Fibronectin and cell shape in vivo: studies on the endometrium during pregnancy.

The rat endometrium during pregnancy was used as a model system to study fibronectin in vivo. Fibronectin distribution on stromal fibroblasts, as determined by indirect immunofluorescence staining, was studied in relationship to cell shape during decidual transformation. Fibroblasts of the estrus endometrial stroma were elongated cells with a fibrillar pattern of fibronectin on their surfaces. During days 1-6 of pregnancy, as these elongated cells acquired a round morphology, fibronectin changed first to a patched distribution on the cells'a surfaces and then disappeared. The change in fibronectin was specific for the fibroblasts since over the same time period there was no decrease in fibronectin found associated with blood vessels or in the epithelial-stromal basement membrane. These results support the proposed relationship between cell surface fibronectin and cell shape that has been inferred from in vitro experiments. After implantation, fibronectin distribution was studied in relationship to the position of the conceptus. In the stroma proximal to the implanting conceptus, fibronectin was absent except around blood vessels, which may help explain how decidual tissue could act as a barrier to trophoblast invasion. Finally, fibronectin distribution was studied in the uterus after parturition. Debris in the uterine lumen was coated with fibronectin, which may be important in the rapid removal of this material by phagocytic cells. Also, fibronectin associated with the epithelial-stromal basement membrane was reorganized after reepithelialization had occurred.

Regulation of expression of male-specific rat liver microsomal 3 beta-hydroxysteroid dehydrogenase.

In the steroidogenic pathways present in the gonads and adrenal cortex, 3 beta-hydroxysteroid dehydrogenase isomerase (3 beta HSD) is a key enzyme which controls the formation of delta 4-3-ketosteroids from delta 5-3 beta-hydroxysteroids. Herein, we used an antibody against human placental 3 beta HSD and a rat testicular 3 beta HSD cDNA probe to study the expression of rat liver 3 beta HSD mRNA and protein. Rat liver microsomal 3 beta HSD activity has been previously reported to exhibit a significant sex difference, with much higher activity in the male. We have shown an age-dependent increase in levels of immunoreactive 3 beta HSD through the time of maturation of the male rat. The immunoreactive protein, of similar molecular size to the human placental and rat testicular 3 beta HSD, was localized to the microsomal fraction of liver and was concentrated in pericentral locations. Immunoreactive protein was not detected in liver of immature (before 25 days of age) rats of either sex or in adult female liver. Northern blot analysis of liver and testicular RNA with a rat testicular 3 beta HSD cDNA probe revealed the presence of a 1.6-kilobase mRNA species in addition to the major 2.1-kilobase mRNA species in adult male liver, neither of which was detected in immature or adult female liver RNA. Hypophysectomy of female rats or treatment with testosterone implants caused induction of liver 3 beta HSD protein, while continuous infusion of GH to male rats decreased the level of 3 beta HSD protein. Similarly, the levels of the mRNA species were decreased after GH treatment. Using [3 alpha-3H]dehydroepiandrosterone as substrate for 3 beta HSD activity, we determined the apparent Km for liver microsomal NAD(+)-dependent 3 beta HSD activity to be 20 microM in both adult male and female liver and was much greater than the Km of rat Leydig tumor 3 beta HSD activity (0.2 microM). Liver 3 beta HSD activity was inhibited by trilostane, a proven inhibitor of gonadal and adrenal 3 beta HSD activity. A rat liver 3 beta HSD cDNA was isolated from a male liver cDNA library that was closely related to the type II 3 beta HSD form of rat ovary but different from type III liver 3 beta HSD. The enzyme obtained upon expression of this cDNA had properties characteristic of male-specific NAD(+)-dependent liver microsomal 3 beta HSD (i.e. high apparent Km for dehydroepiandrosterone) and distinct from those of the high affinity gonadal type I 3 beta HSD.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of steroidogenic enzymes in the bovine placenta and fetal adrenal glands throughout gestation.

The expression of cholesterol side-chain cleavage cytochrome P450 (P450scc), 17 alpha-hydroxylase cytochrome P450 (P45017 alpha), and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) was studied in bovine placenta and fetal adrenal glands throughout gestation. The levels of expression of these enzymes were much lower in the placenta than in the adrenals by Western and Northern analyses. The levels of P450scc, however, remained relatively constant in bovine placenta and fetal adrenal glands at all gestational stages studied. In contrast, P45017 alpha expression was higher in both the placenta and the fetal adrenal glands during the early stages of pregnancy, but declined markedly in both tissues through the period of midgestation. The expression of P45017 alpha increased markedly in the fetal adrenal glands in late gestation. The levels of 3 beta HSD were extremely low in placental tissues, but were higher in the fetal adrenals, where they were found to be slightly elevated in early and late gestation compared to those in midgestational stages. Immunocytochemical examination of the levels of P45017 alpha and 3 beta HSD in the fetal adrenal glands correlated with the results of Western and Northern analyses. In addition, the morphology and distribution of these two enzymes in the developing bovine fetal adrenal glands indicated that while the early activated gland is functional relative to the ability to secrete steroids, structural and functional organization more typical of mature adrenal glands is not achieved until the time of activation of the fetal adrenals in late gestation.

Uterine natural killer cells are targets for a trophoblast cell-specific cytokine, prolactin-like protein A.

PRL-like protein A (PLP-A) is a member of the PRL family expressed in trophoblast cells coincident with establishment of the chorioallantoic placenta. The purpose of this investigation was to identify targets for PLP-A. Using an alkaline phosphatase-tagging strategy, we show that PLP-A specifically interacts with a population of natural killer (NK) lymphocytes within the mesometrial compartment of decidua from pregnant and pseudopregnant rats. These observations are supported by the codistribution of PLP-A targets with cells expressing the rat NK cell surface marker, gp42, the absence of PLP-A binding in conceptuses from NK cell-deficient tg epsilon26 mice, and the specific interaction of PLP-A with a rat NK cell line, RNK-16. We have further demonstrated that PLP-A effectively suppresses RNK-16 cell cytolytic activities. Our results provide evidence for a new paradigm of embryonic-maternal communication involving a PLP-A signaling pathway between trophoblast cells and uterine NK lymphocytes.

Cell type-specific expression of 17 beta-hydroxysteroid dehydrogenase type 2 in human placenta and fetal liver.

The enzymatic actions of the 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) isozymes are crucial in steroid hormone metabolism/physiology. The type 1 isozyme catalyzes the conversion of the biologically inactive C18 steroid, estrone, to the active estrogen, 17 beta-estradiol, and the enzyme is predominantly expressed in the syncytiotrophoblast of the placenta and the granulosa cells of the ovary. 17 beta HSD type 2 is highly expressed in placenta, liver, and secretory endometrium and catalyzes the conversion of bioactive estrogens and androgens to biologically inactive 17-ketosteroid counterparts. The expression pattern of 17 beta HSD type 2 protein was determined in human term placenta and fetal liver by immunohistochemical analysis using monoclonal antibodies directed against distinct epitopes of the 17 beta HSD type 2 protein. In placenta, the protein was detected in the endothelial cells of fetal capillaries, but not in cytotrophoblasts or syncytiotrophoblast. There was dichotomous immunostaining seen among pairs of cotyledonary vessels and chorionic vessels. In the liver, on the other hand, staining was detected in the hepatocytes, but not in the cells lining blood vessels. We conclude that the cell type-specific localization of 17 beta HSD type 2 is in accord with the proposed physiological role of the enzyme, namely to protect tissues, in this case the fetus, from bioactive estrogen and androgen.

Cellular localization of membrane metalloendopeptidase (enkephalinase) in human endometrium during the ovarian cycle.

Previously, we found that membrane metalloendopeptidase (MMEP; enkephalinase) is present in human endometrium where it may inactivate certain bioactive peptides such as endothelin. The specific activity of MMEP in endometrium is correlated positively with the level of plasma progesterone. In the present study, we determined the cellular distribution of MMEP immunoreactivity (IR) in samples of endometrium from 31 ovulatory women at different phases of the menstrual cycle and from 5 postmenopausal women. At all times, MMEP-IR was localized in the endometrial stromal cells and was absent from myometrium, epithelia, and vessels. The presence of the enzyme on the stromal cell plasma membrane was demonstrated by immunostaining of freshly isolated endometrial stromal cells. During the endometrial cycle, there were dramatic changes in MMEP-IR in the zona functionalis: In the proliferative phase when plasma progesterone levels were low, MMEP-IR was weak; but after ovulation, during the early and midsecretory phases, when progesterone levels were increasing or high, MMEP-IR was strong. During the late secretory phase, in the stromal areas that were decidualized, contiguous with spiral arterioles and beneath the surface epithelium, most of the MMEP-IR was lost, whereas MMEP in the other stromal areas remained strong. In the premenstrual phase, there was a more generalized decline in MMEP-IR in the stromal cells. MMEP-IR in the zona basalis was weak to moderate and was relatively unchanged during the ovarian cycle; the findings in postmenopausal tissues were similar to those of proliferative phase tissues. These results are consistent with the previous findings of increased MMEP mRNA, protein, and activity in endometrial tissue of midsecretory phase and in cultured endometrial stromal cells in response to progestin. Furthermore, in this study, we demonstrate that in the late secretory phase in decidualizing stroma, MMEP-IR is decreased considerably compared with midsecretory stromal cells. These changes in MMEP-IR are consistent with the possibility that endothelin, also produced in endometrium, is spared inactivation in the premenstrual phase and may act on the spiral arterioles. These findings are supportive of a role for MMEP in paracrine interactions affecting vascular homeostasis in the endometrium.

Alterations in cerebellar germinal cell division induced by graft-versus-host disease.

A systemic immunological syndrome, graft-versus-host disease (GVHD), which does not cause inflammation or cell death in the cerebellum, is shown to retard granule cell production by decreasing the rate of DNA synthesis (S phase) and prolonging mitosis (M), at metaphase. The rate of cell production in diseased animals at postnatal day 14, quantitated by analysis of the rate of labeling of DNA with 3H-thymidine (3H-Tdr), revealed decreased ability to synthesize new DNA. The number of cells taking up 3H-Tdr label per mm2, as detected by autoradiography, was similar in 14-day-old GVHD and control tissue as was the area of the germinal matrix zone and the number of mitotically active germinal cells per mm2 in sagittal sections near the midline. However, because the total volume of the cerebellum was less, the total number of mitotically active cells in the whole cerebellum of 11-, 14-, and 17-day-old diseased animals was less than in littermate controls. Furthermore, DNA synthesis per mitotically active germinal cell was less in diseased animals at each age examined. The mitotic index was unaffected until late in the disease (day 17), suggesting that a prolongation of the cell cycle was responsible for this GVHD-induced decrease in DNA synthesis. Consistent with a prolongation of the cell cycle was the finding that the mitotic figures in 14-day-old GVHD cerebella were mostly metaphase figures, whereas those in control cerebella were, as predicted, mostly prophase. Prolongation of the cerebellar cell cycle in 11- and 14-day-old diseased animals may explain the dramatic decrease in the mitotic index, the thickness of the germinal matrix zone, and the number of germinal cells at postnatal day 17.

Characterization of cDNAs encoding isoforms of hamster 3 beta-hydroxysteroid dehydrogenase/delta 5-->4 isomerase.

Western blot analyses of various hamster tissues reveal high levels of expression of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in adrenal and liver, and moderate levels of expression in kidney. The expression in liver is sexually dimorphic; very high levels of protein are observed in adult male liver but very low levels are seen in the female liver. Three distinct cDNAs encoding isoforms of 3 beta-HSD were isolated from hamster cDNA libraries. The type 1 isoform is a high-affinity dehydrogenase/isomerase expressed in adrenal and male kidney. The type 2 isoform is also a high-affinity dehydrogenase/isomerase expressed in kidney and male liver. The type 3 enzyme is a 3-ketosteroid reductase expressed predominantly in kidney. Sequencing of the clones showed that all three are structurally very similar, although types 1 and 2 share the greatest degree of similarity. Immunohistochemical staining for 3 beta-HSD in the adrenal was found throughout the adrenal cortex. In the kidney staining was confined to tubules, and in the liver, heavy staining was found in hepatocytes. The cloning of cDNAs for 3 beta-HSD from the liver and kidney should help in elucidating the function of this enzyme in these tissues.

Immune privilege in the testis. II. Evaluation of potential local factors.

The significance of several local factors in prolonging allograft survival in the rat testis has been investigated. The lower temperature of this organ was shown to have no effect, because parathyroid gland allografts implanted s.c. in the ear, which is similarly hypothermic, were rejected as promptly as in conventional sites. Conversely, testes secured in the abdominal cavity afforded privilege to grafts just as well as normal testes. With indirect immunofluorescence using a monoclonal antibody, it was shown that the testis' interstitial tissue is well-endowed with cells bearing class II histocompatibility antigens, a prerequisite for antigen-processing capability. When intratesticular allograft recipients were pretreated with estrogen to suppress Leydig cell synthesis of testosterone, most grafts were rejected promptly, suggesting that local steroid secretion is important in testicular immune privilege. Both testes and s.c. sites supported inflammatory reactions to cotton pledgets, indicating that there is no interference with the nonspecific inflammatory process necessary for graft rejection, but more likely with antigen processing itself.

Pregnancy-induced hyporesponsiveness to paternal alloantigens. II. Factors affecting altered expressions of immunity in parous rats.

It was previously demonstrated that female Fischer rats, primiparous by allogeneic DA males, produced only low levels of agglutinating antibody to a challenge skin graft bearing paternal alloantigens, although graft rejection was unimpaired. In this report, the phenomenon of pregnancy-induced hyporesponsiveness was further investigated with regard to factors responsible for its induction and maintenance. Increasing parity by DA males resulted in increased suppression of the Fischer females' agglutinating antibody production postpartum to a challenge skin graft bearing paternal antigens. Moreover, multiparity also resulted in impaired graft rejection, with greater numbers of litters causing more prolonged survival. Either humoral or cellular hyporesponsiveness, or both, persisted for several weeks or even months in many females. Removal of the iliac lymph nodes prior to pregnancy resulted in less severe suppression which was not maintained through the observation period (21 days postgrafting), whereas splenectomy either before mating or on day 7 of pregnancy had no effect. Tubal-ligated females exposed to multiple DA ejaculates, without pregnancy, manifested weakened humoral immunity similar to that of primiparous females, whereas those exposed to (Fischer X DA)F1 hybrid trophoblast multiple times in ectopic sites had not only somewhat suppressed antibody responses but also significantly impaired capacity to reject the challenge allograft. The potential roles of various antigenic stimuli and the complexities of intrauterine antigen processing during gestation are discussed in relation to this naturally induced allosuppression phenomenon.

Increased free plasma corticosterone and adrenal hyperactivity associated with graft-versus-host disease.

This study profiles adrenal function in 14-day-old neonatal and adult rats with graft-versus-host disease (GVHD). Measurements of adrenal function included total serum corticosterone, the relative percentage of free corticosterone (a measure of biologically active hormone), adrenal cholesterol content, adrenal weights, and adrenal histology. Diseased animals in both age groups displayed a shift from the protein-bound to the free corticosterone fraction, with, surprisingly, no change in total serum corticosterone titers. We discuss the possibility that such a shift toward the biologically active, free-hormone pool results in a physiologic hyperglucocorticoidism that might contribute to the progress of GVHD. Compared with littermate controls, diseased adults had enlarged adrenals. In neonates with GVHD, no adrenal hypertrophy was evident, yet decreased adrenal cholesterol content and associated depletion of adrenal lipid droplets--evidence of adrenal hyperactivity--were identified. The GVHD must be responsible for the observed alterations in adrenal function because treatment of affected animals with alloantiserum directed against donor cells halted the disease and elevated the cholesterol content toward control levels. The possible contribution of adrenal dysfunction to the pathogenesis of secondary manifestations of GVHD is discussed.

Reconsideration of the lymphatic drainage of the rat testis.

An extensive investigation of the lymphatic drainage from the rat testis was carried out in an effort to detect any characteristics which might be important in the immunological privilege the testis extends to allografts. Three different methods revealed a consistent and very efficient lymphatic drainage involving primarily the iliac and renal lymph nodes and to a lesser extent the external lumbar, para-aortic, and posterior gastric nodes. Within minutes, dye or labeled cells injected into the testis could be found within regional nodes. Node hypertrophy was induced by injecting the testes of F1 hybrids with parental strain lymphoid cells (a regional graft-versus-host reaction). Besides indicating that efficient filtration occurs in these nodes, this also established that they are a hospitable environment for cellular immune reactions. Similarly, the capacity of allogeneic cells injected into the testis to induce production of humoral alloantibodies by recipients confirms that exposure to antigens via this route does not in itself suppress immune responsiveness.

Immune privilege in the testis. I. Basic parameters of allograft survival.

The fate of solid tissue allografts--skin or parathyroid glands--implanted in the interstitial tissue of the testis was investigated using inbred rats. The results affirm that the testis is an immunologically privileged site despite its efficient lymphatic drainage. Skin allografts survived at least several days longer than orthotopic grafts of similar size, whether major histocompatibility complex (MHC)-compatible or MHC-incompatible, and failed to induce an alloantibody response in most recipients or to prime for secondary antibody responses on rechallenge. Further assessment employed parathyroid grafts that allowed appraisal of their function and its duration, by monitoring serum calcium levels. Most intratesticular MHC-incompatible parathyroids survived for at least twice as long as control grafts in nonprivileged sites, with a median survival time (MST) of 41 days--and one-third of the grafts functioned at 100 days. MHC-compatible grafts fared even better (MST of 60 days), some surviving more than 400 days. Most F1 hybrid grafts survived virtually indefinitely. Splenectomy 5-23 days prior to implantation had a beneficial rather than a detrimental effect on the privilege afforded intratesticular parathyroid allografts. Allograft rejection was accompanied by antibody production in only one-half the animals. Grafts that had undergone functional rejection at the time of recovery usually had an intense mononuclear cell infiltrate, and long-term surviving grafts displayed varying degrees of cellular infiltration among cords of healthy, functional chief cells. Accepted parathyroids were destroyed by active immunization of the host but were unaffected by passively administered alloantibodies. The possible mechanisms controlling graft rejection in this unique privileged site are discussed.

Isolation and characterization of trophoblast from murine placenta.

A discontinuous density gradient centrifugation method, devised to isolate enriched populations of trophoblast from murine definitive placentae, is described. It is concluded that the isolated adherent cells are trophoblast on the basis of the following characteristics: they are fetally derived, as determined by their donor glucose phosphate isomerase phenotype in embryo transfer experiments; epithelial cells, as shown by the presence of cytokeratin filaments and the absence of vimentin; negative for the stage-specific embryonic antigen-I (SSEA-I); and capable of progesterone secretion. Initially, they grew as individual polygonal cells, tending to form tight confluent monolayers with poorly defined intercellular boundaries. They were mono- or binucleate and increased their nuclear size with time. After two days, giant cells appeared to be formed from binucleated cells by nuclear fusion, and multinucleated cells appeared forming syncytia. Some of these cells also seemed to form giant cells. A low percentage (1 to 10 per cent) of contaminating cells, mainly macrophage-like cells, was observed. The isolated cells were a mixture of alkaline phosphatase- (AP-)positive and AP-negative cells, with some of the latter having phagocytic capacity. All were Fc receptor-negative. The possible identity of these cells in relation to trophoblast in the intact placenta is discussed. This method of isolating and characterizing trophoblast cells from the definitive mouse placenta will be a useful tool for studying the biology and immunology of trophoblast.

Transferrin receptor expression in early postimplantation mouse trophoblast and associated tissues.

The expression of the transferrin receptor (TR) was examined on murine trophoblast cells on days 6, 8 and 10 of gestation, using a monoclonal antibody visualized by indirect immunofluorescence on cryostat sections of the implant site. In the day 6 tissues, TR were observed on both the ectoplacental cone (EPC) and mural giant cell trophoblast populations, as well as on the embryonic ectoderm, anti-mesometrial decidual cells, uterine glandular epithelium and myometrium. By the 8th day of gestation, TR expression was weak, or undetectable on trophoblast giant cells (TGC), but remained strong on the proliferating cells of the EPC and embryo. In the definitive placenta (day 10), TR are expressed primarily on the differentiated labyrinthine trophoblast cells involved in the maternal-fetal transfer of iron.

Regulation of interleukin-8 gene expression in human endometrial cells in culture.

In this study, we investigated the regulation of interleukin-8 (IL-8) gene expression in separated endometrial stromal and epithelial cells of human endometrium. This research was conducted as part of an analysis of the role of these cells in regulating the recruitment of leukocytes to the endometrium. Well-characterized model systems were used to study the regulation of endometrial IL-8 gene expression, namely, stromal cells in monolayer culture after first passage and glandular epithelium in primary culture. The levels of IL-8 mRNA and the accumulation of immunoreactive IL-8 in the medium of endometrial stromal cells is culture increased in a time- and concentration-dependent manner upon treatment with IL-1 alpha, tumor necrosis factor-alpha, or serum. The effects of IL-1 alpha plus serum on IL-8 mRNA levels were at least additive. Serum treatment caused a modest stimulation of IL-8 gene transcription (evaluated after 6 h of treatment) in endometrial stromal cells, but serum also acted in these stromal cells to prolong the half-life of IL-8 mRNA by more than 2.5-fold. The regulation of the levels of IL-8 mRNA in endometrial epithelial cells is distinctly different from that in stroma. First, the levels of IL-8 mRNA in non-treated epithelial cells in serum-free medium were much greater than those in stromal cells under similar conditions. Second, whereas the levels of IL-8 mRNA in endometrial epithelial cells also increased in response to serum and to IL-1 in the absence of serum, in the presence of serum, IL-1 treatment caused no appreciable change in the levels of IL-8 mRNA as was the case in endometrial stromal cells.

Lymphatic vessels in the uterine endometrium of virgin rats.

Uteri from four diestrous rats fixed by perfusion were examined histologically for the presence of lymphatic vessels in the endometrium, using 1 micron epoxy sections stained with toluidine blue. For each animal, multiple random sections were prepared from 3 tissue samples. In all sections, lymphatics were readily apparent in the myometrium, being identified by their irregular shape and contents of stained lymph proteins, which contrasted with the clear, empty blood vessels. While similar lymphatics were not seen in the endometrium of most tissue samples, one sample from each of three females revealed a small lymphatic vessel near the bases of the uterine glands, clearly in the endometrium. One of these was followed in serial alternating light and electron microscopic sections. The vessel maintained its relative position to the endometrial glands for a considerable distance, and also joined with two separate branches that appeared along its course. Ultrastructural observations, including a thin endothelium without a continuous basal lamina, the presence of anchoring filaments, and only mononuclear cells and cellular debris in the lumen, confirmed the vessel's identification as a lymphatic. The vessel's size was normally large enough to be followed by light microscopy; however, at some places along its course, the lumen narrowed to such a degree that it was only identifiable ultramicroscopically . These observations refute claims that there are no lymphatic vessels in rodent uterine endometrium, but indicate that they are small and sparsely distributed.

Current trends in reproductive immunology: an overview.

Over the past decade has come general recognition that, directly or indirectly, immunology intrudes into nearly every aspect of mammalian reproduction. Charles Darwin's notion that the profligacy of women led to reduced fertility gained plausibility with the subsequent discovery of the auto- and alloantigenicity of spermatozoa and of testicular material. From this observation arose the reasonable expectation that immunological control of fertility may be feasible. Discovery of the importance of natural transfer of immunity from mother to offspring, the ontogeny of the immune response, and recognition that pregnancy is an almost consistently successful violation of the "laws of transplantation' are only a few highlights or components of the burgeoning, multifaceted field we have come to recognize as the immunology of reproduction. An overview of this subject is here presented with regard to its evolutionary origins, its accomplishments, its current trends, and some of its potentialities. The immunology of reproduction has not developed in isolation; in recent years it has benefited enormously from developments in other fields, and in its turn it has exerted its own impact on other disciplines, especially on transplantation. The present preoccupation of many immunologists with immunoregulation stems largely from independent discoveries in the realm of reproductive immunobiology - the etiology of Rhesus disease and its prophylaxis, and the principle of immunological tolerance, from investigations on the peculiarities of dizygotic twins in cattle.

Murine trophoblast can be killed by allospecific cytotoxic T lymphocytes generated in GIBCO Opti-MEM medium.

Previous work in this laboratory demonstrated that a population of cultured midterm murine trophoblast cells are not susceptible to lysis by allospecific cytotoxic T lymphocytes (CTL) generated by standard in vitro protocols. We now report that this trophoblast population is killed, in an MHC-dependent manner, by allospecific CTL generated in GIBCO Opti-MEM, a modified tissue culture medium designed to maintain cell growth and proliferation in the presence of low concentrations of fetal bovine serum (FBS).

Autoradiographic analysis of lymphocyte migration into the mammary epithelium and milk of lactating female rats.

Adoptively transferred radiolabeled lymphoid cells migrating to the lactating mammary gland were shown to enter the alveolar epithelium and ultimately the milk. Lactating female rats were injected intravenously with [3H]uridine-labeled syngeneic mesenteric lymph node cells, and the distribution of the cells 24 and 48 h later within intestinal and mammary tissues and their presence in milk were assessed autoradiographically. At both time periods, numerous labeled cells were found in the Peyer's patches and mesenteric lymph nodes. The mammary tissue and intestinal mucosa (lamina propria and epithelium) contained fewer labeled cells per unit area than those sites. Labeled cells within the mammary tissue were distributed equally between the alveolar epithelium and the intralobular connective tissue, where they were seen in both blood vessels and in the loose connective tissue. Occasional labeled cells were observed among the mononuclear cells seen in the alveolar lumina, and an average of 4.8% of the cells harvested from milk samples were labeled. Labeled cells within the secretory epithelium and milk always had the morphologic characteristics of mononuclear leukocytes. Thus, at least a portion of the lymphoid cells which have been shown to migrate to the mammary tissue in increased numbers during lactation actually enter and traverse the epithelium and contribute to the lymphoid component of mammary secretions.