at the spermatocyte stage. The spe-6 gene shows second site non-complementation with an unlinked deficiency, which suggests the presence of interacting gene(s) over that deficiency. I also collaborated on research on the spe-26 gene, but that work is not included in this dissertation.

FER-1 is required for fusion of specialized vesicles, called membranous organelles, with the sperm plasma membrane during Caenorhabditis elegans spermiogeneis. To investigate the role of FER-1 in membranous organelle fusion, I first examined ten fer-1 mutations and found that they all cause the same defect in membrane fusion. FER-1 and the ferlin protein family are membrane proteins with four to seven C2 domains which commonly mediate Ca2+-dependent lipid-processing events. Most of the fer-1 mutations fall within these C2 domains, showing that they have distinct, non-redundant functions. I found that membranous organelle fusion requires intracellular Ca2+ and that C2 domain mutations alter Ca2+ sensitivity. This suggests that the C2 domains are involved in Ca2+ sensing and further supports their independent function. Using two immunological approaches we found three FER-1 isoforms, two of which may arise from FER-1 by proteolysis. By both light and electron microscopy these FER-1 proteins are localized to membranous organelle membranes. Together, these results suggest that the ferlin family members may share a conserved mechanism to regulate cell-type specific membrane fusion.In Chapter III, I present additional results toward studying the function of FER-1 using several broad-based approaches. First, I present a bioinformatics analysis of FER-1 C2 domains and the preliminary results of their calcium-dependent phospholipid binding capabilities. Second, preliminary interactions found with individual FER-1 functional domains by a yeast-two hybrid screen are discussed. Lastly, I present results from a candidate-gene approach to identify additional regulators of MO fusion, the sperm-specific synaptobrevins.

Apoptosis in testicular germ cells has been demonstrated in many species. Features of apoptosis reported in other species were used to confirm use of the TUNEL assay in stallion testes. Eight stallions with normal testicular size and semen quality were evaluated to determine the germ cell types and stages where apoptosis most commonly occurs. Mean numbers of TUNEL-positive germ cells per 100 Sertoli cell nuclei were highest in stages IV and V of the seminiferous epithelial cycle corresponding to meiotic divisions of primary spermatocytes and mitotic proliferation of B1 and B2 spermatogonia. Round and elongated spermatids were labeled less frequently by the TUNEL assay. To examine the relationships between germ cell apoptotic rate and spermatogenic efficiency, seminal traits were assessed to classify stallions into normal or reduced quality semen groups. Apoptotic rates were higher for stages IV-VI and stage VIII seminiferous tubules in stallions with reduced semen quality. Daily sperm production (DSP) per gram and per testis were lower for stallions with reduced semen quality. Regression analyses revealed negative linear relationships for germ cell apoptotic rate with DSP/g, DSP/testis, daily sperm output, progressively motile sperm and morphologically normal sperm in ejaculates. Mean circulating concentrations of inhibin were lower for stallions ejaculating reduced quality semen. Apoptotic rate was negatively correlated with concentrations of inhibin and estradiol-17b and positively correlated with concentrations of LH and FSH. To study germ cell apoptosis and formation of the Sertoli cell barrier during the initiation of spermatogenesis, tubule development was classified based on lumen score. Formation of a seminiferous tubule lumen was consistent with events leading to development of a Sertoli cell barrier. A primary wave of apoptosis removed early differentiating germ cell types prior to the formation of a tubule lumen facilitating both the formation of a tubule lumen and a Sertoli cell barrier. A second wave of apoptosis occurred after the formation of a lumen but before seminiferous tubule cross-sections contained a full complement of germ cells. In conclusion, apoptosis is an essential mechanism during normal spermatogenesis. Apoptosis also accounts for low numbers of normal sperm seen in the ejaculates of some stallions.

Spermatogenesis, the process of male gamete (i.e. spermatozoa) production, requires tight hormonal regulation in order to proceed successfully. The importance of androgens (like testosterone and 5α-dihydrotestosterone) to the regulation of spermatogenesis is well recognized. However, more recently, the importance of estrogens (like 17β-estradiol), has also been demonstrated. These sexual steroids act through ligand-activated transcription factors, estrogen receptors α and β (ERα and ERβ) and androgen receptor (AR), respectively. Most actions of these hormones are achieved through the regulation of target genes. The two ERs have different and sometimes opposing effects on the regulation of target genes, and estrogenic action will ultimately depend on interplay between them, when co-expressed in the same cell. The expression of ERα and ERβ in human testis has been strongly debated and a definite answer to whether only ERβ or both ERs are expressed is pivotal to understanding the estrogenic actions in human spermatogenesis. Several splice variants for ERα and ERβ have been described in testis, playing important roles in the regulation of their prototype receptors. In contrast, only one AR variant has been described so far in human testis, although the existence of more variants responsible for regulatory and non-classical actions is highly expected. The definition of the estrogen and androgen regulated transcriptome is of pivotal importance to understand the precise roles of sexual steroid hormones in testis. The main objectives of this thesis were to clarify the expression of ERα and ERβ in human testis, search for alternatively spliced AR variants, and to identify and characterize novel estrogen and androgen regulated genes with a potential importance in the control of spermatogenesis. The results presented herein demonstrate unequivocally that both ERα and ERβ are expressed in human testis, and clarify their cellular distribution. The existence of alternatively spliced testicular AR variants was confirmed with detection of four new AR forms in human testis, two of them conserved along the vertebrate evolutive line indicating a relevant functional importance. Conserning the sex steroid regulated transcriptome, two novel genes were identified, one regulated by estrogens and the other by androgens. Apoptosis inhibitor and modulator of DNA-damage response Aven was identified as a novel estrogen target gene in testis. Its expression was for the first time characterized in human and rat testis, as well as its cellular distribution to Sertoli and germ cells. Perhaps more importantly, it was shown that the expression levels of Aven in human testis are positively correlated with quality of spermatogenesis. Concerning androgen regulated gene, the expression of Regucalcin (RGN) in response to 5α-dihydrotestosterone was characterized, and RGN shown to be expressed by all cells in rat and human testis. Regucalcin is involved in the control of intracellular calcium concentration and regulation of cell proliferation and apoptosis, processes whose regulation is of pivotal importance in the control of spermatogenesis. Estrogens and androgens are well recognized as germ cell survival factors and are known to regulate control mechanisms for testicular apoptosis. Therefore, we believe that both Aven and RGN are involved in mechanisms of germ cell survival, which are controlled by androgens and estrogens. In conclusion, this thesis has contributed to increase the knowledge about estrogenic and androgenic action in testis. The “new actors cast” to the drama that is the hormonal control of spermatogenesis open new storylines in the research of mammalian spermatogenesis and perheaps male fertility.

Thesis (Ph.D.)--Kent State University, 2007.
Title from PDF t.p. (viewed Mar. 12, 2009). Advisor: Srinivasan Vijayaraghavan. Keywords: sperm, testes, spermatogenesis, protein phosphatase, knock out, spermatid. Includes bibliographical references (p. 115-129).

Thesis (Ph.D.)--East Carolina University, 2009.
Presented to the faculty of the Department of Anatomy and Cell Biology. Advisor: Ann O. Sperry. Title from PDF t.p. (viewed June 12, 2010). Includes bibliographical references.

Soma-germline interactions play conserved essential roles in regulating cell proliferation, differentiation, patterning, and homeostasis in the gonad. In the Drosophila testis, the JAK-STAT, Hedgehog, BMP and EGF pathways are used to mediate soma-germline communication via paracrine signalling. In this thesis, I aim to shed light on the role of juxtacrine signalling, mediated by gap junction proteins, during early spermatogenesis in the fly testis. My analysis demonstrates that gap junctions also mediate direct, bi-directional signalling between the soma and germline. When gap junctions between the soma and germline are disrupted, germline differentiation is blocked and germline stem cells are not maintained. In the soma, gap junctions are required to regulate proliferation and differentiation. Gap junctions are present between germline and somatic stem cells, as well as between differentiating cells by ultrastructural analysis. Localization and RNAi-mediated knockdown studies reveal that gap junctions in the fly testis are heterotypic channels containing Zpg (Inx4) and Inx2 in the germline and in the soma, respectively. Preliminary structure-function analysis of Zpg reveals that the C-terminus of the protein is essential for its function. Furthermore, gap junctions in the testis may be important for mediating calcium signalling. Overall, my results show that bi-directional gap junction-mediated signalling is essential to coordinate the soma and germline to ensure proper spermatogenesis in Drosophila. Moreover, I show that stem cell maintenance and differentiation in the testis are directed by gap junction-derived cues.
Medicine, Faculty of
Graduate

The conjugation of ubiquitin to proteins is catalyzed sequentially by a cascade of members of three classes of enzymes – ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin protein ligase (E3). Polyubiquitinated protein substrates are selectively targeted for degradation by the proteasome. Removal of ubiquitin from ubiquitinated substrates is catalyzed by deubiquitinating enzymes (DUB). In this thesis, I have explored functions of three specific enzymes of the ubiquitin system, the E2 UBC4-testis, the HECT E3 EDD/Rat100 and the deubiquitinating enzyme USP19, in mammalian spermatogenesis or muscle wasting. UBC4-testis is a rodent testis specific E2 enzyme that is induced in round spermtids. Mice lacking the UBC4-testis gene had a delay in postnatal development during the first wave of spermatogenesis but ultimately had in adulthood normal fertility and testis weights, spermatid number, protein content, rate of ubiquitination and quantity and quality of sperm. When subjected to the heat stress of experimental cryptorchidism, the profile of germ cell degeneration was not significantly different from that of wild type mice. Our data suggest that UBC4-testis has a specific function in promoting the evolution of the first wave of spermatogenesis; however, some coexisting isoforms of UBC4 may serve redundant complementary functions in later stages of spermatogensis. EDD/Rat100 is a UBC4 dependent E3 that is highly expressed in rat testis. The poly(A)-binding protein (PABP) is a translation initiation factor that is negative regulated by the PABP-interacting protein 2 (Paip2). Both PABP and EDD/Rat100 share a PABC domain, through which they can interact with Paip2. EDD/Rat100 can ubiquitinate Paip2 in vitro. Under normal in vivo conditions, the abundant PABP may sequester Paip2 from ubiquitination by EDD/Rat100. In PABP-depleted cells, Paip2 is free to interact with EDD/Rat100, which leads to Paip2 ubiquitination and degradation by the proteasome. Degradation of Paip2 may then restore the activity of PABP and therefore maintain its homeostasis. Thus, the turnover of Paip2 in the cell is mediated by EDD/Rat100 but is regulated by PABP. In addition, six proteins that were copurified from immunoprecipitation of EDD/Rat100 in rat testis have been elaborately studied, but none of them was identified as a bona fide substrate of EDD/Rat100. Finally, I studied USP19, a 150 kDa DUB that is induced in atrophying skeletal muscle. A modest increase of expression of USP19 was observed in early differentiation of L6 cells. TNF-α can increase expression of USP19 in L6 myotubes. SiRNA mediated silencing of USP19 expression can increase expression of MHC in L6 myotubes in a myogenin dependent manner. And silencing of USP19 can partially reverse the TNF-α or DEX stimulated catabolism of MHC. Thus, USP19 can regulate synthesis of myofibrillar proteins through modulating transcriptional factor in myotubes. These data demonstrate that the ubiquitin system not only mediates the increased protein breakdown but is also involved in the decreased protein synthesis in atrophying skeletal muscle.
L'attachement de l'ubiquitine à un substrat protéique est catalysé par une série de réactions en chaîne impliquant trois classes d'enzymes- l'enzyme d'activation de l'ubiquitine (E1), l'enzyme de conjugaison de l'ubiquitine (E2) et l'enzyme de ligation de l'ubiquitine (E3). Les substrats protéiques polyubiquitinés sont spécifiquement reconnus par le protéasome pour être dégradés. L'enlèvement de l'ubiquitine présent sur les substrats ubiquitinés est catalysé par les enzymes de déubiquitination (DUB). Dans cette thèse, j'ai étudié les fonctions de trois enzymes du système ubiquitine : l'enzyme E2 UBC4-testis, l'enzyme E3 EDD/Rat100 et l'enzyme de déubiquitination USP19. Leurs rôles dans la spermatogénèse et l'atrophie musculaire ont été étudiés chez les mammifères.UBC4-testis est une enzyme spécifiquement exprimée dans les testicules de rongeurs et est induite dans les spermatides ronds. Des souris ayant le gène UBC4-testis inactivé démontrent un délai dans le développement postnatal durant la première vague de spermatogénèse. Par contre, arrivées à l'âge adulte, ces mêmes souris démontrent une fertilité et un poids testiculaire normal. Aussi, nous avons observé des données normales pour le nombre de spermatides, le contenu protéique, le taux d'ubiquitination ainsi que pour la quantité et la qualité des spermatozoïdes. Lorsque les testicules de ces souris sont soumis à un stress de température en effectuant une cryptorchidie expérimentale, le profil de dégénérescence des cellules germinales n'était pas significativement différent de celui de souris normales ayant subit le même traitement. Nos résultats suggèrent donc que UBC4-testis exerce un rôle dans l'évolution de la première vague de la spermatogénèse mais il est possible que d'autres isoformes UBC4 puissent exercer des fonctions redondantes dans les étapes tardives de la spermatogénèse.EDD/Rat100 est une enzyme E3 qui est dépendante de l'enzyme E2 UBC4 et qui est fortement exprimée dans les testicules de rat. La protéine de liaison au Poly(A) (PABP) est un facteur d'initiation à la traduction qui est négativement régulé par une protéine interagissant avec PABP appelée Paip2. PABP et EDD/Rat100 ont en commun un domaine appelé PABC qui peut interagir avec Paip2. EDD/Rat100 est capable d'ubiquitiner Paip2 in vitro. Dans des conditions normales in vivo, PABP, qui est en abondance, pourrait séquestrer Paip2 pour être ubiquitiné par EDD/Rat100. Dans des cellules qui ont des niveaux de PABP réduits, Paip2 est libre d'interagir avec EDD/Rat100 et est donc ubiquitiné et dégradé par le protéasome. La dégradation de Paip2 peut finalement rétablir l'activité de PABP et par conséquent maintenir son homéostasie. Ainsi, le taux de renouvellement de Paip2 dans la cellule est géré par EDD/Rat100 mais est régulé par PABP. De plus, six protéines copurifiées par immunoprécipitation avec EDD/Rat100 dans des extraits de testicules de rat ont été minutieusement étudiées mais aucune d'entre elle n'a été identifiée comme étant un substrat bona fide de EDD/Rat100.Finalement, j'ai analysé USP19, une enzyme de déubiquitination qui est induite dans les muscles squelettiques dans des conditions d'atrophie musculaire. Une modeste augmentation de l'expression de USP19 a été observée dans des cellules L6 en début de différentiation. TNF- peut augmenter l'expression de USP19 dans les cellules L6. Lorsque les niveaux d'USP19 sont réduits par ARN interférent dans les myotubes L6, l'expression de MHC est augmentée de façon dépendante de la myogénine. Aussi, en diminuant les niveaux d'USP19, la dégradation de MHC stimulée par TNF- ou par DEX peut partiellement être renversée. Donc, USP19 peut réguler la synthèse de protéines myofibrillaires en modulant la transcription dans des cellules musculaires L6. Ces résultats démontrent que le système ubiquitine n'agit non seulement sur la dégradation protéique dans les muscles squelettiques atrophiés mais aussi en diminuant la synthèse protéique.

In the Drosophila testis, sperm are derived from germline stem cells (GSCs) which undergo a stereotyped pattern of divisions and differentiation. The somatic cells at the tip of the testis form the hub, which is the niche for both the somatic cyst stem cells (CySCs) and GSCs. The hub expresses Upd, a ligand for the JAK/STAT pathway that has roles in the maintenance of CySCs and GSCs. Male mutants of upd3, another ligand of the JAK/STAT pathway, become sterile much earlier than the wild-type, leading to the hypothesis that similar to upd, upd3 also promotes the self-renewal of stem cells in testis. It was found here that upd3 is also expressed in the hub, and that mutants of upd3 have fewer CySCs and GSCs. Using a GFP reporter of the JAK/STAT pathway, it was found that the JAK/STAT pathway is not only activated in the stem cells, consistent with its known function in the maintenance of stem cells, but is also activated in the elongated cyst cells that encapsulate late stage differentiating spermatids. The reduction of JAK/STAT activity in the somatic cyst cells led to impaired spermatid individualization, a late stage of spermatogenesis during which the syncytial spermatids are separated. The impairment of individualization was shown by the loss of three characteristic structures: individualization complexes (ICs), cystic bulges (CBs), and waste bags (WBs). The failure of IC formation implies STAT activity is required for the initiation of individualization, and the loss of CBs and WBs suggests STAT activity is required for the progression of individualization. Activation of caspases in elongated spermatids is known to be required for individualization. The reduction of JAK/STAT activity in cyst cells almost completely eliminated the activation of two effector caspases: drICE and DCP-1. It was concluded that JAK/STAT activity in somatic cyst cells promotes individualization by stimulating caspase activity in spermatids. The JAK/STAT pathway is not only required for the maintenance of stem cells at the tip, but also required for individualization away from the tip during late differentiation, thus is reutilized in Drosophila spermatogenesis.

Thesis: Ph. D., Massachusetts Institute of Technology, Computational and Systems Biology Program, 2016.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Mammalian spermatogenesis includes two types of cell divisions. First, germ cells undergo transit-amplifying mitotic divisions, which enable prodigious output of mature spermatozoa. Second, they undergo reductive meiotic divisions to produce haploid gametes. In this thesis, I examine gene expression and regulation during the mitotic and meiotic phases of spermatogenesis. Chapter 2 describes how RA-STRA8 signaling regulates two key transitions: spermatogonial differentiation, which begins the transit-amplifying mitotic divisions, and meiotic initiation, which ends them. First, in mice lacking the RA (retinoic acid) target gene Stra8, undifferentiated spermatogonia accumulated; thus, Stra8 promotes spermatogonial differentiation as well as meiotic initiation. Second, injection of RA into wild-type males induced precocious spermatogonial differentiation and meiotic initiation; thus, RA acts instructively on germ cells at both transitions. Finally, competencies of germ cells to undergo spermatogonial differentiation or meiotic initiation in response to RA were found to be distinct and periodic. Chapter 3 describes a novel method for isolating precise populations of mitotic and meiotic germ cells from the mouse testis. We first synchronize germ cell development in vivo, and perform histological staging to verify synchronization. We then separate these germ cells from contaminating somatic and stem cells by FACS, to achieve ~90% purity of each distinct germ cell type, from the stem cell pool through mid/late meiotic prophase. Utilizing this "3S" method (synchronize, stage, and sort), we can robustly and efficiently separate germ cell types that were previously challenging or impossible to distinguish, with sufficient yield for transcriptomic and epigenetic studies. Chapter 4 presents a systematic comparison of the male and female gene expression programs of meiotic prophase. We performed transcriptional profiling of postnatal testes synchronized in precise stages of meiotic prophase, and compared to the same stages in the fetal ovary. We identified 260 genes up-regulated during both male and female prophase; this shared gene set represents a core meiotic program, composed of known and potential novel meiotic players. We also identified over two thousand genes that are up-regulated during meiotic prophase specifically in the male. These comprise both a male-specific meiotic program, and a preparatory program for cellular differentiation of spermatozoa.
by Katherine A. Romer.
Ph. D.

Spermatogenesis involves a complex series of cell-cell interactions which are probably mediated by secreted proteins. The primary objective of the studies described in this thesis was therefore to identify specific proteins which change in relative abundance in the early stages of toxicant-induced damage to spermatogenesis and which might have potential use as markers of such damage. Identification of such proteins might pin-point the possible biochemical causes of the toxic effects on the testis, and also give insight into normal control mechanisms in spermatogenesis. The chemicals used in these studies were meta-dinitrobenzene (m-DNB), nitrobenzene (NB) and methoxyacetic acid (MAA). The effect of severe disruption of spermatogenesis, induced by short-term local testicular heating, was assessed in order to establish whether protein changes comparable to those seen following toxicant exposure could be identified. Within 4 hours of treatment, stage-specific changes in the incorporation of ³⁵S-methionine into both secreted proteins and intracellular proteins were deserved. Analysis by 2-D SDS PAGE identified 8 proteins which were affected adversely following heat treatment, all of which had been affected by toxicant exposure. In conclusion, the studies presented in this thesis have identified proteins which have potential use as markers of early toxicant-induced damage to spermatogenesis. Studies to date in the rat have identified proteins in peripheral blood which derive from the Sertoli cells and germ cells and the expectation is that most if not all ST-secreted proteins will appear in blood. Therefore the logical next step will be to determine whether any of the proteins identified in the present studies are detectable in peripheral blood and whether the amounts change following toxicant exposure.

Les microtubules sont des éléments du cytosquelette, composées d'hétérodimères de tubuline de type α et β. Ils jouent un rôle important dans plusieurs processus cellulaires, dont le transport cytoplasmique, la mobilité et la division cellulaire. Cependant, les mécanismes par lesquels les microtubules sont adaptés à ces rôles très différents restent largement méconnus.Les modifications post-traductionnelles de la tubuline pourraient contribuer à la diversité de fonction des microtubules. Parmi celles-ci, la polyglutamylation pourrait jouer un rôle important en changeant d'une manière importante les propriétés des microtubules, et les adaptant ainsi à leurs différents rôles. La polyglutamylation correspond à l'addition de longues chaînes latérales d'acides glutamiques aux extrémités C-terminales des tubulines α et β. Ces régions sont des sites connues d'interactions de la tubuline avec ses protéines associées (MAP) et les moteurs moléculaires. Au cours de mes travaux, j'ai étudié le rôle de polyglutamylation de la tubuline dans le développement des spermatozoïdes. En utilisant la souris et la drosophile comme organismes modèles, j'ai montré que le changement de niveau de polyglutamylation de la tubuline dans les spermatozoïdes, soit par une régulation positive soit par régulation négative, provoque des anomalies structurales des spermatozoïdes et entraîne une stérilité des mâles. J'ai également étudié la rôle de la katanine, un enzyme coupant les microtubules, effecteur potentiel de la polyglutamylation. J'ai montré qu'en absence de la katanine, la production des cellules germinales est gravement compromise chez les mâles, provoquant également une stérilité. Pris ensemble, ces résultats démontrent que la régulation de polyglutamylation de la tubuline est indispensable pour le développement correct des spermatozoïdes et que son effet pourrait être médié par la katanine, enzyme dont l'activité pourrait dépendre de la polyglutamylation de la tubuline.Durant mes travaux, j'ai également développé une nouvelle technique de production des microtubules différentiellement glutamylés. En utilisant comme matière primaire de la tubuline de cerveau porcin, hautement polyglutamylée, j'ai réalisé une déglutamylation produisant ainsi une tubuline déglutamylée. En utilisant les deux types de microtubules (avec ou sans polyglutamylation), il est possible de tester si les interactions entre les microtubules et les MAP dépendent de la polyglutamylation de la tubuline
Microtubules are essential cytoskeletal elements composed of α- and β-tubulin heterodimers. They are involved in a number of cellular processes, including intracellular transport, cell motility and cell division. However, how microtubules can adapt to all these different functions remains largely unknown. One of the mechanism, which could contribute to microtubule diversity are posttranslational modifications of tubulin. Among tubulin modifications polyglutamylation has a high potential for changing microtubule properties and thus adapting them to different roles. It consists of addition of long glutamate side chains to multiple glutamate residues located in the C-terminal tail of both α- and β-tubulin, which are known as interaction sites for many microtubule associated proteins (MAPs) and molecular motors. In my studies I focused on the role of polyglutamylation in sperm development. Using mice and Drosophila as model systems, I showed that changing the levels of this modification, either by up- or downregulation, results in the assembly of structurally abnormal sperm and causes male sterility. In addition, I also addressed the role of one of the potential effectors of polyglutamylation, a microtubule-severing enzyme called katanin. I demonstrated that in the absence of katanin the production of male germ cells is severely compromised leading to male sterility. Taken together my data suggest that proper balance of tubulin polyglutamylation is essential for sperm development and that its effects may be mediated by katanin whose activity has been proposed to be dependent on tubulin polyglutamylation. Moreover, during my PhD project I developed a method for production of differentially glutamylated microtubules. Using porcine brain tubulin, which is known to be highly glutamylated, as a starting material I perform deglutamylation to obtain the non-glutamylated version of it. Obtaining these two types of tubulin allows now to directly testing whether the interactions between microtubules and the MAPs of interests are dependent on tubulin polyglutamyaltion

Approximately 131 million Chlamydia trachomatis infections occur annually. The rates of infection are similar between males and females, but female infections have been more widely characterised. C. trachomatis causes infertility in men and women but little is known about the male disease. This study investigated the ability of chronic infections to cause male infertility. In mice, Chlamydia colonises specialised testicular cells, resulting in partial destruction of the testicular structure and production of anti-sperm-antibodies. This correlates with decline in sperm health and the egg-binding ability needed for fertilisation. When infected male mice were bred with healthy female mice, their offspring had reduced viability and health, including the offspring's sperm. This suggests that Chlamydia may have a multigenerational effect on fertility. These results were supported by the detection of replication-capable Chlamydia in human testicular biopsies from men attending IVF clinics.