The impacts of the paternal genome and proteins transferred to the oocyte through spermatozoa cannot be neglected during mammalian embryonic development. Studies over the past 40 years suggest that sperm chromatin alterations (such as DNA fragmentation induced by either chromatin condensation errors, apoptosis and/or oxidative stress) might be negatively associated with fertilization and early embryonic development [1, 2], [3] [4].However, precise molecular mechanisms by which sperm chromatin integrity and sperm proteins impact early embryonic development still remain unclear. Therefore, the objectives of this study were 1) determine DNA fragmentation induced by apoptosis its relationship with male fertility in spermatozoa from bulls with varying fertility, and 2) identify expression dynamics of Protamine 1 and examine chromatin structure in spermatozoa from bulls with varying fertility. To accomplish our goals we determined 1) the DNA damage, phosphatidylserine (PS) translocation, and expression of pro- and anti-apoptotic proteins (BAX and BCL-2) as well as 2) the expression and localization of Protamine 1 (PRM1) with chromatin condensation and protamination in sperm from bulls with varying fertility. Our results demonstrated that the most relevant fertility markers might be the percentage of necrotic spermatozoa detected by flow cytometry and live spermatozoa determined via eosin-nigrosin staining and that there was no relationship between apoptosis and male fertility. While BCL-2 was not expressed, BAX was identified in bovine spermatozoa. However, the expression of BAX did not differ among groups. In addition, defective chromatin condensation and protamination errors were significantly increased in sperm from low fertility bulls, while the expression of PRM1 was significantly abundant in high fertility bulls. Bull fertility was negatively correlated with protamination errors and defective chromatin condensation, and it was positively correlated with the expression of PRM1. We concluded that defective sperm DNA condensation, not abortive apoptosis, might be the major reason of male infertility in bulls and that sperm chromatin stability differs among bulls with varying fertility. Improper chromatin packaging during spermatogenesis might be caused by the limited expression and/or mislocalization of PRM1. Thus, inadequate chromatin dynamics were associated with bull infertility, which might lead improper fertilization.

Male fertility, ability to fertilize and activate the egg and support early embryo development, is crucial for mammalian reproduction and development. Testis specific histone 2B (TH2B) of sperm, protamines (PRM1/2), and posttranslational modifications of histone 3 (H3K27me3 and H3k27ac) are involved in spermatogenesis and male fertility. However, molecular and cellular mechanisms by which TH2B regulates histone to protamine replacement is poorly defined. Immunocytochemistry, western blotting, flow cytometry, computer-assisted sperm analysis (CASA) and bioinformatic approaches were applied to analyze sperm from Holstein bulls with different in vivo fertility. Results from the immunocytochemistry experiments showed that while TH2B and H3K27me3 were localized predominantly at the equatorial and post acrosomal (localized as a crown around the sperm head) parts, respectively. The H3K27ac was also detectable in the bovine sperm head. Signal intensities of TH2B (mean ± SEM) were higher in sperm from the low fertility bulls (220.56 ± 9.20) as compared to those from the high fertility bulls (198.39 ± 10.0). Signal intensities of H3K27me3 (16.25 ± 1.69) were significantly different than those of H3K27ac (4.74 ± 0.88) in bull spermatozoa. Using the bioinformatic tools, including Clustal Omega, Cytoscape, Emboss Dotmatcher, InterProScan, and STRING, we demonstrated that TH2B has the conserved histone H2B domain which has a strong association with proteins involved in chromosome organization and histone ubiquitination. Intensities of PRM1 and PRM2 were significantly associated with one another (p < 0.0001), but neither were significantly associated with fertility. Results from CASA revealed significant differences between high and low fertility bulls regarding average sperm pathway velocity, amplitude of lateral head displacement and straightness (p < 0.05). The interacting proteins of H3 are involved in subcellular processes such as regulation of H3K27 methylation, nucleosome assembly, regulation of DNA replication, and chromatin assembly. These results are significant because they help advance fundamental knowledge in sperm physiology involving epigenetic and genetic determinants. The new knowledge can be used to enhance reproductive biotechnology to improve fertility. In addition, the data generated using the unique bull model can be applied to study mammalian reproduction and development due to similarities in genetics and physiology between bovine and other mammals.

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.

Introdução: Em torno de 15% dos casais apresentam o diagnóstico de infertilidade, sendo que 50% se devem a fatores masculinos. Diversos testes de função espermática são propostos para a avaliação da fertilidade, mas o espermograma é o principal exame para o diagnóstico de infertilidade masculina. O valor prognóstico das características seminais como concentração, morfologia e motilidade como marcadores de infertilidade masculina é confundido. A avaliação desses e a classificação quanto à normalidade dos mesmos permanece sendo tema de discussão frequente. Objetivos: Comparar as diferentes classificações de parâmetros seminais (OMS 1999, OMS 2010 e TMSC) e avaliar as características de pacientes em terapia de reprodução humana assistida que se relacionam com esses parâmetros. Métodos: Foi realizado estudo longitudinal, retrospectivo, baseado em revisão de banco de dados para avaliar as características e parâmetros seminais e comparar as mesmas conforme as classificações da OMS de 1999, OMS de 2010 e TMSC de 477 homens submetidos a investigação de infertilidade ou tratamentos de reprodução assistida na clínica Generar – Reprodução Humana, entre 2011 e 2015. Resultados: 401 pacientes tiveram alguma alteração pela OMS 1999, 223 pela OMS 2010 e 200 pela TMSC. O critério que mais alterou a condição de uma amostra seminal de anormal em 1999 para normal em 2010 foi a morfologia. Conclusão: Os parâmetros se tornaram menos rígidos de 1999 para 2010 alterando significativamente a proporção de indivíduos que deixaram de ser classificados como inférteis. A classificação baseada no TMSC pode ser útil na indicação de alguns tratamentos, mas não pode definir um indivíduo como fértil ou infértil em função de não levar em consideração a morfologia espermática. / Background: Approximately 15% of couples presenting the diagnosis of infertility, and 50% of cases are due to male factors. Several sperm function testing is proposed for the evaluation of male fertility, but sperm is the first test for the diagnosis of male infertility causes. The prognostic value of the seminal characteristics as concentration, morphology and motility as male infertility markers is often confused. Evaluation of semen parameters and classification for normality remains frequent topic of discussion. Objectives: Compare the different classifications of seminal parameters (OMS 1999, WHO 2010 and TMSC) and evaluate the characteristics of patients treated in assisted reproduction therapy relating to these parameters. Methods: Retrospective study based on chart review evaluated 477 semen samples from men undergoing investigation or infertility treatments in assisted reproduction between 2011 and 2015. Results: 401 patients were considered abnormal by the WHO 1999, 223 by WHO 2010 and 200 for TMSC. The criteria that most changed the classification was sperm morphology. Conclusion: The parameters have become less rigid 1999 to 2010 significantly changing the proportion of individuals who are no longer classified as infertile. The classification based on TMSC can’t define an individual as fertile or infertile regardless due to not take into account the sperm morphology, but may be helpful when it comes to the indication of the intrauterine insemination.

Introdução: Em torno de 15% dos casais apresentam o diagnóstico de infertilidade, sendo que 50% se devem a fatores masculinos. Diversos testes de função espermática são propostos para a avaliação da fertilidade, mas o espermograma é o principal exame para o diagnóstico de infertilidade masculina. O valor prognóstico das características seminais como concentração, morfologia e motilidade como marcadores de infertilidade masculina é confundido. A avaliação desses e a classificação quanto à normalidade dos mesmos permanece sendo tema de discussão frequente. Objetivos: Comparar as diferentes classificações de parâmetros seminais (OMS 1999, OMS 2010 e TMSC) e avaliar as características de pacientes em terapia de reprodução humana assistida que se relacionam com esses parâmetros. Métodos: Foi realizado estudo longitudinal, retrospectivo, baseado em revisão de banco de dados para avaliar as características e parâmetros seminais e comparar as mesmas conforme as classificações da OMS de 1999, OMS de 2010 e TMSC de 477 homens submetidos a investigação de infertilidade ou tratamentos de reprodução assistida na clínica Generar – Reprodução Humana, entre 2011 e 2015. Resultados: 401 pacientes tiveram alguma alteração pela OMS 1999, 223 pela OMS 2010 e 200 pela TMSC. O critério que mais alterou a condição de uma amostra seminal de anormal em 1999 para normal em 2010 foi a morfologia. Conclusão: Os parâmetros se tornaram menos rígidos de 1999 para 2010 alterando significativamente a proporção de indivíduos que deixaram de ser classificados como inférteis. A classificação baseada no TMSC pode ser útil na indicação de alguns tratamentos, mas não pode definir um indivíduo como fértil ou infértil em função de não levar em consideração a morfologia espermática. / Background: Approximately 15% of couples presenting the diagnosis of infertility, and 50% of cases are due to male factors. Several sperm function testing is proposed for the evaluation of male fertility, but sperm is the first test for the diagnosis of male infertility causes. The prognostic value of the seminal characteristics as concentration, morphology and motility as male infertility markers is often confused. Evaluation of semen parameters and classification for normality remains frequent topic of discussion. Objectives: Compare the different classifications of seminal parameters (OMS 1999, WHO 2010 and TMSC) and evaluate the characteristics of patients treated in assisted reproduction therapy relating to these parameters. Methods: Retrospective study based on chart review evaluated 477 semen samples from men undergoing investigation or infertility treatments in assisted reproduction between 2011 and 2015. Results: 401 patients were considered abnormal by the WHO 1999, 223 by WHO 2010 and 200 for TMSC. The criteria that most changed the classification was sperm morphology. Conclusion: The parameters have become less rigid 1999 to 2010 significantly changing the proportion of individuals who are no longer classified as infertile. The classification based on TMSC can’t define an individual as fertile or infertile regardless due to not take into account the sperm morphology, but may be helpful when it comes to the indication of the intrauterine insemination.

Près de 15% des couples sont confrontés à des problèmes d'infertilité. Dans près de la moitié des cas, une composante masculine est retrouvée, avec souvent une anomalie des paramètres du spermogramme montrant une diminution de la qualité du sperme. L'étiologie de la grande majorité des infertilités masculines reste inconnue et une origine génétique est probablement responsable d'une proportion importante des troubles de la spermatogénèse. Ce travail comporte deux parties: dans la 1ère partie, l'analyse d'une large cohorte de patients (n=87), nous a permis d'identifier deux nouvelles mutations du gène AURKC. La mutation [c.36-2A>G] a été identifiée uniquement à l'état hétérozygote chez deux frères et le 2ème variant identifié [p.Y248*]: est une mutation récurrente retrouvée chez 11 patients non apparenté d'origine maghrébine et européenne. La 2ème partie de notre étude a été réalisée sur 20 patients infertiles présentant un phénotype homogène d'anomalies flagellaire de type flagelles courts, absents et de calibre irrégulier associé a une asthénozoospermie. Nous avons appliqué la stratégie d'homozygotie par filiation qui a permis de mettre en évidence deux régions d'homozygoties communes: la 1ère région, située sur le chromosome 3, est commune à 9/20 patients et la 2ème sur le chromosome 20 commune à 13/20 patients. Trois gènes candidats présents dans ces régions ont été sélectionnés : les gènes KIF9, SPAG4 et DNAH1. Le séquençage du gène DNAH1 a permis de mettre en évidence des mutations de type faux-sens [c.3877G>A], run-on [c.12796 T>C] et d'épissage [c.5094+1G>A] [c.11958-1G>A]. L'absence de la protéine DNAH1 a pu être mise en évidence par immunomarquage sur les spermatozoïdes d'un patients porteur de la mutation [c.11958-1G>A] et confirme la dégradation du transcrit muté par NMD également observé. Les analyses par microscopie électronique sur les spermatozoïdes d'un patient de la cohorte ont permis de mettre en évidence des anomalies de la structure de l'axonème. Cette étude précise le diagnostic d'infertilité masculine et élargit les connaissances sur les gènes impliquées dans la spermatogenèse. / About 15% of couples are confronted with infertility problems. In half of the cases, a male factor component is found, often with abnormal semen parameters. The etiology of the large majority of male infertility remains unknown and genetic origin is probably responsible of a significant proportion of spermatogenesis disorders. This work comprises two parts: in the first part, the analysis of a large cohort of patients (n = 87), allowed us to identify two new mutations in AURKC gene. A splice site mutation [c.36-2A> G] was identified in only two brothers and the second variant identified [p.Y248*] is a recurrent mutation found in 11 unrelated patients. The second part of our study was carried out on 20 infertile patients with flagellar abnormalities associated with asthenozoospermia. We have applied the strategy of homozygosity by descent who has bring out two regions of homozygosity: the first region, located on chromosome 3, is common for 9/20 patients and the second one, located on chromosome 20, is common for 13/20 patients. Three candidate genes present in these regions were selected: KIF9, SPAG4 and DNAH1. Sequencing of DNAH1 gene has bring out three type of mutations: missense mutation [c.3877G> A], run-on mutation [c.12796 T> C] and splice site mutation [c.5094 +1 G> A] [c.11958-1G> A]. The absence of dnah1 protein has been shown by immunostaining of spermatozoa of a patient carrier the mutation [c.11958-1G> A] and confirms the degradation of the mutated transcript by NMD. An electron microscopic analysis of spermatozoa of one patient of the cohort reveals axoneme abnormalities. This study clarifies the diagnosis of male infertility and broadens the knowledge of the genes involved in spermatogenesis.

Introdução: Em torno de 15% dos casais apresentam o diagnóstico de infertilidade, sendo que 50% se devem a fatores masculinos. Diversos testes de função espermática são propostos para a avaliação da fertilidade, mas o espermograma é o principal exame para o diagnóstico de infertilidade masculina. O valor prognóstico das características seminais como concentração, morfologia e motilidade como marcadores de infertilidade masculina é confundido. A avaliação desses e a classificação quanto à normalidade dos mesmos permanece sendo tema de discussão frequente. Objetivos: Comparar as diferentes classificações de parâmetros seminais (OMS 1999, OMS 2010 e TMSC) e avaliar as características de pacientes em terapia de reprodução humana assistida que se relacionam com esses parâmetros. Métodos: Foi realizado estudo longitudinal, retrospectivo, baseado em revisão de banco de dados para avaliar as características e parâmetros seminais e comparar as mesmas conforme as classificações da OMS de 1999, OMS de 2010 e TMSC de 477 homens submetidos a investigação de infertilidade ou tratamentos de reprodução assistida na clínica Generar – Reprodução Humana, entre 2011 e 2015. Resultados: 401 pacientes tiveram alguma alteração pela OMS 1999, 223 pela OMS 2010 e 200 pela TMSC. O critério que mais alterou a condição de uma amostra seminal de anormal em 1999 para normal em 2010 foi a morfologia. Conclusão: Os parâmetros se tornaram menos rígidos de 1999 para 2010 alterando significativamente a proporção de indivíduos que deixaram de ser classificados como inférteis. A classificação baseada no TMSC pode ser útil na indicação de alguns tratamentos, mas não pode definir um indivíduo como fértil ou infértil em função de não levar em consideração a morfologia espermática. / Background: Approximately 15% of couples presenting the diagnosis of infertility, and 50% of cases are due to male factors. Several sperm function testing is proposed for the evaluation of male fertility, but sperm is the first test for the diagnosis of male infertility causes. The prognostic value of the seminal characteristics as concentration, morphology and motility as male infertility markers is often confused. Evaluation of semen parameters and classification for normality remains frequent topic of discussion. Objectives: Compare the different classifications of seminal parameters (OMS 1999, WHO 2010 and TMSC) and evaluate the characteristics of patients treated in assisted reproduction therapy relating to these parameters. Methods: Retrospective study based on chart review evaluated 477 semen samples from men undergoing investigation or infertility treatments in assisted reproduction between 2011 and 2015. Results: 401 patients were considered abnormal by the WHO 1999, 223 by WHO 2010 and 200 for TMSC. The criteria that most changed the classification was sperm morphology. Conclusion: The parameters have become less rigid 1999 to 2010 significantly changing the proportion of individuals who are no longer classified as infertile. The classification based on TMSC can’t define an individual as fertile or infertile regardless due to not take into account the sperm morphology, but may be helpful when it comes to the indication of the intrauterine insemination.

Over three decades ago, W.H.O. declared infertility as a public health issue; due to its impact on millions of people worldwide. While cases of infertility could be multifactorial (affecting both male and female), 50% of cases are due to male factor infertility and this is mostly characterised by reduced sperm motility (asthenozoospermia). Assisted Reproduction Technology (ART) is the only treatment option available for this condition. Over 20 years ago, non-selective phosphodiesterase inhibitors (PDEi), such as pentoxifylline, were shown to enhance motility of human spermatozoa; however, contradictory results and stimulation of premature acrosome reaction has precluded their clinical use. Advancement in our knowledge have now made it clear that human sperm express several different PDEs and these are compartmentalised at different regions of the cells. By using type-specific phosphodiesterase inhibitors, differential modulation of sperm motility can be achieved without affecting other sperm function such as acrosome reaction. Additionally, by enhancing sperm function through PDE inhibition, there is a possibility of increasing IVF rates. The objective of this thesis is to: (1) examine the effect of phosphodiesterase inhibitors on spermatozoa in order to identify compounds that have clinically relevant enhancement of human sperm motility; (2) identify the signalling pathway(s) involved in the motility enhancing effects of identified compounds by targeting the modulator and mediator of cyclic nucleotides; (3) develop an animal IVF model to assess effects of Ibudilast on fertilization; and (4) optimise high performance liquid chromatography (HPLC) techniques for routine detection of cyclic nucleotides in sperm cells. A two phase drug screening approach was used to systematically and comprehensively screen series of compounds in order to identify those that have clinically relevant enhancement of human sperm motility. In phase 1, 6 compounds (out of 43 compounds) were found to have strong effects on poor motility samples, with magnitude of response ≥60% increase in percentage total motility. Additionally, these compounds significantly enhanced sperm penetration into cervical mucus substitute (p≤0.05), and they did not affect sperm acrosomal integrity nor cause externalisation of phosphatidylserine (p=0.6 respectively). 63% of IVF samples treated with compounds #26, #37 and #38 had significant increase in percentage total motility. For ICSI samples, compounds #26, #37 and #38 were the most effective. In respect to total motility, 88%, 81% and 79% of samples treated with these compounds showed significant increases in total motility, and 94%, 93% and 81% of samples showed significant increases in percentage of progressive cells, respectively. Analysis of the signalling pathways, using PKA, sGC and PKG inhibitors, showed that chosen PDE inhibitors were working predominantly through PKA signalling pathways. Additionally, this study revealed that this pathway is needed for the maintenance of basal progressive motility and hyperactivation in human sperm. Animal IVF studies showed that addition of Ibudilast (compound #26) during sperm-oocyte incubation leads to higher IVF rates. Lastly, this study used an HPLC system to detect cAMP in boar sperm. This was done to explore if HPLC system can be used for high throughput detection of cyclic nucleotides in mammalian sperm.

At least 30 million men are infertile around the world, identifying male factor infertility as a global health issue. In the past 70 years, evidence of a significant general decline in sperm quality has been reported, prompting concerns about the implications for reproductive health. Over the same period, there have been substantial changes in human lifestyles. New technologies, such as mobile phones and wi-fi, have been proposed to have a negative impact on a range of health outcomes, from an increased risk of cancer to a decrease in fertility. However, these links remain controversial. Over the last 30 years, the introduction of assisted reproductive technologies (ART) has offered infertile patients, particularly men with severe male factor infertility, a successful treatment option. However, miscarriage rates associated with fertility treatment can be as high as 30% and how this risk had changed over time was unclear. In addition, there are natural fluctuations in human health, including seasonal changes to birth rates. However, the clinical implications of these fluctuations need to be established. In this thesis, using an integrated approach that combined epidemiological research with laboratory investigations, I show that sperm quality is negatively affected by exposure to RF-EMR from mobile phones and wi-fi. I also identified a seasonal summer increase in sperm motility and morphology that followed patterns of seasonality in birth rates and in the success of assisted conception cycles. I showed that although the number of successful conceptions from ART has increased over time, there has been an equal increase in miscarriage rates. Male reproductive health continues to be under-researched when compared with the female, this inequality needs to be addressed in order to understand the causes of the decline in male fertility and the relationship this has with subsequent reproductive success.

Sperm function has been studied in multiple research fields as it is essential to male fertility. In previous studies a variety of sperm traits have been examined as an assessment of sperm function. Among those traits, sperm viability, sperm motility and sperm metabolism are often commonly examined. However, sperm function can be influenced by both environmental and genetic factors. Specifically, nuclear genome has been demonstrated to play a role in sperm function, especially in sperm competitive capacity. There are increasing evidence for effects of mitochondrial genome on sperm function. Mitochondrial genetic variance has been suggested to affect sperm length and sperm viability in seed beetle and sperm metabolism in rodent. Given the coordinated collaborations between nuclear and mitochondrial genomes in OXPHOS, replication and transcription of mitochondrial genome as well as intergenomic signalling, potential mitonuclear effects on sperm function are expected even though empirical evidence so far remains less. A recent review summarised all the previous work on environmental effects on sperm and found that various factors affects sperm function but largely neglected in ecology and evolution. In the study, we used D. melanogaster as a model to disentangle both genetic and environmental components of sperm function at sperm cell, ejaculate and offspring levels. We found environmental effects on sperm function in D. melanogaster. Specifically, sperm incubation buffers affect sperm viability in chapter 2 and dietary PUFAs influence sperm volume and metabolism in chapter 4. Nuclear effects were found on sperm viability, sperm quality and male fertility in chapter 3. Mitochondrial genome was found to have an effect on sperm function, i.e. sperm viability and sperm quality differed among mitochondrial haplotypes examined. In addition, sperm function was further modified by the interaction of nuclear and mitochondrial genomes in ageing male. Sperm quality and fertilization success were suggested to be dependent on age-related mitonuclear interaction in chapter 3. Moreover, we examined the mitonuclear coadaptation hypothesis in the function of D. melanogaster sperm. No evidence for mitonuclear coadaptation hypothesis was found for sperm function in D. melanogaster as there were no difference between coadapted and non-coadapted lines in sperm traits examined. Lastly, we found that sperm viability, sperm quality and sperm metabolic rate cannot predict male fertility in D. melanogaster as correlation analysis revealed no relationship between them. Our experiment explored and disentangled the genetic and environmental components of sperm function at multiple levels in D.melanogaster systematically. Our results suggested that both mitochondrial and nuclear genome as well as the interaction between them play a role in sperm function in D. melanogaster. In addition to genetic components, our findings revealed environmental components of Drosophila sperm and suggested that it was phenotypic plastic.