Characterization of equine zona pellucida glycoproteins by polyacrylamide gel electrophoresis and immunological techniques

Miller, Carlin J.; Fayrer-Hosken, R.A.; Timmons, Therese M.; Lee, V. H.; Caudle, A.B.; Dunbar, Bonnie S.

doi:10.1530/jrf.0.0960815

Cited by 25 publications

(15 citation statements)

References 19 publications

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“…Dolci et al [49] and Miller et al [50] investigated the involvement of carbohydrates in the hardening of the ZP of mouse oocytes. The characterization of ZP glycoproteins in the horse by PAGE and immunoblotting has been reported by Miller et al [51,52]. Recently, Dobrinsky et al [53] found galactose-binding proteins in the periacrosomal plasma membrane of equine spermatozoa, which could mediate the attachment to oviductal cells useful for maintaining the fertilizing ability of the sperm cell.…”

Section: Discussionmentioning

confidence: 91%

Effects of Fetuin on Zona Pellucida Hardening and Fertilizability of Equine Oocytes Matured In Vitro1

Dell’Aquila

Felici

Massari

et al. 1999

Biology of Reproduction

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In vitro fertilization (IVF) has had poor success in the horse, a situation related to low rates of sperm penetration through the zona pellucida (ZP). Zona pellucida hardening (ZPH) is seen in mouse and rat oocytes cultured in serum-free medium. The hardened ZP is refractory to sperm penetration. Fetuin, a component of fetal calf serum, inhibits ZPH and allows normal fertilization rates in oocytes cultured in the absence of serum. We evaluated whether fetuin is present in horse serum and follicular fluid (FF) and whether fetuin could inhibit ZPH in equine oocytes matured in vitro, thus increasing sperm penetration during IVF. The presence of fetuin in equine serum and FF was confirmed by immunoblotting. Oocytes submitted to in vitro maturation (IVM) in medium containing fetuin were used for ZPH assay or IVF. Intracytoplasmic sperm injection (ICSI) was carried out as a control procedure. The presence of fetuin during IVM did not affect the rate of maturation to metaphase II. Maturation of oocytes in the presence of fetuin reduced ZPH in a dose-dependent manner. After both IVF and ICSI, there was no significant difference in oocyte fertilization between fetuin-treated and untreated oocytes. The fertilization rate was significantly higher after ICSI than after IVF, both in fetuin-treated and in untreated oocytes. In conclusion, fetuin reduced ZPH in equine oocytes but did not improve sperm penetration during IVF. This implies that, in the horse, "spontaneous" ZPH is unlikely to be the major factor responsible for inhibiting sperm penetration in vitro.

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Section: Discussionmentioning

confidence: 91%

Effects of Fetuin on Zona Pellucida Hardening and Fertilizability of Equine Oocytes Matured In Vitro1

Dell’Aquila

Felici

Massari

et al. 1999

Biology of Reproduction

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“…The loss of the zona pellucida in horse embryos around day 8 ( fig. 1) can be determined by the loss of the ZPs which are visible at day 8 (66 kD, pI 6.0; 80 kD, pI 5.0-5.3) [Miller et al, 1992], but are not detectable at day 10 ( fig. 4).…”

Section: Embryonic Coats -A Mailbox Of Early Embryo-maternal Signallingmentioning

confidence: 99%

Early Embryonic Coats: Morphology, Function, Practical Applications

Herrler

Beier²

2000

Cells Tissues Organs

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Mammalian egg and embryo coats are primarily represented by the zona pellucida which, however, undergoes biochemical and structural changes during fertilization and embryo development. It serves several functions, from ovulation until shortly before implantation. Initially the zona pellucida induces sperm-oocyte interaction, acrosome reaction and prevents polyspermy. Later, it prevents disaggregation of the noncompacted blastomeres and the premature attachment to the oviductal and endometrial surface. Additionally, it protects the embryo against toxins and xenobiotics, as well as bacteria, viruses and phagocytes. As the embryo is covered by the zona pellucida until immediately before implantation, all signals of embryo-maternal signalling have to pass the zona and are detectable within it. Logically we may define the zona pellucida as a mailbox of the embryo-maternal signalling, especial for investigating these messages. Oviductal, uterine and embryonic proteins are incorporated into the zona pellucida as embryonic development goes on and change the zona’s morphological and biochemical properties. Whether a protein is able to penetrate the zona, whether it accumulates within the zona or whether it is rejected by the zona depends on its biochemical properties. Three specific proteins have been detected within the embryonic coats (IGFBP3, HB-EGF, P19). New insights into the physiology of the zona pellucida might present new achievements in the in vitro culture of embryos, and present new aspects as to how to prevent zona hardening. Furthermore, knowledge of the zona proteins enables the development of immunocontraceptive vaccines. Consequently, the zona pellucida is not only significant with regard to fertilization, but also during early embryonic development. Investigations of the zona pellucida will yield new insights into early embryo-maternal signalling which in turn may lead to improvements in classic IVF and new approaches to in vitro culture.

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“…Loss of terminal sialic acid from mammalian glycans can increase binding of some proteins to exposed galactose or mannose residues, and increased expression of some uterine lectins have been demonstrated in early pregnancy (Gray et al 2005). In the horse, various proteins in and around the early conceptus have been described in uterine fluid (Zavy et al 1982, McDowell et al 1990, Mü ller-Schö ttle et al 2002, the zona pellucida (Miller et al 1992), the capsule (Stewart et al 1995, Herrler & Beier 2000, Herrler et al 2000b, and yolk-sac fluid (Crossett et al 1996), but both the range of proteins characterized and definition of their developmental times of appearance and disappearance in the conceptus itself remain limited. To further investigate the role of the capsule in the success and failure of pregnancy in horses, we have started to identify proteins that are associated with the capsule and change during the process of fixation at about day 16.…”

Section: Introductionmentioning

confidence: 99%

Changes in major proteins in the embryonic capsule during immobilization (fixation) of the conceptus in the third week of pregnancy in the mare

Quinn

Hayes²,

Waelchli³

et al. 2007

Reproduction

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During the third week of pregnancy, the equine conceptus is enclosed within a capsule, the glycan composition of which changes at around day 16 (ovulationZday 0) when the conceptus becomes immobilized (fixed) in the uterine lumen. Our objective was to characterize the process of fixation by identifying changes in major capsule-associated proteins. Individual equine conceptuses (nZ55) were collected transcervically by uterine lavage between days 13.5 and 26.5. Major proteins extracted from capsules were compared with those in fluids from the uterus and yolk sac by SDS-PAGE. Until day 14, a major capsule-associated protein that migrated at w10 kDa was identified by N-terminal sequencing as equine b2 microglobulin (b2M). During fixation, b2M in the capsule underwent limited proteolysis to an w8 kDa form lacking nine amino acids from the N terminus, and was subsequently degraded. Expression of b2M mRNA was detected in the yolk-sac wall tissues and endometrium between days 13.5 and 17.5. During this period, b2M in the capsule was evidently not part of a complex with major histocompatibility complex class 1 heavy a chain bands because these were undetectable in the capsule and uterine lavage. Uterocalin (p19) was detected in uterine lavage and capsule throughout fixation, but in yolk-sac fluid only before fixation. These studies indicate that intact b2M is a major protein associated with the embryonic capsule before fixation, after which it undergoes limited proteolysis to a truncated w8 kDa form that remains in the capsule after the conceptus is immobilized.

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Characterization of equine zona pellucida glycoproteins by polyacrylamide gel electrophoresis and immunological techniques

Cited by 25 publications

References 19 publications

Effects of Fetuin on Zona Pellucida Hardening and Fertilizability of Equine Oocytes Matured In Vitro1

Effects of Fetuin on Zona Pellucida Hardening and Fertilizability of Equine Oocytes Matured In Vitro1

Early Embryonic Coats: Morphology, Function, Practical Applications

Changes in major proteins in the embryonic capsule during immobilization (fixation) of the conceptus in the third week of pregnancy in the mare

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