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Background: Huntington's disease is induced by CAG expansion in a single gene coding the huntingtin protein. The mutated huntingtin (mtHtt) primarily causes degeneration of neurons in the brain, but it also affects peripheral tissues, including testes. Objective: We studied sperm and testes of transgenic boars expressing the N-terminal region of human mtHtt. Methods: In this study, measures of reproductive parameters and electron microscopy (EM) images of spermatozoa and testes of transgenic (TgHD) and wild-type (WT) boars of F1 (24-48 months old) and F2 (12-36 months old) generations were compared. In addition, immunofluorescence, immunohistochemistry, Western blot, hormonal analysis and whole-genome sequencing were done in order to elucidate the effects of mtHtt. Results: Evidence for fertility failure of both TgHD generations was observed at the age of 13 months. Reproductive parameters declined and progressively worsened with age. EM revealed numerous pathological features in sperm tails and in testicular epithelium from 24- and 36-month-old TgHD boars. Moreover, immunohistochemistry confirmed significantly lower proliferation activity of spermatogonia in transgenic testes. mtHtt was highly expressed in spermatozoa and testes of TgHD boars and localized in all cells of seminiferous tubules. Levels of fertility-related hormones did not differ in TgHD and WT siblings. Genome analysis confirmed that insertion of the lentiviral construct did not interrupt any coding sequence in the pig genome. Conclusions: The sperm and testicular degeneration of TgHD boars is caused by gain-of-function of the highly expressed mtHtt.

Background: Huntington disease (HD) is a fatal neurodegenerative disorder involving reduced muscle coordination, mental and behavioral changes, and testicular degeneration. In order to further clarify the decreased fertility and penetration ability of the spermatozoa of transgenic HD minipig boars (TgHD), we applied a set of mitochondrial metabolism (MM) parameter measurements to this promising biological material, which can be collected noninvasively in longitudinal studies. Objective: We aimed to optimize methods for MM measurements in spermatozoa and to establish possible biomarkers of HD in TgHD spermatozoa expressing the N-terminal part of mutated human huntingtin. Methods: Semen samples from 12 TgHD and wild-type animals, aged 12-65 months, were obtained repeatedly during the study. Respiration was measured by polarography, MM was assessed by the detection of oxidation of radiolabeled substrates (mitochondrial energy-generating system; MEGS), and the content of the oxidative phosphorylation system subunits was detected by Western blot. Three possibly interfering factors were statistically analyzed: the effect of HD, generation and aging. Results: We found 5 MM parameters which were significantly diminished in TgHD spermatozoa and propose 3 specific MEGS incubations and complex I-dependent respiration as potential biomarkers of HD in TgHD spermatozoa. Conclusions: Our results suggest a link between the gain of toxic function of mutated huntingtin in TgHD spermatozoa and the observed MM and/or glycolytic impairment. We determined 4 biomarkers useful for HD phenotyping and experimental therapy monitoring studies in TgHD minipigs.

The ubiquitin-proteasome system regulates many cellular processes through rapid proteasomal degradation of ubiquitin-tagged proteins. Ubiquitin C-terminal hydrolase-L1 (UCHL1) is one of the most abundant proteins in mammalian oocytes. It has weak hydrolytic activity as a monomer and acts as a ubiquitin ligase in its dimeric or oligomeric form. Recently published data show that insufficiency in UCHL1 activity coincides with polyspermic fertilization; however, the mechanism by which UCHL1 contributes to this process remains unclear. Using UCHL1-specific inhibitors, we induced a high rate of polyspermy in bovine zygotes after in vitro fertilization. We also detected decreased levels in the monomeric ubiquitin and polyubiquitin pool. The presence of UCHL1 inhibitors in maturation medium enhanced formation of presumptive UCHL1 oligomers and subsequently increased abundance of K63-linked polyubiquitin chains in oocytes. We analyzed the dynamics of cortical granules (CGs) in UCHL1-inhibited oocytes; both migration of CGs toward the cortex during oocyte maturation and fertilization-induced extrusion of CGs were impaired. These alterations in CG dynamics coincided with high polyspermy incidence in in vitro-produced UCHL1-inhibited zygotes. These data indicate that antipolyspermy defense in bovine oocytes may rely on UCHL1-controlled functioning of CGs.

To increase the developmental competence of bovine oocytes isolated from small, medium, and large follicles (2–3, 3–4, and 4–6 mm in diameter, respectively), we tried to modify the conditions for their in vitro culture. The first step involved conditions maintaining at least for 48 hr a reversible inhibition of the germinal vesicle breakdown (GVBD) and the second step stimulated the resumption of meiosis and completion of nuclear and cytoplasmic maturation during the subsequent 20–22 hr of culture. The effectiveness of this model depended mainly on the medium composition (reduced NaHCO3, substitution of serum with serum albumin, addition of antioxidants (curcumin), increased viscosity by agar, the reduction of oxygen concentration (within 6%–8%), the reduction of the proportion between the number of cumulus-oocyte complexes (COCs), and the reduction of the amount of a medium (within 6–7 μl per COC) to amplify the GVBD-inhibitory effect of oocyte surrounding granulosa cells. The COCs were incubated in clumps of 6–7 COCs. The effectiveness and reversibility of GVBD inhibition depended also on the duration of COCs isolation. The full reversibility of the GV block was controlled morphologically and also by measuring histone H1 and MAP kinase activities. The two-step versus one-step (24 hr) maturation technique was evaluated by the percentage of total and hatched blastocysts at day 9. When compared with one-step maturation, the two-step culture showed a slightly increased proportion of total and hatched blastocysts developed from growing follicles, mainly from the smallest category (13.9% vs. 7.1% and 9.2% vs. 3.3% for total blastocysts and hatched, respectively). However, the two-step culture of oocytes from large regressing follicles substantially reduced the blastocyst yield (9.7% vs. 39.1% and 4.9% vs. 26.7% for total blastocysts and hatched, respectively). The transfer of ten blastocysts (developed after two-step culture) to ten recipients resulted in seven pregnancies. Mol. Reprod. Dev. 71: 197–208, 2005. © 2005 Wiley-Liss, Inc.

The aim of this study was to describe the dynamic changes in the localization of the key nucleolar protein markers, fibrillarin, B23/nucleophosmin, C23/nucleolin, protein Nopp140, during the final stages of bovine oocyte growth. All these proteins were present in the large reticulated nucleoli of oocytes from the small-size category follicles ( 3-mm follicles), all studied nucleolar proteins were detected in the small compact nucleoli. The cap structure, attached to the compact nucleolus surface, was positive for UBF and PAF53 (subunit of RNA polymerase I). The UBF-positive cap showed a close structural association with p130. It is concluded that, during the process of oocyte nucleolus compaction, UBF and PAF53, proteins involved in the rDNA transcription, are segregated from fibrillarin and Nopp140, proteins essential for early steps of pre-rRNA processing. The observed changes may reflect the transition from pre-rRNA synthesis to pre-rRNA processing as an analysis of the relative abundance of the developmentally important gene transcripts confirmed. In addition, discovered structural association between UBF and p130 suggests a role for pocket proteins in ribosomal gene silencing in mammalian oocytes.

BACKGROUND Some promising treatments for Huntington's disease (HD) may require pre-clinical testing in large animals. Minipig is a suitable species because of its large gyrencephalic brain and long lifespan. OBJECTIVE To generate HD transgenic (TgHD) minipigs encoding huntingtin (HTT)1-548 under the control of human HTT promoter. METHODS Transgenesis was achieved by lentiviral infection of porcine embryos. PCR assessment of gene transfer, observations of behavior, and postmortem biochemical and immunohistochemical studies were conducted. RESULTS One copy of the human HTT transgene encoding 124 glutamines integrated into chromosome 1 q24-q25 and successful germ line transmission occurred through successive generations (F0, F1, F2 and F3 generations). No developmental or gross motor deficits were noted up to 40 months of age. Mutant HTT mRNA and protein fragment were detected in brain and peripheral tissues. No aggregate formation in brain up to 16 months was seen by AGERA and filter retardation or by immunostaining. DARPP32 labeling in WT and TgHD minipig neostriatum was patchy. Analysis of 16 month old sibling pairs showed reduced intensity of DARPP32 immunoreactivity in neostriatal TgHD neurons compared to those of WT. Compared to WT, TgHD boars by one year had reduced fertility and fewer spermatozoa per ejaculate. In vitro analysis revealed a significant decline in the number of WT minipig oocytes penetrated by TgHD spermatozoa. CONCLUSIONS The findings demonstrate successful establishment of a transgenic model of HD in minipig that should be valuable for testing long term safety of HD therapeutics. The emergence of HD-like phenotypes in the TgHD minipigs will require more study.

In this paper the effects of capacitation and fertilisation stimulating compounds (heparin, caffeine, glucose, D-penicillamine, bovine serum (BOS), bovine serum albumin (BSA), polyvinyl alcohol (PVA)) were analysed in several in vitro fertilisation protocols. Attention was paid to the rate of penetrated oocytes, kinetics of penetration and to polyspermic fertilisation. Cryopreserved bovine sperm and in vitro matured bovine oocytes were used throughout all the fertilisation experiments. As detected in the first 8 h fertilisation experiment with non-incubated sperm, the supplementation of medium with heparin, BOS and glucose supported the fertilisation rate most effectively (100%), including the kinetics of pronuclei formation (52.4%). The absence of BOS resulted in a decreased fertilisation rate (62.7%) as well as a delay in pronuclei formation (13.6%), similar to that after substitution of heparin with caffeine (73.0% and 25.4%, respectively). The penetration rate in the control medium with BOS (without heparin and caffeine) was surprisingly high, especially in medium without glucose (62.2%). The positive effect of glucose on sperm penetration was observed mainly in a chemically defined medium with PVA. High polyspermy rates were observed throughout all experiments in the media containing heparin or caffeine and BOS as the macromolecular component. D-Penicillamine was not shown to be a fertilisation-stimulating molecule. However, as detected in the second experiment in which oocytes were fertilised with 5 h incubated sperm, its positive effect on the prolongation of a fertile life span of cryopreserved spermatozoa was significant. The presence of either caffeine or heparin in the fertilisation medium (FM) with BOS during sperm incubation induced tyrosine phosphorylation of an approximately 90 kDa protein, detected after 5 h of sperm incubation. The absence of BOS reduced tyrosine phosphorylation of this protein in fertilisation medium with heparin. The percentage of motile spermatozoa and those with intact acrosomes were monitored throughout all experiments.

Contents Proper conditions for the successful induction of a functional two step in vitro culture system for agricultural or clinical applications are hereby described in form of a two-step protocol. The first inhibitory culture of oocytes in media supplemented with specific cdk inhibitors (roscovitine, BL I) appears essential to promote appropriate oocyte-cumulus cell interactions and ensure production of ova competent to undergo normal embryogenesis. This inhibitory period should last for several days, depending on the origin of the follicles, and the degree of accumulation and storage of mRNAs and proteins essential for the acquisition of developmental competence in the oocytes. The full reversibility of the inhibitory step secures that during the second maturation step, oocytes reach metaphase II with correctly arranged microtubuli in the meiotic spindle and well-expanded cumuli.

The effect of D-penicillamine on the fertile life span of frozen-thawed bull spermatozoa was studied in Experiment 1. After thawing, the washed spermatozoa were incubated for 8 h in fertilization medium with 1 mg/mL polyvinyl alcohol (PVA) and 0.5 mg/mL D-penicillamine. The addition of cumulus-free oocytes together with 10% of bovine serum (BOS) + 4 IU/mL heparin to the 8-h incubated spermatozoa resulted in high fertilization and polyspermy rates (97/103, 94.2% and 49/97, 50.5%, respectively). When BOS was substituted with 3 mg/mL of BSA, the fertilization and polyspermy rates decreased to 33/91 (36.3%) and 3/33 (9.1%), respectively. The total absence of fertilization was observed after substitution of proteins with PVA. The 8-h sperm incubation in fertilization medium without D-penicillamine and following fertilization in medium + different capacitation supplements resulted in the total absence of or a very low fertilization rate. In Experiment 2, the first set of cumulus-oocyte complexes (COC) and cumulus-free oocytes were fertilized for 8 h with nonincubated spermatozoa. The second set of COC and cumulus-free oocytes were fertilized in the same wells with spermatozoa after removal of the first set. The fertilization rate for the first set of COC and cumulus-free oocytes was 65/67 (97%) and 73/73 (100%), respectively, with 21/65 (32.3%) and 32/73 (43.8%) polyspermy, respectively. In the second set, the high penetration rate (67/73, 91.8%) was observed only for COC, while that for cumulus-free oocytes (19/76, 25%) was significantly lower (P < 0.01). In Experiment 3, the fertilization rate of COC in medium + PVA + 10 or 100 IU/mL heparin was 69/92 (75%) and 52/74 (70.2%), respectively, with no polyspermy. In medium + BSA + 10 or 100 IU/mL heparin, the fertilization rate of COC was 64/72 (88.9%) and 70/79 (88.6%), respectively, with polyspermy at 2/64 (3.1%) and 7/70 (10%), respectively. In medium + BOS + 10 or 100 IU/mL heparin, the fertilization rate was 79/82 (96.3%) and 79/80 (98.7%), respectively, with a significantly (P < 0.05) higher polyspermy rate (34/79, 43% and 30/79, 38%, respectively). The vitality and capacitation changes of frozen-thawed and 8-h incubated spermatozoa were assessed by Hoechst and chlortetracycline (CTC) staining.

In this study, butyrolactone I (BL I), a potent and specific inhibitor of cyclin-dependent kinases, was shown to block germinal vesicle (GV) breakdown (GVBD) in bovine oocytes in a concentration-dependent manner; GVBD was almost totally inhibited over the course of 24-48 h of culture when 100 microM BL I was included in tissue culture medium 199 containing either polyvinyl alcohol or BSA. Correlated with this inhibition was the failure of either p34(cdc2) kinase or mitogen-activated protein (MAP) kinase to become activated, and it was unlikely that BL I directly inhibited MAP kinase, since 100 microM BL I did not inhibit MAP kinase activity present in extracts obtained from metaphase II-arrested bovine eggs that possess high levels of MAP kinase activity. Nevertheless, the formation of highly condensed bivalents was observed in 78% of the BL I-treated GV-intact oocytes. This result suggests that chromosome condensation during first meiosis in bovine oocytes does not require the activity of either p34(cdc2) kinase or MAP kinase. Treatment of BL I-arrested oocytes with okadaic acid (OA) did not result in either the activation of p34(cdc2) kinase or MAP kinase, or inducement of GVBD. The BL I-induced block of GVBD for 24 h was reversible, and a subsequent 24-h culture resulted in 90% of oocytes reaching metaphase II with emission of the first polar body. Correlated with the progression to and arrest at metaphase II was the full activation of both p34(cdc2) and MAP kinases. The reversibility after 48 h of culture in BL I was partially decreased when compared to that achieved after an initial 24-h culture. Fertilization in vitro of these eggs resulted in a high incidence of both sperm penetration and pronucleus formation (88% and 70%, respectively).

Two principal kinases, p34cdc2 kinase and MAP kinase play a pivotal role in maturation of mammalian oocytes. In the porcine and bovine oocytes both kinases are activated around the time of germinal vesicle breakdown (GVBD). Butyrolactone I (BL I), a specific inhibitor of cdk kinases, prevents effectively and reversibly resumption of meiosis in the porcine and bovine oocytes. Neither p34cdc2 kinase nor MAP kinase are activated in oocytes inhibited in the GV stage. The bovine oocytes maintained for 48 h in the medium supplemented with BL I, progress subsequently to metaphase II in 91%, their cumuli expand optimally and after in vitro fertilization they possess two pronuclei. When the cdc2 kinase is blocked in the porcine oocytes by BL I, MAP kinase, activated by okadaic acid treatment, is able to substitute cdc2 kinase and induce GVBD. The histone H1 kinase activity sharply decreases in the metaphase II oocytes treated by BL I and one or two female pronuclei are formed. These data indicate that BL I is a useful tool either for the two step in vitro culture of mammalian oocytes or for their activation in nuclear transfer experiments.

SummaryWe have investigated the fertilisation competence, protein synthesis, histone H1 kinase and myelin basic protein (MBP) kinase activities in three categories of bovine oocytes (derived from three size categories of follicles: M–medium, 2.5–5.0 mm; S–small, 1.5–2.5 mm; T – tiny, 1.0–1.5mm). In contrast to more or less normal meiotic maturation (85.6%) and fertilisation (70.8%) of M oocytes cultured for 24h, the fertilisation of M oocytes cultured for 40h was associated with increased rates of retarded male pronuclear development and retention of the second polar body. The S and T oocytes cultured for 24h or 40h were mostly arrested at defective late diakinesis - metaphase I (77.5–100%) stage. After fertilisation of S and T oocytes cultured for 24h no polar body was extruded and formation of one, three or four female pronuclei, together with mostly normal male pronuclei, was observed. The fertilisation of S and T oocytes after 40h culture resulted in a higher number of female and a decreased number of male pronuclei. A major change in the pattern of protein synthesis was associated with the resumption of meiosis. There were no significant differences in the profile of protein synthesis between oocyte categories in all groups either matured or fertilised. The H1 kinase activity reached comparable increased levels in oocytes of all categories matured for 24h and decreased during the 40h culture, most significantly in M oocytes. The MBP kinase activity was at approximately the same high level in all categories of oocytes after 24h of culture and remained stable until 40h. The fertilisation after 24h of culture resulted, in M oocytes, in low levels of both H1 and MBP kinase activities; in S oocytes, only H1 kinase was completely inactivated while MBP kinase activity decreased to some extent; in T oocytes, both H1 and MBP kinase activity decreased. Fertilisation of all oocyte categories after 40h culture resulted in complete inactivation of both these kinases to their basal levels.

The effect of exogenous bovine recombinant somatotropin (bST) on the recruitment of follicles and quality of oocytes was investigated in the bovine. Single injections of recombinant depot form of bST were given to nine heifers and seven cows at the time of synchronized estrus (day 0). Nine heifers and six cows served as controls. Three days after bST injection the animals were slaughtered, their ovaries dissected and follicles separated into two categories according to diameter (2–5 and 5–10 mm). A total of 196 (53.2%) healthy oocyte-cumulus complexes (OCC) was isolated and selected from 369 follicles (diameter 2–5 mm) of bST treated heifers and 137 (49.8%) OCC from 275 follicles of bST treated cows. After 25–28 h of culture, 164 (44.4%) and 124 (45.1%) oocytes from follicles of heifers and cows, respectively, had developed to the metaphase of the second meiotic division (MII). From control heifers, 122 (35.5%) OCC derived from 344 follicles (2–5 mm) and from control cows 87 (39.7%) OCC derived from 219 follicles were similarly selected for maturation in vitro and 109 (31.7%) and 78 (35.6%) oocytes in control heifers and cows, respectively, developed to the MII stage. The numbers of follicles developed in the experimental and control groups were similar (P > 0.05) but differences were evident in the healthy OCC and oocytes maturing in the treated versus control groups of heifers and cows (χ2 detected at 1% and 5% levels, respectively). The injection of bST resulted in significantly increased levels of growth hormone (GH) in peripheral blood and follicular fluid (FF), the GH levels in blood plasma correlating with the levels in FF. The level of insulin-like growth factor I (IGF-I) in sera from bST treated animals was significantly higher than in sera from control animals (353.6 vs. 155.5 ng ml−1). In conclusion, recombinant bST positively affected (at day 3) the proportion of OCC and oocytes maturing to MII.

Previously, we showed that the gonadotropin-induced expansion of bovine cumulus oophorus occurs concomitantly with the rearrangement of microfilaments (MFs) inside cumulus cell cytoplasm (Sutovský et al., 1993: Biol Reprod 49:1277-1287; Sutovský et al., 1994: Reprod Nutr Dev 34:415-425) and that cumulus expansion in cattle is accompanied by the increased expression of extracellular matrix (ECM) glycoproteins laminin and type IV collagen as well as of their actin-linked membrane receptors, integrin subunits alpha-6 and beta-1 (Sutovský and Motlik: 1994). The present study was undertaken to determine the spatial and temporal relationship between cytoskeletal rearrangement and ECM synthesis during cumulus expansion. Using electron microscopy and confocal (LSCM) and conventional fluorescence microscopy, we compared the expression of the above integrins and ECM proteins and the rearrangement of cytoskeleton in the gonadotropin-stimulated bovine oocyte cumulus complexes (OCCs) with those exposed to gonadotropin stimulation and to ECM synthesis inhibitor 6-diazo-5-oxo-L-norleucin (DON), or MF-disorganizing drug cytochalasin B (CB). In control OCCs, the 24-hr culture in the presence of follicle stimulating hormone/luteinizing hormone (FSH/LH) caused the expansion of cumuli oophori and an extensive rearrangement of MFs in the cytoplasm of cumulus cells. Concomitantly, we observed an increased deposition of laminin and type IV collagen in the intercellular spaces among cumulus cells. The redistribution of microtubules (MTs), intermediate filaments (IFs), and integrin chains alpha-6 and beta-1 also occurred at this time. The addition of 20 micrograms/ml of CB prevented cumulus expansion and accumulation of laminin and type IV collagen in the OCCs. Moreover, cytochalasin treatment blocked the redistribution of MTs and IFs, and caused the disorganization of MFs and dispersion of integrins in cumulus cells. In contrast, the distribution of integrins and cytoskeletal elements was not affected when we blocked cumulus expansion and ECM protein accumulation by DON. These data suggest that F-actin acts upstream of ECM synthesis in the cascade of events leading to the expansion of bovine cumulus ooophorus.

Mouse preovulatory oocytes, zygotes, parthenogenetically activated pronuclear oocytes, and early embryos, as well as hamster zygotes, were analyzed, by autoradiography, for the distribution of either “maternal” or newly synthesized RNAs. Early mouse embryos were also examined for the distribution of newly replicated DNA. Special attention was attributed to NLBs in oocytes or to NPBs in early embryos. In mouse oocytes, [5-3H]uridine radioactivity accumulated (after a 2-hr pulse) in vitro, in addition to other nuclear compartments, in the central compact material of the NLBs. There was no cytoplasmic labeling. In all parthenogenetic pronuclear embryos developed from similarly labeled oocytes, this label was distinctly detectable in the central compact material of the NPBs; less intensive labeling was seen in the nucleoplasm and cytoplasm. On the contrary, the central compact part of the mouse NPB did not show labeling in DNA after a continuous culture with [6-3H]thymidine. In mouse and hamster pronuclear zygotes, convincing evidence was obtained for a lack of any newly synthesized nucleic acids in the compact material of NPBs using 4- to 10-hr culture with [8-3H]adenosine. Based on these data, it was shown that the NLBs of oocytes or NPBs of early embryos probably contain RNAs synthesized during the last stages of antral follicle oocyte differentiation. This unique pathway of RNAs in the oocyte—embryo system may explain the specific morphology of both oocyte and early embryo “nucleoli.” © 1995 Wiley-Liss, Inc.

Background Large-animal minipig transgenic model of Huntington’s disease (HD), carrying N-terminal part of human mutated huntingtin, was developed in Libechov and is expected to resemble to clinical picture of HD patients. Phenotyping of this model started immediately in presymptomatic stage. Despite of ongoing discussion, whether the glycolytic or mitochondrial metabolism (MM) is crucial for sperm ATP production; we decided to use spermatozoa as a promising biological material to assess MM parameters in HD. Aims The aims of this study were: to optimise methods for MM measurement in minipig spermatozoa; to assess its MM and to establish possible biomarkers of HD. Methods Semen samples were obtained repeatedly (12 boars, two generations, aged 12–65 months). Mitochondrial energy generating system (MEGS) capacity was measured by radiolabeled substrates oxidation, respiration by polarography and citrate synthase (CS) by spectrophotometry. Results Three possibly interfering factors were statistically analysed: the effects of HD; generation and ageing. Eight analysed parameters were incident to distinct generation. These parameters were influenced also by the effect of ageing. MEGS incubation with [1–14C] pyruvate+carnitine+ADP was affected significantly by all analysed effects. Four MM parameters were significantly diminished in HD spermatozoa: [U-14C]malate+pyruvate+malonate+ADP (I2) and [1–14C] pyruvate+ADP incubations, I2/CS ratio and complex I-dependent respiration might be considered as biomarkers of HD. Conclusions Used methods are suitable for MM analysis in minipig spermatozoa. Interestingly, spermatozoa can preferentially utilise pyruvate by sperm-specific isoform of lactate dehydrogenase. Our results in pyruvate-containing MEGS-incubations might indicate both MM and glycolytic defect in porcine HD spermatozoa, but further analyses are needed. Support The Project Contract No. MSMT-28477/2014 “HUNTINGTON” 7F14308 and NPU 1609 (MSMT).

The normalcy of nuclear differentiation in 8-cell bovine embryos derived by in vitro procedures from oocytes isolated from antral follicles of three different size categories was studied using autoradiographic localization of RNA synthesis and fine-structure nuclear morphology. In the few embryos derived from the oocytes from the small category of follicles (1-2 mm) the unlabeled nuclei prevailed. In contrast, the oocytes isolated from the more progressed follicles (medium and large size: 2-8 mm) yielded embryos with only slight, if at all detectable, differences to the picture expected in in vivo developed normal embryos. The diverging features probably concerned a slower onset of gene transcription, its consecutive pattern of localization as well as less pronounced progress of chromatin condensation and nucleolar differentiation. The assessment of these morphological changes and characteristics for individual embryos was obscured by marked differences in the status of the nuclei of particular blastomeres forming an embryo. Here the differences were seen in the fragmentation of nuclei, in the degree of chromatin condensation and in the absence of the nucleolus-associated chromatin in some of the blastomeres. It is suggested that most of the embryos from medium and large follicles have a comparable differentiation pattern of nuclear function development as embryos developing normally in vivo.

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-199 with porcine follicular fluid (50%) and follicle stimulating hormone (FSH) (0.25 IU ml−1) for 44 h at 38.5°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) × 106 spermatozoa ml−1 gave the best fertilisation results (87%), whereas the concentration below 1 × 106 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 ubiquitin-proteasome system regulates many cellular processes through rapid proteasomal degradation of ubiquitin-tagged proteins. Ubiquitin C-terminal hydrolase-L1 (UCHL1) is one of the most abundant proteins in mammalian oocytes. It has weak hydrolytic activity as a monomer and acts as a ubiquitin ligase in its dimeric or oligomeric form. Recently published data show that insufficiency in UCHL1 activity coincides with polyspermic fertilization; however, the mechanism by which UCHL1 contributes to this process remains unclear. Using UCHL1-specific inhibitors, we induced a high rate of polyspermy in bovine zygotes after in vitro fertilization. We also detected decreased levels in the monomeric ubiquitin and polyubiquitin pool. The presence of UCHL1 inhibitors in maturation medium enhanced formation of presumptive UCHL1 oligomers and subsequently increased abundance of K63-linked polyubiquitin chains in oocytes. We analyzed the dynamics of cortical granules (CGs) in UCHL1-inhibited oocytes; both migration of CGs toward the cortex during oocyte maturation and fertilization-induced extrusion of CGs were impaired. These alterations in CG dynamics coincided with high polyspermy incidence in in vitro-produced UCHL1-inhibited zygotes. These data indicate that antipolyspermy defense in bovine oocytes may rely on UCHL1-controlled functioning of CGs.

Kinex antibody microarray analyses was used to investigate the regulation of 188 protein kinases, 24 protein phosphatases, and 170 other regulatory proteins during meiotic maturation of immature germinal vesicle (GV+) pig oocytes to maturing oocytes that had completed meiosis I (MI), and fully mature oocytes arrested at metaphase of meiosis II (MII). Increases in apparent protein levels of protein kinases accounted for most of the detected changes during the GV to MI transition, whereas reduced protein kinase levels and increased protein phosphorylation characterized the MI to MII transition. During the MI to MII period, many of the MI-associated increased levels of the proteins and phosphosites were completely or partially reversed. The regulation of these proteins were also examined in parallel during the meiotic maturation of bovine, frog, and sea star oocytes with the Kinex antibody microarray. Western blotting analyses confirmed altered expression levels of Bub1A, IRAK4, MST2, PP4C, and Rsk2, and the phosphorylation site changes in the kinases Erk5 (T218 + Y220), FAK (S722), GSK3-beta (Y216), MEK1 (S217 + S221) and PKR1 (T451), and nucleophosmin/B23 (S4) during pig oocyte maturation.

The vitality of bovine oocytes stored in isolated follicles was examined. The aim of this work was to prolong the time of in vitro manipulation of oocytes before their maturation and develop a new alternative of oocyte "capacitation" to improve the quality of in vitro produced embryos. Follicles were dissected from the ovaries of slaughtered cows; subsequently, follicles were divided according to their diameter into three categories (2-3, 3-4 and 4-6 mm), and stored at 17-18 degrees C for 24 or 48 h in a modified tissue culture medium-199 (TCM-199) with reduced pH. After that time, the cumulus-oocyte complexes (COCs) were isolated, matured, fertilized, and embryos cultured in vitro for a total of 9 days. The percentage of total blastocysts, and hatched blastocysts developed from oocytes, initially kept ("capacitated") for 24h at 17-18 degrees C, within follicles of 3-6mm size categories, were significantly higher than that oocytes of the control [of control oocytes] (44.9 and 30.3% versus 36.2 and 20.4%, respectively). The oocytes of follicles stored for 48 h at 17-18 degrees C already had decreased developmental capacity. Interesting data were obtained when COCs of the 3-4 and 4-6 categories were additionally divided into two subgroups according to their presumed developmental history (originating from the supposed growing "fit" in contrast to the supposed regressing "unfit" follicles). The higher improvement in the rate of hatched blastocysts from 24h stored oocytes was observed only in the subgroup originated from "fit" COCs (15.3 versus 25.0%, and 20.0 versus 34.4%, in the 3-4 and 4-6mm categories, respectively). The transfer of 26 blastocysts (developed of follicles kept for 24h at 17-18 degrees C) to 26 recipient heifers resulted in 18 pregnancies. Storage of follicles at 17-18 degrees C in vitro resulted not only in recovery of higher numbers of blastocysts of better quality but also facilitated the safe transport of follicles for a long distance. The extended, time of follicle storage before the proper oocyte maturation allowed also the synchronization of an appropriate number of recipient animals according to the number of isolated follicles.

The aim of this study was to describe the dynamic changes in the localization of the key nucleolar protein markers, fibrillarin, B23/nucleophosmin, C23/nucleolin, protein Nopp140, during the final stages of bovine oocyte growth. All these proteins were present in the large reticulated nucleoli of oocytes from the small-size category follicles (1 mm). The entire nucleolus exhibited strong positivity for UBF (upstream binding factor, RNA polymerase I-specific transcription initiation factor), which displayed a dotted staining pattern. In contrast, protein p130 was diffusely distributed throughout the nucleus and excluded from nucleoli. In oocytes approaching the late period of growth (2-3-mm follicles), UBF localization shifted to the nucleolar periphery. Double staining of UBF-p130 revealed a gradual accumulation of p130 at the periphery shell around the nucleolus. In fully grown oocytes (3-mm follicles), all studied nucleolar proteins were detected in the small compact nucleoli. The cap structure, attached to the compact nucleolus surface, was positive for UBF and PAF53 (subunit of RNA polymerase I). The UBF-positive cap showed a close structural association with p130. It is concluded that, during the process of oocyte nucleolus compaction, UBF and PAF53, proteins involved in the rDNA transcription, are segregated from fibrillarin and Nopp140, proteins essential for early steps of pre-rRNA processing. The observed changes may reflect the transition from pre-rRNA synthesis to pre-rRNA processing as an analysis of the relative abundance of the developmentally important gene transcripts confirmed. In addition, discovered structural association between UBF and p130 suggests a role for pocket proteins in ribosomal gene silencing in mammalian oocytes.

In this study a specific inhibitor of cyclin-dependent kinases (cdks), butyrolactone I (BL I), was used for activation of pig and cattle metaphase II (MII) oocytes. BL I at a concentration of 100 μM was able to induce activation of both pig and cattle MII oocytes in a manner dependent on exposure time; however, precise timing of BL I exposure was required for the best activation results. The optimum activation rates were obtained when cattle MII oocytes were treated for 5 h with BL I and subsequently for 3-11 h in control medium, and pig MII oocytes for 8 h in BL I and then for 8-16 h in control medium; the percentage of activated oocytes after such treatment varied between 55% and 74% and between 53% and 81% for cattle and pig oocytes, respectively. Shorter exposures to BL I led to re-entry of the oocytes to the metaphase state in 35-50% of oocytes, the remaining oocytes forming a pronuclear stage; longer exposure to BL I led to increased numbers of oocytes being abnormal or degenerated. The behaviour of histone H1 kinase and mitogen activated protein (MAP) kinase, also measured during the experiment, reflected the morphological changes in the oocytes: both were inactivated after BL I treatment, though the inactivation of histone H1 kinase occurred 2 h ahead of that of MAP kinase. However, in the oocytes treated for a shorter time with BL I, with the reoccurrence of condensed chromatin in proportion of the oocytes cultured in control medium after BL I treatment, both kinases became reactivated. Taken together, these results suggest the possibility of using BL I for activation and cloning experiments in both species.

Nuclear bodies occuring during the 2-cell stage of bovine embryos (obtained either by in vitro fertilisation of in vitro matured ovarian oocytes, or collection after fertilisation and cleavage in vivo) were studied using ultrastructural cytochemistry and immunocytochemistry to determine whether their occurrence may be linked with the onset of embryonic transcription. In addition, the species-specific ultrastructural features of the interchromatin structures of the 2-cell bovine embryo were displayed. Three different types of nuclear bodies were distinguished: (i) nucleolus precursor bodies (NPBs), (ii) loose bodies (LBs) and (iii) dense bodies (DBs). In order to determine their possible functional significance, we considered parallels between these three nuclear entities and interchromatin compartments reported in other cells. As detected by their preferential ribonucleoprotein staining, all types of nuclear bodies contained ribonucleoproteins. In contrast to the other types of nuclear bodies studied, NPBs contained argyrophilic proteins but in no case they did show morphological features of functional nucleoli. Both compact and vacuolated forms of NPBs were seen in both in vivo and in vitro embryos, sometimes simultaneously in the same nucleus. LBs and DBs reacted with antibodies to Sm antigen, indicating the presence of a group of nucleoplasmic, non-nucleolar small nuclear ribonucleoproteins (snRNPs). The immunoreactivity for Sm antigen was more intense and homogeneous in DBs than in LBs. DBs were seen in both categories of embryo. A possible kinship of DBs with the sphere organelle known from oocytes of different animal species or the prominent spherical inclusions of the early mouse embryo nuclei is suggested. The last type of intranuclear body, the LBs, showed a composite structure. Their granular component, occurring in clusters and displaying immunoreactivity for Sm antigen, was similar to interchromatin granules and was therefore named IG-like granules. Another component forming the LBs showed a much finer structure and a lower immunoreactivity with anti-Sm antibodies. We suggest that this amorphous component may be related to the IG-associated zone. All three types of intranuclear bodies were often seen close together, suggesting their possible mutual functional relationship. From these and other observations we conclude that the intranuclear bodies in 2-cell bovine embryos correspond, with the exception of the NPB, to similar structures/compartments supposed to accumulate inactive spliceosomal components in certain phases of somatic cell nucleus functions. Accordingly, the occurrence of such nuclear bodies does not represent cytological evidence for RNA synthesis. In contrast to this, an important morphological feature revealing the status of the bovine 2-cell embryo is the vacuol-isation of the NPB.

Bovine oocytes originated from follicles of two different size categories (medium (M), 3-6 mm and small (S), 1-2 mm) were cultured for 24 and 70 h, respectively, in a meiosis-inhibiting medium (MIM) supplemented with 100 microM butyrolactone I (BL I). At the end of culture, cumulus oocyte complexes (COC) from M and S follicles were labeled by 3H-uridine for 30 min. The autoradiography (ARG) of semi-thin sections of COC showed the labeling of the germinal vesicles (GV) as: (1) The COC of the M category labeled immediately after isolation from follicles showed only weak labeling (+) of the GV. The COC of the S category labeled immediately after their isolation showed mostly intensive labeling (4+,3+) of the GV. (2) When the COC were labeled after 24 and 70 h of culture in MIM, no labeling was observed in the M category. The S category of oocytes showed the slightly decreased labeling (3+,2+) after 24 h and negligible labeling after 70 h of culture. The pattern of very intensive labeling of granulosa cell nuclei of all mentioned groups was practically not changed during the whole 70 h period of culture in both categories. The nucleolar ultrastructure of S category oocytes revealed time dependent changes from the reticular fibrillogranular structure present in freshly isolated oocytes. The several fibrillar centers before the culture changed to the fibrillogranular appearance with few large and a number of small vacuoles and an exclusively fibrillar area after 24 h of culture. Finally, nucleoli acquired a mostly exclusively fibrillar structure with one large fibrillar center after 70 h of culture. In the second experiment, the meiotic maturation of COC of S category was inhibited in MIM for 48 h. The subsequent 24 h culture in a medium with BOS and gonadotropins resulted in 81.0% oocytes matured to metaphase II (M II). Only 27.1 and 11.3% of the control S oocytes cultured in a medium, with BOS and gonadotropins directly after isolation, reached M II after 48 or 72 h of culture, respectively. The two-step culture increased significantly the meiotic competence of cattle oocytes isolated from small antral follicles.

Huntington9s disease (HD) is an inherited neurodegenerative disorder, characterised by psychiatric changes, cognitive disonance and motoric dysfunctions. The mutation responsible for this fatal disease is an abnormally expanded polyQ repeat within the gene encoding the huntingtin (htt) protein. Unfortunately, there is no specific cure available to stop disease progression. Numerous drugs have been developed to produce benefits in this disease, however, preclinical tests are required for confirmation of their effectiveness in treating HD. There are different animal models, each model with its own advantages and limitations, but there is still a huge demand for large animal models for testing the possibilities of HD therapies. As the extensive neuroanatomical and physiological studies indicate, a close similarity of the miniature pig brain to the human brain exists in term of neurochemical and neurotransmitter repertoire. Hence we chose this model for the generation of a transgenic model of HD. Lentiviral constructs encoding N terminal truncated (548aa) htt with 145 polyQ repeats under the control of human HD promoter were microinjected into the one cell stage embryos. The offspring born after embryo transfer were genotyped and one female piglet was transgenic. Both mutant htt incorporation into genome and its mRNA expression were proved using PCR. Western blot analysis confirmed the presence of the mutant htt protein. The transgenic gilt was mated with its brother to produce F1 generation. Three of eight newborn piglets are transgenic. The suitable and publicly acceptable large animal model of HD that can serve to validate the efficacy of treatment approaches targeted to modulate HD was generated. Mutant htt gene incorporated into porcine genome is not only transcribed into mRNA but is also expressed at the protein level. HD transgenic miniature gilt successfully conceived and delivered eight healthy offsprings. Three male piglets are transgenic in the litter.

This study investigated the capacity of healthy oocytes derived from follicles of different size to undergo normal fertilization and early embryonic development in vitro and full-term development in vivo. Ovaries were collected from a local abattoir and dissected and classified as follows: group A, >4–8 mm (large); group B, >2–4 mm (medium); and group C, >1–2 mm (small). Oocytes were isolated by puncturing the follicular wall and pressing of the follicle. Only healthy-looking cumulusoocyte complexes (COC) were used for in vitro maturation. Oocytes were fertilized in vitro by frozen/thawed semen from one bull. Approximately one-fourth of all oocytes was fixed and stained 15–20 h after fertilization, to determine penetration rates. The remaining eggs were transferred to culture medium and were cultivated for up to 9.5 days. Cleavage was observed 65 h and 7 days after fertilization. Expanded, hatching, and hatched blastocysts were fixed and stained after 9.5 days of culture. A total of 86 blastocysts derived from group A and B oocytes was nonsurgically transferred to synchronized recipients 7–8 days after onset of culture. A total of 6.624 follicles were dissected from 265 ovaries, and 1,485 oocytes were isolated from 1,671 group A follicles, 3,509 oocytes from 3,862 group B follicles, and 965 oocytes from 1,091 group C follicles. The fertilization rate, rate of normal fertilization, rate of polyspermy, and rate of other abnormal fertilization features were as follows: group A, 84.9%, 43.2%, 34.1%, 7.6%; group B, 83.6%, 44.8%, 31.1%, 7.8%; and group C, 61.7%, 13.1%, 33.7%, 19.1%. The cleavage rate and the percentage of embryos with 5–8 cells after 65 h amounted in group A to 68.9% and 36.9%; in group B, 64.3% and 32.7%; and in group C, 19.7% and 0.4%, respectively. The percentage of blastocysts on days 7 and 9.5 as well as the rate of hatched blastocysts were 27.7%, 24.8%, and 21.7%, respectively, in group A; 20.9%, 19.2%, and 16.8%, respectively, in group B; and zero in group C. Transfer of 38 blastocysts of group A to 19 recipients led to 10 pregnancies; transfer of 48 blastocysts to 24 recipients of group B resulted in 12 pregnancies. It is concluded that blastocysts from follicles ranging from >2 to 8 mm in size have a similar developmental potential. Oocytes from 1- to 2-mm follicles, however, have a significantly lower competence to undergo in vitro maturation and fertilization and, under the conditions of our experiments, completely lack the capability to cleave beyond the 8-cell stage.

The extracellular matrix (ECM) of porcine mature oocytes was revealed by transmission electron microscopy (TEM) after treatment with tannic acid and ruthenium red. Present in the perivitelline space (PVS) and on the surface of the zona pellucida (ZP), it appeared to be composed of thin filaments and granules at the interconnections of the filaments, which were interpreted respectively as hyaluronic acid chains and bound proteoglycans. In order to determine whether this material is produced by the corona cells (the same ECM was found also on the surface of the zona pellucida and between cumulus cells) or by the oocyte itself, the synthesis of glycoproteins and glycosaminoglycans was checked by autoradiography on semi-thin and thin sections observed by light and electron microscopy. Immature oocytes within or without cumulus cells, were incubated with L [3H-] fucose or L [3H-] glucosamine – precursors respectively of glycoproteins and hyaluronic acid or hyaluronan (HA) bound to proteoglycans – for various times (with or without chase) and at different stages during in vitro maturation. In the first case, incorporation was found in both cumulus cells and ooplasm (notably in the Golgi area for 3H-fucose) and labeled material accumulated in the ECM of the PVS and of the ZP surface. Labeling in the PVS with both precursors was maximum between metaphase I (MI) and metaphase II (MII) and was partially extracted by hyaluronidase but not by neuraminidase. Tunicamycin, an inhibitor of glycoprotein synthesis, significantly decreased the amount of 3H-fucose labeled molecules in the PVS and increased the incidence of polyspermic penetration during subsequent in vivo fertilization. Since cumulus-free oocytes also secreted 3H-glucosamine containing compounds, both oocyte and cumulus cells probably contribute to the production of the ECM found in the PVS of mature oocytes. ECM and particularly its HA moiety present on both sides of the ZP may constitute a favourable factor for sperm penetration.

Zona-free hamster eggs have been fertilized in vitro with boar spermatozoa in a medium enriched by arginine-3H and the sites of localization of newly synthesized arginine-3H–labeled proteins have been investigated using fine-structure autoradiography. It was confirmed that such proteins are synthesized during fertilization and that they accumulate to a notable degree in decondensing sperm chromatin as well as in the chromatin of the female pronucleus and also of the second polar body. A similar process did evidently take place also in defective pronuclei, characterized by a core of a still condensed chromatin and by remaining nuclear membrane. In such male pronuclei the highest concentration of the label was seen just on the border of the condensed chromatin, on the expected site of nuclear protein exchange. It is supposed that, at least in this experimental system, any morphologically detectable sperm decondensation is accompanied immediately by a shift from sperm basic nuclear proteins to other nuclear proteins.

In the course of in vitro fertilization of zona-free hamster and pig eggs by boar and guinea-pig spermatozoa it was observed that homologous and heterologous eggs fused together, forming cell hybrids between two or more cells. The fusogenic activity was attributed to spermatozoa and this was the hypothesis tested. The fusogenic activity (coinciding with sperm penetration activity) was dependent on the duration of sperm preincubation, which may be regarded as capacitation in vitro. Fusion occurred only after 3 hr of sperm preincubation and a narrow optimum was detected at 4–4.5 hr. Fusion of eggs was also dependent on sperm concentration. A relatively high proportion of fusions was observed at a sperm concentration of 4.0 × 104 per ml and an optimum was attained at a concentration of 5.0 × 105 per ml. The first fusions were observed at 90 min after semination. After 3 hr more than a half of the eggs reacted, and by 20 hr of incubation 80% of ova were fused. The fusability of eggs was tested and found to occur at 14 hr after ovulation. The fusion process was also studied using transmission electron microscopy. It is supposed that the process of egg fusion may be caused either by a similar mechanism to sperm-egg fusion, or by products released during the sperm acrosome reaction.

General labeling of ripe mouse spermatozoa was obtained two weeks after six injections of L-arginine-5-3H monohydrochloride, administered within two days. Besides tail labeling a very intensive nuclear labeling was obtained. It is presumed that this nuclear labeling represents almost exclusively tritiated arginine incorporation into basic nuclear protein. Mouse oocytes were fertilized in vitro with such labeled spermatozoa. It was found that the nuclear label is lost very early during male pronucleus formation, probably concomitantly with sperm chromatin decondensation. This might indicate total loss of the basic spermatozoal nuclear protein before male genome activation.

The development of fertilized ova in explanted parts of oviducts was studied from 12 to 36 hours after fertilization, and from 36 to 84 hours after fertilization. No egg mortality during cultivation, nor any slowing of development could be detected in either experiment. It may be presumed that the mortality of mouse ova during the period before implantation has no essential effect on the resulting fertility. During cultivation of oviducts for 144 hours, there occurred in most blastocysts herniation of their walls resulting in a formation similar to the blastocyst, and a migration of a disorganized mass of cells, some of which resembled the giant trophoblastic cells. After transplantation of ova fertilized in vitro to recipients, in four cases there occurred nidation and subsequent normal development of the embryo.