Genetic Aspects of Male Infertility

Summary: Genetic factor appears as the most prominent cause for elimination of early embryos, fetuses and reason for spontaneous abortions. Still, there are technical difficulties for good pre-implantation diagnosis on chromosomal level in gametes. Artificial insemination in vitro has to be very carefully supervised and evaluated on genetic level. It is discussed the possible rise of genetic defects associated with the age of parents. Teratozoospermia seems to present complex diagnostic issue as well as the therapeutic challenge.
 

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Effects of Obesity and Age on the Results of Ovulation Induction

Summary: There are various factors affecting the results of controlled ovarian stimulation. Obesity and age of the patients are being considered as important contributors in this treatment. In obese women hormonal disorders occur more frequently than the normal weight women. There is a correlation between the menstrual disorders as well as infertility and androidal type of obesity. Fertility significantly decreases in women above 35 years of age. This may be associated with the lower number of oocytes retrived in this group after the routine ovulation protocols. However further studies are required to confirm these data.

Key Words: infertility, ovarian stimulation, obesity

Assessment of Human Embryos Quality in IVF-ET Programme

Summary: The overall live birth rates following IVF-ET treatment have remained low. Most of IVF-ET centres replace two – three selected embryos on the second day of culture at the 4 cells stage. The assessment of embryos in this stage is based on: key morphological features and cleavage rate. The quality of replaced embryos and their potential for implantation are of considerable importance. The embryos which appear to be good and show appropriate morphological grade and growth will be selected for transfer on this basis, however in fact it will be cytogenetically abnormal and may contribute to implantation failure. The main challenges of current medicine of reproduction is to elaborate methods in order to optimise conditions of obtaining and identifying embryos with full developmental and implantation potential. In this article the author presents the strategy of sequential assessment of the quality of embryos in IVF-ET programme i.e. assessment of oocytes, pronuclear stage embryos, cleavage stage embryos and blastocystes stage. In the future the embryos selected in this way probably should give acceptable pregnancy rate and low incidence of multiple pregnancy rate. The single embryo transfer in IVF-ET programmes will hopefully become reality.

Słowa kluczowe: human embryos, zygotes, blastocysts, scoring of human embryos.
 

Genetic Aspects of Early Pregnancy Loss

Summary: Genetics aspects of miscarriages (5% frequency of total miscarriages) can be categorised as: I. karyotypic abnormality or II. defect in one or more genes. I. In autosomal and sex chromosomal abnormalities appear: 1) letal miscarriages sex independent a) in preimplantation period estimate mainy during IVF-ET procedure (about 25%), b) early primary abortion (5% genetic defected fetuses) when half of lost conceptus presented trisomies, less than 10% monosomy X and triploidy and 3% tetraploidy,  c) II trimester primary abortion (20% from 2.5% miscarriages),  d) reccurent abortion (with 3% of genetic factor); 2) X-linked genetic disorder lethal in males as incontinentia pigmenti or Rett syndrome; 3) defects of trophoblast as molar pregnancy or chorion mosaicism. II. The groups of molecular mutations  were discussed: 1) in haemostatic disorders with predisposition to thrombosis of placental bed vessels a) Factor V Leiden  (G1691A), b) Factor II prothrombin gene (G20210A); 2) associated with hyperhomocysteinemia, the risk factor for venous thrombosis a) autosomal recessive inherited deficiency of cystathionine b-synthase, b) homozygosity for the 677C->T mutation in the methylenetetrahydrofolate reductase gene (T/T genotype), 3) mutant allele of mannan binding protein responsible for its deficiency caused infection-induced fetal loss.

Key Words: abortus, chromosomal aberration, mutations

Gonadoliberin-Dependent Transcription Activity of Genes Encoding Luteinizing Hormone Subunits in the Pituitary Gland

Summary: Gonadoliberin (GnRH) is a crucial regulator governing the specific expression of the genes encoding gonadotropin subunits (?,LHb, FSHb) in the pituitary gland. Three proteins: SP-1, steroidogenic factor 1 (SF-1) and early growth response protein (EGR-1) form a tripartite element in the rat luteinizing hormone b-gene regulating its GnRH-dependent transcription activity. Localized on the promotor of LHb gene the CArG box element (position -443, sequence: CCATTTTTGG) is responsible for the gene responsiveness for pulsatile GnRH stimulation. GnRH regulates a subunit gene transcription activity through two ETS binding protein domains localized on the promoter of this gene.

Key Words: gonadoliberin, transcription activity, b, LHb subunits genes
 

Role of Opioids in the Local Regulation of Secretory Functions of the Porcine Pituitary and Ovary

Summary: Summary: In the article, data pertaining to in vitro immunoreactive b-endorphin secretion by the porcine anterior pituitary cells and various types of ovarian cells: luteal, granulosa and theca are presented. The role of opioids in the local regulation of LH secretion by the pituitary and steroid hormones by the ovary in the pig has been also described.

Key Words: opioids, b-endorphin, pituitary, ovary, pig.
 

Mechanism of Prolactin Action in Female Reproductive Tract

Summary: Prolactin regulates many diverse biological processes in ovary and uterus. Particular variants of prolactin can bind different forms of the prolactin receptor, activate various cellular signaling pathways and regulate a diverse set of genes. The specific prolactin action depends on tissue, species and reproductive period. Despite the recent effort of many research groups, most of the details concerning the relationship between biological function of prolactin and the specific signal transduction pathway remains to be explored.

Key Words: prolactin, ovary, uterus, receptors, signal transduction
 
 

Involvement of Noradrenergic System in the Corpus Luteum Function

Summary: Data from our original studies on the involvement of catecholamines in secretory function of corpus luteum (CL) are summarised. Short-lasted stimulation of b-receptors  in CL increases progesterone (P4) and ovarian oxytocin (OT) secretion within a few minutes, whereas blockade of b-receptors decreases basal secretion of P4 by 20–40%. Both  b1- and b2-receptors take part in this process. Amount of these receptors are positively correlated with peripheral concentrations of P4 and negatively with concentrations of NA and dopamine (DA) in luteal tissue. CL is able to synthesise NA from DA as a precursor, and this suggests the presence of dopamine b-hydroxylase in this tissue. NA stimulates the post-translational processing of OT and synthesis of P4 by increase activity of cytochrome P450scc and 3b-HSD. Moreover, NA affects gene expression for StAR protein. Short-lasted changes in the ovarian blood flow evoked by NA does not impair CL function. These data indicate that CL itself is able to regulate own function, which is essential in short-lasting stress in pregnant females.

Key Words: noradrenaline, b-receptors, corpus luteum, progesterone, oxytocin
 

Leptin and Reproduction

Summary: Leptin was discovered in 1994 as a product of ob gene. Primarily, leptin is synthesized by white adipose tissue, and in addition in ovary, placenta, and lactating mammary gland. Leptin acts on the level of the hypothalamic nuclei, participating in regulation of energy balance and reproductive function via modulation of hypothalamic-pituitary function. In genetically obese and infertile ob/ob mouse, administration of exogenic leptin rescues their fertility, both females and males. In normal rats, leptin stimulates gonadoliberine (LHRH) release from the hypothalamus and luteinizing hormone (LH) from the pituitary gland. Furthermore, the distinct stimulatory role of central leptin action is realized at puberty, and during inhibition of gonadotrophins release due to fasting or dietary energy restriction. Leptin also directly influences ovary function. However, exact role of leptin action in physiology and pathophysiology in murine and human reproductive system needs to be further elucidated.

Key Words: leptin, leptin receptors, LHRH, LH, FSH, puberty, ovary
 

The Involvement of GABA_A Receptors in the Control of Gonadoliberin Release in Anestrous Ewes

Summary: To examine the role of g-aminobutyric acid receptors A (GABA_A ) in the control of gonadoliberin (GnRH) release, the extracellular concentrations of GnRH, b-endorphin, catecholamines and their main metabolites MHPG and DOPAC were quantified during local infusion of muscimol and bicuculline (agonist and antagonist of GABA_A receptors respectively) to the nucleus infundibularis-median eminence (NI/ME) and the medial preoptic area (MPOA) in anestrous ewes. Stimulation of GABA_A receptors in NI/ME significantly attenuated GnRH release with concomitant increase of b-endorphin, dopamine, MHPG and DOPAC levels. Blockade of the GABA_A  receptors in this structure of the hypothalamus did not affected GnRH and noradrenaline release but inhibited b-endorphin release and decreased dopaminergic activity. In MPOA, infusion of muscimol increased noradrenergic activity and decreased dopaminergic activity. We did not observed any changes in GnRH and b-endorphin levels, but luteinizing hormone (LH) levels in the blood plasma were significantly decreased. Blockade of GABAA receptors with bicuculline had no evident effect neither on catecholaminergic system activity nor b-endorphin-like immunoreactivity. These results suggest, that GABA may suppress GnRH release directly through GABA_A receptors located on the axon terminals or on perikaria of the GnRH neurons in NI/ME and MPOA or indirectly by GABA_A receptors located on b-endorphinergic and catecholaminergic neurons.

Key Words: GABA_A receptors, GnRH, ewe, hypothalamus,
 

Molecular Pathways Involved in Human Menstruation

Summary: Endocrine changes occurring during the menstrual cycle influence the morphology and function of epithelial and stromal cells of the endometrium preparing them for implantation. If conception does not occur, the corpus luteum regress and hormonal changes lead to menstruation. Mechanism of tissue shedding during menstruation involving changes in cellular membranes function, vessels reactions, immune cell paracrine stimulation and induction of apoptosis are discussed in view of experimental data.

Key Words: endometrium, menstruation, cell adhesion, apoptosis.
 

Polispermic Fertilization in Birds – the Role of DNases in Degradation of Supernumerary Spermatozoa

Summary: Birds exhibit physiological polyspermy, i.e. numerous spermatozoa enter the cytoplasm of an oocyte during normal fertilization, but only one sperm nucleus participates in formation of a zygote nucleus, whereas the accessory sperm nuclei degenerate at the early cleavage stages. The fate of supernumerary sperm nuclei and the mechanism responsible for their degradation are not clear. The results of our in vitro studies showed, in oocytes of the Japanese quail, the existence of high DNase I and II activities which are able to digest not only naked phage  l DNA but also DNA contained in quail sperm. We suppose that the enzymes participate in degradation of DNA of supernumerary sperm entering the oocyte during polyspermic fertilization in birds. Thus they could be one of the factors of the late block to polyspermy in the cytoplasm of avian eggs. The presence of high DNase activities in avian oocytes might be also responsible for difficulties in obtaining transgenic birds by microinjection of exogenous DNA into the cytoplasm of fertilized ovum.

Key Words: birds, oocytes, polyspermy, DNase

Role Of Acid Glycosidases in the Fertilization Process

Summary: In mammalian testes, acid glycosidases (EC 3.2.1.) are found in lysosomes of somatic and germ cells. Their acrosomal forms appear at the stage of spermatogenesis in reproductive cells, while their lysosomal forms are discarded. Maturation of spermatozoa takes place in the epididymis. In this process, surface glycoproteins of the spermatozoon can be eliminated, masked and modified by proteinases, glycosyltransferases and acid glycosidases, which are synthesized and secreted by epithelial cells. It has also been suggested that acid glycosidases are involved in binding the spermatozoon to surface glycoproteins of the zona pellucida of an ovum. More puzzling questions than conclusive evidence appear from studies on the role of acid glycosidases in the fertilization process. Recent studies have provided conclusive evidence to answer the question why acid glycosidases, although optimally active in the acid pH range, occur and can function in the neutral pH environment of the reproductive tract. It turned out that acid glycosidases become active in the neutral environment in the presence of their specific (natural) substrates, and that this process can be additionally conditioned by the degree of association/dissociation between enzymatic subunits.

Key Words: acid glycosidases, fertilization

Analysis of the Growth Hormone Gene Expression in Bovine Embryos

Summary: Early embryonic development is controlled by both maternal and embryonic factors. Oocyte maturation, fertilisation and subsequent development are characterised by the lack of an effective transcription thus these processes are fully controlled by factors of maternal origin. The stability of maternally derived transcripts depends on the length of the poly (A) tail that may be elongated or shortened. The embryonic genome activation is a species dependent phenomenon, it takes place after few blastomere divisions and is accompanied by gradual decrease of maternal RNA. Several studies showed significant differences in gene expression profiles between in vivo and in vitro produced embryos. Growth hormone produced in the pituitary gland is known to stimulate postnatal growth. It`s functions in reproduction and embryonic development  is still not clear. It was shown that the GH gene is expressed in bovine oocytes and preimplantation bovine, murine and rat embryos. GH receptor mRNA was localised in cattle ovary, uterus and placenta as well as in bovine and murine embryos. Transcripts for several growth factors (eg. IGF-I, IGF-II) were also detected in embryos. A positive effect of the exogenous GH on bovine and murine oocytes matured in vitro was also shown. The expression of the GH is mainly regulated by the pituitary transcription factor Pit-1. The last findings demonstrated the presence of Pit-1 mRNA in bovine oocytes, zygotes and embryos up to the 8 cell stage. These results may suggest that growth hormone may act in a paracrine or autocrine way in early embryonic development.

Key Words: embryo, GH, Pit-1, genome activation, gene expression regulation.
 

Role of Homeotic Genes in the Development of Mammary Gland

Summary: The mammary gland in its development undergoes several stages and is regulated by hormones, interaction of epithelial cells with extracellular matrix and mezenchymal cells. Molecular mechanisms of most of these processes are poorly understood. Homeotic genes, particularly Muscle Segment Homeobox (Msx) genes, regulate epithelial-mesenchymal interactions and are essential for development of limb, tooth and placenta. Msx transcription factors regulate gene expression directly by binding to their DNA-binding sites or indirectly – by protein-protein interactions with others transcription factors. Recently performed investigations showed that all proteins forming transcription complexes of Msx2 are expressed in the mammary gland. Their expression is tightly correlated with growth and branching of the milk ducts. That allows suggesting that Msx participate in signal transduction from estrogen and progesterone to target genes controlling growth and branching of milk ducts. Recently, experiments aiming to identify these genes are in progress.

Key Words: mammary gland, homeotic genes, Msx.

High and Low Molecular Weight Acid Phosphatases of the Male Reproductive Tract

Summary: The different molecular forms of acid phosphatases occurring in the tissues and fluids of the male reproductive tract have been identified. The defined glycoprotein character of acid phosphatases, differing in the amount and structure of the oligosaccharide units, determine their microheterogeneity and molecular properties. The role of protein tyrosine phosphatases and their diagnostic significance in the patophysiology of accessory sex glands have been defined. These studies have characterized acid phosphatases of boar reproductive system.

Key Words: acid phosphatases, protein tyrosine phosphatases, male, reproductive tract, seminal plasma
 

Somatic Cloning: Perspectives for Animal Breeding, Pharmacy, Medicine, and for Preservation of Endangered Mammalian Breeds and Species

Summary: Although the efficiency of somatic cloning does not exceed 1–2% of born animals in relation to reconstituted oocytes, the cloning is being used to obtain transgenic animals producing human therapeutic proteins in milk or urine, to rescue endangered breeds and species of mammals, and even to restitute some that are already extinct. Work is in progress aimed at obtaining – through somatic cloning – the organs for xenotransplantation, as well as differentiated cells creating perspectives for cell therapy of many illnesses. The doubts are discussed, which have emerged from up to now experiments and concern the mechanisms underlying methods of cloning (oocyte activation, the choice of G0/G1 phase of the cell cycle of donor nucleus) and the causes of developmental abnormalities in cloned animals as well as the age of cloned specimen.

Key Words: somatic cloning, cell cycle, animal breeding, developmental abnormalities, rare.and endangered breeds and species
 

Improvement of Reproductive Potential by In Vitro Culture of Preantral and Early Antral Ovarian Follicles in Mammals

Summary: The ovary of mammals contains thousands of oocytes which are enclosed in the preantral follicles. Since >99.9% of ovarian oocytes undergo atresia, it would be of great practical benefit if these follicles, destined to become atretic, could be rescued by a long-term in vitro culture leading to provide an abundant source of gametes and allowing for maximal improvement of the reproductive potential of female. The first studies on preantral follicles were performed in mice and rats, and the pioneer article was published by Grob in 1964. In the mouse, research on the isolated preantral follicles has led to follicle growth and antrum formation, ovulation in vitro and even to a few live young after in vitro maturation and fertilization of the oocytes recovered from in vitro cultured preantral and even  primordial follicles. The successful results obtained in mouse are encouraging for development of methods for culture of preantral follicles in farm animals. In the article are discussed techniques of follicle recovery, in vitro culture systems, methods for evaluation of oocyte growth and competence for maturity and the recent progress in development of methods in farm animals.

Key Words: ovary, follicles, long-term culture, oocyte growth and competence
 

Recent Advances in Mammalian Oocyte Cryopreservation and Perspectives of their Application in Medicine and Veterinary

Summary: There are many possible practical applications of mammalian oocyte cryopreservation, providing the methods are efficient. Of practitioners interest is organizing banks of oocytes in order to conserve genetic resources, and to increase an impact of female genetic potential in animal breeding. Human medicine needs en established technology of oocyte cryopreservation, to improve moral and logistic circumstances of artificial (in vitro) infertility treatment. Experiments on cloning of animals needs a great number of cytoplasts, which might by easier prepared if sufficient number of oocytes is collected. However, many years of experiments on mammalian oocyte cryopreservation gave acceptable results only in the case of mouse, rabbit and (to some extent) human. Even considering limited ratio of bovine embryos that developed after in vitro fertilization of frozen or vitrified oocytes and some few calves born (primarily in Japan) it was difficult until recently to treat the general efficiency of bovine oocyte cryopreservation as satisfactory. A real breakthrough in bovine oocyte survival was achieved when minimum sample size vitrification methods was introduced. These methods which are referred in this paper to as an „open” vitrification system, gave high survival rate of fragile embryos (pig, hamster, cleavage stage bovine embryos) and especially, when combined with a gentle pre-equilibration of cells, gave a high survival rate of vitrified bovine oocytes.

Key Words: vitrification, minimum sample size, oocyte, pre-equilibration, bovine, human
 

Transplantation of Cells in Tumor Therapy

Summary: One of the promising treatment modalities is to use tumor cells as a form of tumor vaccine. Isolated tumor cells are usually transfected with genes encoding antigen presenting MHC molecules, costimulatory CD80/CD86 and/or cytokines stimulating the immune response. Dendritic cells fed with tumor antigens are also being used for vaccination. Another therapeutic regimen is based on application of T cytotoxic cells able to kill tumor cells.

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Transplantation of Chondrocytes

Summary: Chondrocytes express molecules of the major histocompatibility complex class I and II and probably also tissue specific antigen(s). Transplants of isolated syngeneic chondrocytes originating from hyaline or elastic cartilage produce cartilage similar to that from which they originate, but with less regular distribution of cells. Allogeneic chondrocytes also produce cartilage which is infiltrated by lymphocytes and macrophages and gradually resorbed. Furthermore, they cause both cellular and humoral systemic response of  recipients. Autogeneic chondrocytes taken from no-weight-bearing areas of articular cartilage are recently used for the treatment of localized articular cartilage defects. Autogeneic elastic chondrocytes found clinical application in reconstruction of auricular cartilage in children with poorly developed external ear.

Key Words: hyaline cartilage, elastic cartilage, chondrocyte transplantation.
 

New Transplantological Aspects of Early Human Hematopoietic Cells

Summary: Hematopoietic Stem Cells (HSC) are able to reconstitute hematopoiesis after transplantation. The first step in hematopoietic reconstitution is a homing of early hematopoietic cells to the bone marrow microenvironment. The mechanisms which regulate homing of hematopoietic stem cells after transplantation involve orchestrated action of several adhesion molecules, proteolytic enzymes and chemotactic factors. For example, it is well known that early hematopoietic cells isolated from mobilized peripheral blood engraft faster after transplantation if compared to cells harvested directly from the bone marrow. We explain this observation by the fact that mobilized peripheral blood-derived early hematopoietic cells are highly covered with platelet-derived microparticles, which enhance both adhesion and homing of transplanted HSC into bone marrow.

Key Words: CD34+cells, hematopoietic transplants, microparticles, peripheral blood platelets.
 

Justyna DRUKAŁA, Kazimierz CIEŚLIK

The Use of In Vitro Cultured Keratinocytes for Transplantation

Summary: Conventional methods of skin wound healing are not sufficiently efficient and there is a need to construct skin equivalents. The ability of carrying out large-scale cultures from small human tissue biopsies leads to the practical use of these achievements in medicine. Our experience in this field concerns the use of cultured autologous keratinocytes for healing of burns and trophic leg ulcers. Keratinocytes seeded on fibrin glue induced efficiently wound closure and healing.

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Some Applications of Cells in Biotechnology

Summary: In biotechnology, biological systems are used for production of substances biologically active as well as for construction of modified cell products. In the article are presented some examples of artificial organs and drug delivery systems as well as materials used in those applications.

Key Words: microencapsulation of cells, hollow fiber encapsulation.