Topography of immunoreactive surfactant protein A (SP-A) in the vaginal epithelium of nine postmenopausal women. Immunocytochemistry was carried out, as described in the Materials and methods, using Vector Red as the chromogenic substrate for the alkaline phosphatase reporter and haematoxylin to stain nuclei. (a) Panels 1–8: tissue sections from from four donors; panels 1, 3, 5 and 7 (upper panels) at lower magnification and panels 2, 4, 6 and 8 at higher magnification illustrate the large differences in thickness of vaginal epithelium of women not receiving oestrogen. Panels (a)7 and (a)8, as well as (f), show thickening of the epithelium (acanthosis) and keratinization of the superficial layer, a response to chronic trauma resulting from prolapse of the vagina. The most obvious structural difference between pre- and postmenopausal vagina is in the intermediate layer. It is much thinner and constitutes a much less distinct layer in the postmenopausal that in the oestrogenized premenopausal vaginal epithelium. Nevertheless, SP-A-immunopositive cells, although more patchy than in premenopausal vaginal mucosa, can be seen in the deep intermediate layer in all specimens and there are areas showing strong immunostaining for SP-A in the superficial layer. (b) Panels 1 and 2 show the more oestrogenized appearance of vaginal epithelium of one of the subjects prescribed equine oestrogens. It shows a better developed intermediate layer than in sections from the other 11 postmenopausal subjects and therefore a much clearer separation of the immunopositive cells in the deep intermediate layer and the immunopositive superficial layer. Panels (c)–(f) exemplify the marked variation in SP-A distribution in postmenopausal subjects.

Vaginal surfactant protein A (SP-A) transcripts demonstrated by reverse transcription–polymerase chain reaction (RT–PCR). Total RNA (100 ng) from lung (lane 1) or vaginal mucosa (lanes 2–4) was reverse transcribed. PCR reactions were primed with oligonucleotides that span exons 1–4 and recognize sequence common to SP-A1 and SP-A2. The faint band marked by the small white arrow is identical in size to genomic SP-A.

Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) identification of surfactant protein A (SP-A) tryptic peptides in vaginal fluid. A piece of gel corresponding to the spot shown in Fig. 5 was digested with trypsin and peptides were analysed by MALDI-TOF. Of the complete SP-A amino acid sequence depicted, the peptides shown in bold italics and underlined were identified in the trypsinized gel piece.

Topography of immunoreactive surfactant protein A (SP-A) in premenopausal vagina. (a) SP-A identified by immunocytochemistry (ICC), using Vector Red as the chromogen. (b) Preimmune and secondary antibody controls for ICC for SP-A using alkaline phosphatase reporter, and bromo-chloro-indolyl-phospahate (BCIP) and nitroblue tetrazolium (NBT) as the substrate and chromogen, respectively. (c) Correlation of localization of immunoreactive SP-A with glycogen. (a) Panel 1: at low magnification, SP-A (red reaction product) is seen in two distinct layers: in the basal portion of the intermediate layer, composed of rounded, metabolically active cells; and in the superficial layer, composed of flattened dead cells with pyknotic nuclei. Note the absence of immunostaining in the more superficial cells of the intermediate layer. The arrow points to one of the papillae comprising a core of subepithelial cells [shown in panels 2 and 3 of (a) by the letter ‘p’] surrounded by basal and parabasal cells. (a) Panels 2 and 3 show panel 1 at successively higher magnifications: panel 2 highlights the virtual absence of immunostaining in the more superficial portion of the intermediate layer, while panel 3 highlights the contrast between the strongly immunostaining cells in the intermediate layer and the minimal or no immunostaining of cells in the basal layer. The arrow points to the layer of cells demarcating the epithelium from the subepithelial layer (lamina propria). (b) Panel 1: immunoreactive SP-A (blue/black reaction product of BCIP/NBT) in a section from which glycogen had been removed by pretreatment with α-amylase. Note the similarity in localization of immunoreactive SP-A in the deep intermediate layer with that in sections containing glycogen (a, panel 1). (b) Panels 2 and 3: the absence of immunostaining in preimmune serum (panel 2) and secondary antibody (panel 3) controls. (c) Panels 1, 2 and 3: comparison of the topography of immunoreactive SP-A with that of glycogen. (c) Panel 1: section immunoreacted for SP-A after digestion of glycogen with α-amylase (blue/black reaction product of BCIP/NBT. (c) Panels 2 and 3: glycogen, stained magenta with pararosanaline, using periodic acid-Schiff (PAS) as the chromogenic dye, is seen throughout the intermediate layer. (c) Panel 4: elimination of staining for glycogen with PAS in a section pretreated with α-amylase. The stain for glycogen using pararosaniline in the PAS reaction, unlike basic fuchsin dye, does not stain the amylase-resistant intercellular material (see the Discussion). Sections were counterstained with haematoxylin in panels 1, 2 and 3 of (a), and in panels 2, 3 and 4 of (c). Images were recorded with a Nikon DXM 1200 digital camera using Nomarski optics and Nikon ACT1, version 2, software.

Northern blot analysis of surfactant protein A (SP-A) transcripts in vaginal mucosa. Total RNA (20 µg) from lung (lane 1) and vagina (lane 2) was separated by a 1% agarose formaldehyde gel and transferred to a GeneScreen membrane. The membrane was hybridized with a ³²P-labelled anti-sense SP-A RNA probe and exposed for 10 min (lung) or 36 hr (vagina) at −80°.

Two-dimensional gel electrophoresis immunoblot of vaginal fluid proteins. The blot was immunostained with rabbit anti-human surfactant protein A (SP-A) primary antibody and goat anti-rabbit secondary antibody conjugated with horseradish peroxidase, followed by detection with enhanced chemiluminescence. The arrow indicates the spot that was excised from a parallel silver stained gel and partially digested with trypsin. The resulting tryptic peptides were analysed by mass spectrometry.