Expression of human oviductin in an immortalized human oviductal cell line  Ling Ling, M.Sc., Yin-Lau Lee, Ph.D., Kai-Fai Lee, Ph.D., Sai-Wah Tsao, Ph.D., William S.B. Yeung, Ph.D., Frederick W.K. Kan, Ph.D.  Fertility and Sterility  Volume 84, Pages 1095-1103 (October 2005) DOI: 10.1016/j.fertnstert.2005.06.006 Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 (A) Electron photomicrograph showing an OE-E6/E7 cell from a sample of cells that were trypsinized, fixed in glutaraldehyde, and then embedded in Epon. The cell is endowed with microvilli on the cell surface (arrow). Inside the cytoplasm, the presence of an ovoid nucleus (Nu), numerous mitochondria (Mi), rough endoplasmic reticulum (rER), Golgi apparatus (Gol), and secretory granules (G) are evident. Magnification, ×8,000. (B) High-magnification electron photomicrograph showing the Golgi region, where a condensing vacuole (CV) or immature granule and secretory granules (G) can be seen in the vicinity of stacks of Golgi saccules (GS). MV = multivesicular bodies; Mi = mitochondria; rER = rough endoplasmic reticulum. Magnification, ×10,000. Ling. Expression of human oviductin in OE-E6/E7 cells. Fertil Steril 2005. Fertility and Sterility 2005 84, 1095-1103DOI: (10.1016/j.fertnstert.2005.06.006) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Coamplification of reverse transcriptase-polymerase chain reaction (RT PCR) products for β-actin and human oviductin. The first group of reactions shows coamplification of β-actin (543 bp) and human oviductin (395 bp) using our own newly designed human oviductin primers (lane 1). The second group of reactions shows coamplification of β-actin and human oviductin using the oligonucleotide primers as previously published (30) for β-actin (543 bp) and human oviductin (410 bp, if present; lane 3). Q-solution, a PCR additive for amplification of templates that are GC rich or have extensively secondary structure (provided in the Qiagen Taq PCR Core Kit) was added to one reaction (lane 4). There are two negative controls in which the template was replaced with distilled water (lanes 2 and 6). As well, a reaction in which no reverse transcriptase was added served as a negative control for the RT reaction (lane 5). A 100-bp DNA ladder was used as a molecular marker in both groups. Ling. Expression of human oviductin in OE-E6/E7 cells. Fertil Steril 2005. Fertility and Sterility 2005 84, 1095-1103DOI: (10.1016/j.fertnstert.2005.06.006) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Reverse-transcription polymerase chain reaction (RT-PCR) analysis of human oviductin showing RT PCR products for human oviductin using previously published primers (lane 1) and primers designed by our laboratory (lane 2). Lane 3 shows the OE-E6/E7 cDNA template without adding primers. A 100-bp DNA ladder was used as a molecular weight marker. Ling. Expression of human oviductin in OE-E6/E7 cells. Fertil Steril 2005. Fertility and Sterility 2005 84, 1095-1103DOI: (10.1016/j.fertnstert.2005.06.006) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 Nested polymerase chain reaction (PCR) analysis of human oviductin (254 bp). Lane 1 shows the negative control, which contained distilled water instead of the DNA template. Lane 2 shows the result of the nested PCR analysis of human oviductin (254 bp). Here, a new set of primers was designed within the region flanked by the set designed in our laboratory used in producing Figure 3. The RT-PCR product (395 bp) visualized in Figure 3 was used as the template. Ling. Expression of human oviductin in OE-E6/E7 cells. Fertil Steril 2005. Fertility and Sterility 2005 84, 1095-1103DOI: (10.1016/j.fertnstert.2005.06.006) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 5 Top row of panels (from left to right) shows, respectively, immunostaining for hOGP and Con A binding sites and colocalization of hOGP with ER–Golgi (marked by Con A lectin) in the perinuclear region of OE-E6/E7 cells. Middle row shows the absence of immunoreaction in the OE-E6/E7 cells when a preabsorbed antibody was used. An absence of immunostaining for hOGP also is noted in the endometrial HEC-1 cells that were used as a negative control. Bar = 20 μm. Ling. Expression of human oviductin in OE-E6/E7 cells. Fertil Steril 2005. Fertility and Sterility 2005 84, 1095-1103DOI: (10.1016/j.fertnstert.2005.06.006) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions