Because administration of hCG was unable to stimulate the trans-barrier flux of IgG anionized with succinic anhydride, citraconylation was performed under conditions that blocked a smaller number of the lysine residues (—25%), producing a molecule similar in size and net negative charge to Ial. This anionized IgG molecule possessed a net negative charge (pi -6.3-6.5; Fig. ЗА) and was administered to eCG-primed prepubertal mice via the tail vein and allowed to circulate for 3 h. Immunocytochemistry of these mouse ovaries demonstrated that the citraconylated IgG behaved in a manner similar to that of native Ial. Unlike the IgG anionized with succinic anhydride, the IgG anionized with CA was excluded from the developing follicle in the absence of an ovulatory stimulus (Fig. 3B) but was able to enter the developing follicle after administration of hCG (Fig. 3C).
Charge Density of Larger and Smaller Proteins Had No Effect on Trans-Barrier Flux
Earlier studies demonstrated that large proteins (> 500 kDa) were excluded from the follicle even with an ovulatory stimulus and that very small proteins (< 100 kDa) freely entered the follicle before the ovulatory surge. Therefore, assays using a2-M and BSA were performed to determine whether or not the modification of charge density could affect the trans-barrier flux of larger and smaller proteins. a2-M (pi —6.2) is approximately 700 kDa and is normally excluded from developing follicles before and after hCG administration (Fig. 4, В and C). By following the cationization protocol described earlier, the charge density of human a2-M was modified to a high net positive charge density (pi ~8.5; Fig. 4A). Buy Asthma Inhalers Online
The cationized a2-M was injected into eCG-primed prepubertal mice via the tail vein and allowed to circulate for 3 h. Immunocy-tochemistry of these mouse ovaries demonstrated that even with a high positive charge density, cationized a2-M was still unable to pass through the blood-follicle barrier (Fig. 4D). Although Shalgi et al. demonstrated the presence of albumin in human follicular fluid obtained before an ovulatory stimulus, studies using mouse follicular fluid have been limited. Therefore, BSA (66 kDa), which possesses a high net negative charge density (pi ~4.6; Fig. 5A) was injected into eCG-primed prepubertal mice and allowed to circulate for 3 h. Immunocytochemistry of mouse ovaries obtained from these mice demonstrated that this small negatively charged protein was not excluded from the follicle of eCG-primed mice (Fig. 5B). Thus the trans-barrier flux property of this small protein is unaffected by its high negative charge density.
FIG. 3. Immunolocalization of native and anionized rabbit IgG in the mouse ovary. A) Isoelectrofocusing gel showing the pi of protein standards (lane 1), native rabbit IgG (lane 2), rabbit IgG anionized with CA (lane 3), and rabbit IgG anionized with CA but with only —25% of the lysine residues blocked (lane 4). B) Ovary section from an eCG-primed mouse injected with 250-300H,g of less anionized IgG and probed with anti-IgG (n = 11 animals). C) Ovary section from an eCG-primed mouse injected with 250-300 |xg of less anionized IgG, stimulated with hCG, and probed with anti-IgG (n = 10 animals). Bar = 400 jim.
FIG. 4. Immunolocalization of native and cationized human a2-M in the mouse ovary. A) Isoelectrofocusing gel showing the pi of protein standards (lane 1), cationized a2-M (lane 2) and native a2-M (lane 3). B) Ovary section from an eCG-primed mouse injected with 250-300 |xg of native a2-M and probed with anti-a2-M (n = 9 animals). C) Ovary section from an eCG-primed mouse injected with 250-300 jxg native a2-M, stimulated with hCG, and probed with anti-a2-M (n = 9 animals).
FIG. 5. Immunolocalization of native BSA in the mouse ovary. A) Isoelectrofo-cusing gel showing the pi of protein standards (lane 1) and BSA (lane 2). B) Ovary section from an eCC-primed mouse injected with 250-300 jig native BSA and probed with anti-BSA (n = 6 animals). Bar = 200 |xm.