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Turquoise killifish had to adapt to the inhospitable conditions in which they live, especially drying temporal water bodies, which means certain death. The life sprint of the representatives of this genus is at its peak within one-month post-hatching, when both sexes have fully developed gonads and can reproduce. This rate comes with a high cost, as the killifish gonads begin to show signs of tissue degradation and germ cell apoptosis as early as three months post-hatching. Germ cell loss increases with age. A description of the development and degradation of the gonads in males and their breeding was elaborated. For the evaluation of aging-specific changes, immunochemical methods were used, focusing on the binding of specific antibodies against target epitopes and their visualization using fluorescence microscopy. Procedures for histological specimens have also been described, both for classical light and fluorescence microscopy.

As the problems with the reproduction of livestock and humans starting to increase, the need for knowledge of mechanisms involved in regulating the correct process of gametogenesis and embryogenesis also rises. For the experimental part of this work, two components from two different, mostly separately explored fields, i.e. gasotransmitters and epigenetic mechanisms, which are necessary for the correct process of gametes production and early embryonic evolution were chosen. Hydrogen sulfide was chosen from the series of gasotransmitters, and histone deacetylase Sirtuin 1 (SIRT1) was chosen as its possible substrate. Confirmation of the presence of these components was carried out on oocytes and embryos of laboratory mice (Mus musculus).

Changes in the characteristics of spermatogenic and oogenic cells during gametogenesis may reflect a corresponding alteration in aspects of components such as calcium, which plays prominent roles in regulating a broad range of physiological events in animal reproduction. Basic information regarding distribution of intracellular calcium in different germ cells may provide better understanding of processes of reproduction in fish. The monthly testicular development in the cultured breeding stock of sterlet, Acipenser ruthenus, using histological and serum sex steroid was studied. Results showed four distinct phases including resting, pre-spawning, spawning and post-spawning. Hormonal profiles of 11-ketotestosterone (11-KT) showed peak, which indicated a seasonal pattern of gonadal development. The 11-KT concentration increased considerably during the spermatogenesis (pre-spawning phase) and remained quite high throughout the pre-spermiation period. In the final phase of testicular development (spawning phase), the 11-KT markedly dropped. This study provides basic knowledge of the reproductive biology in male sterlet and a complete guide for gonadal development, heretofore lacking in previous studies of sturgeon gonadal development. The intracellular distribution of calcium during different developmental stages of spermatogenesis was studied in sterlet, A. ruthenus, using a combined oxalate-pyroantimonate technique. The distribution of calcium was described in spermatogonium, spermatocyte, spermatid, and spermatozoon stages. Although calcium is appeared in the form of deposits in limited areas of the early stages cells (spermatogonium and spermatocyte), it is present as an unbound form in larger areas of spermatids and spermatozoa especially nucleus, which probably reflects changes in its physiological function and homeostasis of calcium during male gamete production. Similar to sterlet sturgeon, ultrastructural distribution of calcium during different developmental stages of spermatogenesis was described in a model organism, zebrafish (Danio rerio), using a combined oxalate-pyroantimonate technique. The subcellular distribution of intracellular calcium was detected as deposits mainly in the cytoplasm and the nucleus of the spermatogonium and spermatocyte. Interestingly, large amount of calcium was transformed from isolated deposits into an unbound pool (electrondense mass) within the nucleus of the spermatid and the spermatozoon. The alteration of intracellular calcium at different stage of D. rerio spermatogenesis can be related to specific function of each germ cell types during male gamete development. Unbound calcium in the nucleus of mature spermatozoon can be used for condensation of chromatin and induction of calcium wave during egg activation and fertilization. Using a combined oxalate-pyroantimonate technique, the subcellular distribution of calcium deposits during stages of oogenesis in zebrafish was described. Calcium deposits were localized at different organells within the egg during oocyte development. At the first stage of oocyte development (primary growth), calcium deposits were localized in the cytoplasm, mitochondria, nucleus, and follicular cells. At the cortical-alveolus stage, calcium particles were transported from follicular cells into the cortical alveoli. In the main stage of oocyte development (vitellogenic stage), some cortical alveoli were compacted and transformed from flocculent electron-lucent to electron-dense objects with the progression of the stage. Calcium deposits were transformed from larger to smaller particles, coinciding with compaction of cortical alveoli. As a conclusion, results of this study provide new and convergent insight into information about the regulation and functional roles of calcium during fish gametogenesis which simply describe facilitate release of calcium from related organelles (nucleus and cortical alveoli) as main internal stores of calcium for calcium transport.

It is now more than 35 years since the first world test-tube baby, Louise Brown, was born (1978) in England and it is estimated that since then more than 4 000 000 of children were produced by in vitro fertilization (IVF) worldwide. The initial success of IVF was less than 20% in best clinics, but now it reaches about 40%. This is a consequence of introduction of new methods, standardization and exploitation of new manipulation and culture media, as well as the incorporation of research results. Nevertheless, the most important still remains the skill and experience of IVF clinics and IVF laboratories staff, especially their ability to critically evaluate the quality of biological material and to decide which cure and treatment are the best one. At least, some biological material (immature and low quality oocytes) can be used for training and also for some experiments aiming to explain some questions, which are not yet fully understood (for example aneuploidies in human oocytes and embryos). In addition, this training can facilitate the introduction of new progressive approaches and may also improve indirectly the quality of infertility treatments. The first part of thesis is focused on the quality evaluation of oocytes collected by aspiration from follicles of stimulated patients. For labeling...

PLK1 belongs to the extended family of serine/threonine kinases controlling the cell cycle. It is well known for its role in the control of mitosis and contributes also to the regulation of meiotic division. On a basis of Live Cell Imaging (LCI) experiments we can describe the phenotype of the oocytes with PLK1 inhibited by small molecular inhibitor BI2536. PLK1 inhibition leads to delayed nuclear envelope breakdown (NEBD) and chromatin condensation (CC) and also causes desynchronization of NEBD and CC; in contrast to control oocytes, PLK1 inhibited oocytes break down their nuclear envelope with chromatin almost fully condensed. Also duration of these two early nuclear events is prolonged in oocytes with inhibited PLK1. In contrast to somatic cells, PLK1 inhibition in mouse oocytes does not prevent assembly of spindle with two distinct poles but affects the final spindle volume. Similar to somatic cells, mouse oocytes with PLK1 inhibited from the beginning of the meiotic maturation stay arrested in metaphase I but in the case of mouse oocytes, this block is not dependent on Spindle Assembly Checkpoint (SAC) persisting activity. When mouse oocytes are synchronized on metaphase I/anaphase I transition by proteasome inhibition and then PLK1 kinase activity is inhibited, about 2/3 of the oocytes stay arrested...