1. Hameeda Naimi
Barry Hinton: Department of Cell Biology
4 May 2015
Phenotyping Csk knockouts in the epididymis
Introduction: The goal for this semester was to continue analyzing the effects of Csk knockouts on
development of the initial segment of the epididymis using data as a starting point for further
investigation on this topic. The formal hypothesis investigated concerned whether Csk knockout
mice displayed greater cell vascularization and/or cell proliferation through lack of inhibition of SRC
family tyrosine-kinases, key initiators of the ERK pro-proliferative signaling pathway.
Background:
The Wolffian duct or epididymis is a key structure in the male reproductive system as it provides a
luminal environment critical in the maturation of spermatozoa. Specifically, experiments blocking
luminal factors from entering the epididymis, prevented development of epithelial cells in the initial
segment, an area now suggested crucial to male fertility (1). Previous experiments have also shown
that luminal fluid factors activate the pro-proliferative and pro-differentiating ERK pathway. A group
of cytoplasmic non-receptor Src family tyrosine kinases (SFKs) serve as positive signal
intermediaries between luminal fluid factors and the Erk pathway; Src family kinases activity is
blocked by C-terminal Src Kinase (Csk), serving a critical tumor suppressor role under normal
conditions. The previous semester’s Csk-knockout mouse immunofluorescence preliminary analyses
suggested increased vasculogenesis, proposing potential evidence that reduced inhibition of Src
family kinases permit greater differentiation through ERK pathway signaling components. This
semester’s focus involved proper examination of factors related to the possibility of up-regulated
vasculogenesis in the initial segment, an inquiry that might provide a more holistic understanding of
epididymal development.
Procedures:
• Determining proper analysis of vascularization: Various techniques were researched
however three antibodies were ultimately chosen on the basis of their involvement in
vascularization. Platelet derived growth factor (PDGF) has been noted in enhancing capillary
growth in epididymal tissue (2). Platelet endothelial cell adhesion molecule (PECAM) has
been used to evaluate the degree of tumor angiogenesis and finally, Paxillin, a focal adhesion
scaffolding protein, has been connected with directing capillary endothelial cell motility (3).
See the conclusions section below for further discussion regarding the applicability of the
antibodies for future experiments.
• Immunofluorescence: Tissue samples from epididymides dissections, both control and
knockout, were fixed in 4% PFA in PBS overnight at 4°C, embedded in paraffin, and
sectioned. Slides were then deparaffinated and rehydrated. Antigen unmasking was
completed through an antigen unmasking solution (Vector Laboratories) for 10 min on high
in a 1,300-W microwave and cooled for 1hr at room temperature (RT). Slides were washed in
TBS and then incubated in blocking solution comprised of 10% normal goat serum (Vector
Laboratories), 0.5% cold-water fish skin gelatin (Sigma), and TBS for 70 min at RT. Slides
were then incubated overnight at 4°C in blocking solution with their respective primary
antibodies. The following day, slides were washed in TBS and incubated for 1.5hr at RT with
blocking solution and secondary antibodies. Upon subsequent washing in TBS, slides were
mounted with Prolong Anti-fade reagent (Molecular Probes) with DAPI for nuclear staining
and viewed under a Zeiss microscope. Images can be seen in Figure 2 (A-H) and Figure 3 (A-
F).

2. The following primary antibodies were purchased from Cell Signaling Technology: Phospho-
p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (no.4370s, 1:200 working dilution),
Phospho-c-Raf (Ser259) antibody (no.9421, 1:50 working dilution), PDGF Receptor β
(28E1) antibody (no.3169, 1:100 working dilution), and Phospho-Paxillin (Tyr118) antibody
(no.2541, 1:50 working dilution). The following primary antibodies were purchased from
Santa Cruz Biotechnology: Phospho-p38 (D-8) antibody (no.sc-7973, 1:100 working
dilution) and Pecam-1 (M-20) antibody (no.sc-1506, 1:50 working dilution). The following
antibody was purchased from EMD Millipore: Anti-collagen Type IV antibody (no. AB769,
1:50 working dilution). The following secondary antibody was purchased from Life
Technologies: Alexa Fluor (no.594, 1:200 working dilution).
• ImagePlus Analysis: Perhaps the greatest technique learned and implemented in this
semester’s study, ImagePlus proved a valuable resource in analyzing and quantifying the
intensity of protein labeling with regards to vascularization. Measurements of vascularized
area and total area allowed for statistical analyses between control and KO animals. A
schematic of the technique is demonstrated below for future guidance (Figure 1. A-D).
Figure 1. Basic protocol of ImagePlus Segmentation and Masking: Scale is set and original image
is segmented (A). The segmented image is then replaced by a new mask (B). System set algorithm
automatically counts all segments, highlighted in green (C). User can filter out tiny, nonspecific
labeling to not be included as part of final count; notice that final count is significantly more exclusive
than initial count (D).
A
B
C
D

3. Results:
Figure 2:
Immunofluorescence
staining of control (A, C,
and E) and conditional
knockout (B, D, and F)
epithelial cells at 8wks.
Phospho-c-Raf showed no
observable changes in
expression level between
control (A) and KO animals
(B). Phospho-p44/42
MAPK and phospho-P38
showed similarly
unobservable changes
between control (C and E)
and KO animals (D and F).
A
Control
Csk
KO
C
B
D
pRaf
RI
pErk
RI
RI
RI
pP38
RI
RI
E
F

4. A
Control
Csk
KO
B
C
D
E
F
Figure 3:
Immunofluorescence
staining of control (A, C, E,
and G) and conditional
knockout (B, D, F, and H)
epithelial cells at 8wks.
Platelet-derived growth
factor-beta (PDGF-β)
displayed observable and
statistically significant (p <
0.05) expression in KO (B)
over control animals (A).
Pecam-1 showed increased
non-specific tagging in KO
(D) over control animals
(C). Phospho-Paxillin
showed no observable
differences between control
(E) and KO animals (F).
Anti-collagen Type IV
showed seemingly
increased expression levels
between control (G) and
KO animals (H).
G
H
PDGF-β
Pecam-1
p-Paxillin
Col-4
RI
RI
RI
RI
RI
RI
RI
RI

5. Conclusions/Discussion:
Contrary to what was expected, preliminary immunofluorescence analyses did not
demonstrate any observable differences in phospho-p44/42 MAPK expression levels between control
and KO animals. Similarly unobservable differences were indicated in expression levels for control
and knockout animals in both phospho-c-Raf and phospho-P38 analyses.
ImagePlus analysis was focused to those antibodies specific to angiogenesis: PDGF-β,
Pecam-1, phospho-Paxillin, and Col-4. Scaled measurements of the vasculature area, total area, and a
fraction of the two areas (vasculature area/total area) was taken only for PDGF-β at 4 and 8 weeks.
Pecam-1 staining did not demonstrate efficient labeling and therefore data collected was temporarily
excluded from the current investigation. Furthermore, phospho-Paxillin could not be statistically
included as more replicates were necessary for calculations. PDGF-β, while it displayed no
significant changes (p > 0.05) for control and KO animals at 4 weeks did exhibit significant changes
(p < 0.05) among control and KO animals at 8 weeks. This preliminary investigation suggests a time-
sensitive control on angiogenesis although further analyses will have to be well considered before
such a conclusion can be made with regards to increased initial segment vascularization.
Acknowledgements: A big thank you to Professor Hinton, Bingfang Xu, and Angela Washington for
their patience and guidance throughout these past two semesters of research. J

6. References
1. Xu, Bingfang et al. “Testicular Lumicrine Factors Regulate ERK, STAT, and NFKB Pathways in
the Initial Segment of the Rat Epididymis to Prevent Apoptosis.” Biology of Reproduction 84.6
(2011): 1282–1291. Web.
2. Sato, N. et al. “Platelet-Derived Growth Factor Indirectly Stimulates Angiogenesis in Vitro.” The
American Journal of Pathology 142.4 (1993): 1119–1130. Web.
3. Alexandra E. German, Tadanori Mammoto, Elisabeth Jiang, Donald E. Ingber, and Akiko
Mammoto. Paxillin controls endothelial cell migration and tumor angiogenesis by altering
neuropilin 2 expression. J Cell Sci 2014 127: 1672-1683; Advance Online Article February 12,
2014, doi: 10.1242/jcs.132316. Web.