REPRODUCTIONRESEARCH The direct measurement of embryogenic volume and nucleo-cytoplasmic ratio during mouse pre-implantation development Catherine E M Aiken, Peter P L Swoboda, Jeremy N Skepper and Martin H Johnson Department of Anatomy, Downing Street, Cambridge CB2 3DY, UK Correspondence should be addressed to M H Johnson; Email: [email protected] Abstract After fertilization, the mammalian conceptus undergoes cleavage, a process of cell proliferation in the absence of interphase growth. It is not known when cleavage ends and gives way to fully replicative cell cycles with a stable nucleo-cytoplasmic ratio. We have used two-photon excitation and confocal microscopy to measure directly volumes and nucleo-cytoplasmic ratios of whole murine concepti and their individual constituent blastomeres during pre-implantation development up to the early uterine attachment stage (day 5). We show that the total cytoplasmic volume of the conceptus remains constant during pre-implantation development, and that the average nucleo-cytoplasmic ratio increases exponen- tially throughout the same period. Data from individual blastomeres show that both volume and nucleo-cytoplasmic ratio diverge in the inner and outer subpopulations evident from the 16-cell stage (fifth developmental cycle) onwards. Cells from emergent outer trophoblast populations are larger and have smaller nucleo-cytoplasmic ratios than those from emer- gent inner pluriblast populations. Moreover, the nucleo-cytoplasmic ratio of the trophoblast appears to be stabilizing, suggesting that for this subpopulation cleavage may end at the 16–32-cell transition. Putative hypoblast and epiblast cell subpopulations within the pluriblast were not distinguishable by volume or nucleo-cytoplasmic ratio. Embryonic stem cell volume was higher than that of either cell subpopulation of expanded blastocysts, and their nucleo-cytoplasmic ratio was similar to that of trophoblast cells. Reproduction (2004) 128 527–535 Introduction although the S phase does occupy most of each of the first five developmental cell cycles, G phases are present, even Most proliferative cell cycles involve cell growth and though short and variable, at least some cdk:cyclin oscil- chromosomal replication during interphase followed by lations occur, and there is evidence for some checkpoint division to smaller daughter cells during M-phase, such activity (Streffer et al. 1980, Howlett et al. 1985, Smith & that both cell numbers and total cell mass increase Johnson 1986, Chisholm 1988, Moore et al. 1996, Fulka (O’Farrell 2004). Recent work has explored how growth is et al. 1999, Bemark et al. 2000, Takai et al. 2000, Waclaw coupled to cell cycle progression so as to control the size & Chatot 2004). Cleavage in the mouse also differs from of the maternal cell and thereby its mitotic offspring other organisms in that the oocyte is smaller than many (Kellogg 2003, Fingar et al. 2004). However, some mitotic others (60–100 mm diameter), early cleavage cell cycles cells do not show interphase growth, for example the are longer (12–24 h), transcriptional activation occurs period of post-fertilization development in many animal soon after fertilization (Telford et al. 1990, Schultz 2002), species, traditionally called ‘cleavage’ cell cycles. Clea- development occurs within the nutritive female genital vage is a process by which a large oocyte, laden with tract, and early development is primarily concerned with developmental information and energy supplies, is con- the elaboration of a system of membranes for tapping verted to a multi-cellular embryo or larva. It is not known maternal resources (Johnson & Selwood 1996, Johnson & how growth is suppressed during cleavage. Cleaving cells Day 2000, Johnson & McConnell 2004). in some organisms are characterized by absence of the G1 In this paper, we derive quantitative data on cleavage and G2 phases, and it has been suggested that this feature for the pre-implantation period of development. We is important as it offers no opportunities for growth check- describe the direct measurement of total conceptus points to function. However, during mouse cleavage, volume and nucleo-cytoplasmic ratio and examine q 2004 Society for Reproduction and Fertility DOI: 10.1530/rep.1.00281 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/28/2021 08:17:48AM via free access 528 C E M Aiken and others whether the emergent cell lineages differ in cell volume viewing chamber. In the majority of experiments, triple and nucleo-cytoplasmic ratio. staining was used. Hoechst-stained concepti were rinsed thoroughly in protein-free PBI (phosphate buffered medium 1; supplied courtesy of Sheila Barton, Department of Materials and Methods Physiology, University of Cambridge, Downing Street, Recovery and handling of eggs and concepti Cambridge, UK) and placed in 1 ml of PBI in the centre of a 30 mm petri dish to the base of which had been fixed a Female CD1 and MF1 mice (Harlan, Bicester, UK) of 3–6 No. 1 coverslip using Sylgard (Dow Corning). The cover- weeks were housed in groups of between five and ten. slips were pre-coated with either poly-L-lysine (1% in Procedures were carried out under Home Office license PBS) or (mostly) phytohaemagglutinin (0.1–0.2 mg/ml; and after local ethical review. Ovulation was induced by Sigma) followed by washing with PBI. After concepti had intraperitoneal injection of PMS (10 iu; Folligon; Sigma) firmly adhered to the coverslip, 1 ml M2 þ 4% BSA was followed after 46–48 h by intraperitoneal injection of added. Whole concepti were imaged with their zonae human chorionic gonadotropin (10 iu; Chorulon; Sigma). intact at 37 8C using a Leica TCS-SP-MP two-photon exci- Females were paired with individually housed CD1 stud tation and confocal microscope. Visualization was by males (Harlan) and mating assessed by inspecting for a sequential two-photon excitation and confocal vaginal plug. Concepti were retrieved from the oviducts or microscopy, using a picosecond pulsed tunable Tsunami uteri between day 0 (day of positive vaginal plug) and laser (Spectra Physics, Mountain View, CA, USA), tuned to early day 5 (when attachment is initiated) by flushing with 775 nm to excite Hoechst and Calcein and the 568 nm M2 þ 4% (w/v) BSA culture medium (M7167 embryo line of a Krypton laser to excite FM4-64. A water immer- tested; Sigma) and stored until use under oil (M8410 sion £ 63 lens with a numerical aperture of 1.2 and a embryo tested; Sigma). Recovery and transport were at cover-glass correction collar were used. Emitted fluor- 37 8C. All concepti were imaged live. When removal of escent light was captured in discrete windows (Hoechst the zona was required, brief exposure to acid Tyrode’s 400–480 nm, FM4-64 600–700 nm, and Calcein 505– solution (made in the laboratory) was used. Intact inner 545 nm). The confocal pinhole was set to 1 Airy disk cell masses (ICMs) were recovered from blastocysts by equivalent for the objective lens in both confocal and two- immunosurgery (Solter & Knowles 1975). Concepti were photon imaging. A Z-series of images was collected from recovered from uteri 98 h post-human chorionic gonado- each subject, capturing an image every 1 mm. Every image tropin and sorted into expanded, semi-expanded, nascent was used for three-dimensional (3D) reconstruction, while and pre-nascent blastocysts. Zona-intact blastocysts were every second image was used for stereology. incubated for 30 min in a 1:4 dilution of rabbit anti- trophoblast antibody (a gift from Azim Surani, Department of Physiology, University of Cambridge, Downing Street, Analytical procedures Cambridge, UK), washed through four changes of The Cavalieri principle (Gunderson & Jenson 1987) was M2 þ 4% BSA, and incubated in a 1/5 dilution of guinea- used to estimate volumes of concepti, ICMs, single cells pig complement (Sigma) for 20 min. The blastocysts were and their nucleo-cytoplasmic components. Images on then transferred to M2 þ 4% BSA for 30 min, and the screen (521 £ 521 mm) were overlain with quadratic grids ICMs removed by gentle pipetting through a micropipette. with a spacing of 20 mm for cytoplasm and 10 mm for Disaggregation of concepti was achieved by one of two nuclei. The number of intersections overlying nuclei and methods. Up to the 16-cell stage, zona-free concepti were cytoplasm was counted. The near-spherical shape of the exposed for 10–30 min to calcium-free M2 þ 6% BSA concepti meant that less than the accepted maximum of (made in the laboratory). From the 16-cell stage onwards, 200 points per object from 10–15 sections could be zona-free concepti were exposed to 0.1% trypsin (con- counted, giving an error coefficient of 5–10% (Roberts taining 0.04% EDTA; Handyside 1980) in calcium-free et al. 1993, 1994). The volume of the conceptus (exclud- M2 þ 4% BSA. Embryonic stem (ES) cells (CES3 on ing blastocoelic volume) or its constituent cytoplasm or 129/Sv background; a gift from Azim Surani) were stained nuclei could then be calculated. For single-cell analysis, and imaged, and underwent volumetric analysis using the Z-stacks were captured through the conceptus and indi- same techniques as for concepti. vidual cells within these images were chosen for analysis. Where cell subpopulations were analysed, random selec- Imaging concepti tion of cells was attempted. However, not all cells within Nuclei were stained by incubating concepti in M2 þ 4% the total image were delineated clearly enough to obtain BSA containing 10 mg/ml Hoechst 33258 (Sigma; 1 mg/ml an accurate volume. Data from these cells are not stock) for 2–4 h. Cytoplasm was stained by a 15 min incu- included, and so a degree of non-random selection cannot bation in the calcium dye Calcein AM (0.5 mg/ml in be excluded.