Microtubule turnover in ooplasm biopsy reflects ageing phenomena in the parent oocyte.

Oviductal oocytes retrieved from superovulated B6D2F1 mice at 13.5, 16 and 19 h after human chorionic gonadotrophin (HCG) (groups A, B and C respectively, n = 382) were micromanipulated to obtain 12-20 mum sized ooplasm biopsy fragments. Experiments were divided into three sets. Ooplasmic microtubule dynamics were studied in ooplasm biopsy specimens and parent oocytes (set 1) and ooplasm biopsy specimens (set 2), whilst zona pellucida dissolution time, cortical granule loss and spindle/chromatin morphology using confocal microscopy were also studied in parent oocytes (set 2). Oocytes withstood oocyte biopsy with a high survival rate (98.2%) and the biopsied oocytes underwent successful fertilization and development (set 3). An absolute one-to-one correlation was seen between the oocyte biopsy specimens and the parent oocytes in terms of ooplasmic microtubule dynamics (set 1), and increased ooplasmic microtubule dynamics in oocyte biopsy specimens paralleled ageing phenomena in the parent oocytes (set 2). Zona pellucida dissolution time was significantly lower in parent oocytes from group A versus groups B (P = 0.032), and C (P < 0.001). (Groups A, B, C include minimal, moderate, increased ooplasmic microtubule dynamics in oocyte biopsy specimens respectively.) Oocyte cortical granule loss and spindle/chromatin abnormalities were mainly seen in group C (P < 0.001). Oocyte biopsy can thus be applied to judge age-related changes in the parent oocytes.

Inositol 1,4,5-trisphosphate receptor function in human oocytes: calcium responses and oocyte activation-related phenomena induced by photolytic release of InsP(3) are blocked by a specific antibody to the type I receptor.

Type I inositol 1,4,5-trisphosphate-sensitive receptors (InsP(3)R) are expressed in human oocytes and may be involved in operating the Ca(2+) release triggered by the fertilizing sperm. This study examines the contribution of type I InsP(3)R in operating Ca(2+) release in human oocytes secondary to InsP(3) itself, using a specific function-blocking antibody in conjunction with photolytic release of microinjected InsP(3). Intracellular Ca(2+) responses were assessed in oocytes microinjected with only caged InsP(3) in experiment set A, while in experiment sets B and C, sibling oocytes were injected with caged InsP(3) and the blocking antibody or a corresponding volume of medium, prior to flash photolysis. In experiment set C, certain fertilization-related phenomena (cortical granule exocytosis and chromatin configurations) were assessed using optical sections and three-dimensional image reconstructions obtained from a confocal laser scanning microscope. In experiment set A, photolytic release of InsP(3) triggered a Ca(2+) response (increase from approximately 100 to 220 nmol/l followed by an exponential recovery, n = 8) and a wave in the oocytes that spread from the stimulation point to the opposite pole. In set B, photolytic InsP(3) release generated Ca(2+) responses in control oocytes (n = 9), but not in the antibody-injected oocytes (n = 7). In set C, cortical granule exocytosis and anaphase chromosome configurations were noted in the control oocytes after flash photolysis (n = 6). These changes were completely absent in antibody injected oocytes as their cortical granules were intact and the chromosomes were in metaphase. These oocytes had also lacked Ca(2+) responses as in set B (n = 5). This study demonstrates the functional presence of type I InsP(3)R-operated Ca(2+) channels in human oocytes and further suggests an active role of InsP(3) in triggering the Ca(2+) rise and secondary activation phenomena at fertilization.

Presence and dynamic redistribution of type I inositol 1,4,5-trisphosphate receptors in human oocytes and embryos during in-vitro maturation, fertilization and early cleavage divisions.

We studied the presence and distribution of the intracellular calcium channel regulating type I inositol 1,4,5-trisphosphate receptors (IP3R) in human immature and mature oocytes, pronuclear zygotes and cleaved embryos using a specific antibody. Two approaches were used: (i) fluorescence immunocytochemistry using a confocal laser scanning microscope (CLSM) and (ii) Western blotting. With confocal microscopy, the receptors were found in the oocytes, fertilized zygotes as well as cleaved embryos at all stages studied. The pattern and distribution of the receptor staining in the oocytes changed gradually from a diffuse granular patchy one at the germinal vesicle (GV) stage to a reticular and predominantly peripheral one through the metaphase I and metaphase II (MII) stages. After fertilization, the distribution changed gradually to both, peripheral and central in the zygotes and early 2-4-cell embryos and predominantly perinuclear in the 6-8-cell embryos. Furthermore, an overall increase in the staining intensity was observed from GV to MII stage oocytes and from zygotes to 6-8-cell embryos. We also studied the spatial distribution of the receptor in detail by constructing three-dimensional images from the serial optical sections obtained on the CLSM. Peculiar peripheral aggregates of receptor clusters were noted in the MII stage oocytes, zygotes and some blastomeres from early cleaved embryos. Finally, Western blots performed on the extracts of 72 in-vitro matured oocytes and 50 spare cleavage stage embryos showed positive bands at approximately 260 kDa. These findings coincide with and thus possibly represent the dynamic changes occurring in the cellular Ca2+ release systems through oocyte maturation, fertilization and early embryogenesis. Thus, type I IP3R are likely to play a role during these stages of early development in the human.

Effect of post-ovulatory age and calcium in the injection medium on the male pronucleus formation and metaphase entry following injection of human spermatozoa into golden hamster oocytes.

The occurrence of parthenogenetic activation is a major hurdle in obtaining sperm chromosome metaphases after heterospecific intracytoplasmic sperm injection (ICSI) of golden hamster oocytes with human spermatozoa. We addressed two potential contributors to parthenogenetic activation namely, post-ovulatory age of the oocyte and Ca2+ content of the injection medium. In serial experiments, hamster oocytes were retrieved at 11.5, 13, 16 and 21 h after the ovulatory dose of human chorionic gonadotrophin (HCG) and microinjected with human spermatozoa suspended alternately in a regular (1.9 mM Ca2+) or a Ca2+-free medium. A progressive decrease in the rates of male pronucleus (MPN) formation and metaphase entry and increase in the rates of parthenogenetic activation without male pronucleus occurred with increasing post-ovulatory age. The favourable influence of Ca2+-free injection medium on the mean rates of MPN and metaphase entry was restricted to the relatively older oocytes (MPN 16 h: 49.5 versus 32.3%, P< 0.008; 21 h: 22.2 versus 11.1%, P< 0.001; metaphase entry 16 h: 36.8 versus 25.1%, P< 0.02; 21 h: 13.3 versus 5.2%, P< 0.01 in the Ca2+-free and regular groups respectively). Our data confirm the increased activation sensitivity with post-ovulatory ageing and its adverse influence on the MPN formation and metaphase entry after heterospecific ICSI of hamster oocytes.

In-vitro maturation of human germinal vesicle stage oocytes: role of cumulus cells and epidermal growth factor in the culture medium.

In-vitro maturation (IVM) of oocytes is a promising technique to reduce the costs and avert the side-effects of gonadotrophin stimulation for in-vitro fertilization (IVF). The pregnancy rates from oocytes matured in vitro are much lower than those of in-vivo stimulation cycles indicating that optimization of IVM remains a challenge. Therefore, we investigated the effect of supplementation of the medium with gonadotrophins, oestradiol and epidermal growth factor (EGF) and the effect of retaining or removing the cumulus cells on nuclear and cytoplasmic maturation of immature oocytes. Human germinal vesicle (GV) oocytes obtained after gonadotrophin stimulation for intracytoplasmic sperm injection (ICSI) were cultured in a complex defined medium either supplemented with gonadotrophins, oestradiol and physiological concentrations of EGF (2 ng/ml) or gonadotrophins and oestradiol alone. The cumulus cells were either removed or kept intact. In GV stage oocytes cultured without cumulus (group I) significantly more oocytes reached the metaphase II (MII) stage at 30 h in media supplemented with EGF (64.3 versus 33.9%, P < 0.003). For oocytes cultured with intact cumulus (group II), more oocytes reached MII at 30 h than in group I, but there was no difference in medium with or without EGF supplementation (81.8 and 79.8% respectively). Cytoplasmic maturation of MII oocytes was judged from their capability to activate and fertilize after ICSI. In group I, the rates of activation and normal fertilization were similar. However, in group II, significantly more oocytes underwent normal fertilization in the EGF-supplemented than the unsupplemented group (71.7 versus 45.6%, P < 0.05). The cleavage rates of the fertilized oocytes were similar in the sibling oocyte subgroups cultured with or without EGF supplementation, but the overall cleavage rates were higher in cumulus-intact compared to cumulus-denuded oocytes (88.9 versus 47.8%, P < 0.001). Thus, supplementation of the maturation medium with EGF and maintenance of the cumulus during culture improve the nuclear and cytoplasmic maturation of human oocytes in vitro.

Chromatin decondensation, pronucleus formation, metaphase entry and chromosome complements of human spermatozoa after intracytoplasmic sperm injection into hamster oocytes.

Obtaining karyotypes from human spermatozoa after microinjection into Syrian golden hamster oocytes is difficult and the hitherto reported results are unsatisfactory. This may be related to the injection and culture technique or to the high susceptibility of the hamster oocytes to undergo parthenogenetic activation or both. Therefore, we investigated the hamster oocyte-human sperm microinjection model using the following two approaches: (i) application of contemporary techniques for injection (touching the sperm tail) and culture (hamster embryo culture medium, HECM-3, 10% CO2) and (ii) omission of Ca2+ from the injection medium. Thus, in the first series of experiments, 252 hamster oocytes were injected with human spermatozoa. Among the 219 (87%) oocytes that survived the injection procedure, the mean percentages of male pronucleus formation [two pronuclei (2PN), two polar bodies (PB)], mitotic metaphase entry and sperm chromosome spreads were 41.4, 27.8 and 18.2% respectively. Analysis of the oocytes which failed to develop the male pronucleus following injection revealed that most of them had developed only the hamster female PN while the sperm nuclei were either intact or swollen (partially decondensed), indicating that failure of oocyte activation was not the likely reason for the failure of male PN formation in these oocytes. In the next series of experiments, sibling oocytes were alternately injected with spermatozoa suspended either in the regular (1.9 mM Ca2+) or Ca2+-free injection medium (experiment set 2, n=278). A significant improvement was noted in the mean percentages of oocytes with 2PN, 2PB, metaphase entry and sperm chromosome spreads in the Ca2+-free group versus the regular group (2PN, 2PB: 51 versus 36.6%, metaphase entry: 36.3 versus 26.9% and sperm chromosome spreads: 28 versus 20.4%; all P < 0.04). Thus, parthenogenetic activation appears to be one of the contributing factors for the failure of male PN formation after heterospecific hamster ICSI. From these experiments it can be concluded that application of the advanced injection and culture techniques and omission of Ca2+ from the injection medium are promising for the routine application of the hamster oocyte microinjection for karyotyping of human spermatozoa with poor fertilizing capacity.

Establishment of a human squamous cell carcinoma cell line of the upper aero-digestive tract.

A human squamous cell carcinoma (SCC) cell line has been established from the surgical specimen of an untreated, upper aero-digestive tract tumour, diagnosed as a squamous carcinoma, grade III, of the pyriform fossa. The tumour tissue was grown as a xenograft in an athymic nude mouse and was designated as NT-8. Histological examination of the surgical specimen and the nude mouse tumour showed that the two were identical. NT-8 was subsequently passed by subcutaneous injections into nude mice. After the 6th passage in nude mouse, the tumour was cultured in vitro where it grew as an epithelial cell line, with a typical cobblestone appearance. This cell line was designated as NT-8e. Both the primary tumour as well as xenograft and the cells in culture have retained several common morphological and biochemical characteristics. Immunological markers for epithelial cells including epithelial membrane antigen and cytokeratins were seen in all three, confirming the epithelial lineage. Characterization of the NT-8e cell line including growth parameters, anchorage-independent growth and tumorigenicity in nude mice, chromosome counts and DNA content by flow cytometry have been carried out.

Umbilical cord blood as a source of CD34 positive haematopoietic stem cells.

In this paper we have used a monoclonal antibody to CD34 an antigen expressed solely on stem cells, and stem cell colony assays to show that umbilical cord blood has nearly the same number of functional stem cells as compared to normal bone-marrow. The number of CD34+ve cells in cord blood being 2 to 2.7 per cent, whereas bone-marrow had 3 to 3.5 per cent. The multi-potent colony forming cells (CFU-GEMM) were 60 +/- 18 in cord blood per 2 x 10(5) mononuclear cells (MNCs), whereas normal bone-marrow had 70 +/- 10 per 2 x 10(5) MNCs. Enrichment of these stem cells on Percoll gradients was successful for normal bone-marrow but not for cord blood.

Enhancement of haemopoietic stem cell adherence to bone-marrow derived stroma by phyto-haemagglutinin treated leucocyte conditioned medium (PHA-LCM).

Stem cell adhesion to bone-marrow derived stroma, plays a crucial role in haemopoiesis. However, there is very little information as to the nature of the adhesion molecule. In this paper we have shown that human bone-marrow derived stroma can be established in tissue culture. This stroma is able to adhere human bone-marrow mononuclear cells including the multipotent stem cell, viz. CFU-GEMM. Their adherence increases when the stroma is treated with lymphokines in the form of PHA-treated leucocyte conditioned medium (PHA-LCM). Triton X-100 extracts of the untreated and PHA-LCM treated stroma were analysed on single dimension PAGE. It was observed that PHA-LCM treated stromal extracts showed two extra bands and an increase in the density of a band of approximately 14 kDa. Whether these changes have anything to do with the increased adhesion of stem cell is not yet known.