Equine Zona Pellucida Research Articles

Visco-Hyperelastic Characterization of the Equine Immature Zona Pellucida.

This article presents a very detailed study on the mechanical characterization of a highly nonlinear material, the immature equine zona pellucida (ZP) membrane. The ZP is modeled as a visco-hyperelastic soft matter. The Arruda–Boyce constitutive equation and the two-term Prony series are identified as the most suitable models for describing the hyperelastic and viscous components, respectively, of the ZP’s mechanical response. Material properties are identified via inverse analysis based on nonlinear optimization which fits nanoindentation curves recorded at different rates. The suitability of the proposed approach is fully demonstrated by the very good agreement between AFM data and numerically reconstructed force–indentation curves. A critical comparison of mechanical behavior of two immature ZP membranes (i.e., equine and porcine ZPs) is also carried out considering the information on the structure of these materials available from electron microscopy investigations documented in the literature.

169 Use of stored zonae pellucidae from young and old mares to study sperm–oocyte binding

Natural and standard IVF requires sperm binding to the zona pellucida (ZP); however, difficulties in obtaining equine ZP limit research in this area. In a previous study, an in vitro assay was used to assess ZP sperm binding for oocytes collected from old and young mares’ dominant follicles, with similar numbers of sperm bound per ZP (means of 45.5 and 58.1 sperm per ZP; P=0.12) (Lupole et al. 2010 Anim. Reprod. Sci. 121, 252-253). We hypothesised that ZP from uncleaved equine oocytes after intracytoplasmic sperm injection (ICSI) could be used to study ZP sperm binding. For this study, oocytes from mares >20 years (old, n=13 oocytes) and 4 to 12 years (young, n=16 oocytes) were collected by transvaginal, ultrasound-guided aspirations and injected with a single sperm. Only oocytes that failed to cleave into at least 2 cells within 2 days after ICSI were used. The oocytes were stored in hyperosmotic salt solution at 4°C for 2 years. The sperm binding assay was performed similar to the previous study (Lupole et al. 2010). Oocytes were removed from salt solution and placed into TALP and incubated for 1h at 38.5°C in 6% CO2 and air. Following incubation, oocytes were moved to a 45-µL drop of TALP. Frozen-thawed sperm (100,000 total sperm) from one stallion of good fertility were added and incubated for 2h. Oocytes were then washed through 4 drops of TALP using a pipette tip with a 175-µm inner diameter to remove loosely bound sperm. Oocytes and tightly bound sperm were stained in 4’6-diamidino-2-phenyindole (DAPI; 10µg mL−1) for 15min. The oocytes were secured on a glass slide under a coverslip that was supported by a mix of paraffin and petroleum jelly and viewed at 400× magnification using an epifluorescence microscope (Nikon Eclipse E800, Nikon, Tokyo, Japan). The number of tightly bound sperm was recorded. Sperm counts for age groups were compared using Student’s t-test, with data presented as mean±s.e.m. Sperm binding was not different between groups (59.2±16.9 for old mares and 36.8±10.6 for young mares; P=0.25). As previously observed with oocytes that had not been injected with sperm and minimal time in salt storage, we found no significant effect of mare age on sperm binding to the ZP. In addition, numbers of sperm bound to the ZP were similar between projects. Our findings suggest that equine oocytes that fail to cleave after ICSI could provide a valuable source of ZP for research. Further work is ongoing to determine the effect of time that oocytes are stored in salt solution on sperm binding.

Effects of components of semen extenders on the binding of stallion spermatozoa to bovine or equine zonae pellucidae

The effects of semen extender components on the ability of stallion sperm to bind to the zona pellucida (ZP) and the suitability of using bovine ZP for a ZP-binding assay for stallion sperm were investigated in a series of experiments. In Experiment I, binding of stallion sperm to both bovine and equine ZP was significantly increased when a skim milk-based extender (EZM) was used. In Experiment II, a threefold increase in sperm binding to ZP was observed when sperm were diluted in EZM compared with diluents, which contained no milk (TALP, LAC, and EmCare). In Experiment III, centrifuging the sperm through Percoll did not increase sperm binding to the ZP but did remove any positive effect of EZM on sperm-ZP binding. In Experiment IV, exposure of either sperm or ZP to EZM before co-incubation did not increase sperm binding to ZP. In Experiment V, sperm diluted in TALP containing skim milk, EZM, or INRA96 bound more efficiently to the ZP than sperm diluted in TALP without milk proteins. In Experiment VI, sodium caseinate, native phosphocaseinate, and caseinoglycopeptide increased sperm binding to the ZP. In conclusion, diluents containing milk or milk proteins markedly enhanced the number of sperm bound to both equine and bovine ZP.

Effect of mare age on gene expression of equine zona pellucida proteins and sperm binding

Effect of mare age on gene expression of equine zona pellucida proteins and sperm binding

Equine zona protein synthesis and ZP structure during folliculogenesis, oocyte maturation, and embryogenesis

In the equine, the zona pellucida (ZP) is the major barrier to successful in vitro fertilization. Therefore the aim of our studies was to analyze species-specific features of the equine ZP in regard to structure and glycoprotein ZPB and ZPC expression sites during oocyte development and embryogenesis. The equine ZP revealed high immunological cross-reactivity to porcine ZPB and ZPC. In the ovary, the distribution of ZPB and ZPC was co-localized and correlated with the developmental stage of the follicle. ZPB and ZPC expression started in the oocyte of the late primordial and primary follicle. In the secondary follicle, both the oocyte and the cumulus cells contributed to ZPB and ZPC synthesis. After in vivo maturation the oocyte stopped ZPB and ZPC production whereas the cumulus cells continued synthesis. Contrary, in vitro matured (IVM) cumulus-oocyte-complexes (COCs) revealed a reverse expression pattern. This was correlated to alterations in the distribution, number, and size of pores in the ZP. In the zona, N-acetylglucosamine residues were co-localized with ZPC. The acellular glycoprotein capsule surrounding early equine embryos was negative for ZPB and ZPC. Our results imply that in the horse ZPB and ZPC glycoprotein expression is differentially regulated during folliculogenesis, oocyte maturation, and embryogenesis. Contrary to the bovine and porcine, zona protein synthesis during in vivo maturation is completely overtaken by the cumulus cells implying that in the horse these cells are crucial for zona integrity. During IVM, the cumulus cells lose their ability to synthesize glycoproteins leading to alterations in the zona structure.

Hardening of equine zona pellucida after incubation in vitro either in a mares' oviduct or in culture medium

Hardening of equine zona pellucida after incubation in vitro either in a mares' oviduct or in culture medium

Induction of Acrosome Reactions in Stallion Sperm by Equine Zona Pellucida, Porcine Zona Pellucida, and Progesterone1

Induction of Acrosome Reactions in Stallion Sperm by Equine Zona Pellucida, Porcine Zona Pellucida, and Progesterone1

Equine immunocontraception using porcine zona pellucida: A new method for remote delivery and characterization of the immune response

Equine immunocontraception using porcine zona pellucida: A new method for remote delivery and characterization of the immune response

Large-scale isolation of equine zonae pellucidae

A simple and rapid method is described for the large-scale isolation of cumulus cell-free, zona pellucida-intact equine oocytes. Aspiration of palpable antral follicles present on frozen-thawed equine ovaries was accomplished using a constant vacuum source. The resultant follicular fluid, oocytes, and particulate matter were then filtered through a series of nylon screens of alternating mesh openings in combination with sodium citrate-containing buffer to a final volume of approximately 20 ml. This fluid was transferred to scored Petri dishes and a stereomicroscope was used to locate the oocytes for futher processing or storage. The methodology described is inexpensive, time-efficient, and the recovery rate is similar to or better than other methods previously described for equine oocyte recovery. Collected oocytes are adequate for biochemical evaluation of the equine zona pellucida (EZP) as well as sperm-egg binding assays.

Carbohydrate and protein analysis of the equine zona pellucida

Carbohydrate and protein analysis of the equine zona pellucida

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

This study was designed to explore the composition of the equine zona pellucida (EZP) by one- and two-dimensional polyacrylamide gel electrophoresis (1D- and 2D-PAGE), silver staining and immunoblotting techniques. Antral follicles palpable on frozen-thawed equine ovaries were aspirated with a needle and syringe, and the resultant follicular fluid, cellular material and oocytes were pooled. Oocytes were placed in Petri dishes, moved by narrow-bore pipette to droplets of phosphate-buffered saline (PBS) and mechanically cleaned of cumulus cells. The EZP from these collected oocytes was solubilized, and then analysed by 1D- and 2D-PAGE. Silver stained 2D-PAGE of the EZP revealed the presence of three EZP glycoprotein families of apparent molecular mass ranges of 93-120 kDa, 73-90 kDa and 45-80 kDa. Immunoblot analysis of EZP glycoproteins resolved by 2D-PAGE using rabbit antisera against pig zonae pellucidae (R alpha HSPZ) confirmed the presence of three EZP glycoprotein families and established the existence of common epitopes between equine and porcine ZP glycoproteins. Further immunodetection using 2D-PAGE-separated glycoproteins illustrated that the 45-80 kDa family is recognized by the monoclonal antibody R5, developed against the porcine ZP glycoprotein of molecular mass 55-120 kDa. Guinea-pig antiserum against endo-beta-galactosidase-treated rabbit ZP 55 kDa glycoprotein (R55K), which specifically recognizes the rabbit ZP glycoprotein with the lowest molecular mass, also recognized the EZP 45-80 kDa glycoprotein family. Guinea-pig polyclonal antisera developed against total heat-solubilized rabbit ZP (GP alpha HSRZ) recognized the 73-90 kDa EZP glycoprotein family exclusively. After heat solubilization and treatment of EZP with endo-beta-galactosidase to remove polylactosaminoglycans, silver stained 1D-PAGE again demonstrated the presence of three glycoproteins with apparent molecular masses of 60, 75 and 90 kDa. The partially deglycosylated 60 kDa equine glycoprotein is recognized on immunoblot by the monoclonal antibody R5; the 75 kDa EZP glycoprotein is recognized by GP alpha HSRZ; and all three EZP glycoproteins separated by 1D-PAGE are recognized by R alpha HSPZ. These data add further support to the concept of cross-species zona pellucida glycoprotein antigenicity.

Equine zona pellucida and capsule: some physicochemical and antigenic properties.

The capsule which surrounds the pre-attachment equine embryo has been compared with the zona pellucida (zp) that it replaces, as well as with the rabbit blastocyst coverings, by means of physicochemical and immunological methods. Trypsin solution at pH varying between 7.5 and 9.0 completely solubilized the capsule, as did Na borohydride. However, solutions of pH 2.0 or 12.0, urea, high temperature (65 degrees C, 60 min or 80 degrees C, 30 min), mercaptoethanol and dithiothreitol were able to solubilize the zp but not the capsule at the concentrations used. Indirect immunofluorescence on cryostat sections and whole mounts of fresh or frozen-thawed material showed that 1) common antigens are shared by equine, porcine and bovine zp; 2) day 7 to day 15.5 capsule reacted with anti-capsule-serum but not with anti-zp-serum except for a few patches on the surface of the capsule; 3) anti-capsule-serum, but not anti-zp-serum, reacted with the capsular material recovered along with a broken day 27.5 conceptus; 4) anti-capsule-serum does not react with rabbit blastocyst coverings; 5) anti-capsule antibodies can be absorbed from the anti-capsule-serum by uterine proteins from either pregnant or non-pregnant mares; and 6) the capsule does not contain mouse laminin-like material.