Histology of ovaries of female rabbits immunized with deglycosylated zona pellucida macromolecules of pigs

Abstract

Female rabbits (n = 36, 6 per group) were immunized with: (i) solubilized isolated porcine zona pellucida (SIZP), which contains ZP1, 82 kDa; ZP3 alpha, 55 kDa; and ZP3 beta, 55 kDa; (ii) a purified preparation of ZP3 alpha and ZP3 beta (ZP3); (iii) purified endo-beta-galactosidase digested glycoproteins ZP3 alpha-(EBGD) and (iv) ZP3 beta-(EBGD) (each about 30% deglycosylated); (v) chemically deglycosylated core proteins ZP3 alpha-(DG) and (vi) ZP3 beta-DG (each greater than 92% deglycosylated). Rabbits injected with saline (n = 6) or Freund's adjuvant (n = 6) served as controls. Rabbits were bled weekly to monitor titres. Every six weeks two animals from each group (n = 16) were selected for unilateral oophorectomy followed by histological examination. Sections were scored for numbers of primary, secondary and tertiary follicles. Anti-ZP3 titres developed in all treatment groups and correlated with carbohydrate content (peak per cent [125I]-labelled ZP3 binding by radioimmunoassay: SIZP 71.9 +/- 1.2, ZP3 70.0 +/- 2.5, ZP3 alpha-EBGD 60.9 +/- 5.3, ZP3 beta-EBGD 56.4 +/- 5.0, ZP3 alpha-DG 56.4 +/- 4.0, ZP3 beta-DG 53.5 +/- 4.3) (means +/- SEM). Animals immunized with SIZP, ZP3 and ZP3 beta-EBGD showed a statistically significant reduction in the number of primary, secondary and tertiary follicles compared with controls (P less than 0.01, MANOVA), whereas animals immunized with ZP3 alpha-EBGD, ZP3 alpha-DG and ZP3 beta-DG did not (P greater than 0.05, MANOVA). These results demonstrate that immunization with purified ZP3 alpha macromolecules (ZP3 alpha-EBGD, ZP3 alpha-DG) or ZP3 beta-DG does not produce histopathological changes in ovaries. Such deglycosylated ZP macromolecules represent potential target antigens for immunocontraceptive development.

Antifertility effects of porcine zona pellucida-3 immunization using permissible adjuvants in female bonnet monkeys (Macaca radiata): reversibility, effect on follicular development and hormonal profiles

“…One of the animals (Fig. 2) (Jones et al, 1992). In primate models, the use of deglycosylated ZP3 in baboons (Dunbar el al, 1989) showed decreased concentrations of oestrogen followed by a cessation of ovulation, whereas deglycosylated ZP3ß in mar¬ moset monkeys (Paterson et al, 1992) (Millar et al, 1989), whereas the same peptide in complete Freund's adjuvant (CFA) given to (C5 7B1/6 A/J) Fj mice causes severe autoimmune oophorites (Rhim el al, 1992 (Mahi-Brown et al, 1985) showed that the antibody response generated by using alum was low, but sufficient to protect the animal from pregnancy in the first cycle after immunization.…”

Structure and Function of the Proteins of the Mammalian Zona pellucida

“…Studies using purified deglycosylated native or recombinant ZP proteins have identified specific ZP immunogens which elicit antibodies that inhibit fertility and sperm binding to ZP without altering ovarian follicular development [Millar et al, 1989;Aitken et al, 1990;Sacco et al, 1991;Jones et al, 1992;VandeVoort et al, 1995]. These are in contrast to studies in which antibodies to different ZP antigens alter ovarian follicular development or cause autoimmune oophoritis Jones et al, 1992;Rhim et al, 1992;VandeVoort et al, 1995;Aitken et al, 1996].…”

Contraceptive potential of porcine and feline zona pellucida A, B and C subunits in domestic cats

“…ZP protein was prepared as described previously ( Jones et al 1992) using pig ovaries from a local abattoir (Perth, WA, Australia). Briefly, ovaries were passaged through a descending series of molecular sieves and zonae solubilized and concentrated (!100) using an ultrafiltration cell.…”

“…The extraction procedure was modified from Jones et al (1992). Briefly, ovaries were homogenized and passed through a series of molecular sieves, centrifuged at 1800 g for 12 min at 4 8C and subjected to three rounds of freezing and thawing.…”

“…The immunization of females against ZP results in the formation of ZP-specific antibodies, which bind to native autologous ZP and offer contraception by blocking sperm binding to receptors. Studies have also shown ovarian distruction, either permanent or temporary, with possible correlation to ZP-specific antibody responses in the pig, rabbit, dog and squirrel monkey , Jones et al 1992, Kerr et al 1999, Srivastava et al 2002.…”