Semen activates the female immune response during early pregnancy in mice

Abstract

Insemination elicits inflammatory changes in female reproductive tissues, but whether this results in immunological priming to paternal antigens or influences pregnancy outcome is not clear. We have evaluated indices of lymphocyte activation in lymph nodes draining the uterus following allogeneic mating in mice and have investigated the significance of sperm and plasma constituents of semen in the response. At 4 days after mating, there was a 1b7-fold increase in the cellularity of the para-aortic lymph node (PALN) compared with virgin controls. PALN lymphocytes were principally T and B lymphocytes, with smaller populations of CD3(+) B220(lo), NK1.1(+) CD3(-) (NK) and NK1.1(+) CD3(+) (NKT) cells. CD69 expression indicative of activation was increased after mating and was most evident in CD3(+) and NK1.1(+) cells. Synthesis of cytokines including interleukin-2, interleukin-4 and interferon-gamma was elevated in CD3(+) PALN cells after exposure to semen, as assessed by intracellular cytokine fluorescence-activated cell sorting, immunohistochemistry and quantitative reverse transcriptase polymerase chain reaction. Matings with vasectomized males indicated that the lymphocyte activation occurs independently of sperm. However, in contrast, males from which seminal vesicle glands were surgically removed failed to stimulate PALN cell proliferation or cytokine synthesis. Adoptive transfer experiments using radiolabelled lymphocytes from mated mice showed that lymphocytes activated at insemination home to embryo implantation sites in the uterus as well as other mucosal tissues and lymph nodes. These findings indicate that activation and expansion of female lymphocyte populations occurs after mating, and is triggered by constituents of seminal plasma derived from the seminal vesicle glands. Moreover, lymphocytes activated at insemination may help mediate maternal tolerance of the conceptus in the implantation site.

Figure 1

Insemination increases cell numbers in PALN. (a) Female B6 mice were killed before mating or 4 days after mating with BALB/c (allogeneic) or B6 (syngeneic) males and the numbers of cells retrieved from PALN were determined. Data are mean (± SEM) number of PALN cells. The number of mice in each group is given in parentheses. Data were compared by independent samples t-test. ^a,bDifferent superscripts represent statistical significance between groups. (b) The proportion of CD3⁺ and B220⁺ lymphocytes in PALN of virgin and mated B6 mice characterized by flow cytometry. Quantitative data are given in Table 2. The data shown are representative of 10 independent experiments.

Figure 2

PALN T lymphocytes are activated following insemination. CD69 expression was assessed by flow cytometry in (a,b) CD3⁺ lymphocytes, (c,d) B220⁺ lymphocytes and (e,f) NK1.1⁺ cells from PALN of virgin mice (left hand panel; a,c,e) and day 4 mated mice (right hand panel; b,d,f). The data shown are representative of at least 10 independent experiments.

Figure 3

Cytokine synthesis assessed by intracellular FACS is increased in PALN T lymphocytes after insemination. (a) PALN cells from virgin mice (left hand panel) and day 4 mated mice (right hand panel) were stimulated for 6 hr with PMA and calcium ionophore in the presence of monensin, then stained for intracellular IL-4, IL-5, or IFN-γ. The data shown are representative of at least 10 independent experiments. (b) The percentage of cytokine-producing cells in CD3⁺ lymphocytes from PALN of day 4 mated mice was determined using the level of production in stimulated cells from virgin mice to set the baseline level. Data are mean ± SEM number of IL-4-, IL-5- and IFN-γ-positive cells. PALN from n = 25 virgin and n = 24 day 4 mated mice were assessed. Data were compared by independent samples t-test. *P = 0·04; **P = 0·003.

Figure 4

Cytokine synthesis assessed by immunohistochemistry is increased in PALN lymphocytes after insemination. Frozen sections of PALN tissue taken from virgin or day 4 mated mice were labelled with rat anti-IFN-γ or anti-IL-4 antibodies followed by biotin-conjugated rabbit anti-rat IgG. (a) IFN-γ-producing cells were sparsely scattered within the PALN of virgin mice, and (b) were considerably increased in abundance following mating. (c) IL-5-producing cells were rare in the PALN of virgin mice, and (d) were increased following mating. Staining was absent in tissues from both virgin (e) and day 4 mated mice (f) probed with an isotype-matched control antibody. Scale bar = 100 μm. (g) Stained cells were quantified by manual counting and are expressed as the mean ± SEM number of positive cells per section in virgin and day 4 mated PALN sections. One to three PALN sections from n = 10 virgin and n = 8 day 4 mated mice were examined. *P = 0·02.

Figure 5

Cytokine synthesis assessed by quantitative real-time RT-PCR is increased in PALN lymphocytes after insemination. cDNA was prepared from whole PALN from virgin or day 4 mated mice and mRNAs for (a) IL-2; (b) IL-4; (c) IL-5 and (d) IFN-γ were quantified using primers sets and reaction conditions detailed in Table 1. Data are mean ± SEM relative mRNA expression in arbitrary units, where data are normalized to β-actin mRNA expression and expressed as per cent of the mean of data from virgin PALN tissues. PALN tissue from n = 6 virgin and n = 6 day 4 mated mice was evaluated. *P = 0·03.

Figure 6

Seminal fluid is necessary for lymphocyte activation in PALN following mating. The effect of sperm and seminal vesicle fluid on the immune response elicited in the PALN following mating was evaluated using surgically altered male mice. Females were mated with intact BALB/c males or with BALB/c males rendered sperm deficient by vasectomy (Vas) or seminal vesicle deficient (SV^–) by surgical removal of the seminal vesicles. (a) The mean ± SEM number of cells retrieved from PALN. (b) The percentage of cytokine-producing cells in CD3⁺ lymphocytes from PALN of day 4 mated mice was determined by flow cytometry using the level of production in stimulated cells from virgin mice to set the baseline level. Data are mean ± SEM number of IL-4- and IFN-γ-positive cells. Data were compared by independent samples t-test. The number of mice per group are shown in parentheses. *P < 0·02.

Figure 7

Activated PALN cells can home to uterine tissue. PALN lymphocytes from day 4 mated donors were labelled with ¹²⁵IdUR and adoptively transferred to day 6 pregnant recipients. ¹²⁵IdUR associated with each tissue was determined by gamma-counting. The relative density of donor cells recruited into each tissue is given as mean ± SEM relative tissue content (as defined in the Materials and methods) from n = 17 recipient mice. LN = lymph node; IP = implantation site.