Altered Splenocyte Function in Aged C57BL/6 Mice Prenatally Exposed to Diethylstilbestrol

Figures & data

TABLE 1 Effects of DES on body weight and organ weight

Organ	Gender	Oil	DES
Terminal body weight (g ± SEM)	Female	24.50 ± 1.10	25.30 ± 1.30
	Male	34.10 ± 1.30	34.50 ± 1.20
Reproductive organs
Uterus weight (g ± SEM)	Female	0.10 ± 0.02	0.12 ± 0.02
Uterine weight: Body weight × 100 (±SEM)		0.41 ± 0.06	0.47 ± 0.07
Ovaries weight (g ± SEM)	Female	0.01 ± 0.001	0.02 ± 0.001
Ovaries weight: Body weight × 100 (±SEM)		0.06 ± 0.004	0.06 ± 0.005
Seminal vesicules weight (g ± SEM)	Male	0.53 ± 0.07	0.60 ± 0.06
Seminal vesicules: Body weight × 100 (±SEM)		1.57 ± 0.22	1.74 ± 0.20
Testes weight (g ± SEM)	Male	0.20 ± 0.006	0.19 ± 0.005
Testes weight: Body weight × 100 (±SEM)		0.56 ± 0.03	0.59 ± 0.02

C57BL/6 mice were exposed to either DES (0.25 μg) (n_female = 7, n_male = 11) or oil (n_female = 10, n_male = 8) during prenatal development. At 12 months of age, mice received 1 DES dose (30 μg/kg BW, subcutaneous). Six days later, the mice were inoculated with T. gondii antigen IP. Mice were euthanized at 14 months of age with body weight and organ weight were determined.

FIG. 1. IFNγ levels in serum 6 hours after administration of soluble T. gondii protein. C57BL/6 mice were exposed to either DES (0.25 μg) (n_female = 7, n_male = 11) or oil (n_female = 10, n_male = 8) during prenatal development. One year later, mice were given 1 subcutaneous injection of DES (30 μg/kg BW). Mice were then challenged with 20 μg of soluble proteins derived from the R.H. strain of T. gondii. Serum samples were collected 6 hours after the T. gondii antigen administration. Mean data are presented in pg/ml ± SEM. Differences between genders and treatment groups were considered significant when p < 0.05. ^a denotes significant difference between females given prenatal oil and females given prenatal DES treatment. ^b denotes significant difference between females given prenatal DES and males given prenatal DES treatment.

FIG. 2. IgG levels in serum six hours after T. gondii antigen administration. C57BL/6 mice were exposed to either DES (0.25 μg) (n_female = 7, n_male = 11) or oil (n_female = 10, n_male = 8) during prenatal development. One year later, mice were given one subcutaneous injection of (30 μg/kg bw). Mice were then challenged with 20 μg of soluble protein antigen derived from the R.H. strain of T. gondii. Serum samples were collected 6 hours after the T. gondii antigen administration. Mean data are presented in pg/ml ± SEM. Differences between genders and treatment groups were considered significant when p < 0.05. ^a denotes significant difference between females given DES and females given oil prenatal treatment (p = 0.001).

FIG. 3. Secreted IFNγ levels after 24 hour stimulation with ConA. C57BL/6 mice were exposed to either DES (0.25 μg) (n_female = 7, n_male = 11) or oil (n_female = 10, n_male = 8) during prenatal development. At 1 year of age, all mice were given 30 μg DES/kg BW. Two months later, mice were euthanized to collect splenocytes. Splenocytes were cultured with an optimal concentration of ConA (10 μg/ml). The supernatants were collected after 24 hours of culture and analyzed for IFNγ levels by ELISA. Mean data are represented in pg/ml ± SEM. Differences between gender and prenatal treatments were considered significant when p < 0.05. ^a denotes significant difference between females given DES and females given oil prenatal treatment.

FIG. 4. Splenic lymphocyte count of adult C57BL/6 mice prenatally exposed to oil or DES treatment. C57BL/6 mice were exposed to either DES (0.25 μg) (n_female = 7, n_male = 11) or oil (n_female = 10, n_male = 8) during prenatal development. At 1 year of age, all mice were given 30 μg DES/kg BW. Immediately after harvesting the spleen, splenocyte suspensions were counted on a CASY-1 Cell Counter and Analyzer System (Scharfe System GmbH, Reutingen, Germany). Data presented as mean cellularity ± SEM. Differences between gender and prenatal treatments were considered significant when p < 0.05. ^a denotes significant difference between females given DES and females given oil prenatal treatment.

TABLE 2 Percent expression of CD4 and CD8 splenocytes from DES or oil prenatally exposed mice immediately after harvest

Splenic T-cell Subsets	Female		Male
	Oil	DES	Oil	DES
CD4^+CD8^−	21.5 ± 2.3	20.9 ± 2.0	18.5 ± 2.3	20.2 ± 2.0
CD4^−CD8^+	8.3 ± 2.0	8.6 ± 1.8	10.5 ± 2.0	9.8 ± 1.8
CD4^+CD8^+	1.2 ± 0.2	1.3 ± 0.2	1.2 ± 0.2	1.0 ± 0.2
CD4^−CD8^−	69.1 ± 4.3	69.4 ± 3.7	69.9 ± 4.3	68.5 ± 3.7

C57BL/6 mice were exposed to either DES (0.25 g) (n_female = 6, n_male = 6) or oil (n_female = 6, (n_male = 6) during prenatal development. At 1 year of age all mice were given 30 μg/kg BW DES. Two months later, mice were terminated and splenocytes were stained for T-cell subset markers CD4 and CD8 immediately after harvest. The data are presented as mean ± SEM.

FIG. 5. CD28⁺ CD90⁺ unstimulated and ConA stimulated splenocytes from female mice given prenatal oil or DES treatment. C57BL/6 mice were exposed to either DES (0.25 μg) (n_female = 6) or oil (n_female = 6) during prenatal development. At 1 year of age, all mice were given 30 μg DES/kg BW. Splenocytes were stained for CD28 co-stimulatory marker expressed on T-cells (CD90). The top panel (Figure 5A) shows representative data of female splenocytes. The bottom panel (Figure 5B) shows data presented as mean ± SEM of CD28 expression on T-cells cultured in the presence or absence of ConA for 24 hours in female mice. Differences between prenatal treatments were considered significant when p < 0.05. The relative percentages of CD28 expression on T-cells in media cultures included (Female_oil = 49.4 ± 7.7, Female_DES = 40.7 ± 7.1, Male_oil = 49.4 ± 7.0, Male_DES = 48.5± 7.3). ^a denotes significant difference between cultured splenocytes stimulated with ConA from females given DES and females given prenatal treatment.

TABLE 3 Relative percentages of CD4⁺CD25⁺ splenocytes after 24-hour incubation from female and male mice exposed to either oil or DES prenatally

	Female				Male
	Media		ConA		Media		ConA
Splenocyte Subset	Oil	DES	Oil	DES	Oil	DES	Oil	DES
CD4^+CD25^+	1.5 ± 0.3	1.8 ± 0.4	14.3 ± 2.5	12.2 ± 2.2	0.9 ± 0.2	0.7 ± 0.3	23.3 ± 2.1	22.3 ± 2.4

*Denotes a significant difference between female splenocytes cultured with ConA and male splenocytes cultured with ConA C57BL/6 mice were exposed to either DES (0.25 μg) (n_female = 6, n_male = 6) or oil (n_female = 6, n_male = 6) during prenatal development. At 1 year of age all mice were given 30 μg/kg BW DES. Splenocytes were stained for regulatory T-cells CD4⁺CD25⁺ immediately after harvest. The data are presented as mean ± SEM. Differences between gender and prenatal treatments were considered significant when p < 0.05.