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J Tesarik

Publications and source records attributed to J Tesarik.

At least 73 records · Page 4Linked to original sources

Developmental changes in calcium content of ultrastructurally distinct subcellular compartments of preimplantation human embryos.

The ultrastructural localization of mobilizable Ca2+ in different subcellular compartments of human oocytes and preimplantation embryos was studied using the potassium-pyroantimonate technique and transmission electron microscopy; the specificity was confirmed by chelation experiments and X-ray microanalysis. In unfertilized oocytes, Ca2+ was detected in small vesicles beneath the plasma membrane as well as in other forms of smooth endoplasmic reticulum (SER) and in mitochondria but not in cortical granules. In pronuclear zygotes and blastomeres of cleaving embryos, Ca(2+)-rich vesicles were no longer present close to the plasma membrane, and the entire periphery was poor in Ca(2+)-containing organelles which, however, were abundant in the perinuclear region. The uneven Ca2+ loading of SER and mitochondria from the pronuclear stage onwards suggests that Ca2+ release from both these types of organelle contributes to the embryonic Ca2+ signals. During mitosis, less Ca2+ was detected with organelles, but the antimonate reaction product was more abundant in the cytosol. These data suggest that, in addition to different forms of SER, mitochondria also act as a source of mobilizable Ca2+ in preimplantation human embryos. The previously described developmental and cell cycle related changes in the characteristics of Ca2+ signals are associated with the redistribution and structural reorganization of these organelles.

Antimony↗

Regulation of protein tyrosine phosphorylation in human sperm by a calcium/calmodulin-dependent mechanism: identification of A kinase anchor proteins as major substrates for tyrosine phosphorylation.

Signal transduction pathways regulate various aspects of mammalian sperm function. When human sperm were incubated in a medium supporting capacitation, proteins became tyrosine-phosphorylated in a time-dependent manner. This phosphorylation was inhibited by genistein, a protein tyrosine kinase inhibitor. Phosphorylation was also reduced when sperm were incubated either in the presence of increasing concentrations of extracellular Ca2+ or in a medium containing the Ca2+ ionophore A23187. This Ca2+-induced dephosphorylation was calmodulin-dependent, suggesting that calcineurin was involved. In this regard, the calcineurin inhibitor deltamethrin inhibited the Ca2+ ionophore-induced dephosphorylation. A limited number of Mr 80,000-105,000 polypeptides were the most prominent phosphotyrosine-containing proteins present in human sperm. Unlike mouse sperm, which contains a tyrosine-phosphorylated isoform of hexokinase, a phosphotyrosine-containing hexokinase in human sperm was not detected. Most of the tyrosine-phosphorylated proteins were Triton X-100-insoluble and were localized to the principal piece of the flagellum, the region where the cytoskeletal fibrous sheath is found. Prominent phosphotyrosine-containing proteins of Mr 82,000 and 97,000 were identified as the human homologues of mouse sperm AKAP82, the major fibrous sheath protein, and pro-AKAP82, its precursor polypeptide, respectively. These proteins are A Kinase Anchor Proteins, polypeptides that sequester protein kinase A to subcellular locations. Taken together, these results suggest that protein tyrosine phosphorylation may be part of a signal transduction cascade(s) regulating events pertaining to capacitation and/or motility in mammalian sperm and that an interrelationship between tyrosine kinase and cAMP signaling pathways exists in these cells.

Alkaloids↗

The occurrence and identification of round spermatids in the ejaculate of men with nonobstructive azoospermia.

OBJECTIVE: To evaluate the occurrence of round spermatids in the ejaculate of men suffering from nonobstructive azoospermia as a prerequisite for the therapeutic use of round spermatid injection into oocytes. DESIGN: Prospective study. SETTING: University-based diagnostic and research laboratory. PATIENT(S): Azoospermic men previously thought to be definitively infertile whose cases were revisited in view of the novel method of fertilization by round spermatid injection into oocytes. INTERVENTION(S): Smear preparations of cells from patients' ejaculates were stained by three different methods: Papanicolaou, fluorescein-labeled Pisum sativum agglutinin binding, and antiacrosin antiserum immunolabeling. MAIN OUTCOME MEASURE(S): Search for round spermatids in smear preparations of cells from azoospermic ejaculates. RESULT(S): Of 124 azoospermic men examined, 36 (69%) had round spermatids in the ejaculate. the quantity of round spermatids was not related to serum FSH concentration. CONCLUSION(S): Variable quantities of round spermatids can be detected in the ejaculate of most patients with nonobstructive azoospermia. High serum FSH levels do not predict the absence of spermatids.

Acrosin↗

Combined use of proacrosin immunocytochemistry and autosomal DNA in situ hybridisation for evaluation of human ejaculated germ cells.

The recently reported human pregnancies and births after fertilising oocytes with round spermatids recovered from the ejaculate of men with non-obstructive azoospermia have underscored the need for a more accurate evaluation of the nuclear and cytoplasmic maturation status of ejaculated germ cells. In this study we describe our first experience with a method combining the immunocytochemical visualisation of proacrosin with autosomal DNA fluorescence in situ hybridisation (FISH) to assess ejaculated germ cells from patients with a spermiogenesis defect. The proacrosin immunoreactivity, analysed with the use of the monoclonal antibody 4D4, has been detected in cells of round spermatid size presenting a haploid FISH figure as well as in larger cells whose ploidy corresponds to primary and secondary spermatocytes. These observations are in agreement with previously published results obtained, with the use of the same antibody, by immunocytochemical analysis of histological sections of testicular tissue. All the cells of round spermatid size possessing proacrosin immunoreactivity were found to be haploid by FISH. On the other hand, some of the haploid cells of round spermatid size did not possess proacrosin immunoreactivity. The structural pattern of proacrosin immunoreactivity was highly variable both in spermatids and in younger spermatogenic cells. These data show that cell size is the main criterion to be used for the identification of ejaculated round spermatids, whereas the presence of the developing acrosome represents only an auxiliary criterion. The scoring of acrosomal development in ejaculated spermatids may be useful as part of pre-treatment diagnosis before the inclusion of infertile couples in a spermatid conception programme.

Acrosin↗

Calcium responses of human oocytes after intracytoplasmic injection of leukocytes, spermatocytes and round spermatids.

Oocyte activation in mammals involves the action of a soluble sperm factor (SSF) that enables oocytes to develop a characteristic series of Ca2+ spikes (Ca2+ oscillations). SSF is also likely to be responsible for the Ca2+ oscillations driving oocyte activation after intracytoplasmic sperm injection [ICSI]. With an appropriate injection technique, Ca2+ oscillations do not develop spontaneously after ICSI but can be triggered by subsequent treatment of sperm-injected oocytes with Ca2+ ionophore. Here we show that Ca2+ oscillations, quite similar to those developing after ICSI, can be triggered by the ionophore treatment in human oocytes previously injected with human round spermatids. In contrast, oocytes injected with earlier spermatogenic cell (primary and secondary spermatocytes) and with non-germ cells (polymorphonuclear leukocytes) did not develop Ca2+ oscillations after the ionophore challenge although the subsequent injection of SSF did induce typical Ca2+ oscillations in these oocytes. Disintegration of the plasma membrane of the injected cells was detected in all cases by transmission electron microscopy. Thus, the absence of the typical oscillatory Ca2+ response in spermatocyte-injected oocytes was due to the actual deficiency of SSF in the spermatocytes rather than to a defective responsiveness of the injected oocytes or to the failure of SSF release into the oocyte cytoplasm. The ability of human round spermatids to induce a response to calcium in oocytes that is similar to that induced by mature spermatozoa may be important for normal embryonic development after spermatid conception.

Calcium↗

Developmental changes in calcium dynamics, protein kinase C distribution and endoplasmic reticulum organization in human preimplantation embryos.

Developmental changes in the Ca2+ dynamics of human zygotes and preimplantation embryos were related to changes in the distribution of endoplasmic reticulum (ER) and protein kinase C (PKC). The fertilization-induced Ca2+ oscillations were typically observed over > 5 h, were ryanodine-sensitive and showed a periphery-to-centre propagation of Ca2+ waves. At the same time, ER and PKC were accumulated in the cell periphery. After the appearance of pronuclei, ryanodine-sensitive Ca2+ oscillations of lower amplitude and frequency were observed until the pronuclear breakdown. However, Ca2+ waves then began in the perinuclear region, in the area of ER and PKC accumulation and spread towards the cell periphery. During the second to fourth cell cycle, small sinusoidal Ca2+ fluctuations were observed; sparse higher-amplitude Ca2+ spikes, superimposed on these basal fluctuations, appeared shortly before cell division. The sinusoidal Ca2+ fluctuations were asynchronous in individual blastomeres and disappeared progressively in arrested embryos. The direction of Ca2+ wave propagation and the distribution of ER and PKC were similar to the situation observed in pronuclear zygotes. In contrast to the zygotes, ryanodine did not arrest the Ca2+ oscillations but augmented their amplitude and frequency. These data suggest that human pre-embryos use different mechanisms of Ca2+ signalling in the early post-fertilization period, during the pronuclear development and during cleavage.

Calcium↗

Single cell analysis of tyrosine kinase dependent and independent Ca2+ fluxes in progesterone induced acrosome reaction.

In this study we developed a single cell analysis protocol with which protein tyrosine kinase (PTK)-dependent and independent Ca2+ fluxes occurring in human spermatozoa in response to progesterone were evaluated. By recording the fluorescence emitted by fluo-3-loaded spermatozoa using a confocal laser scanning microscopy system it was possible not only to monitor relative changes in the intracellular free Ca2+ concentration ([Ca2+]i) but also to determine the time at which the acrosomal exocytosis began. The addition of progesterone produced a rapid transient [Ca2+]i increase in 35% of spermatozoa. In approximately 10% of spermatozoa, this initial [Ca2+]i increase was followed by a secondary [Ca2+]i increase beginning 2-10 min after the progesterone addition and leading to the acrosomal exocytosis in most of these spermatozoa. On the other hand, a rapid triggering of exocytosis during the initial [Ca2+]i increase was a relatively infrequent observation. The inhibition of PTK with genistein or herbimycin A did not influence the initial progesterone-induced [Ca2+]i increase but inhibited the secondary [Ca2+]i increase and the ensuing acrosomal exocytosis. The initial PTK-independent Ca2+ response could be induced by progesterone in both non-capacitated and capacitated spermatozoa, whereas the ability to generate the secondary, PTK-dependent response developed during in-vitro capacitation.

Acrosome↗

The role of ryanodine-sensitive Ca2+ stores in the Ca2+ oscillation machine of human oocytes.

This study was undertaken to localize ryanodine-sensitive Ca2+ stores in human oocytes and to evaluate their role in the Ca2+ oscillations responsible for oocyte activation at fertilization. The addition of ryanodine provoked a Ca2+ discharge from stores localized throughout the ooplasm with the exception of the cortical and subcortical peripheral regions. The ryanodine-induced discharge was typically followed by a short series of Ca2+ oscillations that only involved the cytoplasmic region populated by the ryanodine-sensitive stores. In contrast, the Ca2+ oscillations induced by the thiol reagent thimerosal or by spermatozoa at fertilization were of a much longer duration and also involved ryanodine-insensitive stores. Presumably, these ryanodine-insensitive stores are sensitive to inositol 1,4,5-trisphosphate (InsP3). The addition of ryanodine to oocytes during ongoing thimerosal- or sperm-induced Ca2+ oscillations inhibited the oscillations. These data suggest a co-operation between the ryanodine-sensitive and ryanodine-insensitive stores in maintaining the sperm-induced Ca2+ oscillations. In this two-store oscillation model, each periodic [Ca2+]i increase is triggered by a Ca2+ discharge from the peripheral, InsP3-sensitive stores inducing Ca(2+)-induced Ca2+ release from the ryanodine-sensitive stores. However, the pacemaker frequency of the Ca2+ discharges from the InsP3-sensitive stores is conditioned by the actual physiological state of the ryanodine-sensitive stores.

Calcium↗

Effects of protein kinase C activation and inhibition on sperm-, thimerosal-, and ryanodine-induced calcium responses of human oocytes.

Previous data have shown that protein kinase C (PKC) participates in the mechanism of sperm-induced calcium oscillations in mammalian oocytes, but the actual role of this enzyme in the oscillation mechanism is still unknown. In this study we show that drugs modulating PKC activity disturb the oscillations induced by spermatozoa, thimerosal and ryanodine, but in a different way for each of the three oscillogenic agents. Moreover, PKC inhibition interferes with the return of the intracellular free calcium concentration to basal values during the sperm- and ryanodine-induced calcium oscillations, but not during the thimerosal-induced calcium oscillations. When the PKC-modulating drugs were applied before any of the three oscillogens, the subsequent calcium oscillations were also disturbed. However, the first calcium spike induced by spermatozoa and thimerosal was little influenced by PKC activation or inhibition. On the other hand, ryanodine failed to produce any calcium response when the PKC activity was clamped to a high level. These data suggest that sustained high PKC activities impede calcium oscillations by interfering with the opening of the ryanodine-sensitive calcium release channel, whereas sustained low activities of the enzyme paralyse the channel in the open state.

Alkaloids↗

Spermatid injection into human oocytes. I. Laboratory techniques and special features of zygote development.

Spermatid injection into the oocyte cytoplasm has been shown recently to yield viable human embryos developing to term after transfer to the mother. This study provides details of the laboratory techniques related to round spermatid injection (ROSI) and elongated spermatid injection (ELSI) and focuses on some special features of zygote development associated with the use of these types of sperm precursor cells for fertilization. A spermatid-enriched fraction was obtained by centrifugation of cells from azoospermic ejaculates through a discontinuous Percoll gradient column. Individual round or elongated spermatids were identified in this fraction and injected deep into oocytes. Oocyte activation was boosted by a vigorous aspiration of the ooplasm at the time of injection. The fertilization rates after ROSI and ELSI were 45 and 44% respectively. A single large syngamy nucleus was detected in 36% of the zygotes that previously showed two normal-sized pronuclei. This condition did not appear to delay the first cleavage division. These observations underscore the importance of distinguishing the syngamy nucleus of diploid zygotes from the female pronucleus of haploid, parthenogenetically activated eggs.

Cell Separation↗

Spermatid injection into human oocytes. II. Clinical application in the treatment of infertility due to non-obstructive azoospermia.

We have reported recently the first birth after intrauterine transfer of embryos obtained by injection of round spermatids into oocytes in cases of unexpected azoospermia. Here we provide a complete documentation of the series of 11 cases in which this novel method of infertility treatment was employed. In four of these cases, elongated spermatids were identified in the ejaculate, and it was decided to perform elongated spermatid injection (ELSI). In the other six cases, only round spermatids were present, and round spermatid injection (ROSI) was done. In one case, ROSI was given preference to ELSI because of a very poor viability status of elongated spermatids present in the ejaculate. Fertilization of at least one oocyte was achieved in 10 of the 11 treatment cycles; the fertilization rate in these 10 cycles ranged between 7 and 100% with a mean value of 45%. All of the two-pronucleated zygotes cleaved and were transferred to the patient's uterus. A singleton pregnancy was achieved in two ROSI cycles. Both pregnancies developed uneventfully and resulted in the birth of normal infants. These data show the intra-ooplasmic injection of spermatids obtained from the ejaculate may become the treatment of first choice in patients with non-obstructive azoospermia.

Adult↗

Fertilization of oocytes by injecting spermatozoa, spermatids and spermatocytes.

The feasibility of fertilization by injecting spermatozoa into oocytes has increased significantly the possibilities for treatment of severe male infertility. However, the rapidity of human application has raised some concern about potential health hazards for the progeny. Human pregnancies and births have also occurred with the use of immature spermatozoa and spermatids, and normal offspring have been born after the injection of secondary spermatocytes into mouse oocytes. This short review deals with the problems that may arise from the injection technique and from the use of deficient or immature sperm cells for fertilization, with particular attention to human applications. Tests for screening parents and follow-up of children are suggested to control the main suspected risk factors.

Animals↗