Analysis of Osteopontin at the Maternal-Placental Interface in Pigs1

Abstract

Noninvasive, epitheliochorial placentation in the pig follows a prolonged preimplantation period characterized by migration, spacing and elongation of conceptuses, and secretion of estrogen for maternal recognition of pregnancy. Osteopontin (OPN) is an extracellular matrix protein that binds integrins to promote cell-cell attachment and communication. OPN appears to play a key role in conceptus implantation and maintenance of pregnancy in sheep; however, a role for OPN in the porcine uterus has not been established. Therefore, this study examined OPN expression and function in the porcine uterus and conceptus (embryo/fetus and associated extraembryonic membranes). Northern and slot blot hybridization detected an increase in endometrial OPN expression between Days 25 and 30, and levels remained elevated through Day 85 of pregnancy. In situ hybridization localized OPN mRNA to discrete regions of the uterine luminal epithelium (LE) on Day 15 of pregnancy and to the entire LE thereafter. Glandular epithelial (GE) expression of OPN mRNA was first detected on Day 35 of pregnancy and increased through Day 85. Both 70- and 45-kDa forms of OPN protein were detected in cyclic and pregnant endometrium by Western blotting. OPN protein was localized to the LE and GE by immunofluorescence; however, only the 70-kDa OPN was detected in uterine flushings. OPN protein was present along the entire uterine-placental interface after Day 30 of pregnancy. In addition, OPN mRNA and protein were localized to immune-like cells within the stratum compactum of the endometrium in both Day 9 cyclic and pregnant gilts. Incubation of OPN-coated microbeads with porcine trophectoderm and uterine luminal epithelial cells induced Arg-Gly-Asp (RGD)-dependent integrin activation and transmembrane accumulation of cytoskeletal molecules at the apical cell surface as assessed by immunofluorescence detection of talin or alpha-actinin as markers for focal adhesions. These results suggest that OPN, expressed by uterine epithelium and immune cells, may interact with receptors (i.e., integrins) on conceptus and uterus to promote conceptus development and signaling between these tissues as key contributors to attachment and placentation in the pig.

Seminal Plasma Proteins as Potential Markers of Relative Fertility in Boars

“…In the present experiment, Western blot analysis with an antiserum against human OPN identified 3 immunoactive bands at 70, 12, and 9 kDa. A 70-kDa form of OPN has been reported in uterine flushings in the pig (Garlow et al, 2002) and appears to be homologous to SP-1, a stallion seminal plasma osteopontin (Brandon et al, 1999). Although the 70-kDa osteopontin species identified in the present study of boar seminal plasma is homologous to the bull (55 kDa) and stallion (70 kDa) OPN forms, the relative amount of OPN-70 did not differ among boars.…”

“…OPN has been detected in both female Garlow et al, 2002) and male (Cancel et al, 1999;Rodríguez et al, 2000;Luedtke et al, 2002) reproductive tracts. OPN has been identified as a seminal plasma protein that is positively associated with fertility in the bull (55 kDa, pI 4.5) (Cancel et al, 1997(Cancel et al, , 1999 and the stallion (SP-1, 72 kDa, pI 5.6) (Brandon et al, 1999).…”

Detection of osteopontin on Holstein bull spermatozoa, in cauda epididymal fluid and testis homogenates, and its potential role in bovine fertilization

“…More recently it has been detected in many tissues and fluids, including urine (Sorenson et al 1995), milk (Sorensen & Petersen 1993, Sorensen et al 2003, kidney (Xie et al 2001), uterine endometrium of rabbits (Apparao et al 2003), sheep (Johnson et al 2003, Kimmins et al 2004, cows (Kimmins et al 2004) and pigs (Garlow et al 2002), the bovine oviduct (Gabler et al 2003), and on the luminal surfaces of epithelial cells of human gastrointestinal and reproductive tracts, gall bladder, pancreas, lung, breast, urinary tract, salivary glands, and sweat glands (Brown et al 1992). OPN may be glycosylated, phosphorylated and sulfated, and its expression and post-translational modifications are tissue-specific and regulated by many hormones and growth factors (Denhardt & Guo 1993).…”

Molecular mechanisms of membrane interaction at implantation

“…During the mid-to late-secretory phase of the menstrual cycle, OPN has been identified on the apical surface of uterine luminal and glandular epithelial cells, particularly in the vicinity of pinopodes (Quenby et al, 2007). The expression of OPN subsequently extends into the decidualizing stroma and embryonic trophectoderm (Omigbodun et al, 1997;Quenby et al, 2007), a distinctive pattern of events that has also been identified in other mammals, including pigs, rabbits, and sheep (Johnson et al, 1999(Johnson et al, , 2003bGarlow et al, 2002;Apparao et al, 2003;White et al, 2005). Moreover, while knockout OPN mice are fertile, they exhibit reduced pregnancy rates during mid-gestation, suggesting peri-implantation pregnancy loss (Weintraub et al, 2004), highlighting a functional role for OPN during pregnancy.…”