Basement of healthy embryo development comes from quality of oocytes and spermatozoa. Today, when percentage of couples suffering infertility together with assisted reproductive therapy (ART) is increasing, understanding to gamete biology and heritable epigenetic code is crucial. The study is focused on promising epigenome based markers that could serve as indicators of gamete quality for either their screening or selection for ART. Accordingly selected markers were used for the investigation of environmental pollutant bisphenol S (BPS) effect on gametes quality. To obtain these aims, we have used human semen samples, boar semen samples and ICR mice gametes. Samples were analyzed by flow cytometry, immunocytochemistry and western blot analysis. All experimental work was in accordance with Ethics committee University Hospital in Pilsen and approved experimental designs for appropriate experimental animal project. In the study, we detected the dimethylation of histone H3 on lysine K4 (H3K4me2) as potential epigenetic marker of sperm quality and chromatin immaturity. Secondly, we observed the role of the gasotransmitter hydrogen sulphide (H2S) as anti-capacitating agents, slowing down capacitation possibly through post-translational modification of proteins. Thirdly, SIRT1 histone deacetylase was...

In order to evaluate the short term metabolic effects of gossypol on the testes as well as any possible effects on the secondary sex organs, Balb C mice were injected subcutaneously with various doses of gossypol (0.25‐25.0 mg/kg body weight) in corn oil for 10 days. Wet weights of several different secondary sex reproductive organs decreased during gossypol treatment. However, wet weights of the testes during treatment remained equal to or greater than control values. Following 10 days of gossypol treatment, incorporation of [3H]thymidine or [3H]amino acids into trichloroacetic acid precipitable macromolecules was inhibited in the seminal vesicles and ventral prostates normalized to either DNA or wet weight. Treatment with gossypol also had an inhibitory effect on epididymal sperm count at the two highest doses. These results demonstrate that gossypol will decrease sperm count at high dose levels after treatment of male mice for as short as 10 days. However, its overall effects are not limited to the testes and spermatogenesis but, in addition, it has dramatic inhibitory effects on protein and nuclei acid metabolism in the secondary sex organs.

This study was conducted to investigate the effect of platelet activating factor (PAF) on the acrosome reaction and fertilizing capacity of spermatozoa, and development of the resulting embryos in the rabbit. Rabbit spermatozoa were exposed to PAF, Iyso-PAF, or high ionic strength medium (HIS) prior to subzonal sperm injection (SUZI) into 326 mature oocytes, or morphological assessment of the acrosome reaction. The rates of fertilization and blastocyst formation were compared among the three treatment groups. Acrosome reaction was assessed by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) staining and electron microscopy. PAF-treated spermatozoa fertilized the oocytes at a significantly higher rate (56.1%) than did lyso-PAF-(36.8%, P< 0.01) or HIS- (38.2%, P < 0.05) treated spermatozoa. The embryos produced by PAF-treated spermatozoa showed significantly higher blastocyst formation rates (34.0%) than lyso-PAF- (8.6%, P < 0.050) or HIS-(8.8%, P< 0.05) treated spermatozoa. FITC-PSA staining demonstrated a significantly higher incidence of acrosome reaction in PAF-treated spermatozoa (45.8%) than in Iyso-PAF- (28.0%, P < 0.01) or HIS- (34.9%, P < 0.01) treated spermatozoa. Acrosome reaction of PAF-treated spermatozoa was also confirmed by electron microscopy. PAF treatment of spermatozoa enhances fertilizing capacity for SUZI possibly by augmenting the acrosome reaction. Enhanced embryonic development was also found in the oocytes fertilized by SUZI of PAF-treated spermatozoa.

Space flight has been shown to have many adverse effects on various systems throughout the body. Because the opportunity to place research animals on board a Space Shuttle or the International Space Station is infrequent, various techniques have been designed to simulate the effects of microgravity in Earth based laboratories. A commonly used technique is known as antiorthostatic suspension, also often referred to as hind limb suspension. In this technique the hind portion of the animal is raised so that its hind limbs are non-weight bearing. This places the animal in roughly a 30° head down tilt position. This results in cephalic fluid shifts similar to those seen in actual space flight. This technique has also been shown to mimic other physiological parameters that are affected during space flight. This study examined testicular tissue from rats subjected to a 7 day antiorthostatic suspension. This tissue was acquired through a tissue sharing program and some of the experimental animals were injected with Interleukin 1 receptor antagonist (IL-1ra) which was hoped to ameliorate some of the effects of antiorthostatic suspension. The injection of IL-1ra was not expected to have any effect on testicular tissue, however this tissue was included in the morphological and statistical analysis to conduct a more complete study. All tissues were embedded in paraffin, sectioned, and stained using standard H&E staining. The tissue was then qualitatively ranked according to the "health" of the seminiferous tubules. Our findings indicate that 7 days of antiorthostatic suspension had adverse effects on the tissue that comprises the walls of the seminiferous tubules. It has long been known that antiorthostatic suspension has deleterious effects on testicular tissue, however this research indicates that these effects occur much faster than indicated by previous researchers. This is a significant finding because it indicates that meaningful earth based studies in this area can be carried out in a shorter time span. This could result in more studies per year as well as saving money by avoiding longer than necessary animal suspensions. This is especially important as we enter an era when, without Space Shuttle, flight opportunities will become scarce. These antiorthostatic suspension studies indicate that space flight, even short duration spaceflight, may have harmful effects on the seminiferous tubules and blood-testis barrier of astronauts.

Forensic analysis of DNA from sexual assault kits is a laborious process. These samples may be a mixture of sperm and male or female epithelial cells (E-cells). Generally, it is the sperm cells that are of greatest forensic value. Since its introduction in 1985 by Gill, Jefferys and Warrett, differential extraction has remained an essential pre-PCR extraction procedure adopted by most forensic laboratories for the preferential lysis of E-cells and isolation of sperm cells/male fraction prior to DNA profiling. The differential extraction procedure operates based on the packaging of DNA in these two types of cells. The E cells are first lysed by sodium dodecyl sulfate (SDS) and Proteinase K which leaves the sperm cells intact. The mixture is centrifuged leaving E-cell DNA in the supernatant and sperm cells in the pellet. After several wash steps to remove residual E cell DNA, the sperm fraction is then subjected to lysis using SDS, proteinase K, and dithiothreitol (DTT). DTT reduces the disulfide bonds present in the sperm nucleus, thereby releasing sperm cell DNA. The traditional Gill method of differential extraction, while proven to be highly effective in providing two separate fractions for a simplified interpretation of profiles, is a labor intensive and time-consuming process, requiring approximately six hours of an analyst’s concentration. In a casework scenario where an evidence sample is of a higher E cell concentration compared to sperm cells, it is inevitable to obtain mixture profiles that becomes more difficult to interpret. To mitigate carryover from the female fraction, the sperm cell fraction is usually subjected to multiple wash steps. Furthermore, the resulting fractions must be subjected to additional pre-PCR DNA purification procedures to remove PCR inhibitors such as SDS and Proteinase K which result in varying degrees on DNA loss. Progress has been made over the years to introduce methods that allow for PCR-ready lysates without additional purification steps, often referred to as direct lysis methods. However, none have been proven to be viable options for use in sexual assault samples. Our laboratory has developed a novel differential extraction procedure that is not only time-efficient and less laborious but also utilizes a direct-lysis procedure requiring no further pre-PCR purification for most samples. The novel procedure uses ZyGEM, which contains the thermophilic EA1 protease proven to effectively digest biological samples and produce PCR-ready lysates suitable for downstream nucleic acid amplification, thereby minimizing DNA loss. The procedure uses a multi-enzymatic approach and utilizes the different optimal activity temperatures of the enzymes to perform most of the process in a DNA extraction lab thermocycler, requiring only a single centrifugation for the usual separation of the E-cell fraction and no subsequent washing steps for the sperm cell fraction. It has the potential to be a rapid, robust procedure that can be easily implemented in any forensic laboratory. This thesis will describe the procedure and report progress in the procedure optimization. / 2019-10-24T00:00:00Z

Of central importance to the study of heme proteins are the effects imposed by axial ligand(s) on the heme structure and, therefore, on the overall activity of the protein. In this study, we confirm and extend the spectroscopic characterization of a mutated sperm whale myoglobin in which the proximal Histidine is replaced with a Tyrosine residue (MbH93Y). The MbH93Y, as well as wild-type sperm whale myoglobin and horse erythrocyte catalase (HEC), was purified and characterized by optical absorption and x-ray absorption (XAS) spectroscopies. Optical absorption spectra of HEC and the metmyoglobin, cyanometmyoglobin, reduced, oxy, and carbon-monoxy forms of both sperm whale myoglobin (SWMb) and MbH93Y were identical to previously reported values within the respective errors:. Extended x-ray absorption fine structure (EXAFS) studies revealed that the proximal bond length in MbH93Y was 2.13 ± 0.03 A, compared to 2.14 ± 0.02 A for sperm whale metmyoglobin and 1.90 ± 0.02 A for catalase. Additionally, the sixth coordination site normally occupied in wild type sperm whale metmyoglobin and in catalase at low temperatures was vacant in MbH93Y, a result corroborated by the optical absorption spectra and cyanogen bromide modification of the distal histidine. Measurements were also made on the cyanide complexes of the three proteins as well, among which, (i) the average iron-to-pyrrole nitrogen bond distance for MbH93Y-CN was 1.96 ± 0.015 A compared to 2.00 ± 0.015 A for WT SWMb-CN and HEC-CN and (ii) the proximal bond length in MbH93Y-CN was 2.07 ± 0.02 A, while that of WT SWMb-CN was 2.10 ± 0.02 A and that of HEC-CN was found to be 2.12 ± 0.02 A. Further, upon exposure to 2-molar equivalents of hydrogen peroxide, sperm whale myoglobin formed a Compound II -like spectrum, while the Soret absorbance of MbH93Y was rapidly, significantly, and irreversibly decreased. Furthermore, the dissociation constants for CN- binding to MbH93Y were found to be, on average, approximately three orders of magnitude higher than those of wild-type sperm whale myoglobin and are consistent with the many-fold higher cyanide binding kinetics for wild type, relative to the mutant protein. Finally, the PK. of the mutant was found to be more than three orders of magnitude higher than that of the native protein. Explanations focusing on probable electronic effects of the phenolate oxygen atom in the sperm whale myoglobin pocket are discussed.

Orientador: João Carlos Pinheiro Ferreira / Resumo: O uso do sêmen refrigerado proporciona maiores taxas de prenhez se comparado ao do sêmen congelado. Essa diferença parece estar relacionada às lesões mais severas das membranas espermáticas desencadeadas pelo processo de congelação. Por sua habilidade de se ligar às proteínas ligadoras de espermatozoides e ao íon cálcio, o caseinato de sódio vem sendo estudado como uma substância capaz de inibir a capacitação espermática precoce, uma importante causa de diminuição da taxa de prenhez quando do uso de sêmen congelado. O primeiro objetivo deste estudo foi avaliar a possibilidade de um diluente comercial a base de gema de ovo, destinado à congelação de sêmen bovino, ser empregado para a criopreservação de sêmen bubalino; o segundo objetivo foi investigar o efeito do uso desse diluente, suplementado com caseinato de sódio, na criopreservação de espermatozoides bubalinos, por meio da avaliação dos espermatozoides, por citometria de fluxo, e da cinética espermática, empregando-se o sistema CASA. Na primeira parte do estudo, quando comparados os resultados das avaliações da cinética espermática e integridade das membranas plasmática e acrossomal, observou-se que o processo de congelação seminal promoveu mais danos celulares que o processo de refrigeração. Na segunda parte do estudo, não foram observados efeitos da adição do caseinato de sódio ao diluente a base de gema de ovo. A partir dos resultados do presente estudo foi possível concluir que o diluente a base de gema de ovo testad... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The use of cooled semen results in higher pregnancy rates compared than the use of frozen semen. This result seems to be related to the more severe damages triggered by the freezing process, when compared to those observed during the refrigeration. Due to its ability to bind to sperm-binding proteins and calcium ions, sodium caseinate has been studied as a ubstance capable to prevent early sperm capacitation, a major cause of decreased pregnancy rate after using frozen semen. The first objective of this study was to evaluate if a commercial egg yolk diluent developed for freezing bovine semen could be used for buffalo semen cryopreservation; the second objective was to investigate the effect of this diluent, added with sodium caseinate, during the procedures of buffalo sperm cryopreservation, using flow cytometry and computer-assisted sperm analysis. In the first part of the study, comparing the results of spermatic kinetics and plasma and acrosomal membranes integrity, it was observed that the freezing process resulted in more cell damage than the cooling process. In the second part of the study, no effects of the addition of sodium caseinate to the egg yolk diluent were observed. From the results of the present study it was possible to conclude that the egg yolk-based diluent was suitable for buffalo semen cryopreservation and that the addition of sodium caseinate did not decrease the deleterious effects related to seminal cryopreservation. / Mestre

Parthenogenetic development (PD) in Chinese Painted quail decreases hatchability and increases early embryonic mortality. The objectives of this study were to determine if PD alters egg albumen ions, gases, and pH (virgin and mated hens) as well as the success of subsequent fertilization in mated quail and if egg storage and incubation temperature increase PD. In virgin hens, PD altered albumen characteristics over incubation. In fact, albumen from mated and virgin hens exhibiting PD showed similar albumen characteristics, and these characteristics were similar to early dead embryos in mated hens. Also, mated hens selected for parthenogenesis had less sperm holes in the perivitelline membrane and a higher percentage of eggs without holes as compared to birds not selected for parthenogenesis. Increasing storage and incubational temperature increased PD and parthenogen size. In conclusion, PD alters egg albumen characteristics, decreases fertility, and can be affected by storage and incubation temperatures.

Alternatives to antibiotic growth promoters have been widely exploited due to concerns about antimicrobial resistance. These feed additives improve growth, in part, by modulating intestinal microbiota. However, their impact on male reproductive performance is not well elucidated. Therefore, the objective of this study was to evaluate the impacts of a yeast fermentation product (YP) and Bacillus subtilis on rooster semen quality and microbiota. Dietary supplementation of YP linearly increased the concentration of yeast and bacteria in semen, whereas it linearly decreased sperm motility, suggesting that bacteria attached to yeast were excreted from the gut, contaminated semen at the cloaca and then decreased sperm movement. However, direct in vitro exposure of semen or dietary supplementation with B. subtilis did not affect semen quality or seminal concentration of this bacterium, likely because Bacillus naturally occur in semen. In conclusion, unlike B. subtilis, dietary YP can alter semen quality by altering semen microbiota.

First, a preliminary study was conducted looking for the optimum sperm concentration to be used for analysis with the Computer-Assisted Sperm Analysis (CASA). Results showed that 75x106 sperm cells/mL is the optimum one. Then, the actions of relaxin on sperm motility were evaluated by determining the effect of relaxin on full motility characteristics of spermatozoa during storage, using CASA; then identifying the relaxin receptors on spermatozoa, and finally establishing actions of relaxin in intraspermatic cAMP content. Motile spermatozoa were selected through percoll gradient and incubated for 1 hour with 4 relaxin concentrations at 37°C, during four days. Relaxin affected sperm motility (P<0.05). This action appears associated with the presence of relaxin receptors RXFP1 and RXFP2 that were found in spermatozoa. However, the cAMP levels were not affected by relaxin (P<0.05). This study indicates a beneficial action of relaxin on sperm motility; however, its mechanism of action requires further research.