In the last 3 years, several studies have shown that xenogeneic transplantation of rodent spermatogonia is feasible. The treatment of infertile patients with spermatogenic arrest using the injection of immature germ cells has yielded only poor results. We attempted to establish a complete spermatogenetic line in the testes of mutant aspermatogenic (W/Wv) and severe combined immunodeficient mice (SCID) transplanted with germ cells from azoospermic men. Spermatogenic cells were obtained from testicular biopsy specimens of men (average age of 34.3 ± 9 years) undergoing infertility treatment because of obstructive and non-obstructive azoospermia. Testicular tissue was digested with collagenase to promote separation of individual spermatogenic cells. The germ cells were injected into mouse testicular seminiferous tubules using a microneedle (40 μm inner diameter) on a 10 ml syringe. To assess the penetration of the cell suspension into the tubules, trypan blue was used as an indicator. Mice were maintained for 50 to 150 days to allow time for germ cell colonisation and development prior to them being killed. Testes were then fixed for histological examination and approximately 100 cross-sectioned tubules were examined for human spermatogenic cells. A total of 26 testicular cell samples, 16 frozen and 10 fresh, were obtained from 24 men. The origin of the azoospermia was obstructive (OA) in 16 patients and non-obstructive (NOA) in 8 patients. The concentration of spermatogenic cells in the OA group was 6.6 × 106 cells/ml, and 1.3 ? 106 cells/ml in the NOA group (p < 0.01). The different spermatogenic cell types were distributed equally in the OA samples, ranging from spermatogenia to fully developed spermatozoa, but in the NOA group the majority of cells were spermatogonia and spermatocytes. A total of 23 testes from 14 W/Wv mice and 24 testes from 12 SCID mice were injected successfully, as judged by the presence of spermatogenic cells in histological sections of testes removed immediately after the injection. However, sections from the remaining testes examined up to 150 days after injection showed tubules lined with Sertoli cells and xenogeneic germ cells were not found. The reason why the two strains of mouse used as recipients did not allow the implantation of human germ cells is probably due to interspecies specificity involving non-compatible cell adhesion molecules and/or immunological rejection.

Previous studies have suggested that both acrosome-intact and acrosome-reacted guinea pig sperm are capable of binding to the zona pellucida of cumulus-free oocytes, but the acrosomal status of guinea pig sperm during penetration of the cumulus has not been reported. We made video recordings of the interaction between capacitated guinea pig sperm and cumulus-invested guinea pig oocytes. The videotapes were analysed to identify sperm with hyperactivated motility and to classify the acrosomal status of sperm during penetration of the cumulus and after binding to the zona pellucida. The resolution of the video recordings was not sufficient to recognise sperm with swollen acrosomes. However, sperm that had completed the acrosome reaction were easily identified. Acrosome-reacted sperm were found adherent to the outer boundary of the cumulus, but were never observed to penetrate the cumulus. The percentage of acrosome-intact, hyperactivated sperm was higher in the cumulus oophorus than in culture medium, suggesting that changes in motility were elicited in response to contact with the cumulus. Fully acrosome-reacted sperm were found adherent to the zona pellucida, and solubilised guinea pig zona pellucida was capable of inducing acrosome reactions in capacitated guinea pig sperm. Acrosome-intact sperm were also observed on the zona, but they were not tightly bound and did not have hyperactivated motility, suggesting that these sperm were not functionally capacitated. Our observations demonstrate that guinea pig sperm penetrate the cumulus matrix in an acrosome-intact state. Although we did not observe sperm undergoing the acrosome reaction, our observations and experimental data suggest that the acrosome reaction of guinea pig sperm is completed on or near the surface of the zona pellucida.

Mitogen-activated protein kinase (MAPK) has been reported to be involved in oocyte maturation in all animals so far examined. In the present study we investigate the expression and localisation of active phosphorylated MAPKs (p44ERK1/p42ERK2) during maturation of pig oocytes. In immunoblot analysis using anti-p44ERK1 antibody which recognised both active and inactive forms of p44ERK1 and p42ERK2, we confirmed that MAPKs were phosphorylatred around the time of germinal vesicle breakdown (GVBD) and the active phosphorylated MAPKs (pMAKs) were maintained until metaphase II, as has been reported. On immunofluorescent confocal microscopy using anti-pMAPK antibody which recognised only phosphorylated forms of MAPKs, pMAPK was localised at the spindle poles in pig mitotic cells. On the other hand, in pig oocytes, no signal was detected during GV stage. After GVBD, the area around condensed chromosomes was preferentially stained at metaphase I although whole cytoplasm was faintly stained. At early anaphase I, the polar regions of the meiotic spindle were prominently stained. However, during the progression of anaphase I and telophase I pMAPK was detected at the mid-zone of the elongated spindle, gradually becoming concentrated at the centre. Finally, at the time of emission of the first polar body, pMAPK was detected as a ring-like structure between the condensed chromosomes and the first polar body, and the staining was maintained even after the metaphase II spindle was formed. The inhibition of MAPK activity with the MAPK kinase inhibitor U0126 during the meiosis I/meiosis II transition suppressed chromosome separation, first polar body emission and formation of the metaphase II spindle. From these results, we propose that the spindle-associated pMAPKs play an important role in the events occurring during the meiosis I/meiosis II transition, such as chromosome separation, spindle elongation and cleavage furrow formation in pig oocytes.

Soybean trypsin inhibitor (SBTI) inhibits the catalytic activity of serine proteases, and has been shown to bind to acrosin, an acrosomal hydrolase which is not exposed on the surface of macaque sperm until after the acrosome reaction. Following activation with caffeine and dibutyryl cAMP, cynomolgus macaque sperm were induced to acrosome react with calcium ionophore A23187 in the presence of SBTI and were fixed for ultrastructural observation. Transmission electron microscopy (TEM) revealed secondary labelling of anti-SBTI-IgG with colloidal gold in association with the acrosomal matrix and fused membranes of sperm undergoing the acrosome reaction, but gold labelling was not observed on acrosome-intact sperm. When SBTI was conjugated with the fluorochrome Alexa 488, labelled (acrosome-reacted) sperm showed bright fluorescence that ranged from a patchy or punctate appearance to solid labelling over the region of the acrosomal cap. Following treatment with ionophore, the percentages of total acrosome-reacted sperm (motile and non-motile) as assessed with Alexa-SBTI, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), and TEM were 54.6%, 51.6% and 61.5%, respectively. Measures of acrosomal status with FITC-PSA and Alexa-SBTI were highly correlated (r = 0.94; n = 3). Macaque zonae pellucidae were co-incubated with activated sperm for 1 min and then rinsed in medium containing Alexa-SBTI and immediately observed with epifluorescence microscopy. The mean percentage of Alexa-SBTI-labelled (acrosome-reacted) motile sperm bound to the zona was 45.7 ± 14 (range: 22–80.4%; n = 4). Fewer than 1% of the motile sperm in suspension surrounding the zonae were acrosome-reacted. Alexa-SBTI had no effect on sperm motility, survival, or zona binding capability.

The aim of this study was to optimise porcine in vitro fertilisation (IVF) with cryopreserved semen with the exploitation of the oviduct secretion. The oocytes were cultured in NCSU37 supplemented with db-cAMP (1 mM), porcine follicular fluid (pFF; 10%), cysteine (0.1 mg/ml) and β-mercaptoethanol (25 μM) for 44 h (the first 20 h with 10 IU/ml hCG and PMSG). The oviductal epithelial cells (OEC) were cultured in TCM-199 medium (with 10% FCS, 0.2 mM pyruvate and 50 μg/ml gentamicin) for 48 h. To determine the effects of OEC and conditioned medium, oocytes were separated into five groups for the last 3 h of maturation and placed in: fresh maturation medium (controls), OEC-cNCSU with OEC in the maturation medium for 24 h; OEC-fNUSU with fresh OEC in maturation medium; cTCM with TCM-199 conditioned with OEC for 48 h; or fTCM with fresh TCM-199. Results indicate that OEC-cNCSU and OEC-fNCSU increase the number of oocytes reaching the two pronucleus (2PN) stage (p < 0.01) and decrease the polyspermy rate (p < 0.01) compared with controls. The rates are significantly lower than controls when cTCM and fTCM were used (p < 0.01). As regards blastocyst rates, an increase was observed in the OEC-cNCSU and cTCM groups (p < 0.05). For the second experiment, spermatozoa were incubated with OEC in IVF medium (mTBM medium supplemented with 0.1% BSA) without caffeine for 4 h prior to IVF. Results indicate that sperm treatment with OEC increases the 2PN rate (p < 0.01) compared with controls and reduces the polyspermy rate (p < 0.01). In conclusion, our study shows that co-incubation of OEC with both oocytes and sperm before IVF reduces polyspermy rates and improves embryo development.

We show that in contrast to metaphase II oocytes, metaphase I oocytes cannot be activated by fusion with the zygote. Fusion of metaphase I oocytes with G2 zygotes was followed by premature chromosome condensation, with 60% of the hybrids becoming arrested at metaphase I, the remainder progressing and arresting at metaphase II. Hybrids of metaphase I oocytes and M-phase zygotes underwent accelerated maturation, but all arrested at metaphase II. In both cases the arrest could be overcome by treatment with the parthenogenetic activators ethanol and cycloheximide. We discuss these findings in relation to the possibility that the metaphase I oocyte contains cytostatic factor activity that is activated by its zygotic partner. Alternatively, the G2 zygote may provide an inhibitor of anaphase, normally never present in the metaphase I oocyte and which is absent from the M-phase zygote.

Pig oocytes at metaphase II were activated by penetration of spermatozoa in cycloheximide-free and cycloheximide-containing fertilisation media. The precise nuclear stage, and the kinetics of degradation of cyclin B1 and dephosphorylation of MAP kinase were assessed after insemination. After maturation culture, 96% of oocytes reached metaphase II. At 6 h after insemination in cycloheximide-free medium, 68% of the oocytes were activated and had progressed to anaphase II or beyond. After 8 h, 89% of the oocytes were activated: a female pronucleus had formed and the heads of penetrating spermatozoa had enlarged and changed to male pronuclei. In the cycloheximide-containing medium, activation of oocytes started earlier than in cycloheximide-free medium. After 4 h, 43% of the oocytes were activated, and the percentage increased to 97% after 6 h. Pig cyclin B1 disappeared in the oocytes at 6 h after insemination in both cycloheximide-containing and cycloheximide-free media. Pig oocytes at metaphase II contained two types of MAP kinase – ERK 1 and ERK 2 – in their active phosphorylated forms. At 8 h after insemination ERK 2 changed to the fast-migrating inactive form in the oocytes cultured in both cycloheximide-containing and cycloheximide-free media, although the shift-down was not complete. The change was delayed by 2 h after the degradation of cyclin B1 molecules. These results demonstrate that degradation of pig cyclin B1 molecules corresponds to the transition of the oocytes from metaphase II arrest to anaphase II/telophase II and was followed by MAP kinase dephosphorylation.

In the present study the role of glycosphingolipids (GSL) in amphibian development was investigated. We analysed the de novo synthesis of neutral GSL and gangliosides through the initial stages of Bufo arenarum embryo development and their participation during gastrulation using 1-phenyl-2-palmitoyl-3-morpholino-1-propanol (PPMP), a potent inhibitor of glucosylceramide synthase. Ganglioside synthesis began at the blastula stage and reached a maximum during gastrulation (stages 10-12) while neutral GSL synthesis showed a slight gradual increase, the former being quantitatively more significant than the latter. Ganglioside synthesis was reduced by 90% while neutral GSL synthesis was inhibited by 65% when embryos at blastula stage were cultured for 24 h in 20 μM PPMP. The depletion of GSL from amphibian embryos induced an abnormal gastrulation in a dose-dependent manner. We found that PPMP had a pronounced effect on development since no embryos exhibited normal gastrulation; their developmental rate either slowed down or, more often, became totally arrested. Morphological analysis of arrested embryos revealed inhibition of the gastrulation morphogenetic movements. Analysis of mesodermal cell morphology in those embryos showed a severe decrease in the number and complexity of cellular extensions such as filopodia and lamellipodia. Mesodermal cells isolated from PPMP-treated embryos had very low adhesion percentages. Our results suggest that glycosphingolipids participate in Bufo arenarum gastrulation, probably through their involvement in cell adhesion events.

The aim of the present study was to determine the presence of the connexins Cx43, Cx32 and Cx26 in Bufo arenarum ovarian follicles during the breeding season as well as to analyse the possible alterations in the meiotic process when connexins are blocked by specific antibodies. Western blot analysis revealed that the Cx43 and Cx32 proteins were present but not Cx26. We demonstrated that the anti-Cx43 and anti-Cx32 antibodies produced the uncoupling of the gap junctions. When these junctions are blocked the maturation process is triggered in the oocytes. We determined that dbcAMP exerts an inhibitory effect on the maturation induced by the uncoupling of the gap junctions when the oocytes are injected or pretreated with this metabolite. We propose the idea that cAMP is the regulatory molecule in meiotic arrest in this amphibian species.

Four porcine sperm plasma membrane proteins were previously identified as putative ligands for the oocyte plasma membrane. The present study examined the binding of these proteins and two additional porcine sperm membrane proteins to oocytes from sheep, mice and hamsters as a first step in assessing potential conservation of these putative sperm ligands across species and across mammalian orders. Plasma membrane vesicles were isolated from porcine sperm, solubilised, and the proteins separated by one-dimensional gel electrophoresis. The 7, 27, 39 and 62 kDa porcine sperm protein bands demonstrating predominant binding of the porcine oocyte plasma membrane on ligand blots, a 90 kDa protein band demonstrating minor binding, and a 97 kDa protein band that did not bind the oocyte plasma membrane probe were electroeluted. Proteins were biotinylated, and incubated with zona-free oocytes. Bound biotinylated protein was labelled with fluorescent avidin and the oocytes examined with a confocal microscope. The 7 kDa, 27 kDa and the 39 kDa proteins bound to the sheep oocytes but not to a majority of the hamster or mouse oocytes. The 62 kDa protein bound to sheep oocytes and mouse oocytes but not to a majority of the hamster oocytes. The 90 kDa protein bound to oocytes from all three species. The 97 kDa protein, which did not recognise the porcine oocyte probe on a Western ligand blot, did not bind to oocytes from any species and served as a negative control. These observations are consistent with significant conservation of molecule and function among species within the same mammalian order. Hence, one species may be a good model for other species from the same order. Only limited conservation of binding activity of porcine sperm plasma membrane proteins to rodent oocytes was observed, suggesting a greater divergence either in molecular structure or in function among species from different orders.