Summary. The effects of a range of commercially available proteases and glycosidases on blastocyst development and hatching were examined on rabbit embryos cultured from the morula stage in a defined medium supplemented with charcoal-treated bovine serum albumin. The proteases tested were trypsin, α-chymotrypsin, thrombin, elastase, plasmin, papain, clostripain, collagenase, Streptomyces griseus protease and cathepsin C. The glycosidases tested were neuraminidase, α-mannosidase, β-galactosidase and hyaluronidase. None of these enzymes appeared to stimulate blastocyst growth. The only enzymes which digested the embryonic investments, the zona and mucin coat, sufficiently to cause complete blastocyst hatching were trypsin and Streptomyces griseus protease at relatively low concentrations (250 ng/ml) and chymotrypsin and elastase at higher concentrations.
M. T. KANE
Studies on the effects of pH on growth of preimplantation mammalian embryos have so far been limited to the mouse. Brinster (1965a) found that blastocysts developed in culture from two-cell mouse ova in media with pH values ranging from 5·87 to 7·78 with an apparent optimum of 6·82. He later found, however, that the optimum pH appeared to depend on the carbohydrate energy source present in the medium (Brinster, 1965b). These studies were carried out using bicarbonate-buffered media.
The work reported here extends the study of these problems to the rabbit, using a synthetic medium similar to that developed by Kane & Foote (1970) for culture of rabbit ova. One-cell rabbit ova were cultured in media of various bicarbonate concentrations. Two experiments were carried out.
Experiment 1 was a preliminary study in which bicarbonate concentrations were varied between 1·875
M. T. Kane
Summary. Fraction V bovine serum albumin (BSA) was dissolved in 5% formic acid and filtered through a molecular filter with a cutoff of Mr 10 000. The freeze-dried filtrate stimulated increased cell division of cultured rabbit morulae to blastocysts (up to 8-fold increase in cell number) and increased blastocyst expansion (> 2-fold). These results show that some samples of commercial BSA contain an embryonic growth factor of a low molecular weight.
M. T. Kane
Summary. Rabbit morulae were cultured to blastocysts in various concentrations of the potential phospholipid precursors, myo-inositol, choline, serine and ethanolamine. Serine (20–2500 μm) had a significant stimulatory effect on blastocyst formation and blastocyst expansion and inositol (3–375 μm) had a significant stimulatory effect on blastocyst expansion. There was no significant stimulatory effect of choline or ethanolamine.
Keywords: blastocyst expansion; rabbit; phospholipid precursors
M. T. Kane
Summary. Commercial samples of bovine serum albumin (BSA) in a complex medium caused growth of 1-cell rabbit embryos to completely hatched blastocysts. Heat treatment of the BSA at 65 or 80°C significantly decreased blastocyst formation and expansion and destroyed the ability to cause blastocyst hatching. Addition of trypsin at levels down to 20 ng/ml caused the formation of hatched blastocysts which degenerated rapidly. The effects of 5 protease inhibitors (ovomucoid trypsin inhibitor, α-1-antitrypsin, TAME, TLCK and soyabean) were tested. Ovomucoid trypsin inhibitor, TAME and TLCK significantly inhibited blastocyst hatching but only at the highest concentration used. These inhibitors also reduced blastocyst formation and expansion, indicating that their effect was not specifically on blastocyst hatching in vitro. It is concluded that hatching of rabbit blastocysts is probably not dependent on protease action.
M. T. Kane
Summary. Rabbit morulae were cultured in vitro for 4 days in a synthetic culture medium supplemented with two different lots of commercial bovine serum albumin (BSA) and two different amino acid formulations in a factorial 2×2 arrangement. One lot of BSA caused complete hatching of a proportion of blastocysts and formation of more than twice as many cells per blastocyst (hatched and unhatched) as that of the second BSA lot which did not cause complete hatching of any blastocysts. The mean cell numbers of hatched blastocysts were more than twice those of non-hatched blastocysts. There was no significant effect of amino acid formulation.
C. Duffy and M. T. Kane
The aims of this study were to investigate in mouse embryonic stem cells (1) the requirement for myo-inositol for cell proliferation, (2) the incorporation of inositol into the phosphoinositides and inositol phosphates of the phosphatidylinositol (PtdIns) signal transduction system and (3) the effect of serum growth factors on PtdIns turnover. Exogenous myo-inositol was not essential for embryonic stem cell proliferation. Lithium, an inhibitor of endogenous inositol recycling, inhibited embryonic stem cell proliferation but this effect was not reversible by the addition of high concentrations of exogenous inositol. [3H]inositol was incorporated into the phosphoinositides, PtdIns, PtdIns4P and PtdIns(4,5)P2 in similar proportions as reported for other cells. [3H]inositol was also incorporated into a fourth lipid, tentatively identified as an inositolglycan. [3H]inositol was also incorporated into a number of inositol phosphates, with the greatest amount of incorporation after 24 h into an inositol pentakisphosphate. After serum starvation for 24 h, the addition of 10% whole or dialysed serum for 2 or 20 min increased (P < 0.05) incorporation into inositol trisand tetrakisphosphates. These results demonstrate the presence of PtdIns system components in embryonic stem cells and increased PtdIns turnover in response to serum growth factors.
M. M. Fahy and M. T. Kane
The effects of lithium, an inhibitor of the recycling of inositol in the phosphatidylinositol cycle, on rabbit blastocyst growth and metabolism of phosphoinositides were investigated. Day 2 rabbit morulae were first cultured for 2 days in basic culture medium and then transferred to medium containing myo-[2-3H]inositol for culture for a further 3 days. At the end of culture, the resulting blastocysts were incubated with LiCl (0, 1, 5, 10, 20 mmol l−1) for 1 h. The blastocysts were then lysed and both the aqueous and lipid portions were analysed for incorporated radioactivity. Thin layer chromatographic separation of the lipid portion indicated that lithium had no significant effect on formation of radiolabelled phosphoinositides. However, high performance anion exchange chromatography indicated that lithium significantly stimulated accumulation of radiolabelled inositol monophosphate and inositol 1,4,5-trisphosphate. This result indicates that the phosphatidylinositol cycle is turning over in rabbit blastocysts. Continuous culture of rabbit embryos for 5 days in media containing LiCl (5, 10, 15 and 20 mmol l−1) significantly decreased blastocyst growth as measured by blastocyst expansion and incorporation of [3H]thymidine. However, supplementing the medium with excess inositol (up to 9375 μmol l−1), in an attempt to increase the intracellular uptake of inositol and thus compensate for the inhibitory effect of lithium on inositol recycling, did not reverse the inhibitory effect of lithium on blastocyst growth.
P. M. Morgan and M. T. Kane
The protein content of rabbit embryos during the first 7 days of development in vivo was determined. The protein content of intact embryos, embryonic cells (intact embryos without mucin coats for developmental stages up to 96 h post-coitum and free of blastocyst coverings for later stages) and blastocyst coverings were determined by the Pierce Micro BCA assay. The mean protein content of intact one-cell or two-cell embryos was 0.16 μg and increased at the four- to six-cell stage with no further increase until the late morula/early blastocyst stages (days 3 to 4). There was a 53-fold increase in protein from the early to late blastocyst stages. The protein content of embryonic cells was stable at a mean value of 0.16 μg until the late morula stage (day 3) and then increased to a mean of 6.85 μg on day 6 and 50.38 μg on day 7. The increase in protein content of intact embryos up to about 72 h appeared to be due solely to an increase in the protein content of the mucin coat. The protein content of the blastocyst coverings increased from a mean of 2 μg on day 5 to a mean of 35 μg on day 7. For blastocyst stages, the total protein content of intact blastocysts and of embryonic cells was correlated with the surface area of the embryos (r 2 = 0.895 and 0.873, respectively) and, thus, an increase in blastocyst size is a true index of blastocyst development.
M. T. Kane and D. R. Headon
Summary. Normal bovine serum albumin (BSA) in a complete medium without energy substrates promoted growth of 1-cell embryos to hatched blastocysts. Defatted charcoal-treated BSA did not promote growth to the blastocyst stage but the addition of pyruvate or palmitic and oleic acids allowed blastocyst growth but not blastocyst hatching. Sodium dodecyl sulphate-gel electrophoresis showed that both the normal and defatted BSA samples were heavily contaminated by proteins other than albumin. Fractionation of the normal BSA on Sephadex G-200 indicated that the property of promoting complete blastocyst hatching was not due to the albumin but was associated with the higher mol. wt fraction of the BSA. Extraction of normal BSA with chloroform appeared to destroy the hatching-promoting ability as neither the residue after extraction nor defatted BSA to which the organic extractate had been added promoted complete blastocyst hatching. It is concluded that commercial BSA may have at least two effects on blastocyst growth: (1) energy provision via albumin-bound fatty acids, and (2) promotion of blastocyst hatching by a non-albumin component.