Benjamin J. Reading, Ph.D.
Postdoctoral Research Associate
North Carolina State
University
Department of Biology
Campus
Office: 377 David Clark Laboratory
Office Phone: (919) 515-3830
Email: bjreadin@unity.ncsu.edu
Email (alt.): ben_reading@unity.ncsu.edu
Education
·
Doctor
of Philosophy (Zoology),
Dissertation: Molecular
and biochemical characterizations of vitellogenesis
in white perch, Morone
·
Master
of Science (Zoology), Southern
Thesis: Evidence for polyfactorial
sex determination in muskellunge (Esox masquinongy).
·
Bachelor
of Arts (Biological Science), Southern
Professional Experience
- 2008-Present: Postdoctoral Research Associate,
- 2003-2008: Graduate Research Assistant,
- 2003: Logan
Hollow Fish Farm,
- 1999-2003: Graduate Research Assistant, Fisheries and Illinois
Aquaculture Center, Southern
Interests
·
Reproductive
Physiology of Fishes, Functional Genomics of Reproduction, Fisheries and
Aquaculture.
Research
Basic: Functional
genomics of oocyte growth, maturation, and lipid
metabolism, with an emphasis on yolk deposition and utilization in fishes. I have a secondary focus in aquaculture and
fisheries management as well.
Applied:
Indicators of gamete quality and biotechnology related to reproductive
dysfunction with an emphasis on manual propagation of cultured fishes and
management of fisheries stocks.
The broad scope of
my research emphasis is on reproductive physiology of vertebrates, specifically
fishes. Species important to fisheries
and aquaculture, such as striped bass, are particularly suited for this and
also serve as valuable model organisms for basic research in biomedicine and
agricultural biotechnology.
Manual
propagation of fishes. (Left) Female striped bass broodstock are injected with human chorionic
gonadotropin (hCG)
to induce ovulation. Ovulated eggs are
then manually stripped from the females and fertilized with sperm from striped
bass males. These fertilized eggs are
then incubated in upwelling jars (Right,
Top) and once fry hatch from the eggs, reared in aquaria (Right, Bottom) until they can be stocked
into outdoor earthen ponds.
I have particular interest in aspects of oogenesis
related to deposition and packaging of components in the oocyte
that are required for embryonic growth and development. As examples these include yolk protein
precursors, lipids, and egg envelope precursors, and the proteins and
mechanisms involved in their production, uptake, processing, and
utilization. To study these processes, I
employ a variety of molecular and biochemical techniques to characterize both
the gene transcript and protein products and the regulation thereof.
B.J. Reading, Ph.D.
Oocyte growth and vitellogenesis in
fishes. (Left)
“Oogenesis” is the process by which primordial germ
cells (oocytes) become ova (eggs) that are ready to
be fertilized. In fishes and other
egg-laying vertebrates, once oogonia become committed
to develop as oocytes, they begin to accumulate
neutral lipids (during early secondary growth) and then proceed to vitellogenic growth (vitellogenesis),
during which time yolk precursors called vitellogenins are taken up and
stored within yolk granules in the oocyte. The yolk serves as a nutrient store for
growing embryos and larvae once the eggs have been ovulated and
fertilized. (Right, Top) Induction of vitellogenesis
starts with production of follicle stimulating hormone (FSH) in the
pituitary. FSH stimulates production of estradiol (estrogen) in the ovarian follicles, and estrogen
in turn induces the liver to produce vitellogenins. Vitellogenins are
released into the blood stream and then specifically incorporated into oocytes by receptor-mediated endocytosis. (Right,
Bottom) Circulating vitellogenins exist as homodimers in the blood.
Once taken up by the oocyte, complete vitellogenins are processed into distinct yolk
proteins. The largest yolk protein is lipovitellin which is important nutritional source of amino
acids and polar lipids to support embryonic development. Phosvitin consists
largely of serine residues which act as carriers of phosphorus and calcium and
may be involved in the stabilization of vitellogenin
structure. Two more yolk proteins,
beta-component and C-terminal component, are known to be released from the
C-terminal end of vitellogenin, however, their
physiological functions are unknown. A
shorter, incomplete type of vitellogenin also exists
that only consists of the lipovitellin yolk
proteins. Vitellogenins
are a family of very large particles (400-600kDa in size) and belong to the
lipoprotein superfamily, since they consist of both
lipid and protein components. Vitellogenins contain approximately 20% lipid by weight and
has been generally believed to be a major transporter of oocyte
lipids, the majority of which are structural lipids (polar,
phospholipids). To date, several types
of vitellogenins have been characterized in a number
of fish species and they have been shown to have specific and distinct
nutritive roles during embryogenesis.
Central
Dogma of Molecular Biology.
Genomics sciences employ a variety of techniques to understand the
functions of genes and their related RNA and protein products.
Complete Curriculum
Vitae [PUT LINK TO CV pdf HERE]
Technical Skills
Biochemistry
and Immuno-biochemistry: Gradient sodium dodecyl-sulfate
(SDS) polyacrylamide gel electrophoresis (PAGE) and native PAGE of proteins; Western blotting (native and
SDS-PAGE); protein purification from acrylamide gel
matrix; protein purification by column chromatography [open and high
performance liquid chromatography (HPLC)] using anion exchange (POROS and MonoQ), gel filtration (Superdex),
and affinity (Affigel) media; preparation and
absorption of polyclonal antisera (pAbs) for immuno-biochemistry;
double immuno-diffusion and Ouchterlony;
receptor-ligand blotting; in-well receptor binding assay; digoxigenin
(DIG) labeling of proteins; competitive enzyme-linked immunosorbent
assay (ELISA); immunocytochemistry.
Molecular Biology: mRNA differential display; Cloning of
full-length complementary DNAs (cDNAs)
from UniZAPII bacteriophage
l cDNA library; in vivo excision of pBluescriptÒ SK(-) phagemid from Uni-ZapTM
XR vector using the ExAssistTM/SOLRTM
system; 5’- and 3’-rapid amplification of cDNA ends
(RACE); nucleic acid probe construction by direct incorporation of DIG and [g-32P]ATP
DNA radiolabeling using T4 polynucleotide kinase for DNA-DNA (Southern, colony, and plaque screening)
hybridization; reverse transcription (RT) of RNA into cDNA
(first strand synthesis); chain termination (dideoxy-nucleotide)
DNA sequencing and interpretation; microsatellite
isolation from an un-enriched sub-genomic library and microsatellite
analyses; AFLP DNA fingerprinting; PAGE and agarose
gel electrophoresis (AGE) of nucleic acids; polymerase chain reaction (PCR) and
degenerate PCR primer design and optimization of PCR conditions; restriction
enzyme digest; molecular cloning of DNA; bacterial transformation, screening,
and culture; purification of nucleic acids from tissue (RNA and DNA) and gel
(DNA) matrices.
Refereed Publications
Heist,
E.J., Jenkot, J.L., Keeney, D.B., Lane, R.L., Moyer, G.R.,
Abstracts and Papers Presented at Scientific Meetings and Symposia
Sullivan, C.V.,
Professional Presentations and Public Forums
Co-Authored Abstracts and Papers
Sullivan,
C.V.,
Hiramatsu, N., Todo,
T., Ito, T., Massaki, K., Kasahara,
A., Amano, H.,
Sullivan, C.V., Williams, V.N.,
Hiramatsu, N., Hiramatsu, K.,
Non-Refereed Publications
McReynolds,
R. and
Teaching
Experience
·
2003-Present:
Supervised several undergraduate and graduate students and trained them
in molecular biology, biochemistry, and immuno-biochemistry
techniques related to research on the reproductive physiology of fishes.
·
2005:
Graduate Teaching Assistant,
·
2004:
Graduate Teaching Assistant,
·
2003:
Graduate Teaching Assistant,
·
1999-2002:
Graduate Teaching Assistant, Southern
·
1999-2001:
Basic Life Support Instructor for the American Heart Association (AHA),
Memorial
Professional Societies and Organizations
American Association for the
Advancement of Science (AAAS),
American Fisheries Society (AFS),
American Fisheries Society (AFS) Fish
Culture Section, and World Aquaculture
Professional Services and Activities
Peer
reviewer for the journals Marine
Biotechnology (Springer) and North
American Journal of Aquaculture (The American Fisheries Society).
Honors and Awards
Lewis L. Osborne Best Student Paper. 2003.
The 41st Annual Meeting of the Illinois Chapter of the
American Fisheries Society, March 4-6, Rend Lake State Park, Illinois (see Abstracts and Papers Presented at Scientific
Meetings and Symposia above).
Brevetted
Second Lieutenant,
Current Research Areas
- Transcriptomics of ovarian genes during the
natural reproductive cycle.
- Follicle atresia
and the role of hatching enzymes.
- Characterization of ovarian
lipoprotein receptors and deposition of lipoprotein nutrients in growing oocytes.
B.J. Reading, Ph.D.
Hypothetical models proposed for maternal nutrient transfer into Morone oocytes involving several
different serum lipoproteins and their related receptor and enzyme systems. Following ovulation and during embryogenesis,
the cargo delivered by LDL and VLDL (non-polar lipids: triacylglycerol,
fatty acids, and cholesterol) deposited during oocyte
growth contribute largely to egg buoyancy, cell and organelle membrane
synthesis, and serve as energy sources during late development, whereas cargo
delivered by vitellogenin (polar lipids and protein)
contribute to egg buoyancy, protein synthesis, cell and organelle membrane
synthesis, and serve as energy sources during early and late embryonic
development. Abbreviations are defined
as follows: A (apolipoprotein
A), ApoB/ER (apolipoprotein
B and E receptor), ApoER (apolipoprotein
E receptor), B (apolipoprotein B), C (apolipoprotein C), CAT (cathepsin),
CD36 (fatty acid transporter CD36), CHOL (cholesterol), E (apolipoprotein
E), EL (endothelial lipase), FA (fatty acid), FAPB (fatty acid binding
protein), HDL (high density lipoprotein), LDL (low-density lipoprotein), LDLR
(LDL receptor), LPL (lipoprotein lipase), LR7 (seven ligand
binding repeat receptor), LR8 (eight ligand binding
repeat receptor), LR8(+) (LR8 receptor containing an O-linked sugar domain), LR8(-) (LR8 receptor lacking an O-linked sugar domain), PL (phospholipid), TAG (triacylglycerol),
VLDL (very low-density lipoprotein), VLDLR (VLDL receptor), Vtg
(vitellogenin), Vtgr (Vtg receptor).
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