Cellular prion protein distribution in the vomeronasal organ, parotid, and scent glands of white-tailed deer and mule deer

Figures & data

Figure 1. Scent glands and non-integumentary tissues sampled from mule deer and white-tailed deer from Canadian forces base wainwright, Alberta.

Figure 2. Relative pixel intensity analysis of PrP^C expression in non-integumentary cranial exocrine clarified 10% (w/v) gland homogenates of mule deer (a, c) and white-tailed deer (b, d). Background-adjusted average pixel intensities of PrP^C bands of the vomeronasal organs (a-b) and parotid glands (c-d) were compared between lanes of SDS-PAGE PVDF membranes probed with anti-PrP Sha31. Unclarified white-tailed deer whole brain homogenate was used for reference. Sample size, mean, 95% confidence intervals, and significance by Mann-Whitney tests are shown.

Figure 3. Relative pixel intensity analysis of PrP^C expression in facial integumentary clarified 10% (w/v) gland homogenates of mule deer (A, C, E) and white-tailed deer (B, D, F). Background-adjusted average pixel intensities of PrP^C bands of forehead (a-b), preorbital (c-d), and vestibular nasal glands were compared between lanes of SDS-PAGE PVDF membranes probed with anti-PrP Sha31. Unclarified white-tailed deer whole brain homogenate was used for reference. Sample size, mean, 95% confidence intervals, and significance by Mann-Whitney tests are shown.

Figure 4. Relative pixel intensity analysis of PrP^C expression in leg integumentary clarified 10% (w/v) gland homogenates of mule deer (A, C, D) and white-tailed deer (B, D, F). Background-adjusted average pixel intensities of PrP^C bands of tarsal (a-b), metatarsal (c-d), and interdigital (e-f) glands were compared between lanes of SDS-PAGE PVDF membranes probed with anti-PrP Sha31. Unclarified white-tailed deer whole brain homogenate was used for reference. Sample size, mean, 95% confidence intervals, and significance by Mann-Whitney tests are shown.

Figure 5. Protein concentration-adjusted PrP^C protein expression in deer exocrine glands. Capillary electrophoresis immunoassay chemiluminescence sample size, mean, and 95% confidence intervals of clarified 10% (w/v) deer gland homogenates prepared in RIPA buffer. Deer facial (a-e) and leg (f-h) tissue and gland homogenate samples were adjusted to final protein concentrations of 1.5 μg/μL for the immunoassay. PrP^C signal was detected by anti-PrP SHA31 antibody.

Table 1. Species and sex influence on PrP^C detection by anti-PrP SHA31 capillary gel electrophoresis assay with total protein concentration-standardized 10% (w/v) clarified gland homogenates

Exocrine	Sample Size						2-Way ANOVA P Values
Tissue	Mule Deer			White-tailed Deer
		Male	Female		Male	Female	Interaction		Species		Sex
Vomeronasal	n = 10	n = 6	n = 4	n = 9	n = 4	n = 5	0.0241	*	0.1322	n.s.	0.5962	n.s.
Parotid	n = 11	n = 6	n = 5	n = 10	n = 6	n = 4	0.0250	*	0.0011	**	0.9608	n.s.
Forehead	n = 10	n = 5	n = 5	n = 11	n = 6	n = 5	0.6200	n.s.	0.3282	n.s.	0.0054	**
Preorbital	n = 11	n = 6	n = 5	n = 12	n = 6	n = 6	0.1997	n.s.	0.0564	n.s.	0.0517	n.s.
Nasal	n = 11	n = 6	n = 5	n = 12	n = 6	n = 6	0.0049	**	0.3594	n.s.	0.6823	n.s.
Tarsal	n = 11	n = 6	n = 5	n = 12	n = 6	n = 6	0.2779	n.s.	0.1540	n.s.	0.4555	n.s.
Metatarsal	n = 11	n = 5	n = 6	n = 12	n = 6	n = 6	0.0050	**	0.0106	*	0.3302	n.s.
Interdigital	n = 12	n = 6	n = 6	n = 11	n = 6	n = 5	0.2029	n.s.	0.0002	***	0.4598	n.s.

PrP^C-associated chemiluminescent baseline-fit corrected peak areas were compared between species and sex.

2-Way ANOVA significance indicators: n.s. p ≥ 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001

Figure 6. Quantified PrP^C protein concentration in deer gland homogenates. Total PrP^C concentrations of individuals, means, and 95% confidence intervals of clarified 10% (w/v) mule deer (MD) and white-tailed deer (WT) facial (a-e) and leg (f-h) gland homogenates prepared in RIPA buffer as determined by SHA31-N5 sandwich ELISA. Protein concentration was calculated using a full-length recombinant deer prion protein standard curve.

Table 2. Mean PrP^C concentrations and 95% confidence intervals of clarified 10% (w/v) gland homogenates as determined by SHA31-N5 sandwich ELISA

Exocrine	PrP^C Concentration (ng/µL)						2-Way ANOVA P Values
Tissue	Mule Deer			White-tailed Deer
		Male	Female		Male	Female	Interaction	Species	Sex
Vomeronasal	4.36 ± 0.84	3.73 ± 1.08	5.30 ± 1.00	4.75 ± 0.82	4.95 ± 0.59	4.49 ± 1.81	0.0579	0.5961	0.2159
	n = 10	n = 6	n = 4	n = 9	n = 4	n = 5	n.s.	n.s.	n.s.
Parotid	4.96 ± 1.04	4.99 ± 1.65	4.92 ± 1.94	8.89 ± 1.45	8.01 ± 2.44	9.95 ± 1.86	0.2116	<0.0001	0.2441
	n = 12	n = 6	n = 6	n = 11	n = 6	n = 5	n.s.	****	n.s.
Forehead	2.28 ± 0.44	1.83 ± 0.23	2.74 ± 0.67	2.23 ± 0.34	2.28 ± 0.34	2.17 ± 0.89	0.0261	0.7878	0.0731
	n = 10	n = 5	n = 5	n = 11	n = 6	n = 5	*	n.s.	n.s.
Preorbital	3.95 ± 0.66	3.75 ± 0.76	4.15 ± 1.40	4.45 ± 0.32	4.51 ± 0.13	4.37 ± 0.91	0.4621	0.1812	0.7143
	n = 12	n = 6	n = 6	n = 11	n = 6	n = 5	n.s.	n.s.	n.s.
Nasal	2.03 ± 0.21	2.16 ± 0.18	1.89 ± 0.49	2.30 ± 0.30	2.10 ± 0.62	2.51 ± 0.24	0.0469	0.0962	0.6493
	n = 11	n = 6	n = 5	n = 12	n = 6	n = 6	*	n.s.	n.s.
Tarsal	5.96 ± 0.92	5.41 ± 1.39	6.63 ± 1.52	7.25 ± 1.39	7.47 ± 2.55	7.02 ± 2.24	0.3013	0.1342	0.6316
	n = 11	n = 6	n = 5	n = 12	n = 6	n = 6	n.s.	n.s.	n.s.
Metatarsal	9.52 ± 1.63	9.21 ± 3.64	9.78 ± 2.28	13.02 ± 1.84	13.40 ± 3.86	12.64 ± 2.26	0.5771	0.0071	0.9359
	n = 11	n = 5	n = 6	n = 12	n = 6	n = 6	n.s.	**	n.s.
Interdigital	6.93 ± 1.50	6.09 ± 2.01	7.76 ± 2.75	10.48 ± 1.23	9.94 ± 1.75	11.12 ± 2.49	0.7841	0.0006	0.1187
	n = 12	n = 6	n = 6	n = 11	n = 6	n = 5	n.s.	***	n.s.

Tissue homogenates were not adjusted for total protein concentration.

2-Way ANOVA significance indicators: n.s. p ≥ 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Figure 7. Tissue structure PrP^C distribution in mule deer and white-tailed deer exocrine glands. Anti-PrP BAR224 immunohistochemistry is contrasted with haematoxylin counterstain. BAR224 and no-primary antibody control immunohistochemistry slides of each gland were developed in the same batches. Scale bars for tarsal glands: 500 µm. All other scale bars: 250 µm.

Figure 8. PrP^C distribution within non-integumentary facial glands. Immunohistochemical BAR224 detection of PrP^C (brown) with haematoxylin counterstaining. Female mule deer A) vomeronasal organ, and B) parotid gland. Structure abbreviations: SA, serous acini; SD, striated duct; SM, submucosa; VL, vomeronasal lumen; VRE, vomeronasal respiratory epithelium; VTS, vomeronasal tubular serous glands.

Figure 9. PrP^C distribution within facial integumentary glands. Immunohistochemical BAR224 detection of PrP^C (brown) with haematoxylin counterstaining. White-tailed male deer A) forehead gland, B) preorbital gland with magnified inset of infiltrating leukocytes, and mule deer female C) lateral vestibular nasal gland. Structure abbreviations: E, epidermis; FC, follicular canal; HS, hair shaft; L, leukocytic infiltrates; M, skeletal muscle; RS, follicular root sheath; SE, sebaceous glands; and SU, sudoriferous glands.

Figure 10. PrP^C distribution within leg integumentary glands. Immunohistochemical BAR224 detection of PrP^C (brown) with haematoxylin counterstaining. White-tailed male deer A) tarsal gland, B) metatarsal gland, and mule deer female C) interdigital gland. Structure abbreviations: APM, arrector pili muscle; DE, dermis elastic layer; E, epidermis; FC, follicular canal; HS, hair shaft; L, leukocytic infiltrates; NHS, nucleated region of the hair shaft; RS, follicular root sheath; SE, sebaceous glands; and SU, sudoriferous glands.