The role of mesenchymal-epithelial transition in endometrial function and receptivity.

OBJECTIVE: During reproductive age of a woman, endometrium undergoes frequent stuctural and functional changes. Abilities of regeneration, remodelation and differentiation are precondition of endometrial receptivity and implantation and development of an embryo. These processes are conditioned by mutual transformation between mesenchymal and epithelial fenotype of endometrial cells: epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). The aim of this study is to present contemporary knowledge of transformation between epithelial and mesenchymal endometrial cells and its influence on human fertility. DESIGN: Review article. SETTING: Department of Obstetrics and Gynecology, Faculty of Medicine, Masaryk university and University Hospital Brno; Department of Obstetrics and Gynecology, University Hospital Faculty of Medicine, Palacky University, Olomouc. METHODS: PubMed was searched for articles in English indexed until February 2019 with terms of „endometrial receptivity“, „embryo implantation“, „endometrial regeneration“, „mesenchymal-epithelial transition/transformation“. RESULTS: It has been proved, that mesenchymal stromal cells participate on regeneration of not only the endometrial stroma, but also of the epithelium. During endometrial decidualisation under influence of ovarian steroids, the MET is under way. Stromal fibroblasts gain the morfological and functional properties of epithelial cells. During implantaion of an embryo, the trofoblast interacts with decidualised endometrium. Epithelial cells transform into mesenchymal (EMT), which mediate the growth of trofoblast. CONCLUSION: Mutual transformation between stromal and epithelial cells in essential for normal function of endometrium and implantation and development of an embryo.

Uterine microbiome and endometrial receptivity.

OBJECTIVE: Structure and correct function of endometrium is necessary for embryo implantation, pregnancy development and childbirth. It is a result of many factors - anatomical and histological structure, hormonal effects and signalling pathways at the molecular genetic level. A little known phenomenon is the presence of microorganisms on the endometrium. Traditionally, the uterine cavity was considered sterile, but new findings have been changing this view fundamentally. The aim of this work is to present new findings on endometrial microbiome and its importance for embryo implantation and development. DESIGN: Review article. SETTING: Department of Obstetrics and Gynecology, Faculty of Medicine, Masaryk University and University Hospital Brno; Department of Obstetrics and Gynecology, University Hospital Faculty of Medicine, Palacky University Olomouc. METHODS: PubMed was searched for articles in English indexed until 30th June 2018 with terms of „uterine microbiome analysis“ and „endometrial receptivity“. RESULTS: The vital information on bacterial colonization of endometrium brought new diagnostic methods for their detection based on ribosomal RNA analysis in 16S subunit, which are capable of detection and exact identification of bacteria that cannot be detected by classical cultivation methods. The endometrial microbiome is assumed to modulate the function of endometrial cells and local immunity system, it prevents growth of pathogenic microorganisms by its presence and production of protective substances. CONCLUSION: Endometrial microbiome seems to be important factor of endometrial receptivity.

Bisphenols in the pathology of reproduction.

OBJECTIVE: Bisphenols are one of the most widespread endocrine disruptors that the population of west world countries is exposed to. Objective of this study is to summarize information about influence of bisphenols on reproduction health. DESIGN: Review article, Setting: Department of Obstetrics and Gynecology, Faculty of Medicine, Masaryk University and University Hospital Brno. METHODS: PubMed was searched for articles in English indexed bisphenol and reproduction up to October 2018. RESULTS: Increased levels of bisphenol A and S have been proven in body fluids and tissues. Bisphenol molecules have effect similar to estrogens therefore they influence hormonal regulation and activity of estrogen receptors. Their negative influence on oocyte maturation, spermatogenesis and development of reproductive system has been shown. Bisphenol S, which has replaced bisphenol A, has comparable negative effects on reproduction. CONCLUSION: Bisphenols are widespread endocrine disruptors that could cause severe fertility disorders of men and women.

Expression of PTX3, HAS2 AND TNFAIP6 genes in relation to real-time proliferation of porcine endometrial luminal epithelial cells in primary cultivation model.

Endometrial cells undergo very specific changes associated with reproductive processes. Cells prepare for embryo development by increasing their volume. Then, if fertilization fails, endometrial cells are liable for apoptosis, preparing new cells that are ready for subsequent processes related to the possibility of embryo implantation and the development of pregnancy. PTX3 and TNFAIP6 are absent or reduced in cultured COCs, resulting in a functional change in COC in vitro. In this work, we want to check how PTX3, HAS2 and TNFAIP6 behave in luminal epithelium primary cell culture. Cells obtained during slaughter from porcine specimens were cultured primarily in vitro for 7 days. Their proliferation patterns were then analysed using RTCA, with the expression of genes of interest evaluated with the use of immunofluorescence and RT-qPCR. The results of these changes in the expression of the genes of interest were analysed on each of the seven days of the porcine luminal primary cell culture. Our study showed the increased level of PTX3, HAS2 and TN¬FAIP6 expression at the same hours of primary culture. Rt-qPCR showed a higher level of expression of the PTX3 gene in the first 72 h, at the end of the lag phase (in the phase of stasis in which the cells adapt to the new environment and often die). In contrast, TNFAIP6 expression increases about 96 hours when the cells are in the full log phase (logarithmic phase growth) and continue this trend in the plateau phase. We did not observe such drastic changes in the HAS2 expression pattern, which leads us to hypothesize that PTX3 and TNFAIP6 are designed to maintain a constant level of HAS2 in the cell throughout its lifetime. The obtained results could become a point of reference for further in vivo and clinical research.

Human Wharton's jelly mesenchymal stem cells: properties, isolation and clinical applications.

Human Wharton's jelly mesenchymal stem cells (WJ-MSCs) exhibit CD29, CD79 and CD105 markers, characteristic for mesenchymal cell lines. Under the influence of the appropriate factors, WJ-MSCs can be dedifferentiated to osteoblasts, chondrocytes, adipocytes, myocytes, cardiomyocytes, glial cells and dopaminergic neurons. Wharton's jelly (WJ) is one of the potential sources of mesenchymal stem cells (MSCs) - obtaining these cells does not raise moral or ethical objections, because the umbilical cord (UC) is a regular waste material. The expression of the OCT-4 and Nanog proteins, which are characteristic for WJ-MSCs may indicate that these cells have retained some embryonic character. The collected data suggests that WJMSCs show increased division and telomerase activity compared to bone marrow MSCs (BM-MSCs). The published results showed no human leucocyte antigen (HLA) class II expression, with the possibility of HLA class I modification by WJ-MSCs, allowing for the transplantation of these cells both within the same and other species - which allows the use of human cells in animal models. The results of selected studies indicate that WJ-MSCs can be an essential element of regenerative medicine of the 21st century.

Insight into "nuclear-cytoplasmic shuttling" as a developmental and differentiational capability of cells in primary culture models.

Shuttling proteins are molecules that can facilitate transport through the nuclear envelope. A very large number of proteins are involved in this process that includes nuclear pore buildup, signal, receptor and enzyme proteins. There are many examples of proteins whose biological activity depends on nucleocytoplasmic transport. Very often they are largely responsible for the proper occurrence of cell division, maturation, development and differentiation. Thanks to the well mastered methods of in vitro cell culture, it is possible to trace the levels of protein expression and their distribution in cells. Advanced molecular techniques allow for precise determination of their displacement in time. Several studies are still being carried out, using primary cultures, to identify the factors that determine the maturation, development and differentiation of cells. In understanding of the detailed mechanisms controlling cell life, the key is not the level of expression of a specific protein, but its distribution in individual cellular compartments.

Transcriptomic profile of cell cycle progression genes in human ovarian granulosa cells.

The ovarian granulosa cells (GCs) that form the structure of follicle undergo substantial modification during the various stages of human folliculogenesis. These modifications include morphological changes, accompanied by differential expression of genes, encoding proteins which are mainly involved in cell growth, proliferation and differentiation. Recent data bring a new insight into the aspects of GCs' stem-like specificity and plasticity, enabling their prolonged proliferation and differentiation into other cell types. This manuscript focuses attention on emerging alterations during GC cell cycle - a series of biochemical and biophysical changes within the cell. Human GCs were collected from follicles of women set to undergo intracytoplasmic sperm injection procedure, as a part of remnant follicular fluid. The cells were primarily cultured for 30 days. Throughout this time, we observed the prominent change in cell morphology from epithelial-like to fibroblast-like, suggesting differentiation to other cell types. Additionally, at days 1, 7, 15 and 30, the RNA was isolated for molecular assays. Using Affymetrix® Human Genome U219 Array, we found 2579 human transcripts that were differentially expressed in GCs. From these genes, we extracted 582 Gene Ontology Biological Process (GO BP) Terms and 45 KEGG pathways, among which we investigated transcripts belonging to four GO BPs associated with cell proliferation: "cell cycle phase transition", "G1/S phase transition", G2/M phase transition" and "cell cycle checkpoint". Microarray results were validated by RT-qPCR. Increased expression of all the genes studied indicated that increase in GC proliferation during long-term in vitro culture is orchestrated by the up-regulation of genes related to cell cycle control. Furthermore, observed changes in cell morphology may be regulated by a presented set of genes, leading to the induction of pathways specific for stemness plasticity and transdifferentiation in vitro.

Balance between epithelial and stromal marker expression and distribution in primary culture model of porcine endometrium during real-time cell proliferation..

The similarity between humans and pigs, when it comes to tissue morphology, makes Sus scrofa not only a good research model, but also a potential source of cells for tissue engineering. Cell samples obtained from the pig donor, could be influenced in vitro, in order to become a source of tissue material for xenotransplantation, reconstructive and regenerative medicine. Significant amounts of data point to especially major similarities in pig and human reproductive systems. Because of that, particular scientific focus is centered on research concerning porcine COCs, theca and granulosa cells in primary cultures. One of the aspects of the reproductive process, that is still largely undiscovered, is the interaction between preimplantation blastocyst and maternal uterine tissues. In this study, we used molecular analysis techniques, such as RT-qPCR and immunocytochemistry, to analyze the expression and distribution of cytokeratin 18 and panCytokeratins 8, 18 and 19 and vimentin in porcine luminal endometrial epithelial cells, coupled with analysis of their behavior in RTCA. The results have confirmed the presence of epithelial, as well as stromal cell markers in the cells, varying in levels at different stages of culture. They have also given insight into the modes of proliferation and differentiation of studied cells in in vitro culture, as well as providing additional proof for the possible mesenchymal transdifferentiation of epithelial cells.

Enzyme linked receptor protein signaling pathway is one of the ontology groups that are highly up-regulated in porcine oocytes before in vitro maturation.

Before being able to fully participate in the processes associated with its function as a female gamete, the oocyte needs to undergo a range of changes to achieve its mature form. These morphological, biochemical and metabolomic processes are induced by the somatic tissues surrounding the oocyte, through the expression of specific transcription and growth factors. The maturation of the oocyte is highly important for the proceedings that lead to successful fertilization, early embryonic development and implantation. Domestic pigs were used as models for our study, with the cumulus-oocyte complexes obtained from the ovaries that were recovered at slaughter. After shedding of the cumulus, oocytes were assessed with BCB test, with the viable ones chosen to undergo in vitro maturation. With the use of expression microarrays, we analyzed gene expression before and after IVM and detected major changes in both genes that were proven to be associated with oocyte maturation before (FOS, VEGFA, CHRDL1, TGFBR3, FST, INSR, ID1, TXNIP, SMAD4, MAP3K1, EIF2AK3 and KIT) and genes not previously linked with reproduction associated processes (MYO1E, PHIP, KLF10 and SHOC2). All the genes were briefly described, with consideration of possible involvement of the newly discovered elements of the transcriptome in the process of oocyte maturation.

[Medroxyprogesteron acetate use to block LH surge in oocyte donor stimulation]. / Vyuzití medroxyprogesteron acetátu k blokáde LH pri stimulaci dárkyní vajícek.

OBJECTIVE: To compare the results of ovarian stimulation with LH surge blockade by medroxyprogesterone acetate or GnRH antagonist in oocytes donors. To present current options of exogenous and endogenous progestins instead of GnRH analogues to block LH surge during ovarian stimulation. DESIGN: Retrospective study of oocyte donor cycles and literature review. SETTING: Department of Obstetrics and Gynecology, Faculty of Medicine, Masaryk university and University Hospital Brno. METHODS: Thirteen oocyte donors (26.8 ± 2.5 years old) were stimulated with recFSH and MPA to block the LH surge during January - October 2017. The results were compared to the previous cycle stimulated with recFSH and GnRH antagonist performed during January -December 2016. Wilcoxon´s pair test was used to test the statistics. A literature search of SCOPUS was carried out. RESULTS: In cycles with MPA blockade the average number of oocytes was 14.5 ± 5.1, in cycles with GnRH anta-gonist blockade 12.0 ± 4.5 oocytes (statistical significance p = 0.025). FSH total dose (1611 ± 327 vs. 1565 ± 322 IU), days of stimulation (9.1 ± 0.8 vs. 8.5 ± 0.9) and maximum estradiol levels (5.9 ± 2.1 vs. 6.0 ± 3.0 nmol/l) were not statistically different. Progestins are effective in blocking the LH surge during ovarian stimulation and do not affect the number and quality of collected oocytes or obtained embryos. Their adverse effect on the endometrial receptivity obstructs the embryo implantation in the same cycle. Such protocol requires total freezing and delayed transfer. Progestins can be used in a variety of stimulation protocols - progestin primed follicular phase stimulation, luteal phase stimulation with endogenous progesteron, double stimulation in follicular and luteal phase of the same cycle "duostim" in low responders. CONCLUSION: Eggs donor ovarian stimulation with MPA resulted in more oocytes than stimulation protocol with GnRH antagonist, the total dose of FSH and the length of stimulation were similar. According to current experiences progestins effectively block the LH surge and do not affect the number and quality of collected eggs and obtained embryos. Their use opens new possibilities of ovarian stimulation protocols and their flexibility. Its main constraint is that it requires total freezing and delayed transfer.

Haematopoiesis: “living in the shadow” of stem cell differentiation.

Haematopoiesis is one of the most well understood stem-cell associated processes. It is a process in which pluripotent hematopoietic stem cells (HSCs) self-proliferate and differentiate into all types of blood cells. The process takes place in marrow of the flat bones in adults, however its location changes several times through embryonic and foetal development. Given the broad range of blood cells and the major differences in their build and function, together with the fact that their numbers need to be maintained within relatively narrow margins in order to maintain homeostasis despite changing environmental conditions, makes the whole process of haematopoiesis highly regulated and depending on a variety of growth factors. When influenced by those, HSCs undergo several irreversible steps, with every next one committing them to an even more specialised fate, ending with all the specific types of mostly short-lived blood cells, that are unable to proliferate on their own and need constant replenishment from the HSC pool. Because the process of haematopoiesis is the only source of all the members of the group of cells performing a range of highly important roles in functioning of the organism, significant damage to the underlying stem cells can cause a range of severe diseases. Many treatments are suggested for managing their symptoms or slowing progress, with bone marrow transplant being one of the only ones that offer possible permanent solution and, despite being a relatively risky procedure, is being widely performed, with the methods constantly improving in order to achieve progressively better results in both treatability and survivability of the patients.

Expression of genes responsible for cell morphogenesis involved in differentiation in porcine buccal pouch mucosal cells during long-term primary culture and real-time proliferation in vitro.

Recently, using experimental animal model, we demonstrated that porcine buccal pouch mucosal cells reflect increased proliferation capability during primary cultivation in vitro. Although the histological structure and morphogenesis in oral cavity is well recognized, the molecular mechanisms which regulate this process still need further investigation. This study was aimed to analyze the molecular marker expression profile involved in morphogenesis and differentiation capacity of porcine buccal pouch mucosal cells during their long-term primary cultivation in vitro. The experiment was performed on buccal pouch mucosal cells isolated from 80 pubertal crossbred Landrace gilts. After collection, the cells were treated enzymatically and transferred into a primary in vitro culture (IVC) system and cultured for 30 days. The cells were collected for RNA isolation after 7, 15 and 30 days of IVC and were checked for their real-time proliferative status using the RTCA system. We found an increased expression of FN1 and SOX9 genes when calculated against ACTB after 7, and 30 days of IVC, (P less than 0.01, P less than 0.001, respectively). The CXCL12 mRNA was down-regulated after 7, 15 and 30 days of IVC, but not statistically significant. Similar expression profile was observed when calculated against HPRT, however, DAB2 was found to be higher expressed at day 15 of IVC, (P less than 0.05). The cell index measured during real-time cell proliferation was substantially increased between 96 h and 147h of IVC and reached the log phase. Since FN1 and SOX9 revealed significant increase of expression after long-term culture in vitro, it is suggested that expression of these differentiation and stemness genes is accompanied by cell proliferation. Moreover, FN1 and SOX9 might be recognized as new markers of buccal pouch mucosal cell proliferation and differentiation in pigs in in vitro primary culture model.

[Fertility preserving methods in women with breast cancer before gonadotoxic therapy]. / Zachování fertility u zen s karcinomem prsu pred gonadotoxickou lécbou.

OBJECTIVE: To present current options and own experiences with ovarian stimulation in young women with breast cancer before gonadotoxic therapy. DESIGN: Review article with own experiencies. METHODS: Literary search, analysis of own experiences with complex therapy in women with breast cancer planning pregnancy, definitions of stimulating protocols, using methods of assisted reproduction. SETTING: Department of Obstetrics and Gynecology, Faculty of Medicine, Masaryk University and University Hospital Brno. RESULTS: Embryo and oocyte cryopreservation are important methods of fertility preservation requiring controlled ovarian stimulation before the start of chemotherapy. Current studies demonstrante effectivenes of this special stimulating protocol and did not find negative side effects such a progression of the breast cancer. Since January 2016 to June 2016 we performed controlled ovarian stimulation and oocyte retrieval in three patients with breast cancer before chemotherapy. We used short stimulation protocol with recombinant FSH, GnRH agonists and letrozole and retrieved 13, 12 and 9 oocytes. We cryopreserved 8 and 6 embryos, one woman prefered freezing of 12 oocytes. During the stimulation estradiol level did not exceed 1,6 nmol/l. CONCLUSION: Neither the normal nor the cancerous cells in the breast react to the gonadotropins FSH, LH, nor to hCG. Conversely, there is a cellular proliferation and an increase in cancer cell lines with oestrogen receptors with exposure to oestrogen, and it is dose - dependent. The special stimulation protocol with FSH, GnRH agonists and letrozole is effective in gain of efficient amount of gamets with minimal increase of estradiole level. Current study reviews did not find higher risk of progression of breast cancer in association with ovarian stimulation. It is important to respect the recomendation of oncology committee and to effectively suppress the estradiol level.

Expression of cell mitotic progression proteins and keratinocyte markers in porcine buccal pouch mucosal cells during short-term, real-time primary culture.

The porcine model is often used in clinical trials. The pig has many fundamental anatomic, physiological and nutritional similarities to humans. Additionally, the European Medicines Agency (EMA) demands the use large animals in clinical studies. Oral mucosa has received special attention due to its regenerative properties. Oral tissue is composed of several types of cells including fibroblasts and keratinocytes. The porcine oral mucosa/buccal pouch mucosa has a cellular structure with defined proliferation and differentiated capability. In this study, we investigated the expression pattern of porcine buccal pouch mucosal cell proliferation and differentiation markers such as Ki-67, proliferating cell nuclear antigen (PCNA), and involucrin. We observed a clear monolayer culture of spindle-shaped, porcine buccal pouch mucosal cells during 168 h of real-time in vitro culture. The RTCA assays revealed parametric and progressive increases in proliferation after 72 h of IVC. We found an altered proliferation index (PI) in the replicated groups of experiments except through the 144-168 h proliferation period. The RT-qPCR results demonstrated a significant increase in Ki-67 and PCNA expression after 48, 120, and 168 h of IVC as compared to other culture periods (P<0.001). The involucrin mRNA displayed increased expression after 168 h of IVC as compared to other periods. We observed a lack of PCR product at 24 h in the case of Ki-67 and both before IVC (0h) and after 24 h of IVC for PCNA mRNA. When we analyzed the three transcripts together, we found the highest expression of involucrin during each of the culture periods. It has been suggested that Ki-67, PCNA, and involucrin may be successfully used as markers of porcine buccal pouch mucosal cell proliferation and differentiation capability in vitro.

The origin, in vitro differentiation, and stemness specificity of progenitor cells.

Since the successful collection of the first progenitor stem cells (SCs), there has been an increased interest in these cells as a model for undiscovered and unlimited potential of differentiation and development. Additionally, it was shown that SC populations display an ability to form pluripotent and/or totipotent cell populations. It was found that human ovarian granulosa cells (GCs) maintain a large capacity for differentiation into several other cell lineages, such as chondrogenic, osteogenic, neurogenic, and adipogenic, particularly during long-term, in vitro culture. In these cases, the specific media supplements that promote various pathways of differentiation, such as leukemia-inhibiting factor (LIF) and/or FSH, are well recognized. However, these are only some examples of the differentiation possibilities of human SCs in vitro and other pathways still require further investigation. Many SC populations, which are directed to differentiate into specific cell types, are also successfully used in several human disease therapies, e.g. leukemia. Moreover, SCs are used for tissue scaffold construction in patients with respiratory and cardiovascular diseases. In this review, the most recent knowledge about the in vitro growth and differentiation capacity of SCs is presented. Furthermore, we discuss the possible worldwide application of SCs in advanced cell and tissue bioengineering. In conclusion, it is suggested that, in the future, SCs will be a basic strategy in human therapy, and their use will open new gates in regenerative and reconstructive medicine in the 21st century.

[Current issues of reproductive medicine in the Czech Republic]. / Soucasná témata reprodukcní medicíny v Ceské republice.

INTRODUCTION: During the 25th symposium of assisted reproduction in Brno was lunch time organised as the lunch table discussion on the selected topics of assisted reproduction. More than 150 specialists reviewed themes related to gynecology and embryology.Discussed topics: Lunch table discussion covered the following topics: (1) Cross-border health care in assisted reproduction; (2) Indication for PGS (preimplantation genetic screening) in the context of actual information; (3) Does ovarian stimulation belong to the ambulance of registering gynecologists? (4) Therapy with clomifen - only for IVF specialists? (5) How and with whom should psychological support be directed during IVF? (6) Stimulation in women with low ovarian reserve; (7) Is basic semen analyses sufficient? (8) Time-lapse systems as relevant markers of embryonic development; (9) How to be oriented with choices of media and consumables in the IVF lab, and (10) "Freeze All" - is this new trend in cryopreservation suitable for all? CONCLUSIONS: Panel conclusions were presented during the afternoon session, which had great attendance, featured lively commentary, and produced some definitive consensus. Certain issues remained inconclusive, and these matters will be the subject of further discussion in the future. Specific summation of all deductions is presented in this paper.

Microfluidic versus molecular assays - different approaches in assessing oocyte developmental competence.

In recent years, molecular techniques have brought about new solutions that focus on the developmental capacity of female oocytes and reproductive performance in the mammalian species. The developmental potency is the ability of oocytes to reach the MII stage following the long stages of folliculo- and oogenesis. The main proteins involved in this process belong to the connexin (Cx) family, which are responsible for the formation of gap junction (GJC) connections between the female gamete and surrounding somatic cells. The Cx are involved in bi-directional transport of small molecules and are therefore responsible for correct oocyte-somatic cell nutrition, proliferation, and differentiation. However, the application of certain molecular techniques often leads to destabilization or destruction of the materials of interest, such as cells or whole tissues. Therefore, the applications of microfluidic methods, which are non-invasive and quantitative, give new opportunities to further this area of biomedical research. Microfluidic research is based on real-time experiments that allow for control and/ or observation of the results during each step. The purpose of this review is to present both positive and negative aspects of molecular-microfluidic methods while describing the role of connexins in oocyte developmental capacity.

Energy Status Characteristics of Porcine Oocytes During In Vitro Maturation is Influenced by Their Meiotic Competence.

The characteristics of energy status in porcine oocytes as related to their meiotic competence and in vitro maturation were studied. Cycling pubertal gilts in the early luteal to early follicular phases of the ovarian cycle were used as oocyte donors. The oocytes recovered from medium (MF) or small follicles (SF) were considered meiotically more or less competent, respectively. A half of oocytes from each category was matured by the standard protocol. The oocytes were examined before or after maturation by confocal microscopy, a bioluminescent cell assay and Western blotting. Four experiments, each in triplicate, were performed to assess both SF and MF oocytes in terms of metabolic units formed by mitochondria and lipids, ATP and lipid consumption and lipid droplets with adipose differentiation-related protein (ADRP) expression. The proportion of oocytes with metabolic units, the mean ATP content and the number of lipid droplets per oocyte, and the relative number of lipid droplets with ADRP expression were significantly higher in the MF compared to SF oocytes before maturation. On the other hand, after maturation, there was an increase in the proportion of oocytes with metabolic units and the relative number of lipid droplets with ADRP expression in the SF compared to MF oocytes. In conclusion, specific differences in energy characteristics between porcine oocytes with different meiotic competence were found. Meiotically more competent oocytes are more advanced in terms of energy reserves before maturation, while meiotically less competent oocytes are more active in replenishing energy stores during maturation.

Mitochondrial patterns in bovine oocytes with different meiotic competence related to their in vitro maturation.

This study was designed to specify chromatin and mitochondrial patterns in bovine oocytes with different meiotic competence in relation to maturation progress, resumption of meiosis, MII onset and completion of maturation. Oocytes with greater or lesser meiotic competence, recovered separately from medium (MF) and small follicles (SF), were categorized according to morphology. Four oocyte categories, healthy and light-atretic MF and healthy and light-atretic SF oocytes were matured and collected at 0, 3, 7, 16 and 24 h of maturation. Specific differences in terms of chromatin and mitochondrial patterns were found among the maturing oocyte categories. Resumption of meiosis was accelerated in light-atretic oocytes, as compared with healthy oocytes, regardless of their meiotic competence. More competent oocytes activated mitochondria twice during maturation, before resumption of meiosis and before completion of maturation, while less competent oocytes did it only once, before completion of maturation. Changes in mitochondrial activity differed in light-atretic compared with healthy in both more and less competent oocytes. Healthy meiotically more competent oocytes formed clusters and produced ATP for the whole time of maturation until its completion, while light-atretic more competent oocytes and healthy less competent oocytes reduced these activities earlier, at MII onset. Contrary to these oocyte categories, light-atretic less competent oocytes increased cluster formation significantly before resumption of meiosis. It can be concluded that bovine oocytes with different meiotic competence and health differed in the kinetics of mitochondrial patterns during maturation.