Regionalization and Lateral Proteins in Unfertilized and Diffusion of Membrane Fertilized Mouse Eggs

The unferti l ized mouse egg has a round and highly villated main body and a "n ipp le" that is unvil lated and buds off on ferti l ization to form the second polar body. Fluorescent markers stain the body more intensely than the nipple, which has been assumed to result from surface amplif ication due to microvill i. Using fluorescence recovery after photobleaching and microfluorescence photometry, we have measured the membrane protein diffusion and concentration on the main body and nipple region of unferti l ized and on fertil ized CD-1 mouse eggs. Two general membrane protein labels were used: rhodamine-labeled succinylated concanavalin A and tr initrobenzene sulfonate visualized with a rhodamine Fab fragment of a sheep anti-tr ini trophenyl. We found that whi le the diffusion coefficient was the same on the nipple and main body, considerably higher recovery was observed on the nipple for both probes. The ratio of intensity of fluorescence on the nipple to main body was significantly lower for the concanavalin A stain than for the tr ini trophenyl stain, indicating that true concentration gradients exist beyond those that result from surface amplification. The effect of ferti l ization was not general. No effect was observed for the concanavalin A stain for either diffusion coefficient or percent recovery. For the tr initrophenyl stain, percent recovery decreased approximately twofold whi le diffusion coefficient increased approximately threefold. The ability of membrane lipids and proteins to move freely in the plane of the membrane is fundamental to membrane function in many cases (20). Considerable experimental effort has been directed at the issue of how the motion of membrane components is altered by membrane transformations such as fertilization and differentiation (6, 9, 14, 21, 22, 26, 35, 38). The development of direct biophysical measures of lateral diffusion of membrane components, such as fluorescence recovery after photobleaching (FPR), 1 has contributed considerably to our understanding of the motion of membrane molecules. Recent experiments have suggested that the lipids in membranes are not mixed homogeneously but rather sequester into an ensemble of microenvironments or domains (16, 17, 29, 35, 38) and, therefore, that the membrane does not have a single bulk viscosity. It is also apparent that plasma membrane protein diffusion is in general too slow to be controlled by lipid Abbreviations used in this paper: C16 diI,3,Y-dihexadecylindocarbocyanine iodide; Con A, concanavalin A; D, diffusion coefficient; DTAF, 4,6-dichloro-s-triazin-2-yl amino fluorescein; FI, fluorescence intensity; FPR, fluor©sccnce recovery after photobleaching; hCG, human chorionic gonadotrophin; %R, percent recovery; S-Con A, rhodamin¢-conjugat©d succinylated Con A; TNBS, trinitrobcnzene sulfonat¢; TNP, trinitrophenyl; TRITC-sFab aTNP, rhodamine-labeled Fab fragment of sheep [gG against TNP. "viscosity." Rather, interactions with other cellular components, such as the cytoskeleton, appear to control membrane protein diffusion (32, 42). The nature both of the lipid domains in membranes and the various interactions that control membrane protein diffusion are just beginning to be understood. An excellent system for the study of the effect of cellular transformation on the organization and motion of membrane components is fertilization of the mammalian egg. As seen in Fig. 1, the unfertilized mouse egg has two regions: a round and highly viUated main body, and a protruding unvillated nipple (8). When the egg is fluorescently tagged for a surface component such as concanavalin A (Con A) receptors, the stain is "polarized" with the main body staining more intensely than the nipple (13). Sperm bind and fuse to the main body of the egg (13), initiating the cortical reaction (10), and rendering the egg refractory to further sperm egg fusion (10, 40, 41). The nipple region subsequently constricts and buds off to form the second polar body (see Fig. 1). Data of Wolf et al. (35, 38) suggest that fertilization in both enchinoderms and mice is accompanied not by a change in bulk membrane viscosity but rather by an alteration in the ensemble of lipid domains. In this paper we consider the effect of fertilization on the diffusion of membrane proteins in the mouse egg. Specifically, we will address three questions: (a) Is THE ]OURNAL OF CELL BIOLOGY, VOI.UME 96 IUNE 1983 1786-1790 1 786 © ]-he Rockefeller University Press • 0021-9525/83/06/1786/05 $1.00 on July 8, 2017 jcb.rress.org D ow nladed fom there a general effect of fertilization on the diffusion of membrane proteins? (b) In the unfertilized mouse egg is there a difference in the diffusibility of membrane proteins on the main body vs. the nipple? (c) Do differences in fluorescence staining between the nipple and main body merely reflect surface amplification due to microvilli or are there true differences in the concentration of membrane proteins in these two regions of a continuous plasma membrane? MATERIALS AND METHODS En7 bryos: Female CDI mice (Charles River Breeding Laboratories, Inc., Wilmington, MA) 3-6 wk of age, were induced to ovulate synchronously by an injection of l0 IU i.p. of pregnant mare's serum (Intervet, Cambridge, United Kingdom) followed 48 h later by 5 IU of human chorionic gonadotrophin (hCG; Intervet). For experiments where fertilized eggs were required, the hormonaUy primed females were mated with CD-I males. The presence of a vaginal plug was taken as an indication of successful mating. At 15-20 h post hCG, unfertilized and fertilized egg masses were popped from the ampullae of excised oviducts into Hanks' balanced salt solution containing 4 mg/ml bovine serum albumin (HBSS plus BSA). The cumulus ceils were removed by a 5-10-min exposure to 0A% wt/vol hyaluronidase (Sigma Chemical Co., St. Louis, MO). Eggs were then washed through three changes of HBSS plus BSA. Zonae pellucidae were removed by a brief exposure ( 10-30 s) to prewarmed acid Tyrode's solution (23). The completeness of this method of zona removal has been demonstrated by Bleil and Wassarman (5). Most unfertilized eggs had one polar body and an observable nipple. Eggs were judged to be fertilized if they contained two pronuclei and /or two or three polar bodies. Fluorescent Labeling of Embryos: Amino groups on the surface of zonafree unfertilized and fertilized eggs were labeled covalemly with I mM trinitrobenzene sulfonate (TNBS) in HBSS plus BSA for 20 rain at 37°C. Eggs were washed through three changes of HBSS plus BSA and incubated for 10 rain at room temperature in a rhodamine-conjugatcd Fab fragment of sheep immunoglobulin G (IgG) directed against trinitrophenyl (TNP) (TRITC-sFab aTNP). To ensure that the rabbit antibody was free of Fc fragments, eggs were labeled first with TNBS, then with the TRITC-sFab ctTNP, a whole sheep IgG aTNP or no antibody, and lastly with a 4,6-dichloro-S-triazin-2-yl amino fluorescein (DTAF)-labeled protein A (which binds to the Fc portions of antibody molecules). No DTAF-protein A fluorescence was seen on embryos when the TRITCsFab ctTNP fragment or no antibody was used, but fluorescence was seen when the intact sheep IgG aTNP was used. Zonafree unfertilized and fertilized eggs were incubated for 10 min at room temperature in rhodamine-conjugated succinylated concanavalm A (S-Con A; 100 ~g/ml in HBSS plus BSA; Vector Laboratories, Inc., Burlingame, CA) and washed through three changes in HBSS plus BSA. As described above, both SCon A and TRITC-sFab aTNP are general membrane protein labels. Mouse embrjos do not exhibit exocellular matrices at this stage (12). Fluorescently-labeled embryos were taken up into 100-/zm pathlength microslides (Vitro Dynamics, Inc., Rockaway, Nff) for examination in the fluorescence microscope and for FPR measurements. Fluorescence Recovery after Photobleaching: Thetechnique for FPR has been described in detail elsewhere (2). FPR provides us with two measures of diffusion: first, the fraction of the componem that is free to diffuse (percent recovery, %R ) and second, the diffusion coefficient (D) of that fraction. Our instrument is similar to that of published designs. It consists of a Lexe195-2 Argon Laser (Lexel Corp., Pain Alto, CA), a beam splitter attenuator similar to that described by Koppel (18), a Leitz Dialux fluorescence microscope with I2, D2, and, N2.1 epiUumination fdter systems, and Leitz MPV photometry system (Kramer Scientific Corp., Yonkers, NY), modified to accept an EM1 9568 pbotomultiplier tube in a Products for Research, Inc. (Danvers, MA) dry-icecooled housing with amplifier discriminator and electronic shutter from EMI. The image plane diaphragm of the Leitz MPV was always set to insure that light was collected only from a single plasma membrane. This procedure is discussed in detail by Wolf and Edidin (34). Photons are counted on a custom buih scaler, which aLso interfaces the instrument to a Technico SS16 computer (Columbia, MD), which stores and analyzes the data on dual 8" floppy disks. Data are fitted by nonlinear least squares programs after Bevington (4) according to algorithms described by Barisas and Leuther (3) and Wolf and Edidin (34). Measurements were made using a Leitz 63 x 1.4 numerical aperture phase piano achromat. The beam exp(-2) radius was determined (28) to be (0.63 =t: 0.10) itm. Bleaching times were ~5 ms at ~10 mW at 514.5 nm. Monitoring intensities were ~1 #W. Typically, we used a counting interval of 500 ms. In all cases it was determined that no major faster components (_> 10 -7 cm2/s) of diffusion were present. Photomicrography: Photomicrographs were made using either phase FIGUR[ 1 (a) Phase-contrast micrograph of unfertil ized mouse egg showing round main body and protruding nipple (upper right). (b) Same egg as in a showing fluorescence staining of TNBS-labeled sites. Considerable less fluorescence is observed on the nipple as compared with the main body of the egg. Egg was labeled for 20 rain in 1 mM TNBS in HBSS plus BSA at 37°C; washed three times in HBSS plus BSA; and incubated for 10 rain in TRITC-sFab aTNP at room temperature and washed three times in HBSS plus BSA. (c) Phase-contrast micrograph of a fertilized mouse egg, showing two pronuclei (center) and second polar body (upper left). (d) Same egg as in c showing fluorescence staining with S-Con A. While the ring staining of the body is intense, staining of the second polar body, which buds from the nipple, is faint. The bright area diametrically opposed to the second polar body is the site of sperm entry. Egg was incubated with S-Con A at 100 #g/ml in HBSS plus BSA for 10 rain at room temperature, then washed three times with HBSS plus BSA. Bar, 10/Lm. X 450. or standard epilumination of the Dialux system. We used Ilford XPI-400 film "pushed" to an effective ASA of 800.