Boar spermatozoa were prepared for intracytoplasmic sperm injection (ICSI) by two different treatments to facilitate sperm chromatin decondensation and improve fertilisation rates after ICSI in pigs: spermatozoa were either frozen and thawed without cryoprotectants, or treated with progesterone. Morphological changes of the sperm heads after the treatments were examined and then the activation of oocytes and the transformation of the sperm nucleus following ICSI were assessed. After freezing and thawing, the plasma membrane and acrosomal contents over the apical region of sperm head were lost in all the spermatozoa. Following treatment with 1 mg/ml progesterone, the acrosome reaction was induced in 61% of spermatozoa. After injection of three types of spermatozoa, non-treated spermatozoa and progesterone-treated (i.e. acrosome-reacted) spermatozoa induced oocyte activation, but frozen-thawed spermatozoa induced oocyte activation at a significantly lower rate. Sixty-two per cent of sperm heads remained orcein-negative for 6 h, however, resulting in delayed sperm chromatin decondensation and low male pronuclear formation in the oocytes injected with a non-treated spermatazoon. Since the treatments of freezing and thawing and progesterone for spermatozoa accelerated the initial change in sperm chromatin and the latter treatment induced oocyte activation earlier, it is considered that the delay in oocyte activation and decondensation of sperm chromatin after injection of non-treated spermatozoa is caused by the existence of the sperm plasma membrane. These results show that progesterone treatment efficiently induces the acrosome reaction in boar spermatozoa without destroying their potency for oocyte activation, and the induction of the acrosome reaction results in the promotion of male pronuclear formation after ICSI.

The sperm glycine receptor/Cl- channel (GlyR) is important to the initiation of the mammalian sperm acrosome reaction by the egg zona pellucida, but its presence in spermatogenic cells has not been demonstrated. Reverse transcriptase-polymerase chain reaction studies confirmed that GlyR beta subunit transcripts similar to those of the neuronal GlyR beta subunit are present in the testis of Swiss Webster mice. In situ hybridisation analysis demonstrated that GlyR beta subunit mRNAs were expressed within the germ cells of seminiferous tubules in those mice.

This study was designed to examine the effects of extracellular reduced glutathione on development of pig embryos, produced by in vitro maturation (IVM) and in vitro fertilisation (IVF), in a chemically defined North Carolina State University (NCSU) 23 medium or in NCSU 23 medium with bovine serum albumin (BSA). Microfilament distribution, as a marker of embryo quality, was also examined by immunocytochemical staining and confocal microscopy. When the inseminated oocytes were cultured in the defined medium containing 0-0.5 mM glutathione, blastocyst formation as observed only in the media with glutathione (8.5-16.0%). Increased numbers of blastomeres were observed in the blastocysts as the concentration of glutathione was increased (18.8±7.2 to 31.0±8.6). In NCSU 23 medium with 4 mg BSA/ml, addition of glutathione at concentrations of 0.125-0.5 mM significantly increased the proportions of oocytes that developed to blastocysts (39.2-52.5%) compared with the control (29.5%). However, no difference was observed in the average cell number in the blastocysts (41.9±15.6 to 49.1±15.5). There were no significant differences in the microfilament distribution in the embryos produced in the defined medium and in the BSA-containing medium. These results indicate that pig embryos produced by IVM/IVF can develop to the blastocyst stage in a defined medium. BSA and glutathione have a synergistic effect on pig embryo development.

In the present study we determined the chromatin organization and fate of introduced mitochondria in porcine embryos following intracytoplasmic injection of pig or mouse sperm cells. At 3, 6, 9 and 12 h following injection of pig or mouse spermatozoa or isolated sperm heads, the oocytes were fixed and stained with propidium iodide. Between 3 and 6 h following injection, both porcine and murine sperm chromatin developed into pronuclei. The male and female pronuclei were apposed within 12 h in porcine oocytes following sperm injection from either source. We also introduced foreign mitochondria from either mouse or pig sperm midpiece into porcine oocytes following sperm injection. While porcine sperm mitochondria rapidly disappeared from the actively developing porcine oocytes, mouse sperm mitochondria remained in the embryos until the 8-cell stage. These results suggest that pronuclear formation and movement occur between 6 and 12 h following sperm incorporation into the cytoplasm, and that foreign mitochondria are selectively removed in a species-specific manner.

Chromatin and microtubule organisation was determined in maturing and activated porcine oocytes following intracytoplasmic sperm injection in order to obtain insights into the nature of sperm chromatin decondensation and microtubule nucleation activity. Sperm chromatin was slightly decondensed at 8 h following injection into germinal vesicle stage oocytes. Sperm-derived microtubules were not seen in these oocytes. Following injection into metaphase I (MI)-stage oocytes, sperm chromatin went to metaphase in most cases. A meiotic-like spindle was seen in the sperm metaphase chromatin. In a few MI-stage oocytes, sperm chromatin decondensed at 8 h after injection, and a small sperm aster was seen. Sperm injection into oocytes at 5 h following activation failed to yield pronuclear formation. Maternally derived microtubules were organised near the female chromatin in these oocytes, and seemed to move condensed male chromatin closer to the female pronucleus. At 18 h after sperm injection into pre-activated oocytes, a condensed sperm nucleus was located in close proximity to the female pronucleus. These results suggest that the sperm nuclear decondensing activity and microtubule nucleation abilities of the male centrosome are cell cycle dependent. In the absence of a functional male centrosome, microtubules of female origin take over the role of microtubule nucleation for nuclear movement.

A monoclonal antibody (6964M) was generated against the envelope component gp69/64 of Xenopus laevis eggs. On indirect immunofluorescence using this antibody, the positive reaction was seen on the surface of both vitelline envelope (VE) and coelomic envelope (CE). On immunoelectron microscopy, gp69/64 was preferentially distributed on the thick bundles forming the edge of the tunnel openings on CE, and this distribution pattern was fundamentally inherited by VE. Counting the number of immunogold particles indicated that VE has about twice as many particles as CE, with a 3-4 times higher density at the animal pole than vegetal pole. The number of sperm bound to CE was small, being approximately one-twentieth of the number of sperm bound to VE. An extremely small number of sperm (< 2 per animal hemisphere) was found to bind to VE* of activated eggs as a background. The sperm binding to CE was inhibited by pretreatment of the envelopes with 6964M or in the presence of purified gp69/64 from VE on insemination, confirming that sperm binding is mediated by gp69/64 exposed on the CE surface. In spite of at most a 2-fold increase in the amount of exposed gp69/64, the sperm binding increased about 20-fold upon CE-to-VE conversion, suggesting that the increase in the amount of exposed gp69/64 is itself insufficient to explain the increase in the number of bound sperm.

Bovine embryos in the early blastocyst/blastocyst stage were analysed by [5-3H]uridine labelling followed by electron microscopic autoradiography. In normal control embryos an intact zona pellucida, evenly developed blastomeres and a transparent perivitelline space were seen. In this group, the blastomeres of the trophoblast and embryoblast showed high homogeneous labelling localised in the nucleoplasm and even more intense labelling in the nucleolus. On the contrary, in addition to evident cytoplasmic disintegration, a clearly different labelling pattern and a low labelling intensity were observed in the nuclei of the segregated cells in the subzonal space and in those free in the blastocoele cavity. A typical nuclear morphological feature of these blastomeres was chromatin marginalisation, similar to that observed in embryos treated with actinomycin D for transcription inhibition. It is concluded that the segregated cells are arrested in their further differentiation.

Tail-cut bovine spermatozoa were microinjected into ooplasmic lipid polarised, in vitro matured bovine oocytes using a piezomicropipette-driving system. No exogenous oocyte activation treatment was used. Of the sperm-injected oocytes, 86.3% were activated, 71.8% cleaved and 22.7% developed to the blastocyst stage. The average cell count of the blastocysts was 122.5 ± 15 and a majority (81.8%) of the blastocysts were cytologically normal (diploid). When transferred to recipient cows, 5 of 8 blastocysts developed to fetuses and 4 of 7 recipients became pregnant. Normal offspring were born.

Interspecific hybrid embryos are useful models for the study of maternal-fetal interactions, transmission pattern of species-specific markers and parental contributions to growth and developmental potential of pre-attachment embryos. In an attempt to investigate the possibility of producing hybrid embryos of domestic cattle (Bos taurus) and water buffalo (Bubalus bubalis), cattle oocytes were exposed to buffalo sperm and buffalo oocytes were exposed to cattle sperm and the cleavage rate and the post-fertilisation features of hybrid embryos up to the blastocyst stage were compared with those of buffalo and cattle embryos. The cleavage rate in buffalo oocytes exposed to cattle sperm was low (40.8%), with only 8.8% of these hybrid embryos reaching the blastocyst stage. Cattle oocytes exposed to buffalo sperm showed 86.3% cleavage, while 25.9% of these attained the blastocyst stage. The speed of development of both types of hybrids was intermediate between that of cattle and buffalo embryos, with hatching occurring on day 7.5 in hybrid embryos, day 8-9 in cattle and day 7 in buffalo. The proportions of cells contributing to the trophectoderm and the inner cell mass were closer to those of the maternal species in both types of hybrid embryos. Our results indicate that cattle-water buffalo hybrid embryos produced using interspecies gametes are capable of developing to advanced blastocyst stages and that their in vitro fate, and developmental potential, are influenced by the origin of the oocyte.

In this work we carried out an ultrastructural analysis of the cell interface between oocyte and follicle cells during the oogenesis of the amphibian Ceratophrys cranwelli, which revealed a complex cell-cell interaction. In the early previtellogenic follicles, the plasma membrane of the follicle cells lies in close contact with the plasma membrane of the oocyte, with no interface between them. In the mid-previtellogenic follicles the follicle cells became more active and their cytoplasm has vesicles containing granular material. Their apical surface projects cytoplasmic processes (macrovilli) that contact the oocyte, forming gap junctions. The oocyte surface begins to develop microvilli. At the interface both processes delimit lacunae containing granular material. The oocyte surface has endocytic vesicles that incorporate this material, forming cortical vesicles that are peripherally arranged. In the late previtellogenic follicle the interface contains fibrillar material from which the vitelline envelope will originate. During the vitellogenic period, there is an increase in the number and length of the micro- and macrovilli, which become regularly arranged inside fibrillar tunnels. At this time the oocyte surface exhibits deep crypts where the macrovilli enter, thus increasing the follicle cell-oocyte junctions. In addition, the oocyte displays coated pits and vesicles evidencing an intense endocytic activity. At the interface of the fully grown oocyte the fibrillar network of the vitelline envelope can be seen. The compact zone contains a fibrillar electron-dense material that fills the spaces previously occupied by the now-retracted microvilli. The macrovilli are still in contact with the surface of the oocyte, forming gap junctions.

During the early preimplantation stages of human embryos, pyruvate and lactate, but not glucose, are the preferred energy substrates. Transport of these monocarboxylates is mediated, in mammalian cells, by a family of transporters, designated as monocarboxylate transporters (MCTs). Human and mouse genetic expression of MCT members 1, 2, 3, 4 and basigin, a chaperone protein of MCT1 and MCT4, was qualitatively analysed using the reverse transcription nested polymerase chain reaction (RT-nested PCR) in immature oocytes (germinal vesicle stage; GV), in non-fertilised metaphase II (MII) oocytes and in embryos from 2-cell stage to blastocysts. Transcripts encoding for MCT1 and MCT2 were present, under a polyadenylated form, in the majority of the human and mouse oocytes and early embryos. MCT3 transcripts were not detected in either human or mouse. MCT4 mRNA was not detected in human oocytes and embryos, but was present in mouse oocytes and embryos. This fact could imply differences in lactate transport and regulation of intracellular pH between human and murine early embryos. Basigin transcripts were present in mouse and human MII oocytes and preimplantation embryos, but were not detected at GV stage. However, using 3' end-specific primers in the RT reaction instead of Oligo(dT)12-18 primers, transcripts encoding for this protein were then detected at GV stage in both species. This result suggests that a regulated polyadenylation process occurs during oocyte maturation for these transcripts. Thus, basigin mRNA can be considered as a marker of oocyte cytoplasmic maturation in human and mouse species.

Potassium (K+) channels are believed to regulate mammalian sperm acquisition of fertilising capacity. However, the molecular identity of these proteins in sperm has not been elucidated. In this report, using immunoconfocal and electron microscopy we show that a minimum of four different classes of K+ channels (Kv1.1, Kv1.2, Kv3.1 and GIRK1) are present and regionally distributed over the surface of mouse epididymal sperm. In addition, the use of reverse transcription polymerase chain reaction on RNA from mouse spermatogenic cells allowed the amplification of multiple transcripts corresponding to the channels identified by immunocytochemistry. Consistent with this, whole-cell patch-clamp recordings showed the expression of at least two different outwardly rectifying K+ currents in spermatogenic cells.