Ovaries and uteri from normal adult female rats at known stages of the estrous cycle were analyzed for the presence of cellular retinol-binding protein (CRBP) and both types of cellular retinoic acid-binding protein (CRABP and CRABP II). Northern and Western blot analysis of the uteri revealed a peak of CRBP during diestrus and a peak of CRABP during proestrus, whereas CRABP II peaked sharply during estrus. Immunohistochemical studies showed CRABP II localized to the luminal epithelium, while both CRBP and CRABP were observed only in the smooth muscle layers of the uterus. In the ovary, CRABP was not detected, while CRBP levels remained relatively constant throughout the cycle and CRABP II peaked slightly during metestrus. CRBP in the ovary was localized to the oocytes, nearby granulosa cells, and some regions of stroma. CRABP II was found predominantly in the granulosa cells of mature follicles and early corpora lutea, as well as some regions of the stroma. These results suggest a need for further studies to assess the role of retinol and its metabolites in normal uterine function and ovarian follicular development.

Vitamin A (retinol) and retinoic acid are necessary for the maintenance of the female reproductive system of higher animals. Our previous work has demonstrated cell specific expression of cellular retinoic acid-binding protein (CRABP) and cellular retinoic-acid binding protein(II) [CRABP(II)] in the uterus of the rat. CRABP(II) expression was shown to be induced in the uterine surface epithelial cells by treatment of prepubertal rats with pregnant mare serum gonadotropin (PMSG). Here we report that, after PMSG treatment, collected uteri had markedly higher levels of retinoic acid than did the uteri of prepubertal rats treated with the control vehicle. Smooth muscle, stromal, and epithelial cells were then cultured from uteri from such animals and provided with retinol or with the retinol/retinol-binding protein complex. Retinoic acid production, analyzed by high-performance liquid chromatography, was observed for the epithelial cells from the uteri of prepubertal animals treated with PMSG, cells previously shown to express CRABP(II) and confirmed here to continue to express it in culture. Little or no retinoic acid was produced by cultured epithelial cells from the prepubertal uteri [shown previously to be negative for CRABP(II)] or by smooth muscle and stromal cells taken from uteri of prepubertal or PMSG-treated rats (shown previously to express CRABP). Retinoic acid production by uterine epithelial cells [and CRABP(II) expression] was also observed if the prepubertal rat was treated with estrogen before cell collection. At no time did cells expressing CRABP exhibit significant retinoic acid synthesis. Thus, this system revealed an important difference in retinoid metabolism between cells expressing CRABP and CRABP(II) and suggests CRABP(II) may participate in retinoic acid production and/or secretion.

Sera from 11 volunteers immunized with a recombinant HIV-1 gp160-expressing vaccinia virus (HIVAC-le; Oncogen/Bristol-Myers Squibb, Seattle, WA) and boosted with baculovirusderived rgpl60 (VaxSyn; MicroGeneSys, Inc., Meriden, CT) were evaluated for functional serum antibodies and their epitopes. Sera obtained prior to boosting had undetectable HIV-1-specific IgG and neutralizing activity, and did not block HIV-1 from binding or fusing to CD4+ MT-2 cells. 14 d after boosting, sera from each volunteer contained HIV-1-specific IgG titers of 1:40 to 1:1,280. Five of these sera also contained neutralizing antibodies, where most or all neutralizing activity was blocked by a synthetic peptide corresponding to amino acids 307-330 ofthe V3 loop ofgpl 20, indicating that neutralizing antibodies were mostly V3 loop-specific. All sera obtained after boosting contained HIV-1 binding/fusion-inhibition antibodies, and a significant portion of their activity was blocked by the V3 loop peptide, a result consistent with the presence of antibodies against the region of the V3 loop that participates in fusion. Three sera with V3 loop-specific neutralizing and fusion-inhibition antibodies were studied further. In competitive antibody binding experiments, antibodies reactive with the conformation-dependent, CD4 binding site ofgpl20 were undetectable in each serum. When evaluated in combination with a monoclonal antibody to the CD4 binding site of gpl 20, two sera demonstrated synergism in neutralizing assays, and all three sera demonstrated synergism in binding/fusion-inhibition assays, further indicating that the functional antibodies were primarily V3 loop-specific. The synergism also suggests that a vaccine that elicits strong serum antibody responses to both regions of gpl20 may improve the potential for inducing protective immunity. (J. Clin. Invest. 1993. 92:840-847.)

The induction of pseudopregnancy by the injection of eCG in rats results in the appearance of cellular retinoic acid-binding protein type II (CRABP[II]) in the granulosa cells of the ovary and the lining epithelium of the uterus within 48 h. This expression pattern is also seen in the normal mature female rat, in which CRABP(II) is expressed in the uterine epithelium during estrus (but not diestrus) and in the granulosa and luteal cells of the ovary. We have previously demonstrated that the uterine epithelial cells from the pseudopregnant rat have gained the ability to synthesize retinoic acid from retinol, in correlation with the induced expression of CRABP(II). If this is true for other sites of CRABP(II) expression, then local production of retinoic acid is intimately connected with various stages of reproduction in the female. Here we report that granulosa cells from the ovary of the eCG-treated immature rat and luteal cells from the ovary of the eCG/hCG-treated immature rat (both of which express CRABP[II]) synthesized markedly higher amounts of retinoic acid when cultured, compared to granulosa cells cultured from the ovary of the prepubertal rat treated with control vehicle. Culturing the granulosa cells from either control or eCG-treated animals had no effect on the expression of CRABP(II) cells. These data are consistent with our hypothesis that CRABP(II) expression is associated with retinoic acid synthesis and strengthen the case that local generation of retinoic acid plays an important role in reproduction.

Two members of the superfamily of small intracellular carrier proteins for lipophilic compounds are cellular retinoic acid-binding protein and cellular retinoic acid-binding protein II [CRABP(II)]. CRABP is found in many adult tissues, whereas CRABP(II) is more restricted and is reported as abundant primarily in skin. Here we report a much greater expression of CRABP(II) in rat corpus luteum than in any other organ/tissue examined, including skin. A rat complementary DNA clone encoding CRABP(II) was isolated and the ovarian expression followed during gonadotropin induction of follicular development in the pseudopregnant rat. The pattern of rat CRABP(II) messenger RNA and protein expression correlated with the appearance of corpora lutea and the rise in progesterone production as the corpora lutea developed, and was similar to the induction of 3 beta-hydroxysteroid dehydrogenase. Immunohistochemical localization revealed that CRABP(II) appeared in luteal cells and was dramatically restricted to their cytoplasmic compartment, with no apparent presence in the nucleus. This suggests that CRABP(II) may be expressed to restrict retinoic acid from occupying nuclear retinoic acid receptors, implying that the differentiation and maintenance of the rat corpus luteum may involve in part a release of certain pathways from retinoid suppression.