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Hameeda Naimi Barry Hinton: Department of Cell Biology 10 December 2014

Protein expression analysis of Csk knockouts in the epididymis

Introduction: The goal for this semester was to analyze the effects of Csk knockouts on development of the initial segment of the epididymis in order so that data collected may add to preceding research and be used for further investigation. The formal hypothesis concerned whether Csk knockout mice displayed greater cell proliferation and/or cell differentiation through lack of inhibition of upstream 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, whose length is 1m in the mouse to 6m in humans (1). This elongated and convoluted epididymal duct provides a luminal environment that serves a critical role in the development and maturation of spermatozoa, where its initial segment (IS) has been suggested crucial to male fertility (2). Under normal physiological conditions, the IS has been shown to express higher levels of the ERK pathway components in relation to other regions of the epididymis. Activated by luminal fluid factors which are propelled into the epididymis from Sertoli cells in the testis, the ERK pathway encourages proliferation and differentiation in the IS through this higher-than-average activity (2). Key to this growth and development is a group of cytoplasmic non-receptor Src family tyrosine kinases (SFKs) that serve as positive signal intermediaries between luminal fluid factors and the Erk pathway. Discovered as oncogenes, the Src family kinases are kept inactive by C-terminal Src kinase (Csk), which phosphorylates C-terminal residues on Src kinases thereby suppressing oncogenic activity under normal conditions (3). In Csk-knockout mouse models, dysregulation of Src kinases is expected to promote up-regulation of Erk pathway components thus contributing to greater cell proliferation and differentiation in the IS of the epididymis. Therefore, proper investigation of IS development and underlying sperm maturation in relation to the regulation provided by Csk is crucial to a more sophisticated understanding of male fertility. Procedures:

o Knockout Mouse Model: Cre/lox recombination was carried out to produce a tissue-specific Csk conditional knockout mouse model. Mice carrying loxP-flanked Csk allele (Cskflox/flox) were first bred with Tg(Rnase10-Cre) mice according to protocols established by The Jackson Laboratory (4). The first-generation progeny, containing heterozygous Csk conditional knockouts, were then bred back to the homozygous loxP-flanked Csk mice. Among the second-generation progeny, 25% were expected to display the experimental genotype: IS homozygous for the loxP-flanked Csk allele and hemizygous for the Cre transgene (Cskflox/flox; Rnas10-CreTg).

o Genotyping: Csk knockout male mice from the second-generation litter were selected through repeated PCR according to protocols established by The Jackson Laboratory (4). For DNA isolation, pre-cut mice tail tips were homogenized in Tris lysis buffer; the homogenate was centrifuged (10,000 x g for 10 min) and the supernatant was added to a master mix composed of MyTaq Red Mix (buffer, dNTPs MgCl2), ddH2O, and primers. The latter were designed for Csk and Cretg as follows: Csk/Flop 1459F (5′-GAGTTCCAGGACAGCCAGAG-3′) and Csk/Flop 14597R (5′- GTCTCCACCTAGCACCGAAC-3′); Cre2 (5′-CAGGCAGGCCTTCTCTGAAC-3′) and Cre2-as (5′-CATTCTCCCACCATCGGTGC-3′). Primer-specific reactions were run for all samples with an added positive and two negative controls. For gel electrophoresis, amplified

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DNA was loaded onto 2% agarose gel for Csk and 1.5% agarose gel for Cre, respectively, the results of which are shown in Figures 1 and 2.

o Microscopy: To examine global morphology of Csk knockouts in comparison to controls, dissected epididymides (completed by mentors) of 8wk mice were fixed in 4% PFA in PBS overnight at 4°C. A dissection microscope (model MZ10 F; Leica) was used to observe and capture images of the testis and epididymides taken from control and conditional KO animals. The images can be seen in Figure 3 (A-C). Fresh tissue from the epididymides was minced dispersing the spermatozoa, which were then plated in BWW medium and viewed with a phase contrast microscope. Movies from various regions of the plate were recorded using the CASA plug-in for ImageJ Software (imagej.nih.gove/ij/). The same software’s Manual Tracking plug-in was implemented to qualitatively capture motility differences between straight and curved/compromised knockout spermatozoa. The movies for control and KO have been uploaded to Collab and stilled images are shown in Figure 4 (A-D).

o 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 5 (A-J). The following primary antibodies were purchased from Cell Signaling Technology: Phospho-p44/42 MAP (Erk1/2) (Thr202/Tyr204) antibody (no.9106s, 1:100 working dilution), Phospho-SRC (Tyr416) antibody (no.2101, 1:100 working dilution), and Phospho-SRC (Tyr527) antibody (no.2105, 1:50 working dilution). The following secondary antibody was purchased from Life Technologies: Alexa Fluor (no.594, 1:200 working dilution).

Results:

Figure 1: PCR of 8 littermates to find Cskflox/flox homozygotes; 1Kb ladder. Green stars: males. Yellow stars: females. Orange arrows: Csk homozygous males.

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Figure 3: Global morphology of Csk KO and control mice. (A) Testes from control and KO animals. (B) IS and efferent ducts (ED) from control and KO animals. (C) Epididymides from control and KO animals; 1: ED, 2: IS, 3A: Proximal caput, 3B: Distal caput, 4: Corpus, 5A: Proximal cauda, 5B: Distal cauda. Csk KO IS appears to show more robust vasculogenesis than controls.

Figure 2: PCR of the same 8 littermates to find hemizygous Cretg individuals; 1 Kb ladder. Green stars: males. Yellow stars: females. Cyan arrow: A2 is the only male heterozygous for Csk and hemizygous for Cretg – this will be the Csk control. Orange arrow: A5 is the only male individual homozygous for Cskflox/flox and hemizygous for Cretg - this is our conditional KO mouse model.

Figure 4: Manual Tracking images of control vs. KO spermatozoa. (A) Straight spermatozoa in control animal. (C) Control displays fairly straight and smooth motility path. (B) Curved spermatozoa in KO animal. More curved spermatozoa were observed overall in KO animal. (D) Circular rather than straight path of KO curved spermatozoa.

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 Conclusions and Discussion: Thus far, our Cre/lox recombination breeding resulted in only one test mouse, A5, displaying both the homozygous Cskflox/flox, as marked in Figure 1, as well as the Cretg, marked in Figure 2. The rest of the litter were either female, A7 and A8; expressed the heterozygote Cskflox/+ genotype, A1, A2, A4, and A6; or expressed the Cskflox/flox genotype but did not express the Cretg, A3 (Figures 1 and 2). It is also important to mention the significance of choosing a mouse model to understand the molecular mechanisms at work within the

initial segment. Practically speaking, mouse models are neat tools for genetic manipulation and modeling of development processes. Specifically, the initial tubular organization of the epididymis progresses quickly to a more complicated and convoluted structure during development, a course observed within mice as well. Thus, it is accepted that mice models of genetic and physiological changes within the Wolffian duct will also help to explain processes within humans (5). Dissection of mouse A5 and subsequent dissection microscopy displayed more robust vasculogenesis in the initial segment of the epididymis than controls (Figure 3B). This increased vasculogenesis, seen through more prominent vessel presence in the IS, suggests possible evidence that inhibition of Csk leading to depressed inhibition of SRC kinases contribute to differentiation through ERK pathway components upregulating vasculogenesis. Although the causes are unknown at the moment, these possible changes in vasculature might have also contributed to the greater number of defective sperm observed in KO animals. Manual tracking methods allowed us to follow

Figure 5: Immunofluorescence staining of control (A, C, and E) and conditional knockout (B, D, and F) epithelial cells at 8wks and nuclear DNA staining of IS regions 1 (G and H) and 2 (I and J). Phospho-SRC (Tyr 527) showed more depressed activity in KO (B) than in controls (A). Phospho-p44/42 MAPK showed no observably significant changes in expression level between control (E) and KO animals (F). Phospho-SRC (Tyr 416), while showing minimal variation in and near the lumen region, displayed no significant changes between control (C) and KO animals (D). Controls in both regions 1 and 2 (G and I) displayed more clumping of nuclear DNA staining than in KO where more nuclear staining was also observed towards the lumen (H and J).

A   B  Control   Csk  KO  

C   D  

E   F  

G   H    

I    

J  

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movement of individual spermatozoa and observe significant differences in flagellar structure; sperm with straight flagella traveled fairly straight pathways whereas those with curved flagella contributed to redundant, circular motion (Figure 4C and 4D). These latter defective sperm that are assumed to have not matured fully might have difficulties progressing through hypotonic medium/female tract and are thus possible contributing factors to male infertility. Preliminary immunofluorescence test detected lower phosphorylation levels for Phospho-Src (Tyr-527), an inhibiting site on Src, in KO over controls (Figures 5A and 5B). This was expected since Csk targets the Src inhibiting site thus knockout Csk can no longer suppress SRC activity. Phospho-Src (Tyr-416), an activation site on SRC, and phospho-p44/42 MAPK did not show particularly variable activity levels between control and knockout (Figures 5C-5F). HE staining showed most nuclei positioned at the base of the epithelial cells in controls (Figures 5G and 5I) over knockouts, which displayed more nuclei positioned towards the lumen of the cell (Figures 5H and 5J). While these initial observations did not prove too helpful in inspecting expression levels, aside from Src Tyr-527, looking forward we have decided to more qualitatively investigate these two through western blot analysis. Concerning immunofluorescence and the significant side effects observed in both control and KO tissue, fixation periods have been adjusted for future research. Finally, further genotyping and collection of KO animals should provide the opportunity to also design quantitative analysis regarding sperm motility, structure, and angulation. Acknowledgements: A big thank you to Professor Hinton and Bingfang Xu for their continuous encouragement and patience – and for being great advocates of exploration and knowledge! J

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References

1. Xu, Bingfang, Ling Yang, and Barry T. Hinton. “The Role of Fibroblast Growth Factor Receptor Substrate 2 (FRS2) in the Regulation of Two Activity Levels of the Components of the Extracellular Signal-Regulated Kinase (ERK) Pathway in the Mouse Epididymis.” Biology of Reproduction 89.2 (2013): 48. PMC. Web. 23 Nov. 2014.

2. 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. PMC. Web. 22 Nov. 2014.

3. Okada, Masato. “Regulation of the Src Family Kinases by Csk.” International Journal of Biological Sciences 8.10 (2012): 1385–1397. PMC. Web. 22 Nov. 2014.

4. Hinton BT, et al. (2011): “How do you get six meters of epididymis inside a human scrotum?” Journal of Andrology 32(6): 558-564. Web. 23 Nov. 2014.

5. Hinton, B. T., Galdamez, M. M., Sutherland, A., Bomgardner, D., Xu, B., Abdel-Fattah, R. and Yang, L. (2011), How Do You Get Six Meters of Epididymis Inside a Human Scrotum?. Journal of Andrology, 32: 558–564. doi: 10.2164/jandrol.111.013029