1998;274:C1708CC1717. residues 209C230 in the second extracellular loop of rat occludin, indeed, reversibly perturbed the Sertoli cell TJ-barrier (9). Most importantly, when administered directly to testes of adult rats intrates-ticular injection, this peptide reversibly disrupted the BTB (9). On the basis of these observations, it is plausible that if this peptide can be conjugated to a delivery vehicle targeted to the testis, it is an excellent candidate to transiently open the BTB for drug delivery without compromising other epithelial barriers. Herein, we statement the use of a deglycosylated mutant of FSH as a testis-specific vehicle for this 22-amino acid occludin peptide. FSH receptors are limited to Sertoli cells in mammals (10C12). It was also reported that deglycosylated FSH possessed relatively little hormonal activity is still but capable of binding onto its receptors (for a review, observe 13). Theoretically, this FSH mutant (FSH) can serve as a specific carrier for the occludin peptide Zaltidine the blood circulation to the testis, where FSH can bind onto its receptor and brings the peptide to close proximity to the BTB. As such, the occludin peptide can induce a short-term BTB disruption and provide a windows for drug delivery. To test this hypothesis, the occludin peptide was conjugated to the FSH mutant by genetic engineering and/or chemical cross-linking techniques, and its effects around the BTB and other TJ barriers were examined. MATERIALS AND METHODS Animals Sprague-Dawley rats (outbreds) were purchased from Charles River Laboratories (Kingston, NY, USA). The use of animals reported herein was approved by The Rockefeller University or college Animal Care and Use Committee with Protocol Figures 97113, 00111, 03017, and 06018. Preparation of recombinant FSH mutant-occludin peptide (FSH-occludin) conjugate The FSH-occludin conjugate was prepared by PCR as detailed in Supplementary Method 1. Additional occludin peptides were chemically conjugated to the N terminus of each of the two subunits of the FSH-conjugate, which was performed at SoluLink (San Diego, CA, USA). In brief, FSH (at a concentration of ~2 mg/ml) was altered with succinimidyl 4-formylbenzoate (SFB) at pH 7.2 with 10 equivalents of SFB to FSH to yield aldehyde group at the N-terminus, Zaltidine the sample was desalted and the aldehydes were quantified (step 1 1, see Fig. 1). The 22-amino acid synthetic occludin peptide was reacted with SANH [acetone 5-(succinimidyloxycarbonyl)-pyridine-2-ylhydrazone] (Merck Biosciences, Darmstadt, Zaltidine Germany) to generate hydrazine group at its N terminus, and the sample was desalted, and hydrazines were quantified (step 2 2). The SFB-modified FSH was then reacted with the activated occludin peptide in a Conjugation Buffer (0.1 M sodium phosphate, 0.15M NaCl, pH SYNS1 6 at 22C) at room temperature overnight (observe Fig. 1). Unreacted hydrazine or aldehyde groups were then capped by 2-sulfobenzal-dehyde, and the conjugate was isolated by gel filtration chromatography. It is noted that SFB is usually a heterobifunctional cross-linker in which its N-hydroxysuccinimide ester (NHS-ester) can react with the amine-containing Lys residues on FSH-peptide conjugate besides the N-terminal amino-groups, to yield additional free aldehydes. The aldehyde can subsequently react with the hydrazine at the occludin peptide to form the stable hydrazone conjugate. Thus, the mass of occludin peptide in the FSH conjugate as shown in Table 1 may be an underestimate. Open in a separate window Physique 1 A schematic illustration for the conjugation of additional 22-amino acid occludin peptide to the N terminus of FSH. FSH was initially altered with SFB (succinimidyl 4-formylbenzoate) to incorporate benzaldehyde moieties to its N-terminus, and likely to the free amino group-containing Lys residues. Then, N-terminal-hydrazido-terephthlate-modified occludin peptides were added to the activated FSH, forming stable FSH-occludin conjugates the hydrazone linkage. TABLE 1 Effects of different doses of FSH-occludin conjugate around the BTB integrity and germ cell loss Zaltidine from your seminiferous epithelium in adult Sprague-Dawley rats i.p. (elongating and round spermatids, and spermatocytes found in tubule lumen (observe Fig. 5). About 600 tubules were examined and scored from three rats. ns, not statistically significant; +, detectable germ cell loss. Measurements of intracellular levels of cAMP The intrinsic biological activity of the FSH-occludin conjugate was assessed by its ability to induce the production of cAMP in cultured rat Sertoli cells native FSH. In short, Sertoli cells were isolated from 20-day-old rat testes and cultured in F12/Dulbeccos altered Eagle medium as explained (14). Cells were plated on Matrigel (BD Biosciences)-coated 24-well plates at a cell density of 0.5 .