This paper examines some of the problems and questions that must be considered in relation to research on the role of growth factors in preimplantation embryos. It reviews and summarizes the large body of work on gene expression of growth factor receptors and ligands in preimplantation embryos and in oviduct and uterine tissue. It also reviews the literature on the effects of gene knockout in preimplantation embryos and concludes with a review of work on the effects of growth factors on cultured embryos.

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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\m=x\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.

This study reports a novel, simple method for culture of mouse follicles which results in follicles with cell numbers similar to in vivo fully grown follicles. Using this method, follicles (180 -240 mm in diameter) were cultured in a 100 ml inverted drop of medium without oil and compared with culture in upright drops with and without a mineral oil overlay. Follicles, isolated from C57BL/6 3 CBA/ca crossbred and MF1 inbred mice, were cultured individually at 37 8C in 96-well round-bottomed suspension cell tissue culture plates for 6 days. Follicles grown in the inverted drop culture system reached a markedly higher final diameter (means6S.E.M.; 471 6 6.0 mm) as compared with the upright with oil (363 6 2.7 mm) and without oil (358 6 4.0) systems. There was no significant effect of mouse strain on follicle diameter. Follicular secretion of oestradiol and lactate into the medium was measured on days 2, 4 and 6 of culture. Secretion of oestradiol per follicle on day 6 was 2.49 6 0.45 ng in the inverted and 0.90 6 0.17 ng in the upright without oil system (P < 0.001). Follicular secretion of lactate on a per unit of follicle volume basis remained constant in the inverted system over days 2, 4 and 6 and was less (P < 0.001) than secretion in both the upright with and without oil systems. Follicle cell proliferation was markedly increased in the inverted as compared with the upright with oil system; the increases in cell numbers were significant on day 3 (P < 0.01) and on all subsequent days (P < 0.001). These results are discussed in relation to the supply of oxygen to the follicle in culture.

A factor of low M(r) with growth-promoting effects on rabbit embryos was extracted and purified from commercial bovine serum albumin (BSA). This embryotrophic factor was extracted from BSA dissolved in formic acid by membrane filtration (membrane cutoff of M(r) 10,000) and then freeze-drying of the filtrate. The extract was purified successively by chromatography on G-10 Sephadex, QAE-Sephadex A-25 anion exchange and high-performance liquid chromatography (HPLC) reverse-phase columns. Mass spectrometry of the active reverse-phase material indicated that the major component in this material had an M(r) of 192. The embryotrophic factor in the low M(r) extract of BSA was shown to be citrate, because: (i) the mass spectra of the active reverse-phase material and citrate were identical, (ii) the activity was eluted at the identical position to citrate on an analytical HPLC anion-exchange column, (iii) the original BSA sample was shown by enzyme assay to be heavily contaminated by citrate and (iv) citrate stimulated cell proliferation and expansion of blastocysts.