Multiparous Mice Research Articles

Skeletal changes in multiparous mice fed a nutrient-sufficient diet containing cadmium

Female mice were given nutrient-sufficient, purified diets containing either 0.25, 5, or 50 ppm Cd. One-half of the females were bred for 6 consecutive 42-day rounds of pregnancy/lactation (PL mice); remaining females were non-pregnant controls (NP mice). PL mice and NP controls were sacrificed after 1, 2, 4, or 6 consecutive rounds of pregnancy/lactation. No consistent, cadmium-dependent decreases in body weight, femur calcium content, or calcium/dry weight (Ca/DW) ratio occured among the NP mice during the 252 days of cadmium exposure. In contrast, significant, cadmium-dependent decreases in body weight (3–11%), femur calcium content (15–27%), and Ca/DW ratio (5–7%) occurred in the multiparous mice exposed to 50 vs 0.25 ppm Cd. In addition, among the PL mice, the effect of cadmium was dose-dependent, with femur calcium contents decreasing significantly as the cadmium exposure level increased from 0.25 to 5 then 50 ppm Cd P < 0.05. Results demonstrate that dietary cadmium exposure had a greater effect on the skeletons of dams exposed to cadmium during the stresses of pregnancy and lactation than in non-pregnant controls. The results provide evidence that the combination of cadmium exposure and multiparity may have played a role in the etiology of Itai-Itai disease in Japan.

Alterations in Immunological Reactivity during Pregnancy in Mice Determined in vitro by Lymphoproliferation Tests

Primiparous and multiparous mice, either syngeneically or allogeneically pregnant, were sacrificed at various stages of pregnancy, and the immune reactivity of cells from different lymphatic organs was analyzed by mixed lymphocyte reaction (MLR) and concanavalin A (Con A)-induced lymphocyte proliferation. In the MLR, spleen cells and cells from the axillary lymph nodes of pregnant animals showed similar changes in their response to allogeneic cells during the course of gestation. In comparison to age-matched virgin controls they had an increased reactivity between the 7th and 11 th day of pregnancy. During the preimplantation period and the last week of pregnancy, their alloreactivity was comparable to that of the controls. Cells from the para-aortic lymph nodes, which drain the uterus, also showed an increased reactivity at mid-gestation, but in the preimplantation period and third week of pregnancy their alloreactivity was even decreased in comparison to the control animals. When T cell immunocompetence was measured by means of lymphocyte transformation induced with Con A, the pattern of reactivity was completely different. Lymphoid cells from all the above-mentioned tissues showed the highest response to Con A during the preimplantation period, constantly weakening towards term.

In vitro replication potential of serially passaged mammary parenchyma from mice with different reproductive histories

Growth properties of multicellular units (organoids) of mouse mammary parenchyma have been analyzed. These intact units grew differently in collagen-matrix cultures than did dispersed cells prepared from them. The latter actively migrated in the collagen matrix and reorganized themselves into multicellular structures before producing three-dimensional protuberances in gel. Terminal unit (end-bud/alveoli)-enriched fractions grew more extensively than did ducts, as predicted from growth patterns in vivo. To assess the growth potential and the relationships between replication history in vivo and replication potential in vitro in mammary parenchyma, intact terminal units from mammary glands of mice of different ages and with different reproductive histories were isolated and their growth characteristics compared. Terminal-unit organoids were cultured in collagen gel matrix and passaged weekly for up to 5 weeks. Morphology, growth rates, and growth fractions were compared among organoids from young virgin, old virgin, monoparous, and multiparous mice. Morphologies observed in various passages of organoids from the groups of mice were similar. Organoids from old virgin and multiparous mice declined in growth rate for four passages and then growth rate increased again during the fifth passage. (However, fifth-passage organoids failed to form tumors if implanted in syngeneic mice in vivo.) Growth of organoids from either old or young virgin mice was less at any given passage than tissue from multiparous mice of similar age. Growth fractions of organoids from old parous mice were the same as those from old virgin mice but reached the same maximum fraction faster. Later passage organoids from the different mouse groups responded morphologically to the hormone combination of estrogen, progesterone, prolactin, and cortisone but did not respond to cholera toxin. These results suggest that an animal's hormonal history (altered profoundly by pregnancy and lactation) may be as important as chronological age in determining subsequent growth potentials of mammary epithelium.

Allogeneic matings and immunization have different effects on nulliparous and multiparous mice.

CBA/J female mice have a high rate of spontaneous fetal loss when mated with DBA/2 males. We have confirmed that this fetal resorption rate can be significantly reduced by immunizing the female with C57BL rather than DBA leukocytes. The current studies have been extended to show the effect of continued immunization into second pregnancies. Three new findings emerge: second pregnancies in unimmunized CBA/J females X DBA/2 males proceed with a low percentage of spontaneous resorptions; continued immunization of multigravid, multiparous mice is associated with a high percentage of late onset fetal resorptions; and comparison of sex ratios between treated and untreated pregnancies showed no significant shift. These results suggest that untreated CBA/J females mated to DBA/2 males can be immunized to trophoblast antigens during first pregnancies, and that such immunization leads to "normal" reproductive outcome in subsequent untreated pregnancies. Persistent immunization with leukocytes appears to increase the percentage of fetal wastage in subsequent pregnancies regardless of whether the mother receives paternal or nonpaternal leukocytes. Intentional chronic immunization with male leukocytes does not influence the sex ratio of viable offspring. These data are discussed with reference to immunotherapy for women who suffer unexplained, recurrent spontaneous abortions.

&lt;I&gt;Pasteurella pneumotropica&lt;/I&gt;の選択培地

Bacteria from the subcutaneous abscesses which appeared in a laboratory colony of DS mouse since October of 1977 were identified as Pasteurella pneumotropica by various biological examinations. The abscess formation was limited to multiparous female mice over 100 day-age, but virgin females were free from the disease. The MIC of various antibacterial substances showed that potassium tellurite, kanamycin and bacitracin were effective to isolate the organism selectively from various infection sites harboring many other species of bacteria. A novel NKBT medium was prepared by adding these antibacterial substances to the heart infusion agar (HIA) supplemented by 10% Fildes digested blood. A fluid culture medium, TGN broth was prepared for multiplication of the organism by adding 10% Fildes digested blood and potassium tellurite to GN broth. To isolate the organism from the pharyngo-larynx a direct application of mucus wiped off the infection site onto the culture medium was sufficient, but pre-multiplication in the TGN broth was required for isolation of the organism from gut contents before inoculation onto the NKBT medium. The pre-cultivation in the TGN broth vastly improved the recovery of the organism especially from feces. Thereby we could easily detect the latent infection of this bacterium without sacrificing animals.

Inhibition by indomethacin of spontaneous mammary tumorigenesis in SHN mice.

This experiment was designed to examine the effects of indomethacin, a potent prostaglandin synthesis inhibitor, on spontaneous mammary tumors in mice. The growth of established mammary tumors and the appearance of new tumors in multiparous SHN mice were significantly suppressed by the subcutaneous implantation of pellets of indomethacin diluted to 1/12 with cholesterol. Furthermore, the same treatment inhibited normal and preneoplastic mammary gland growth in virgin SHN mice. The pattern of estrous cycles, ovarian structure, and plasma prolactin levels were not affected significantly by the treatment. All results have demonstrated that indomethacin inhibits mammary tumorigenesis of mice primarily by route(s) other than the endocrine system under the present experimental conditions. Indomethacin would be the first agent that appears to inhibit the growth of spontaneous mammary tumors of palpable size in mice.

The effects of suppressive activities mediated by cells on the protection of fetus against maternal immune rejection.

Using both mixed lymphocyte reaction (MLR) and graft-versus-host reaction (GVHR) assays, investigations have been made on 60 multiparous mice of late pregnancy, derived from C57BL mating with SWI as well as C57BL mating with BGI. The results demonstrate the presence of suppressor cells both in uterus-draining-lymphnodes (DLN) and in spleen of allogeneic pregnant mice. The suppressive immune regulatory activity mediated by these cells might play an important role in the successful maintenance of fetus allograft. The possible mechanisms about the inducing of the changes in lymphocyte subpopulation are also studied in this paper.

Secretory immunoglobulin binding to bacteria in the mouse uterus after mating

Bacteria of several species were present in the mouse uterus on the morning after mating, as demonstrated by bacteriol cultures and microscopic examination of Gram-stained uterine luminal contents. The similarity between bacteria cultured from the vagina before mating and from the uterus after mating suggested that bacteria were introduced into the uterus from the vagina, possibly by coitus. The bacteria were cleared from the uterus about two days after mating. Immunohistochemical labeling of smears of the luminal contents on the morning after mating demonstrated IgA, IgG, and possibly IgM bound to many of the bacteria. The bacteria were often agglutinated, and there was a correlation between the intensity of immunoglobulin labeling on bacteria and the extent of agglutination. The amount of antibody bound to bacteria in multiparous mice was about the same as in mice that had not been mated previously. We observed both IgG and IgA on bacteria when organisms from vaginal cultures were incubated for 60 min in the uteri of estrogen-primed, virgin, female mice. This indicated that the uterus was the source of at least part of the immunoglobulins bound to bacteria. We did not demonstrate that the immunoglobulins bound to bacteria were specific anti-bacterial antibodies, but the binding persisted through three washing steps and there wasno immunoglobulin binding to sperm in the same preparations. Neutrophils in the uterine lumen on the day after mating contained phagocytosed bacteria. These results suggest that the secretory immune system in the female mouse reproductive tract may play a role in returning the uterus to an aseptic state after mating by at least three mechanisms: direct blocking of attachment sites involved in bacterial binding to mucosal epithelium, agglutination of bacteria and thus reduction in the number of organisms available for binding to the epithelium, and opsonization of bacteria for phagocytosis by neutrophils.

A monoclonal antibody, EC-1, derived from a syngeneically multiparous mouse alters in vitro fertilization and development

A monoclonal antibody designated ‘EC-1’ was derived from a fusion of myeloma cells with lymphoid tissue from a syngeneically multiparous, but otherwise unimmunized, mouse and was selected by screening for reactivity with teratocarcinoma cells. The IgM antibody binds to the cell surface of ova, zygotes, and 2-cell embryos. Binding is not detected on the 4- or 8-cell embryo but reappears on the morula and blastocyst. EC-1 binds to the trophoblast but not to the inner cell mass of in vitro attached blastocysts and the ectoplacental cone of the peri-implantation embryo. In adult tissues, EC-1 binds to the follicular cells of the ovary, the lining epithelium of the pregnant uterus, the interstitial region of the testes and to epidydimal but not testicular sperm. In nongonadal tissues EC-1 binds to an epitope located in some, but not all, regions of connectives tissues associated with basement membrane. The antigen detected by EC-1, as expressed on teratocarcinoma-derived cell line PYS-2, is a large glycoprotein which is sensitive to reduction. EC-1 inhibits in vitro fertilization and partially inhibits in vitro development of in vitro fertilized ova. The possible implications of EC-1 binding and activity are discussed.

Inhibition of mammary tumorigenesis in nulliparous C3H mice by chronic feeding of the synthetic retinoid, N-(4-hydroxyphenyl)-retinamide.

Feeding of N-(hydroxyphenyl)retinamide (4-HPR) (1.0 mM/kg diet) for 39 weeks to 2 month old nulliparous C3H/Crgl mice significantly (p less than 0.01) reduced the incidence of mammary tumors; 98 control mice developed a total of 235 mammary tumors, 100 4-HPR treated mice developed 171 mammary tumors. Feeding the same dietary level of 4-HPR to 4-6 month old multiparous C3H mice for 14 weeks did not significantly influence the development of mammary tumors; 78 control mice developed a total of 115 mammary tumors; 78 4-HPR treated mice developed 130 mammary tumors. Body weight gains were comparable among 4-HPR treated mice and control mice. Significant suppression of mouse mammary gland tumorigenesis in vivo by dietary retinoids (nulliparous C3H mice) has heretofore not been reported.

Antibodies to antigens on teratocarcinoma cells are associated with parity in mice.

Previous studies have demonstrated that the maternal host responds immunologically to antigens of the fetus, including oncofetal antigens (OFA). However, until now, the OFA that have been studied are those associated with the postimplantation embryo. In this paper we provide evidence that the maternal host also responds immunologically to antigens associated with the mouse teratocarcinoma (TC) cell lines F9 and PYS-2, which have been established as models for the preimplantation embryo. In a solid-phase radioimmunoassay using cell membrane extracts as antigen and 125I-protein A to detect binding antibodies, sera from parous C57BL/6 mice had a significantly greater reactivity than sera from age-matched virgin animals to F9 but not PYS-2, splenocyte or fibroblast antigen extracts. This finding was confirmed by surface immunofluorescence (IF) with fluorescein-labeled anti-mouse IgG. IF also detected a small number of multiparous mice with antibodies to PSY-2. Positive serum reactivity did not correlate directly with the number of previous litters. These TC cell lines express at least 2 OFA, F9 and Endo, which are found on preimplantation embryos and sperm, and preimmunization of female mice with F9 cells has been shown to reduce fertility. The parity-induced antibodies detected in this study could react with embryos at early stages of development and contribute to the natural decrease in litter size which occurs in mice after the second litter.

Enhancement by Retinyl Acetate of Hormone-Induced Mammary Tumorigenesis in Female GR/A Mice&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="FN3"&gt;3&lt;/xref&gt;

Feeding of retinyl acetate (82 mg/kg ration) for 13-16 weeks to estrone- and progesterone-treated nulliparous and multiparous inbred GR/A mice resulted in a substantial increase in the incidence of mammary carcinomas. Mammary carcinoma incidence in nulliparous control and retinoid-fed mice in experiment #1 was 22/65 (34%) and 37/65 (57%) (P less than 0.05), respectively; in experiment #2, 27/48 (56%) and 37/48 (77%) (P less than 0.05), respectively. Mammary carcinoma incidence in multiparous control and retinoid-fed mice in experiment #1 was 13/30 (43%) and 23/30 (77%) (P less than 0.05), respectively; in experiment #2, 19/19 (100%) and 19/19 (100%), respectively. The purported chemopreventive activities of retinyl acetate in murine mammary tumorigenesis were not demonstrated in this study; indeed, the vitamin A analog appeared to enhance this oncogenic process in the steroid hormone-treated GR mouse mammary cancer model.

Regulatory T cells in pregnancy. IV. Genetic characteristics and mode of action of early MLR suppressive T cell subpopulations.

The genetic characteristics, surface markers, and mechanisms of action of early-acting, mitomycin-resistant, MLR suppressor T cells from multiparous mouse spleens were studied in MLR of various strain combinations. The cell activity appears to be genetically restricted at the suppressor/reactive cell level, and the major restriction occurs via the IC subregion. However, C3H.OL recombinant pointed toward a possible minor requirement for S-G subregion syngenicity. The suppressor T cell was characterized as an Ia+, Ly1-, Ly2+ subset. The use of Karush double chambers as well as of supernatants of regulated MLR provided evidence that the suppressor cells act via soluble product(s).

Selenium and the genesis of murine mammary tumors.

One hundred forty-seven female Sprague-Dawley rats were divided into 5 groups and at 60 days of age were treated i.g. with 5 mg of 7,12-dimethylbenzanthracene (DMBA) suspended in 1.0 ml of sesame oil. Selenium (Se), as selenium dioxide (SeO2), was administered in the drinking water to 4 of the 5 groups (30 rats/group) at 2 doses (2 and 4 mg/l) from 30-90 days of age (series 1) and from 90-150 days of age (series 2) prior to the onset of palpable mammary tumors. One group of rats (27 rats) served as controls. All rats were palpated weekly for mammary tumors and sacrificed 28 weeks after DMBA treatment. Total number of palpable mammary carcinomas which developed in each group were: controls, 60; series 1, 2 mg Se dose, 27, 4 mg Se dose, 29; series 2, 2 mg Se dose, 24, 4 mg Se dose, 32. Each dose level of Se in each series significantly (P less than 0.05) reduced the incidence of mammary carcinomas. These results provided evidence that Se can inhibit the early promoting phases of polycyclic hydrocarbon induced mammary carcinogenesis in female rats. Two hundred twenty-six nulliparous and 99 multiparous GR mice were treated daily with estrogen and progesterone for 13-16 weeks. Se (SeO2) was administered in the drinking water (2 mg/l) to one-half of these mice. Total number of mammary carcinomas in control nulliparous and multiparous mice were 119 and 90, respectively; in Se treated nulliparous and multiparous mice, 113 and 81, respectively. Se did not significantly effect mammary carcinoma incidence in hormone treated nulliparous and multiparous GR mice.

Immunocytochemical Distribution of Mouse Mammary Tumor Virus Antigens in BALB/cfC3H Mammary Epithelium2

The distribution of mouse mammary tumor virus (MuMTV) antigens was studied in normal, preneoplastic, and neoplastic mammary epithelia from female BALB/cfC3H mice with the use of a polyvalent anti-MuMTV serum and indirect immunoperoxidase techniques. The MuMTV antigens were on the apical surface or in focal cytoplasmic aggregates or diffused throughout the infected cells. As much as 70% of all cells in adenocarcinomas and as much as 100% of all cells in preneoplastic hyperplastic alveolar nodules contained MMTV antigens. Comparable percentages of cells from mammary glands of multiparous mice were MuMTV-positive. Some mammary tissues of nullparous and primiparous mice did not contain detectable MuMTV antigen. The MuMTV antigen-containing cells in lactating mammary glands tended to be in discrete lobuloalveolar clusters surrounded by antigen-negative alveoli. The percentage of MuMTV-positive cells in a given gland was proportional to the amount of virus found in the animal's milk.

Inhibition of mammary tumorigenesis in GR mice with 2‐bromo‐α‐ergocryptine

A high incidence of mammary tumors is found in multiparous GR mice during the 2nd and 3rd pregnancies and in nulliparous GR mice treated with estrone/progesterone. The purpose of this study was to determine if prolactin is a contributing hormone in the genesis of these neoplasms. In one series of experiments, 238 15-week-old nulliparous GR mice were treated with estrone (drinking water, 0.5 mg/liter) plus progesterone (30 mg progesterone pellet with cholesterol, implanted SC once monthly) for a period of 13 weeks. Half of these mice were injected SC once daily with 100 micrograms of the prolactin-suppressing drug 2-bromo-alpha-ergocryptine (CB-154) for the duration of hormone treatment, and the other half were injected SC once daily with 0.9% NaCl solution (controls). In another series of experiments, 87 pregnant GR mice were divided into two groups and injected SC once daily from day 7 to 21 of pregnancy with 0.9% NaCl solution (controls) or CB-154 (100 micrograms/mouse). In the first series, the numbers of mice with mammary tumors and total number of mammary tumors were: controls, 58/119 (49%) and 73; CB-154 treatment, 34/119 (29%) and 37, respectively. In the second series, the numbers were: controls, 39/44 (89%) and 73; CB-154 treatment, 24/43 (56%) and 43, respectively. In both studies, CB-154 treatment significantly (p less than 0.05-0.005) reduced the percentage of mice with mammary tumors and total number of mammary tumors. These results provide evidence that prolactin is a contributing hormone in the genesis of estrone/progesterone and pregnancy-induced mammary tumors in female GR mice.

Immunological resistance to growth of tumours in syngeneic multiparous mice

The hypothesis that BALB/c mice which have experienced a pregnancy are rendered immune to a 3-methylcholanthrene-induced syngeneic tumour was tested by direct tumour cell challenge of multiparous mice and by carrying out cell transfer assays using purified T cells obtained from the spleens of multiparous mice. Control mice were virgin BALB/c mice of the same age. The results showed that mice which have experienced a single multiparous pregnancy are resistant to the growth of the tumour MC 677. In addition, it was shown that purified splenic T cells form multiparous mice inhibited the growth of tumour following implantation of a mixture of tumour cells and splenic T cells to normal recipient mice. Age-matched virgin controls did not show resistance to tumour growth following challenge nor were their lymphoid cells inhibitory to tumour cells. These results demonstrate clearly that pregnancy in mice confers a degree of transplantation resistance against the syngeneic tumour, MC 677 and suggest that certain membrane-expressed oncofoetal antigens are operative in resistance against tumours. They also provide support for a possible immunological link between the observed decreased risk of breast cancer and early pregnancy in humans.

Lower skin tumor incidence in carcinogen-treated multiparous mice

Summary The skin of 12-month-old multiparous mice was treated with beta rays and 4-nitroquinoline-1-oxide. The incidence of malignant tumors (1.3%) was much lower than that observed in identically treated young virgin mice (12.6%).

Analysis of Murine Oncofetal Antigens as Tumor-Associated Transplantation Antigens

In previous studies with in vitro activated cytotoxic T lymphocytes, we have demonstrated the presence of oncofetal antigens (OFA) on a range of murine tumor cells. The present studies with the same tumor lines attempt to determine whether these antigens are also capable of activating lymphocyte responses in vivo. Several experimental designs were followed, each being performed many times (1). Preimmunization of mice with irradiated fetal liver cells and followed by challenge with viable tumor cells did not consistently produce a state of tumor resistance. However, injection of live fetal cells frequently led to enhanced tumor growth (2). The growth of tumors in multiparous mice can be inhibited, in contrast to controls, but this effect was relatively short lived after parturition (3). Preimmunization with fetal cells in vivo did not result in augmented secondary cytotoxic T cell responses in vitro (4). Immunization of mice with irradiated tumor cells frequently led to a state of resistance to the clonal growth of hemopoietic fetal cells, although again the level of resistance was usually relatively weak. From the overall results, we conclude that OFA are relatively poor immunogens on tumor cells or fetal cells in vivo, and in contrast to in vitro responses, do not act as potent tumor transplantation antigens.

Demonstration of tumor-associated immunity with a leukocyte adherence inhibition (LAI) assay.

A modification of the leukocyte adherence inhibition (LAI) assay of Halliday was used to search for immune reactions against tumor-associated antigens of mouse tumors as well as against embryonic antigens in such neoplasms. Soluble antigen extracts were prepared from transplanted (BALB/c) methylcholanthrene-induced sarcomas and carcinomas, from normal BALB/c embryos taken at 14-18 days' gestation and from kidneys and livers of adult BALB/c mice. Peritoneal cells (PC) from mice immunized against syngeneic tumors gave leukocyte adherence inhibition more commonly when exposed to antigens prepared from the same tumor than did PC from normal (untreated) mice or from mice immunized against a different tumor. However, different tumor antigen preparations varied vastly in their ability to give specific adherence inhibition; some preparations consistently gave a high tumor-specific inhibition, while others did not. This may explain why the degree of reactivity observed was low and its tumor specificity not absolute when data obtained with all different antigen extracts were pooled. PC from multiparous mice gave adherence inhibition when exposed to antigenic extracts from syngenic mouse embryos or from tumors, as compared to PC from virgin mice exposed to the same extracts. Furthermore, PC from tumor-immunized mice reacted more commonly against antigen extracts from mouse embryos than did peritoneal cells from normal untreated mice. Adherence inhibition was not observed when PC from tumor-immunized or multiparous mice were exposed to antigen extracts from adult syngeneic livers or kidneys.