The Ovarian Blood Follicle: RESULTS(1)

3 Jan
2013

RESULTS(1)

Cationization of Ial Promoted Its Entry into the Developing Follicle

Previous studies have determined that Ial is necessary for stabilization of the cumulus extracellular matrix and for optimal ovulation. However, Ial has been found to enter developing follicles only after hCG administration or a natural ovulatory surge (Fig. 1C). To test the hypothesis that charge may affect barrier permeability, human Ial was isolated by HPLC and cationized. Ca-tionized Ial protein was isoelectrofocused and found to possess a high net positive charge (pi —9.5, Fig. 1A). Ca-tionized Ial was injected into eCG-primed prepubertal mice via the tail vein and allowed to circulate for 3 h. Immu-nocytochemistry of fixed ovary sections from these mice demonstrated that cationized Ial was able to enter the developing follicles in the absence of an ovulatory stimulus (Fig. ID). Cationization of Ial thus abrogated the requirement of an ovulatory stimulus for its entry into the follicular antrum (compare Fig. 1, В and D). buy diabetes drugs

Anionization of Rabbit IgG Resulted in Its Exclusion from Developing Follicles Until an Ovulatory Stimulus

Further evidence to support the hypothesis that charge is involved in barrier permeability and selectivity was obtained in studies using rabbit IgG. This protein is 155 kDa, and therefore it is similar in size to Ial (220 kDa), but it possesses a net neutral to slightly positive charge (pi —6.57.0; Fig. 2A). Purified rabbit IgG was injected into eCG-primed prepubertal mice via the tail vein and allowed to circulate for 3 h. In contrast to studies with native Ial, immunocytochemistry of the ovaries from these mice demonstrated that rabbit IgG was present within the developing follicles before an ovulatory stimulus (Fig. 2B). To determine whether or not charge might also regulate the permeability of IgG, this molecule was anionized to a pi of —6.1 using succinic anhydride (Fig. 2A). After injection of the anionized IgG via the tail vein of eCG-primed prepubertal mice, immunocytochemistry demonstrated that it was unable to permeate the barrier and enter the developing follicle (Fig. 2C). Indeed this sample of anionized IgG was unable to enter the follicle even after an ovulatory stimulus (Fig. 2D).
Fig1The ovarian blood follicle
FIG. 1. Immunolocalization of native and cationized human lal in the mouse ovary. A) Isoelectrofocusing gel showing the pi of protein standards (lane 1), cationized lal (lane 2) and native lal (lane 3). B) Ovary section from an eCC-primed mouse probed with anti-lal (n = 6 animals). C) Ovary section from an eCC-primed mouse stimulated with hCG and probed with anti-lal (n = 6 animals). D) Ovary section from an eCG-primed mouse injected with 250-300 jxg of cationized lal and probed with anti-lal (n = 8 animals). Bar = 200 |xm.

Fig2The ovarian blood follicle
FIG. 2. Immunolocalization of native and anionized rabbit IgC in the mouse ovary. A) Isoelectrofocusing gel showing the pi of protein standards (lane 1), native rabbit IgC (lane 2), and rabbit IgC anionized with succinic anhydride (lane 3). B) Ovary section from an eCG-primed mouse injected with 250-300 jjug of rabbit IgC and probed with anti-IgG (n = 12 animals). C) Ovary section from an eCG-primed mouse injected with 250-300 |xg of anionized igG and probed with anti-IgG (n = 9 animals). D) Ovary section from an eCG-primed mouse injected with 250-300 |xg anionized IgG, stimulated with hCG, and probed with anti-IgG (n = 10 animals).

top