Demonstration of a glycoprotein derived from the Ceacam10 gene in mouse seminal vesicle secretions

Abstract

CEACAM10 was purified from mouse seminal vesicle secretions by a series of purification steps that included ion exchange chromatography on a DEAE-Sephacel column and ion exchange high-performance liquid chromatography on a sulfopropyl column. It was shown to be a 36-kDa glycoprotein with an N-linked carbohydrate moiety. The circular dichromoism spectrum of CEACAM10 in 50 mM phosphate buffer at pH 7.4 appeared as one negative band arising from the beta form at 217 nm. CEACAM10 was expressed predominantly in seminal vesicles of adult mice. Both CEACAM10 and its mRNA were demonstrated on the luminal epithelium of the mucosal folds in the seminal vesicle. The amount of Ceacam10 mRNA in the seminal vesicle was correlated with the stage of animal maturation. Castration of adult mice resulted in cessation of Ceacam10 expression, while treatment of castrated mice with testosterone propionate in corn oil restored Ceacam10 expression in the seminal vesicle. During the entire course of pregnancy, Ceacam10 might be silent in the embryo. A cytochemical study illustrated the presence of the CEACAM10 binding region on the entire surface of mouse sperm. CEACAM10-sperm binding greatly enhanced sperm motility in vitro.

Fig. 1

Purification of 36-kDa glycoproteins from mouse SVS proteins. A) Fractionation of soluble mouse SVS proteins by ion exchange chromatography on a DEAE-Sephacel column. B) Resolution of fraction III sample from A by ion-exchange HPLC on an SP column. C) Demonstration of the glycoprotein nature. Each of the a-to-d peaks from B were digested with N-glycosidase F. The parent proteins (lane 1, peak a; lane 3, peak b; lane 5, peak c; lane 7, peak d) and their deglycosylated forms (lanes 2, 4, 6, and 8) were identified by SDS-PAGE on a 12% polyacrylamide gel slab. The proteins in the gel were stained with Coomassie brilliant blue

Fig. 2

Identification of the 36-kDa glycoprotein derived from Ceacam10 and its circular dichroism. A) The trypsin-digested sample of peak c from Figure 1B was resolved by reverse-phase HPLC on a C₁₈ column (see Materials and Methods). B) The protein sequence was deduced from the reading frame of Ceacam10 cDNA (GenBank accession number NM_007675). The initial and stop codons are underlined. The potential N-linked glycosylation sites are denoted by open boxes. The deduced protein sequence and the amino acid sequences determined directly from protein analysis for peaks a to d in Figure 1B and peaks 9 and 18 in Figure 2A agree in all positions except that Asn¹¹ from the cDNA-deduced protein was not identified in protein sequencing. The cleavage points for the generation of mature protein are indicated by an arrow. C) Circular dichroism of CEACAM10 in 50 mM phosphate buffer at pH 7.4 at room temperature

Fig. 3

Distribution of Ceacam10 and its protein among the reproductive glands. Total RNA (20 μg) or protein extract (50 μg) prepared from the homogenates of each sexual gland were analyzed by Northern blot procedure (A) or Western blot procedure (B) (see Materials and Methods)

Fig. 4

Ceacam10 expression in the luminal epithelium of the seminal vesicle. A) Immunolocalization of CEACAM10 to the luminal epithelium of the seminal vesicle. Tissue slices were histochemically stained for CEACAM10 with antibody against the protein, biotin-conjugate goat anti-rabbit IgG, and alkaline phosphatase-conjugated streptavidin a. The specimens were stained as in a except that the antibody was replaced by normal serum (b). For contrast, the specimens were further stained with Nuclear Fast Red. Photographs were taken with brightfield illumination. MF, mucosal fold; SM, smooth muscle; LF, luminal fluid. The staining of Nuclear Fast Red is in pink and the signals of CEACAM10 protein, demonstrated by staining of alkaline phosphatase activity, are in dark blue. Bar = 20 μm. B) Demonstration of Ceacam10 mRNA in the luminal epithelial cells of the seminal vesicle. The epithelial cells of MF or SM cells in a tissue slice (8 μm) of mouse seminal vesicle were selectively captured and transferred to films by LCM. The tissue slides before and after LCM were stained and observed (see text for details). A Ceacam10 cDNA fragment (237 bp) or a Gapd cDNA fragment (557 bp) was amplified from the total RNA of MF or SM by reverse transcription-polymerase chain reaction. The level of Gapd mRNA was used as an internal control. Bar = 100 μm

Fig. 5

Developmental profile of Ceacam10 mRNA in embryos and seminal vesicles. Ceacam10 mRNA and Gapd mRNA in the total RNA prepared from mouse seminal vesicles at different ages (A) or in mouse embryos collected on various days postcoitus (B). The RNA messages were measured by Northern blot as described in the text

Fig. 6

Androgen dependence of Ceacam10 mRNA expression in seminal vesicles of adult mice. Northern blot analysis for 1.1-kilobase Ceacam10 mRNA in total RNA from seminal vesicles from normal adult mice (lane 1), adults castrated 3 wk previously and treated only with corn oil (lane 2), and adults castrated 3 wk previously and treated testosterone propionate in corn oil for 8 consecutive days (lane 3). Total RNA (20 μg) was used for each experiment. Gapd mRNA was used as an internal control

Fig. 7

Analysis of sperm motility under the influence of CEACAM10. A) Demonstration of the CEACAM10-binding zone on epididymal spermatozoa. Fresh sperm were incubated with or without CEACAM10 as described in Materials and Methods. The cells on slides were incubated with normal serum (a and b) or affinity-purified anti-CEACAM10 antibody (c and d). The slides were then incubated with rhodamine-conjugated anti-rabbit IgG and observed via light microscopy (a and c) or fluorescence microscopy (b and d). Bar = 10 μm. B) Illustration of CEACAM10 on ejaculated sperm. Freshly prepared cells (see Materials and Methods) on slides were incubated with normal serum (a and b) or the CEACAM10 antibody (c and d) and followed by incubation with rhodamine-conjugated anti-rabbit IgG. The slides were observed via light microscopy (a and c) or fluorescence microscopy (b and d). Bar = 10 μm. C) Freshly prepared mouse spermatozoa in modified Tyrode solution (10⁵ cells/ml) containing 1.8 mM CaCl₂ were incubated alone (○) or in the presence of 90 μM CEACAM10 (•) at 37°C for 0 to 60 min. Cell motility determined at each specified incubation time was expressed as a percentage of control cell motility at time zero. Points are mean ± SD for three determinations. *P < 0.01 in a paired statistical comparison with the corresponding control. Values were evaluated using one-way analysis of variance