Isolation and Partial characterization of Immunoglobulin M - like Protein in Eggs of Rainbow Trout (Oncorhynchus mykiss)
Date
1998
Authors
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Degree Level
Masters
Abstract
An immunoglobulin M (IgM)-like protein was isolated from egg extracts of rainbow
trout (Oncorhynchus mykiss) by both affinity chromatography on Mannan Binding Protein
(MBP) and by selective precipitation and volume exclusion using a proprietary "Gammayolk"
purification procedure. Rainbow trout serum IgM was also isolated using both
procedures. One step affinity chromatography using MBP, yielded relatively high purity
IgM-like molecules as observed by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). Percent recovery from total proteins loaded was
approximately 1% for egg extracts and 3% for serum by MBP, while "Gamma-yolk"
yielded higher recoveries (8% for egg and 28% for serum). However purity of the
preparations were compromised as evidenced by multiple banding under SDS-PAGE.
Total egg and serum protein content were 59.9 ± 5.7 mg/ml (n=36) and 36.7 ± 12.7
mg/ml (n=19) respectively.
Molecular weight of egg immunoglobulin was estimated to be 216 kD under nonreducing
conditions in 3% SDS-PAGE. This was much smaller than the purified serum
IgM which was approximately 882 kD. The 216 kD band corresponds to the monomeric
form of serum IgM which occurs in a tetrameric form in fish.
Molecular weight estimation by 12% SDS-PAGE under reducing conditions and
immunoblotting with monoclonal or polyclonal antibodies raised against trout serum IgM,
discerned the heavy and light chains of the isolated trout egg IgM-like molecule at 87 and
24 kD respectively. This was similar to that observed for seric IgM. However, an
additional light chain with molecular weight of 21kD was observed in the egg preparations
while serum IgM had an additional light chain at 28 kD. The light chains of egg IgM probably occurred as two different isomeric forms, as previously reported for serum lgM
light chain.
Concentration of IgM in the serum and egg of rainbow trout raised in disease-free
aquatic facilities was determined by enzyme-linked immunosorbent assay (ELISA), using
polyclonal goat anti-trout Ig antibodies as capture antisera, and monoclonal antibodies
specific to H chain of trout IgM as detecting antibodies. Female rainbow trout (3-4 kg) had
significantly higher ( p < 0.05) egg IgM (1.03 ± 0.25 mg/ml, n=15) than serum lgM (0.11 ± 0.02 mg/ml, n=15). The concentration of IgM in the ovum and egg was highly correlated
(r = 0.8, p < 0.05) with the concentration of IgM in the maternal serum (n=18 ). Maturation
significantly (p < 0.05) reduced ovum IgM concentration from a mean of 14.5 mg/ml (n=6)
in oocytes whose mean diameter was 1 mm to 3.2 mg/ml (n=6) in pre-ovulatory oocytes
(mean diameter = 4 mm).
The stability or shelf-life of the serum and egg IgM molecules was tested using
SDS-PAGE, western blot and silver staining techniques by monitoring the appearance of
immune reactive bands with time. Egg IgM appeared to be stable at 4°C for at least 10 days,
while serum IgM appeared to be stable for only 6 days.
Localization of IgM in the ova was assessed by immunohistochemistry of cryostat
sections reacted with either monoclonal mouse anti-trout IgM H chain antibodies (4D11), or
polyclonal rabbit anti-male rainbow trout serum IgM antibodies. The lgM-like molecule
was diffusely present in the ovum yolk. Strong immunoreactivity was observed in the inner
margin of ovarian follicles as a thin layer protecting the egg. Immunoreactivity was
observed in ova from small (<1 mm in diameter) to mature eggs, indicating early transfer of serum IgM to the egg.
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Degree
Master of Science (M.Sc.)
Department
Veterinary Anatomy
Program
Veterinary Anatomy