OBJECTIVEThe transfer of good quality embryo in the program of assisted reproduction in the case of azoospermia, dg. Sertolli cells only syndrome (SCO sy) + maturation arrest (MA). Testes were assessed and found to have a high occurrence of Sertolli cells and very low occurrence of germinal cells, which were arrested at the round spermatid level. The histological evaluation was hypospermatogenesis gr. 3 (minimum 1 spermatid/sample).DESIGNCase report.SETTINGLaboratory IVF, Iscare, a. s., Department of Biology and Biochemistry of Fertilization, Institute of Molecular Genetics, Czech Academy of Sciences, Prague.SUBJECT AND METHODThe successful integration of three methods provides a solution for this case of azoospermia. Immunology and histology can more exactly diagnose the degree of azoospermia. Detection and visualisation of spermatids using monoclonal antibodies against sperm proteins predicts the eventual occurrence of spermatogenesis, and histological evaluation confirms these immunological findings. Using the information of both methods it is possible to use special in vitro cultivation of testicular cells and so obtain injectable spermatozoa, or precursors of sperm, for the ICSI method.CONCLUSIONThe probability of acquisition of good-quality embryo in the program of assisted reproduction is higher when these three methods are applied in combination.

OBJECTIVE:The transfer of good quality embryo in the program of assisted reproduction in the case of azoospermia, dg. Sertolli cells only syndrome (SCO sy) + maturation arrest (MA). Testes were assessed and found to have a high occurrence of Sertolli cells and very low occurrence of germinal cells, which were arrested at the round spermatid level. The histological evaluation was hypospermatogenesis gr. 3 (minimum 1 spermatid/sample).DESIGN:Case report.SETTING:Laboratory IVF, Iscare, a. s., Department of Biology and Biochemistry of Fertilization, Institute of Molecular Genetics, Czech Academy of Sciences, Prague.SUBJECT AND METHOD:The successful integration of three methods provides a solution for this case of azoospermia. Immunology and histology can more exactly diagnose the degree of azoospermia. Detection and visualisation of spermatids using monoclonal antibodies against sperm proteins predicts the eventual occurrence of spermatogenesis, and histological evaluation confirms these immunological findings. Using the information of both methods it is possible to use special in vitro cultivation of testicular cells and so obtain injectable spermatozoa, or precursors of sperm, for the ICSI method.CONCLUSION:The probability of acquisition of good-quality embryo in the program of assisted reproduction is higher when these three methods are applied in combination.

The aim was to predict of eventual occurrence of spermiogenesis in testicular biopsies in azoospermic patients using the unique monoclonal antibodies (MoAbs), which detect the presence of acrosomal proteins. Cells or cell remnants from ejaculates were visualized with MoAbs, which in immunofluorescence test specificaly labeled intact sperm acrosome.These results were compared with histological results and with evaluations after TESE in IVF laboratory. In the cases, in which no spermatozoa was received, the biopsy cultivation was used. In positive patients the injectable cells were found either immediately or after 36 h of cultivation. Whilst in negative patient only few injectable cells were found. Histological specimen confirmed these results. Our results show, that MoAbs labelling may predict and suggest possible sperm retrieval.

Micromanipulation techniques are widely used in assisted human reproduction and it is logical to assume that successes with recent animal cloning will invariably raise the question of human cloning along with its related ethical problems. However, it is often overlooked that even in animals many complications are still associated with this technique. The purpose of our article is to highlight and discuss some of these problems in the context of the eventual use of nuclear and/or cytoplasmic transfer techniques in assisted human reproduction.

Bovine oocytes were irradiated at germinal vesicle (GV) or metaphase II (MII) stage, after Hoechst staining, with chromosomally focused UV-C (254 nm) or UV-A ( > 330 nm). UV-C irradiation at GV stage did not inhibit germinal vesicle breakdown (GVBD) or chromosomal condensation; spindle formation was abolished and maturation promoting factor (MPF) levels failed to increase. UV-A irradiation at GV stage caused meiotic arrest at anaphase I; MPF levels were lower than control. UV-C irradiation at MII stage led to subsequent abnormal parthenogenetic activation when MPF levels failed to decrease. A normal male but no female pronucleus was formed at fertilization. UV-A irradiation at MII stage also caused abnormal activation; MPF levels declined normally. A normal male and abnormal female pronucleus formed at fertilization. UV-A irradiation results have implications for oocyte evaluation during development using Hoechst staining. UV-C irradiation is a potential means for oocyte enucleation in nuclear transfer.

Mouse germinal vesicle GV-G2 stage oocytes were fused to very early S-phase blastomeres from four cell stage embryos to test whether oocytes have a mechanism which detects replicating DNA and thereafter arrests meiosis at the GV stage. Our results show that oocytes are unable to recognize replicating DNA in transplanted nuclei and undergo germinal vesicle breakdown (GVBD). GVBD and blastomere nucleus breakdown are only blocked by inhibitors of both oocyte maturation and DNA synthesis. These results show that mouse immature oocytes do not possess a feedback control capable of detecting replicating DNA.

The presence of acidic argyrophilic protein(s) in cumulus cells and oocytes was examined in bovine, ovine, porcine and murine cumulus-oocyte complexes (COCs) using a specific silver-staining method. The COCs were isolated from small (1 mm) or large (3-5 mm) antral follicles; they were fixed immediately after slaughter (first group) or after transfer to the laboratory (ca 60 min, second group) and then silver stained according to Likovskí and Smetana (1981). The argyrophilic proteins were accumulated mainly in foot processes of cumulus cells on oocytes isolated from large bovine follicles. The intensity of the reaction was less evident in small follicles. The intensity of staining gradually disappeared from cumulus cells as the maturation continued. Moreover, we were unable to detect similar strong labelling in mouse, pig and sheep COCs. Such a chronology suggests that argyrophilic protein(s) may play a role in the transition from meiotic arrest to resumption in bovine oocytes. Our results also suggest that preliminary events of maturation may occur just after slaughter, ie a relatively long time before the COCs are isolated and cultured in vitro.

It is demonstrated that the microinjection of cytoplasm from competent, fully grown oocytes can induce germinal vesicle breakdown in meiotically incompetent growing oocytes. Injection of cytoplasm from oocytes at methaphase I had a similar effect. The maturation in injected growing oocytes was arrested at the premetaphase or metaphase I stage. Induction of germinal vesicle breakdown by cytoplasm microinjection occurs in a dose-dependent manner.

Pig zona-free and zona-intact ova were successfully penetrated in vitro by ejaculated boar spermatozoa. The oocytes were obtained from antral follicles (2-5 mm in diameter) of slaughtered gilts and matured in M-1 99 with porcine follicular fluid (50%) and follicle stimulating hormone (FSH) (0.25 IU ml-1) for 44 h at 38.5-degrees-C. In vitro fertilisation (IVF) was carried out with a spermatozoa-rich fraction of a fresh boar ejaculate which had been incubated in M-199 supplemented by 10% inactivated bovine serum. The penetration rate of zona-free oocytes was dependent on the duration of sperm incubation. IVF of zona-intact ova was designed to estimate the optimal spermatozoa concentration for insemination. The concentration of (1-1.5) X 10(6) spermatozoa ml-1 gave the best fertilisation results (87%), whereas the concentration below 1 X 10(6) spermatozoa ml-1 resulted in a lower fertilisation rate (62%) but in a higher number of monospermic eggs (36%) and male pronucleus formation (80%).

The aim of this study was to characterize embryonic nucleologenesis by determining the appearance and localization of acid argyrophilic, basic lysine-rich and histone proteins in 8-cell bovine embryos. Two silver staining techniques, ethanolic phosphotungstic acid (PTA) and immunocytochemical methods using specific antibodies, were applied at the ultrastructural level. The silver-stained proteins were detected at the onset of nucleologenesis on the periphery of the dense nucleolus precursor bodies (NPBs). The amounts of these proteins increased during the transformation of the NPBs into the fibrillogranular nucleolus. At this stage the well-developed dense fibrillar components encircling fibrillar centres showed intense staining. PTA-positive (basic lysine-rich) proteins were present within most nucleolar structures during nucleologenesis as well as in the chromatin. Histones H2B, H3 and H4 were concentrated throughout the chromatin including the nucleolus-associated chromatin. At the onset of nucleologenesis, histones were absent in the NPBs. The first weak histone labelling was detected in the multivacuolated NPBs, both in the fibrous mass as well as inside the vacuoles. Nucleolar histones appeared with the massive penetration of DNA into the NPBs. We suggest that nucleologenesis may serve as a criterion of normal early embryonic development and that the proteins involved in the process of nucleologenesis and transcription could be used as chemical markers of nucleolar function.

Pig oocytes were matured in vitro in a modified M-199 medium for 44 h, subjected to electrical stimulation and scored for activation 6 h later. Sham pulsed oocytes, exposed to electroporation medium and an a.c. field, did not develop the female pronucleus any more frequently than occurs spontaneously (8.3% within 50 h of culture). However, a single d.c. pulse proved extremely efficient in activating pig oocytes. Pulses of 0.75-1.65 kV cm-1 lasting 30 or 100 microseconds activated at least 90% of matured oocytes. The developmental pathway taken by the activated oocytes depended on the parameters of the pulse. The lowest effective stimulation (0.45 and 0.60 kV cm-1 for 30 microseconds) frequently produced oocytes that remained in pre-pronuclear stages of activation (29.4 and 42.3%, respectively). Extrusion of the second polar body and creation of one pronucleus was the most frequent type of activation (in up to 88.2% among the activated oocytes). The strongest stimulations used (1.05-1.65 kV cm-1 for 100 microseconds) often yielded oocytes that failed to extrude the second polar body and formed two or more pronuclei (up to 56.3%). Under optimal stimulation (0.75 kV cm-1), the activated oocytes proceed synchronously to interphase of the first mitotic division. Anaphase II is reached within 30 min and telophase Ii at 1 h after application of the pulse. The second polar body is extruded about 2 h after activation. Well-defined swelling pronuclei were found in oocytes 5-6 h after activation. The relationship between the stage of oocyte maturation and susceptibility to activation was investigated. The period of culture in which the oocytes develop the activation competence (32-36 h of culture) overlapped with the period in which the oocytes complete meiosis (28-38 h). This suggests that ageing in meiotic arrest is not essential for pig oocytes to become activated by electric pulses. Activation of pig oocytes was accompanied by release of cortical granules. In sections of control (metaphase II) oocytes, an average of 7.3 intact cortical granules per 10 microns of overlying cytoplasmic membrane was found. This number dropped to 1.5 in 10 microns within 30 min after the pulse.

The effect of 6-dimethylaminopurine (6-DMAP) on germinal vesicle breakdown (GVBD) and maturation in bovine oocytes was investigated in this study. This puromycin analog has been shown to be an inhibitor of phosphorylation. Whereas GVBD occurred in nearly all oocytes (96.8%, 120/124) in control medium, presence of 6-DMAP (2 mM) blocked this process almost completely, irrespective of the presence (98.3% GV, 349/355) or absence (97.1% GV, 165/170) of cumulus cells. When lower concentrations of 6-DMAP were used (100-500 microM), GVBD was observed in 87.9% of oocytes, but their maturation was arrested at late diakinesis-metaphase I stage. The inhibition of GVBD was fully reversible, but most of the metaphase II plates were abnormal (80%). To assess whether the action of 6-DMAP is different from the inhibitors of protein synthesis, metaphase II oocytes were exposed to either cycloheximide or 6-DMAP, respectively. Whereas in cycloheximide-supplemented medium approximately 80% of the oocytes were activated, parthenogenetic activation was much less frequent after incubation in 6-DMAP (14.5%). Fusion studies showed that, even if GVBD occurs in 6-DMAP supplemented medium, the level of the maturation-promoting factor (MPF) is decreased. These experiments may indicate the importance of phosphorylation for GVBD in cattle oocytes.

The factor(s) produced by porcine cumulus cells (cumulus cell factor (s): CCF) was described as quantitatively inhibiting the maturation of oocytes in vitro (Petr et al, 1989). When 1, 10, 20 or 40 cumulus oocyte complexes (COCs) were cultured in a droplet of medium (vol 10 microliters), germinal vesicle breakdown (GVBD) was observed in 85, 78, 57 or 19% of the oocytes, respectively. GVBD was observed in 82, 84, 80 or 90% of cumulus-free oocytes, respectively, when they were cultured at the same numbers per 10-microliters droplet. When 1, 10, 20 or 40 cumulus-free oocytes were cultured under the same conditions in a medium containing 140 dbcAMP per ml, 61, 63, 60 or 58% of them were observed at GVBD. However, when COCs were cultured in a 10 microliter droplet of medium with 140 micrograms of dbcAMP per ml, GVBD occurred in 64, 42, 9 or 0% respectively. Based on these results, we can conclude that dbcAMP exerted a further inhibitory effect on GVBD in pig oocytes cultured under the influence of inhibitory factor(s) from cumulus cells. On the other hand, dbcAMP was shown to partly overcome the effect of CCF on GVBD in porcine oocytes. This suggestion was based on the finding that a 6-h pre-culture of COCs in a medium with 1,000 micrograms of dbcAMP significantly decreased the subsequent effect of CCF (GVBD: 44%) compared with those pre-cultured in a medium with 140 micrograms of dbcAMP/ml (GVBD:5%) or without dbcAMP (GVBD: 15%).(ABSTRACT TRUNCATED AT 250 WORDS)

Oocyte cumulus complexes were aspirated from 3 to 5 mm follicles of cows prestimulated with 2.000 IU PMSG 24 h before slaughter. Oocytes matured in culture were fertilized in vitro by heparinized freshly ejaculated or epididymal spermatozoa. The cultivation procedure for fertilized eggs was the same as that used for cultivation of oocytes. From 163 matured oocytes, 109 cleaved to the 2-cell stage 24 h after fertilization and after 6 days of cultivation, 18 developed to the late morula and 18 to the blastocyst stages. Eleven blastocyts and 1 late morula were transferred surgically to the uteri of 7 recipient heifers. Two heifers became pregnant: one delivered a bull-calf at term, while the other pregnancy resulted in abortion at the 3rd month. The examination of some embryos by transmission electron microscopy showed an almost normal morphology for most cells. The degenerated cells contained mostly electron-dense residual bodies of unknown origin.

Eight-cell cow embryos were isolated and cultured in vitro in a medium enriched with 200 microCi of [5-3H]uridine for 20 min. Epon ultrathin sections of the embryos were investigated for the nucleolar morphology and for the appearance and localization of the sites of [5-3H]uridine incorporation by means of electron microscopic autoradiography. In addition to this, a general pattern of replicated embryonal DNA distribution was revealed by [methyl-3H]thymidine incorporation and light microscopic autoradiography. The essential phases of the transformation of the small nucleolus precursor body (NPB) into a vast, functionally fully active nucleolus, characterized by typical nucleolar substructural components, are taking place within the eight-cell stage. This process differed in its morphology from the nucleologenetic process in early embryogenesis of other mammals, especially of that in the mouse. The first sign of NPB transformation was the appearance of a large central vacuole followed later on by perinucleolar chromatin penetration into NPB, documented by both morphology and [3H]thymidine autoradiography. In some cases, concentration of dense fibrillar material forming clumps or stalks was seen in the central vacuole. The following rapid nucleolar development was characterized by the formation of secondary vacuoles concomitant with the onset of [5-3H]uridine incorporation into the dense fibrillar component and with the appearance of the first granules in the otherwise fibrillar structure of the nucleolus. During the late eight-cell stage, the still-rounded nucleolus developed features of a reticulated nucleolus known from somatic cells intensively synthesizing rRNA: a dense fibrillar component with associated labeling encircling fibrillar centers and a well-developed granular component. The labeled dense fibrillar component was observed mostly in the central area of the nucleolus; early embryonic NPB dense fibrous material not involved in transcription was disappearing rapidly. At the transition to the 16-cell stage the nucleoli lost their rounded shape because of the accumulation of a large amount of granular component, and they occupied a considerable part of the nucleus. In conclusion, the appearance of the nucleolar vacuole in eight-cell cow embryo is the starting point for following morphogenetic events linked with the onset of transcription.

Mouse oocytes at the germinal vesicle (GV) stage were fused with maturing oocytes in which GVs were no longer visible. The fused cells were fixed at different time-intervals after the initiation of fusion and prepared for scanning electron microscope (SEM) observation. Concomitantly, some fused cells were prepared for light microscope evaluation. Our SEM observations showed no significant differences in surface morphology between immature and maturing oocytes. However, immediately after fusion was initiated, dramatic changes occurred on the surface of the maturing oocytes. The microvilli were shortened or disappeared locally and the plasma membrane was deeply ruffled. One hour after fusion, when the giant cells were nearly spherical, the microvilli reappeared and the ruffling gradually disappeared. In some areas, the microvilli were extremely long. Three hours after fusion, the fused cells were perfectly round and their surfaces were generally covered with microvilli of equal length. No further ruffling was observed. It is suggested that cytoplasmic mechanisms regulate the surface morphology of the oocytes during fusion.

Porcine cumulus oocyte complexes (COCs) were cultured together in 10-microliters droplets of culture medium. When 10 COCs were cultured for 24 h, germinal vesicle breakdown (GVBD) occurred in 81% of them. When more COCs (20 or 40) were put into the same volume of medium the frequency of GVBD gradually decreased. This inhibition was not observed in denuded oocytes. The process of GVBD was adversely influenced when 10 COCs were cultured in cumulus-preconditioned medium. It is concluded that porcine cumulus cells produced a factor inhibiting GVBD. After removing the inhibitory block and extensive washing, GVBD of arrested oocytes was significantly accelerated. The addition of LH or heparin only partially overcame the inhibitory action. This factor produced by porcine cumulus cells negatively influenced maturation of bovine oocytes; however, a similar effect was not demonstrated in the mouse. Our results suggest that a high concentration of porcine cumulus cells exerts a quantitative inhibitory effect upon GVBD of porcine and cattle oocytes cultured in vitro.

The distribution of embryonic DNA in nuclei of blastomeres of early‐preimplantation cow embryos was studied by autoradiography. Two‐, 4‐, 8‐, and 16‐cell embryos were cultured for 18–20 h in the presence of [methyl‐3H]thymidine to ensure that all replicated embryonic DNA was labeled.In nuclei of blastomeres before the onset of transcription, taking place in the progressed 8‐cell cleavage division of the cow embryo1, labeled DNA was distributed excentrically at the nuclear periphery. After transcription started, the distribution of the labeled DNA was uniform throughout the nucleus.The progressive association of DNA with the nucleolus‐precursor body (NPB) also correlated with the expected onset of rRNA synthesis. The penetration of the labeled DNA into NPB was seen to start contemporaneously with the formation of a big central vacuole in NPB, but remained restricted, at this stage, to a few points of contact with the adjacent nucleolus‐associated chromatin. No labeled DNA was detected inside this big vacuole, still showing a uniform nucleoplasmic texture.The inside of the nucleolus was penetrated by DNA in the next step of nucleologenesis when secondary small vacuoles were formed and when the first signs of nucleolar transcription were detected1. These small vacuoles contained a coarse fibrillar component which was a frequent site of labeled DNA detection.This study of shifts of DNA‐containing sites during transition from maternal to embryonic genome expression correlates closely with previous evidence obtained by a study of genome reactivation which was situated in the late 8‐cell cleavage division of cattle early embryogenesis. It is during this stage, according to the present evidence, that the embryonic DNA occupies the whole of the nucleus, probably due to the extension of the DNA molecule at the onset of transcription. The association of rDNA with the nucleolus‐precursor body leading to the activation of rRNA synthesis is a morphologically well defined process which may reveal, in different experimental systems, the transcription capacity of cattle embryonic genome.

The method of polyethylene-glycol-induced fusion of mammalian oocytes was applied to study maturation-promoting factor (MPF) activity. After homologous fusions of one maturing--late diakinesis (LD), metaphase I (MI)--pig or mouse oocyte to one, two, or three immature-germinal vesicle (GV)--oocytes, giant cells were cultured in control or cycloheximide supplemented medium for 3 hours. The occurrence of germinal vesicle breakdown (GVBD) and premature chromosome condensation (PCC) served as a control of MPF activity. In giant cells composed of one maturing and one, two or three immature oocytes, GVBD and PCC were observed in all cases after cultivation in the control medium. In the presence of cycloheximide, the completion of GVBD and PCC remained high when one maturing and one immature oocyte were fused (83.7% and 95.7% of GVBD in pig and mouse, respectively). However, in giant cells composed of one maturing and up to three immature oocytes, all GVs were broken down only occasionally (4.8% and 11.7% in pig and mouse, respectively). These results suggest that in pig and mouse oocytes MPF does not amplify autocatalytically, but requires active protein synthesis for its production.

Bovine oocytes were obtained from: (a) 3–5 mm follicles of slaughtered cows or heifers and matured in vitro for 24 h, (b) preovulatory follicles of superovulated heifers slaughtered 18 h after hCG treatment and (c) oviducts of superovulated and castrated heifers 24, 32 and 36 h after hCG injection. The fertilization in vitro was carried out with heparinized and preincubated epididymal or ejaculated spermatozoa. In vitro matured oocytes, a, were fertilized by epididymal and ejaculated spermatozoa at a frequency of 72.8% and 54.0%, respectively. A higher fertilization rate of eggs fertilized by epididymal spermatozoa resulted in higher incidence of fertilization anomalies (32.6%) as compared to 5.4% in eggs fertilized by ejaculated spermatozoa. The fertilization rate of oocytes from superovulated heifers, b, fertilized by ejaculated spermatozoa increased from 23.8%, in preovulatory oocytes isolated 18 h after hCG injection, to 52.8% and 69.2% in ovulated oocytes isolated 24 and 32 h after hCG treatment, c, respectively. The fertilization rate of aged oocytes isolated 36 h after hCG was 21.9%.