Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Tereza Klapková Supervisor: Assoc. Prof. PharmDr. František Trejtnar, CSc. Title of diploma thesis: Recent knowledge on drug effect on male fertility Among the various types of side effects presented in clinically used drugs, negative effects on male reproductive functions can be find. This issue seems to be important and current especially due to the general trend of the decrease in fertility in men and the increasing drug use in younger age groups. The aim of this diploma thesis was to create an overview summarizing current expert knowledge on the effect of drugs on male fertility. For this purpose, we selected relevant publications in the PubMed database, perform their analysis and create the text ofthe thesis. The review focuses mainly on groups of drugs that are often clinically used and discussed in relation to male fertility, such as drugs acting on the cardiovascular system, antimicrobial drugs, drugs used in pain therapy, antidepressants, antiepileptics, antipsychotics, immunosuppressants and some other drugs. In addition to standard drugs, the review also includes several important natural substances, which are used as adjunctive therapy of various diseases or are important from a...

Numerical analysis of the lengths and positions of the two basal bodies (BBs), lamellar strip (LS) and anterior mitochondrion (AM) relative to each other in mid‐ and late‐stage spermatids of mosses and liverworts reveals the existence of several well denned, but previously unrecognized, features which clearly distinguish the blepharoplasts of the two groups. The ten possible quotients were calculated from measurements of anterior BB lengths, posterior BB lengths, LS lengths, distances between the anterior tips of the BBs and distances between the transition regions of the BBs in mid‐stage spermatids of 9 mosses and 16 hepatics. These critical data may be quickly compiled from a small number of electron micrographs. A Mann‐Whitney rank order t test showed highly significant differences in 6 of the 10 quotients between the moss and liverwort taxa. The primary data for late‐stage spermatids (4 mosses, 6 liverworts) also included the length of the AM. A Wilcoxon signed rank procedure revealed that the relationship between the AM and other blepharoplast components changed significantly between mid‐ and late‐stage spermatids in mosses but not in liverworts. The clear‐cut numerical differences between the blepharoplast components in each group are related to different patterns of development namely (1) bidirectional assembly of the LS in young spermatids of liverworts versus unidirectional (anterior) elongation at the same stage in mosses (2) elongation of the posterior BB over the nucleus in mid‐stage spermatids of mosses and (3) maturational elongation of the AM in mosses. Since the differences between the blepharoplasts of mosses and liverworts become apparent only during the later stages in ontogeny and since the mode of development of basal body stagger, involving the same precisely defined patterns of proximal triplet microtubule extension, is unique to mosses and liverworts, we suggest that the two groups share a common ancestry. The blepharoplasts of all the taxa used in the calculations are illustrated in a simplified form and the ‘average’ blepharoplast for mid‐ and late‐stage spermatids of both mosses and liverworts is reconstructed from all the data presently available on the two groups. The same analysis of the blepharoplasts of hornworts, birlagellate lycopods, and charophytes highlights the differences between these groups and mosses and liverworts. Most striking is the side‐by‐side orientation of the basal bodies in hornworts and charophytes compared with the staggered arrangement in mosses, liverworts and the lycopods.

Ultrastructural observations reveal that the spermatozoids of the hornworts Notothylas and Phaeoceros contain two mitochondria and not one as described previously. Mitochondrial ontogeny and nuclear metamorphosis during spermiogenesis in these plants differ from all other archegoniates. The discovery that the posterior region of the coiled nucleus (when viewed from the anterior aspect) lies to the left of the anterior, in striking contrast to the dextral coiling of the nucleus of spermatozoids of other embryophytes, underlines the isolated nature of the hornworts among land plants. As the blepharoplast develops, the numerous ovoid mitochondria initially present in the nascent spermatid fuse to form a single elongated organelle which is positioned subjacent to the MLS and extends down between the nucleus and plastid. At the onset of nuclear metamorphosis, the solitary mitochondrion has separated into a larger anterior mitochondrion (AM) associated with the MLS and a much smaller posterior mitochondrion (PM) adjacent to the plastid. The PM retains its association with the plastid and both organelles migrate around the periphery of the cell as the spline MTs elongate. By contrast, in moss spermatids, where mitochondria undergo similar fusion and division, the AM is approximately the same size as the PM and the latter is never associated with the spline. As in other archegoniates, except mosses, spline elongation precedes nuclear metamorphosis in hornworts. Irregular strands of condensed chromatin compact basipetally to produce an elongated cylindrical nucleus which is narrower in its mid-region. During this process excess nucleoplasm moves rearward. It eventually overarches the inner surface of the plastid and entirely covers the PM.

During spermatogenesis, histones are largely replaced by transition proteins and protamines in normal stallions. Incomplete nucleoprotein exchange results in the abnormal retention of histones and transition proteins, which is an indicator of poor sperm quality. Equine nucleoprotein exchange has not previously been investigated in detail, so that equine sperm chromatin quality problems, which are often responsible for poor breeding performance of stallions, are not well understood. In order to characterize chromatin remodeling events in stallion spermatogenesis and to identify proteins indicative of sperm chromatin defects, such as excessive amounts of histones, we identified antibodies that recognize equine testis-specific proteins of interest. Immunoblotting of testis and sperm protein lysates and immunofluorescence staining of histological tissue sections were used to identify candidate marker proteins of incomplete sperm chromatin maturation. Results of the study, which represents the first comprehensive characterization of the nucleoprotein exchange during spermatogenesis in the stallion, challenge the paradigm that the main function of histone H4 lysine (hyper-) acetylation (concomitant H4K5 and H4K8 acetylation) is to facilitate nucleosome ejection during spermatid nuclear elongation to allow for transition protein and protamine insertion into the chromatin. That paradigm was based on observations in mice and rats where H4 acetylation in several lysine residues occurs just prior to or during nuclear elongation. In contrast, the equine data presented here show strong acetylation of H4 in K5, K8 and K12 positions immediately after meiosis in round spermatids, independent of nuclear transition protein 1 deposition. Furthermore, results of H4K16 acetylation analyses underline the importance of this mark, which is likely mediated by DNA damage signaling pathways, emphasizing the importance of DNA repair processes for the exchange of nucleoprotein exchange in spermiogenesis and therefore, in extension, for male fertility. In addition, a revised description of the equine spermatogenic cycle is proposed here that is better aligned with human, mouse and rat spermatogenesis. Finally, the testis-specific histone variant TH2B was identified as a potential quantitative marker of equine sperm quality.

Mammals receive more germline mutations from fathers than mothers. While the paternal bias in mutation has historically been attributed to errors in DNA replication during spermatogenesis, evidence suggests that in humans mutational mechanisms independent from cell division may play a more prominent role. Understanding how the ratio of paternal-to-maternal mutations, 𝛼, varies across animals differing in their gametogenic development, physiologies, and habitats can provide unique insights into the processes by which mutation arises in male and female germlines. To these ends, this thesis examines features of paternal mutation bias in dozens of amniote species using a combination of sequencing and evolutionary approaches. A direct way of measuring the strength of paternal mutation bias involves sequencing pedigrees of related individuals and detecting mutations arising in a single generation. In Chapter 2, we applied this approach to measure 𝛼 in olive baboons (Papio anubis) and humans. Strikingly, we estimated that in baboons 𝛼 = 4.5, similar to humans, despite baboons experiencing far fewer spermatogenic cell divisions than humans. A model of mutation based on cell division differences in the two species failed to explain this observation. Our results provide added evidence for non-replicative processes driving paternal bias in mutation and suggest that these causes are likely shared across mammals. In Chapter 3, we expanded our analysis to survey 𝛼 across 42 amniote species. We estimated 𝛼 from putatively neutral substitution rates of sex chromosomes and autosomes and found that in mammals, 𝛼 ranges up to 4 and correlates with generation times. In contrast, birds and snakes harbor a stable 𝛼 of roughly 2. These results are well predicted by modeling sex bias in mutation as a product of an early developmental phase when mutation occurs equally in both parents and a late phase after sexual differentiation when the male germline is more mutagenic. That the paternal mutation bias is widespread and occupies a narrow range of values suggests that it is caused by endogenous damage sources that are similar across species. Through a combination of pedigree sequencing and evolutionary techniques, this work demonstrates how a comparative approach across diverse taxa can shed light on the origins of sex-bias in germline mutation.

Crustacean spermatozoa are among the most peculiarly modified germ cells in the animal kingdom. Many of their striking cytological specializations may be observed in the sperm cells or Emerita analoga, the so-called sand crab. This animal has been used by the writer for studies on spermatogenesis and on spermatozoan behavior during the summer or 1951, 1952, and 1953 at the Pacific Marine Station.

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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.