Cloning and Characterization of Boar Epididymal Secretory Proteins by Homology to the Human

Abstract

Northern blot analysis suggested that the boar epididymis produces closely related counterparts to human epididymal proteins HE1, HE3, HE4, HE5 and HE12. 'Full-length' cloning by nucleic acid and amino acid sequence similarity was achieved by RT-PCR methods in the case of the porcine counterparts of HE3 and HE4, while the homologues of HE5 and HE12, despite their cross-hybridization during Northern blot analysis, have not yet been cloned. The two novel porcine cDNAs were derived from moderately abundant epididymal mRNAs that were 75 and 83% identical to HE3 and HE4 cDNAs, respectively. To emphasize their relationship to the corresponding HEs, they were named Se3 and Se4 cDNAs. Their open reading frames predicted small secretory proteins with 55% (Se3) and 76% (Se4) conserved amino acids. Monospecific antipeptide antibodies to HE secretory proteins identified He3- and HE12-related proteins on Western blots of porcine epididymal fluid and semen. Both Northern and Western analyses indicated that the Se proteins were produced in a regionalized pattern and accumulated in the cauda fluid.

Sperm-binding fibronectin type II-module proteins are genetically linked and functionally related

“…Sperm were immediately prepared for fluorescence microscopy and for protein extraction. Porcine epididymal fluid was collected by micropuncture of epididymal ducts and microperfusion of the luminal fluid (Dacheux et al, 1989;Syntin et al, 1996;Schäfer et al, 2003) and was a generous gift of Dr. Jean Louis Dacheux, INRA, Tours-Nouzilly, France.…”

“…Blots were hybridized as described (Kirchhoff et al, 1996;Schäfer et al, 2003) using 32 P-labelled cDNAs. Probes were labelled by random primer extension (Feinberg and Vogelstein, 1983) using the Prime-It® II Random Primer Labelling Kit (Stratagene) and α-32 P-dCTP (3000 Ci/mmol; Amersham Pharmacia Biotech).…”

“…Alternatively, the SDS-polyacrylamide gels were blotted onto PVDF membranes as recently described (Ekhlasi-Hundrieser et al, 2002). Protein blots were blocked with 1% blocking reagent (Roche, Mannheim, Germany) in TBS overnight at 4°C and sequentially probed with the polyclonal antipeptide antibody for Ce12 (amino acid 103-122; Saalmann et al, 2001) in guinea pig (1:1000 v/v) as described by Schäfer et al (2003), and alkaline phosphatase conjugated anti-guinea pig (1:5000 v/v; Dianova, Hamburg, Germany) in blocking buffer for 60 min at room temperature. Washing steps were performed with TBS containing 0.1% Tween-20.…”

“…In fact, all ungulate species studied thus far express both classes of Fn2-module proteins, i.e. members of the ELSPBP1 family and small Fn2-module proteins, in subsequent regions of the male genital tract (Saalmann et al, 2001;Schäfer et al, 2003;Ekhlasi-Hundrieser et al, 2005).…”

“…the pig and the mouse. The boar, like other ungulates, expressed representatives of both classes of Fn2-module proteins, pB1 as a member of the small Fn2-module protein family (Hadjisavas et al, 1994;Calvete et al, 1997;Plucienniczak et al, 1999), as well as ELSPBP1-homologous proteins (Schäfer et al, 2003). We studied the chromosomal location and expression patterns of the corresponding genes, and described their potential acceptor molecules and topography on the sperm surface.…”