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Modulation of the maternal immune system by the placenta is a mechanism by which the fetus ensures its own survival in a genetically foreign environment. The immunoinhibitor CD274 (also called B7-H1 or PD-L1) is highly expressed in the placenta, positioned to interact with maternal leukocytes. Further, immunoblot analysis of first- and second-trimester placental lysates showed that CD274 expression is low in the first trimester but dramatically rises around the onset of the second trimester. As this coincides with the expected onset of maternal blood flow to the placenta and a corresponding rise in local oxygen tension, we explored the possibility that oxygen regulates CD274 expression in trophoblast cells by culturing term trophoblast cells under oxygen concentrations similar to those found in vivo. Indeed, CD274 protein levels paralleled the in vivo situation: expression increased with rising oxygen concentrations. Furthermore, downregulation of CD274 mRNA by low oxygen was rapid, occurring within 4–12 h. We conclude that oxygen is a potential mediator of CD274 expression in vivo such that it is induced coincidentally on exposure of fetal tissues to maternal blood. Further, the regulation of this immunomodulator by oxygen may implicate its alteration during and involvement in the pathogenesis of complications of pregnancy such as preeclampsia.

The human placenta utilizes both active and passive mechanisms to evade rejection by the maternal immune system. We investigated the pattern of expression of the B7 family of immunomodulatory molecules B7-H1 (PD-L1), B7-2 (CD86), and B7-1 (CD80) at the term maternal-fetal interface. Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that B7-H1 mRNA is abundant in term placenta and that cytotrophoblasts are sources of this message. Immunohistochemistry demonstrated that B7-H1 is constitutively expressed by the syncytiotrophoblast and by extravillous cytotrophoblasts, both of which are juxtaposed to maternal blood and tissue. By contrast, placental stromal cells, including macrophages, lacked the protein. Expression of B7-H1 protein was low in first-trimester placenta compared to second- and third-trimester tissue (P < 0.05) and was enhanced in cultured cytotrophoblasts by treatment with either interferon-gamma or epidermal growth factor (P < 0.05), suggesting that one or both of these mediators regulates B7-H1 expression in the placenta. RT-PCR and immunofluorescence analysis of term placental tissue revealed different patterns of expression of the immunostimulatory protein, B7-2. In contrast to B7-H1, B7-2 mRNA and protein were absent in cytotrophoblast cells but present in maternal macrophages and some fetal macrophages. The B7-1 mRNA and protein were absent at the maternal-fetal interface. These studies document expression of the B7 family proteins at the maternal-fetal interface and demonstrate that B7-H1 is positioned such that it could facilitate protection of fetal cells against activated maternal leukocytes. Conversely, B7-2 was absent on trophoblasts and was appropriately localized to fetal and maternal macrophages, which may participate in antigen presentation.

The expression and function of the human major histocompatibility complex (MHC) class Ia genes, human leukocyte antigen (HLA)-A, -B, and -C, is well-established; they are expressed in most nucleated cells and present endogenous peptides to CD8+ T cells. However, MHC class Ib genes are poorly characterized and have unknown functions. In humans, the best-characterized class Ib gene is HLA-G. This gene has a restricted tissue expression of the mRNA and a unique pattern of protein expression; it is expressed mainly in the extravillous cytotrophoblast cells in the placenta. The function of HLA-G is not clear, but its presence at the maternal-fetal interface suggests a role in protection of the semiallogeneic fetus. Whereas functional studies using in vitro models and transgenic mice provide useful insights regarding the potential function of this molecule, in vivo studies cannot be performed in humans. Nonhuman primates that are closely related to humans phylogenetically contain homologues of HLA-G. T...

Integrins and vascular cell adhesion molecule-1 (VCAM-1) are required for normal placental development. In this study, integrin subunits a4, av, b1, and b3, and VCAM-1 were investigated for expression in uteroplacental units (gestation day [g.d.] 6 and 8) and placentas (g.d. 10, 12, 14, 16, and 18) of Swiss-Webster mice. All subunits and VCAM-1 mRNA (identified by reverse transcriptase polymerase chain reaction [RT-PCR]) and protein (detected by immunofluorescence) were present in all tissues throughout gestation. VCAM-1 was expressed strongly in the ectoplacental cone and trophoblast giant cells, a 4w as expressed strongly by trophoblast giant cells and moderately by spongiotrophoblast and labyrinthine trophoblast, and av was expressed more strongly in the spongiotrophoblast than in the labyrinthine zone. The b1 was more strongly expressed in the labyrinthine than the spongiotrophoblast zone, while b3 and VCAM-1 were essentially equal in the two zones. Trophoblastlike SM9‐1 cells were positive for all of the adhesion molecules when tested by RT-PCR and immunocytochemistry. Adhesion molecule expression in SM9‐1 cells was consistent with expression in the labyrinthine zone. Collectively, the results of this study demonstrate that murine placentas contain mRNA and protein for a4, av, b1, b3, and VCAM-1, and that expression is cell-specific. These results and the identification of an adhesion molecule-expressing trophoblastic cell line should facilitate future studies on the function of adhesion molecules in placental development.

Nitric oxide (NO), a potent and versatile free radical, is synthesized in leukocytes by the inducible form of NO synthase (iNOS). In this study, leukocytes in pregnant mouse uterus were investigated for expression of the iNOS gene. Inducible NOS mRNA, which was identified by reverse transcriptase polymerase chain reaction, was high relative to an invariant mRNA, glyceraldehyde-3-phosphate dehydrogenase, in midgestation uteri (gestation days [g.d.] 10, 12, and 14) but was low in late-gestation uteri (g.d. 16 and 18). Inducible NOS protein, identified immunohistochemically in paraformaldehydefixed uteri taken from g.d. 6 through 18 using rabbit antibodies generated to mouse carboxyl terminus iNOS peptides, was prominent in a few myometrial mast cells at early stages and was strongly expressed from g.d. 6 through g.d. 14 in myometrial macrophage-like cells. Inducible NOS protein was first detected in uterine (u) natural killer (NK) cells at g.d. 8. Signals peaked in this lineage at g.d. 10 and declined thereafter. Uterine leukocytes cultured in vitro expressed the iNOS gene; a hybridoma cell line derived from mouse uNK cells (GWM1-2) contained iNOS mRNA, and cells migrating from mouse metrial gland explants included iNOS+ leukocytes. Large, granular iNOS+ uNK cells were absent from the uteri of homologously mated pregnant TgE26 mice, an NK cell-deficient transgenic mouse strain, but immunoreactive iNOS was detectable in trophoblast, a cell lineage that did not contain immunoreactive iNOS in NK cell-competent Swiss-Webster mice. In TgE26 mothers gestating normal embryos, the same pattern was observed. Collectively, the results of this study demonstrate that iNOS is present in mouse uterine leukocytes including mast cells, macrophage-like cells, and uNK cells, and suggest that in the absence of uNK cells, the placenta synthesizes iNOS. These findings are consistent with the postulate that leukocyte NO contributes importantly to events associated with successful pregnancy that are likely to include relaxation of vascular smooth muscle.

Tumor necrosis factor alpha is a major component of the infection-stimulated cytokine milieux associated with preterm labor and pregnancy termination. As a consequence, this potent factor is thought by some to have exclusively negative effects on the course of pregnancy. Yet in humans and other mammals, messenger RNA hybridizing with tumor necrosis factor alpha cRNA probes and protein detected by anti-tumor necrosis factor alpha have been identified in normal cycling and pregnant uteri, placentas, and embryos, consistent with an important role for tumor necrosis factor in female reproduction and embryonic development. Here, evidence for this intriguing dichotomy is presented, unresolved aspects are discussed, and potential roles for uteroplacental and embryonic tumor necrosis factor are proposed.

The expression of tumor necrosis factor α (TNF) protein in mouse myometrial cells on each day of the estrous cycle and after ovariectomy, with and without replacement of estradiol (E 2 ), progesterone (P 4 ), or E 2 +P 4 , was investigated. TNF protein was assessed by immunocytochemistry. In addition, the numbers of mast cells and the numbers of TNF-containing mast cells were determined under each condition. The total number of mast cells fluctuated during the estrous cycle; and at each stage, essentially 100% of the mast cells exhibited TNF immunoreactivity. In contrast, after ovariectomy and after ovariectomy with E 2 replacement, only ∼50% of the mast cells contained immunoreactive TNF. P 4 treatment resulted in further decline in mast cell TNF whereby after 24 h of P 4 , only 3% of the mast cells were TNF-positive and by 72 h of treatment, no TNF-positive mast cells could be detected. E 2 and P 4 in combination increased the total number of mast cells, and in a pattern reminiscent of normal cycling myometrium, over 90% of the mast cells exhibited TNF immunoreactivity after 24 h of treatment. TNF protein was detectable in muscle cells throughout the estrous cycle, with weak immunostaining observed on proestrus and more intense immunostaining on estrus, diestrus-I, and diestrus-II. After ovariectomy, only light immunostaining was observed in the muscle cells. Immunoreactive TNF was detected in muscle cells after each type of steroid treatment. E 2 and E 2 +P 4 treatments resulted in a biphasic pattern of immunoreactive TNF expression. Immunostaining intensity was increased at 6 h after treatment, decreased after 24 h, and increased again after 72 h. In contrast, the greatest intensity of TNF immunostaining was observed 1 h after P 4 administration and the staining intensity decreased thereafter. These studies reveal 1) the cellular localization of TNF protein in the mouse myometrium, 2) the identification of the mast cell as a major source of uterine TNF, and 2) regulation of myometrial TNF by E 2 and P 4

Macrophages and a special subset of lymphocyte natural killer (NK) cells populate the uteri of cycling humans, mice, and rats. After implantation, major changes take place that have important functional implications. The macrophages and NK cells increase in number, are redistributed into specific uterine compartments, and exhibit markers consistent with cell activation. Activation enhances macrophage and NK cell production of a wide range of pleiotropic, multifunctional polypeptide growth factors, reactive oxygen intermediates, and bioactive lipids. Thus, activated uterine hematopoietic cells are equipped to perform certain immunological and nonimmunological functions within their microenvironments that could have major influences on the course of pregnancy.

In order to map mouse tumor necrosis factor-alpha (TNF) gene expression in detail and to determine whether transcription or translation of the TNF gene is regulated by uterine colony stimulating factor-1 (CSF-1), preimplantation embryos, oviducts, uteri, and uteroplacental units were studied in various strains of mice. These included homozygous osteopetrotic (op/op) female mice, which completely lack CSF-1, and heterozygous (+/op) females, which have normal levels of CSF-1. TNF mRNA was identified in all samples except preimplantation embryos by use of Northern blot hybridization or reverse transcriptase polymerase chain reaction. In situ hybridization and immunocytochemical experiments showed that the TNF gene was expressed in mouse oviduct and uterine epithelial cells, decidual cells, macrophage-like cells, placental trophoblast, and embryos. Despite an absence of CSF-1, TNF gene expression in the uteri, placentas, and embryos of op/op mothers did not differ in any major respect from expression in +/op or other strains of mice. The results of this study therefore indicate that the TNF gene is transcribed and translated in an ordered sequence through mouse gestation, and that maternal CSF-1 is not essential to expression of this cytokine gene. Collectively, these findings are consistent with a major role for TNF in mouse reproduction and development and with a potential compensatory function for this potent polypeptide factor in CSF-1 deficiency.

Ovarian cells that transcribe and translate the gene for tumor necrosis factor alpha (TNF alpha) were identified in the adult cyclic mouse by using in situ hybridization and immunocytochemistry. TNF alpha mRNA was observed in > 97% and protein was contained in approximately 53% of the oocytes of healthy follicles with two or more layers of granulosa cells, but neither was detectable in oocytes of primordial follicles and follicles with a single layer of granulosa cells. In early atretic follicles, only 13% contained TNF alpha protein and 40% contained TNF alpha mRNA. In late stages of atresia, intense immunoreactive TNF alpha was observed in all of the oocytes, but TNF alpha mRNA was present in only 13%. In approximately 85% of follicles, theca and/or granulosa cells exhibited TNF alpha mRNA hybridization signals. Macrophage-like cells within the interstitium were positive for TNF alpha mRNA and protein. In corpora lutea, luteal cells and macrophage-like cells contained TNF alpha message, while only the latter lineage contained immunoreactive TNF alpha. Hybridization signals and immunoreactivity were more intense in older corpora lutea than in corpora lutea of the present cycle. Northern blot analysis revealed a 2.2-kb TNF alpha mRNA in the ovary that was unchanged relative to 28S rRNA (constitutive RNA) during the cycle. Similarly, TNF alpha hybridization signals and immunoreactivity did not appear to change throughout the cycle. These results indicate that TNF alpha gene transcription in the oocyte coincides with the synthesis of immunoreactive TNF alpha and that these complex biochemical processes occur at distinct steps of follicular development in the mouse.(ABSTRACT TRUNCATED AT 250 WORDS)

Although previous studies have shown that the tumor necrosis factor-alpha (TNF) gene is expressed in pregnancy decidua, it has not been determined whether this gene is transcribed or translated in the endometrium prior to implantation. To address this question and to identify cells expressing the TNF gene, samples of normal cycling human endometria were tested for TNF mRNA by in situ hybridization using a biotinylated antisense RNA probe, and the corresponding protein was localized in the same tissues by immunocytochemistry with a monoclonal antibody to TNF. Both TNF message and protein were identified in the endometrium throughout the menstrual cycle, and the same types of cells that contained transcripts also contained protein. As judged by the intensities of the hybridization signals, TNF mRNA increased during the proliferative phases, declined in the early secretory phase, and again rose during mid-to-late secretory phases, suggesting positive associations with levels of female sex hormones that show similar cyclic fluctuations. At the initiation of the cycle, transcripts were primarily localized to glandular epithelial cells whereas by the midproliferative phase, message was also present in stromal cells. Hybridization signals were consistently more intense in functionalis-region than in basalis-region stromal cells, and were frequently stronger in basalis-region glandular epithelia than in functionalis-region glands. These observations document that the TNF gene is expressed in normal cycling endometria, suggest that ovarian hormones may regulate TNF gene transcription, and identify differences in specific endometrial compartments. The findings are also consistent with the postulate that TNF is a local mediator of cellular communications in the human endometrium.

Modulation of the maternal immune system by the placenta is a mechanism by which the fetus ensures its own survival in a genetically foreign environment. The immunoinhibitor CD274 (also called B7-H1 or PD-L1) is highly expressed in the placenta, positioned to interact with maternal leukocytes. Further, immunoblot analysis of first- and second-trimester placental lysates showed that CD274 expression is low in the first trimester but dramatically rises around the onset of the second trimester. As this coincides with the expected onset of maternal blood flow to the placenta and a corresponding rise in local oxygen tension, we explored the possibility that oxygen regulates CD274 expression in trophoblast cells by culturing term trophoblast cells under oxygen concentrations similar to those found in vivo. Indeed, CD274 protein levels paralleled the in vivo situation: expression increased with rising oxygen concentrations. Furthermore, downregulation of CD274 mRNA by low oxygen was rapid, occurring within 4-12 h. We conclude that oxygen is a potential mediator of CD274 expression in vivo such that it is induced coincidentally on exposure of fetal tissues to maternal blood. Further, the regulation of this immunomodulator by oxygen may implicate its alteration during and involvement in the pathogenesis of complications of pregnancy such as preeclampsia.

The human placenta utilizes both active and passive mechanisms to evade rejection by the maternal immune system. We investigated the pattern of expression of the B7 family of immunomodulatory molecules B7-H1 (PD-L1), B7-2 (CD86), and B7-1 (CD80) at the term maternal-fetal interface. Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that B7-H1 mRNA is abundant in term placenta and that cytotrophoblasts are sources of this message. Immunohistochemistry demonstrated that B7-H1 is constitutively expressed by the syncytiotrophoblast and by extravillous cytotrophoblasts, both of which are juxtaposed to maternal blood and tissue. By contrast, placental stromal cells, including macrophages, lacked the protein. Expression of B7-H1 protein was low in first-trimester placenta compared to second- and third-trimester tissue (P0.05) and was enhanced in cultured cytotrophoblasts by treatment with either interferon-gamma or epidermal growth factor (P0.05), suggesting that one or both of these mediators regulates B7-H1 expression in the placenta. RT-PCR and immunofluorescence analysis of term placental tissue revealed different patterns of expression of the immunostimulatory protein, B7-2. In contrast to B7-H1, B7-2 mRNA and protein were absent in cytotrophoblast cells but present in maternal macrophages and some fetal macrophages. The B7-1 mRNA and protein were absent at the maternal-fetal interface. These studies document expression of the B7 family proteins at the maternal-fetal interface and demonstrate that B7-H1 is positioned such that it could facilitate protection of fetal cells against activated maternal leukocytes. Conversely, B7-2 was absent on trophoblasts and was appropriately localized to fetal and maternal macrophages, which may participate in antigen presentation.

The expression and function of the human major histocompatibility complex (MHC) class Ia genes, human leukocyte antigen (HLA)-A, -B, and -C, is well-established; they are expressed in most nucleated cells and present endogenous peptides to CD8+ T cells. However, MHC class Ib genes are poorly characterized and have unknown functions. In humans, the best-characterized class Ib gene is HLA-G. This gene has a restricted tissue expression of the mRNA and a unique pattern of protein expression; it is expressed mainly in the extravillous cytotrophoblast cells in the placenta. The function of HLA-G is not clear, but its presence at the maternal-fetal interface suggests a role in protection of the semiallogeneic fetus. Whereas functional studies using in vitro models and transgenic mice provide useful insights regarding the potential function of this molecule, in vivo studies cannot be performed in humans. Nonhuman primates that are closely related to humans phylogenetically contain homologues of HLA-G. The MHC-G loci in nonhuman primates appear to have diverged from the human HLA-G. However, in the rhesus monkey (Macaca mulatta) and olive baboon (Papio anubis), a novel class Ia-related locus has been described. This gene encodes glycoproteins with characteristics that resemble those of HLA-G, including restricted tissue distribution, alternative splicing of mRNA, truncated cytoplasmic domain, and limited polymorphism. Thus, this molecule may be the functional homologue of HLA-G, and these two species may comprise appropriate models for elucidating the function of HLA-G.

Integrins and vascular cell adhesion molecule-1 (VCAM-1) are required for normal placental development. In this study, integrin subunits alpha4, alphav, beta1, and beta3, and VCAM-1 were investigated for expression in uteroplacental units (gestation day [g.d.] 6 and 8) and placentas (g.d. 10, 12, 14, 16, and 18) of Swiss-Webster mice. All subunits and VCAM-1 mRNA (identified by reverse transcriptase polymerase chain reaction [RT-PCR]) and protein (detected by immunofluorescence) were present in all tissues throughout gestation. VCAM-1 was expressed strongly in the ectoplacental cone and trophoblast giant cells, alpha4 was expressed strongly by trophoblast giant cells and moderately by spongiotrophoblast and labyrinthine trophoblast, and alphav was expressed more strongly in the spongiotrophoblast than in the labyrinthine zone. The beta1 was more strongly expressed in the labyrinthine than the spongiotrophoblast zone, while beta3 and VCAM-1 were essentially equal in the two zones. Trophoblast-like SM9-1 cells were positive for all of the adhesion molecules when tested by RT-PCR and immunocytochemistry. Adhesion molecule expression in SM9-1 cells was consistent with expression in the labyrinthine zone. Collectively, the results of this study demonstrate that murine placentas contain mRNA and protein for alpha4, alphav, beta1, beta3, and VCAM-1, and that expression is cell-specific. These results and the identification of an adhesion molecule-expressing trophoblastic cell line should facilitate future studies on the function of adhesion molecules in placental development.

Nitric oxide (NO), a potent and versatile free radical, is synthesized in leukocytes by the inducible form of NO synthase (iNOS). In this study, leukocytes in pregnant mouse uterus were investigated for expression of the iNOS gene. Inducible NOS mRNA, which was identified by reverse transcriptase polymerase chain reaction, was high relative to an invariant mRNA, glyceraldehyde-3-phosphate dehydrogenase, in midgestation uteri (gestation days [g.d.] 10, 12, and 14) but was low in late-gestation uteri (g.d. 16 and 18). Inducible NOS protein, identified immunohistochemically in paraformaldehyde-fixed uteri taken from g.d. 6 through 18 using rabbit antibodies generated to mouse carboxyl terminus iNOS peptides, was prominent in a few myometrial mast cells at early stages and was strongly expressed from g.d. 6 through g.d. 14 in myometrial macrophage-like cells. Inducible NOS protein was first detected in uterine (u) natural killer (NK) cells at g.d. 8. Signals peaked in this lineage at g.d. 10 and declined thereafter. Uterine leukocytes cultured in vitro expressed the iNOS gene; a hybridoma cell line derived from mouse uNK cells (GWM1-2) contained iNOS mRNA, and cells migrating from mouse metrial gland explants included iNOS/ leukocytes. Large, granular iNOS + uNK cells were absent from the uteri of homologously mated pregnant TgE26 mice, an NK cell-deficient transgenic mouse strain, but immunoreactive iNOS was detectable in trophoblast, a cell lineage that did not contain immunoreactive iNOS in NK cell-competent Swiss-Webster mice. In TgE26 mothers gestating normal embryos, the same pattern was observed. Collectively, the results of this study demonstrate that iNOS is present in mouse uterine leukocytes including mast cells, macrophage-like cells, and uNK cells, and suggest that in the absence of uNK cells, the placenta synthesizes iNOS. These findings are consistent with the postulate that leukocyte NO contributes importantly to events associated with successful pregnancy that are likely to include relaxation of vascular smooth muscle.

Tumor necrosis factor alpha is a major component of the infection-stimulated cytokine milieux associated with preterm labor and pregnancy termination. As a consequence, this potent factor is thought by some to have exclusively negative effects on the course of pregnancy. Yet in humans and other mammals, messenger RNA hybridizing with tumor necrosis factor alpha cRNA probes and protein detected by anti-tumor necrosis factor alpha have been identified in normal cycling and pregnant uteri, placentas, and embryos, consistent with an important role for tumor necrosis factor in female reproduction and embryonic development. Here, evidence for this intriguing dichotomy is presented, unresolved aspects are discussed, and potential roles for uteroplacental and embryonic tumor necrosis factor are proposed.

The expression of tumor necrosis factor alpha (TNF) protein in mouse myometrial cells on each day of the estrous cycle and after ovariectomy, with and without replacement of estradiol (E2), progesterone (P4), or E2 + P4, was investigated. TNF protein was assessed by immunocytochemistry. In addition, the numbers of mast cells and the numbers of TNF-containing mast cells were determined under each condition. The total number of mast cells fluctuated during the estrous cycle; and at each stage, essentially 100% of the mast cells exhibited TNF immunoreactivity. In contrast, after ovariectomy and after ovariectomy with E2 replacement, only approximately 50% of the mast cells contained immunoreactive TNF. P4 treatment resulted in further decline in mast cell TNF whereby after 24 h of P4, only 3% of the mast cells were TNF-positive and by 72 h of treatment, no TNF-positive mast cells could be detected E2 and P4 in combination increased the total number of mast cells, and in a pattern reminiscent of normal cycling myometrium, over 90% of the mast cells exhibited TNF immunoreactivity after 24 h of treatment. TNF protein was detectable in muscle cells throughout the estrous cycle, with weak immunostaining observed on proestrus and more intense immunostaining on estrus, diestrus-I, and diestrus-II. After ovariectomy, only light immunostaining was observed in the muscle cells. Immunoreactive TNF was detected in muscle cells after each type of steroid treatment. E2 and E2 + P4 treatments resulted in a biphasic pattern of immunoreactive TNF expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Macrophages and a special subset of lymphocyte natural killer (NK) cells populate the uteri of cycling humans, mice, and rats. After implantation, major changes take place that have important functional implications. The macrophages and NK cells increase in number, are redistributed into specific uterine compartments, and exhibit markers consistent with cell activation. Activation enhances macrophage and NK cell production of a wide range of pleiotropic, multifunctional polypeptide growth factors, reactive oxygen intermediates, and bioactive lipids. Thus, activated uterine hematopoietic cells are equipped to perform certain immunological and nonimmunological functions within their microenvironments that could have major influences on the course of pregnancy.

In order to map mouse tumor necrosis factor-alpha (TNF) gene expression in detail and to determine whether transcription or translation of the TNF gene is regulated by uterine colony stimulating factor-1 (CSF-1), preimplantation embryos, oviducts, uteri, and uteroplacental units were studied in various strains of mice. These included homozygous osteopetrotic (op/op) female mice, which completely lack CSF-1, and heterozygous (+/op) females, which have normal levels of CSF-1. TNF mRNA was identified in all samples except preimplantation embryos by use of Northern blot hybridization or reverse transcriptase polymerase chain reaction. In situ hybridization and immunocytochemical experiments showed that the TNF gene was expressed in mouse oviduct and uterine epithelial cells, decidual cells, macrophage-like cells, placental trophoblast, and embryos. Despite an absence of CSF-1, TNF gene expression in the uteri, placentas, and embryos of op/op mothers did not differ in any major respect from expression in +/op or other strains of mice. The results of this study therefore indicate that the TNF gene is transcribed and translated in an ordered sequence through mouse gestation, and that maternal CSF-1 is not essential to expression of this cytokine gene. Collectively, these findings are consistent with a major role for TNF in mouse reproduction and development and with a potential compensatory function for this potent polypeptide factor in CSF-1 deficiency.

Although previous studies have shown that the tumor necrosis factor-alpha (TNF) gene is expressed in pregnancy decidua, it has not been determined whether this gene is transcribed or translated in the endometrium prior to implantation. To address this question and to identify cells expressing the TNF gene, samples of normal cycling human endometria were tested for TNF mRNA by in situ hybridization using a biotinylated antisense RNA probe, and the corresponding protein was localized in the same tissues by immunocytochemistry with a monoclonal antibody to TNF. Both TNF message and protein were identified in the endometrium throughout the menstrual cycle, and the same types of cells that contained transcripts also contained protein. As judged by the intensities of the hybridization signals, TNF mRNA increased during the proliferative phases, declined in the early secretory phase, and again rose during mid-to-late secretory phases, suggesting positive associations with levels of female sex hormones that show similar cyclic fluctuations. At the initiation of the cycle, transcripts were primarily localized to glandular epithelial cells whereas by the midproliferative phase, message was also present in stromal cells. Hybridization signals were consistently more intense in functionalis-region than in basalis-region stromal cells, and were frequently stronger in basalis-region glandular epithelia than in functionalis-region glands. These observations document that the TNF gene is expressed in normal cycling endometria, suggest that ovarian hormones may regulate TNF gene transcription, and identify differences in specific endometrial compartments. The findings are also consistent with the postulate that TNF is a local mediator of cellular communications in the human endometrium.

The purpose of the present investigation was to evaluate the in vitro requirements for rat placental cell DNA synthesis. A cell line established from the labyrinth region of midgestation rat chorioallantoic placentas was used to examine the actions of various agents. Transferrin was found to stimulate rat placental cell DNA synthesis and cell proliferation. The effects of transferrin on rat placental cell growth paralleled those observed with fetal bovine serum. Rat placental cells were responsive to both rat and human transferrin. Iron-saturated (bob-) transferrin was a more potent stimulator of rat placental cell DNA synthesis than was iron-free -(apo-) transferrin. Addition of insulin, epidermal growth factor, or insulin-like growth factor-lI to serum-free medium supplemented with rat transferrin did not signIficantly enhance rat placental cell DNA synthesis beyond that observed with only transferrin. The results demonstrate that a population of cells exists within the rat cborioallantoic placenta that are highly responsive to transferrin.

Cyclic adenosine 3’,S’-monophosphate (cAMP) has been implicated in the control of placental function. The present investigation was designed to evaluate the actions of cAMP analogues on the control of rat placental development. Two model systems were used to assess the actions of cAMP in the placenta: 1) a rat placental cell line and 2) rat labyrinth placental explants. Elevation of intracellular cAMP via treatment with cAMP analogues, 3-isobutyl-J -methylxanthine, forskolin, or cholera toxin inhibited placental cell DNA synthesis whereas treatment with an analogue to cyclic guanosine 3’,5’-monophosphate was without effect. The inhibitory actions of dibutyryl cAMP on DNA synthesis were at least partially reversible and were not the result of metabolic toxicity. Dibutyryl cAMP had dramatic effects on the organization and morphology of placental cells growing in vitro and diminished the ability of the placental cells to grow following transplantation into allogeneic hosts. Dijferentiatio n-associated characteristics of rat placental cells were also affected by cAMP, cAMP analogues stimulated placental cell progesterone release and inhibited placental cell alkaline phosphatase activity. Dibutyryl cAMP had effects on placental labyrinth explants similar to its effects on the placental cell line. Dibutyryl cAMP inhibited explant outgrowth while stimulating explant release of progesterone. In summary, cAMP effectively modulates the growth and differentiation of rat placental cells in vitro.

The purpose of the present investigation was to evaluate the in vitro requirements for rat placental cell DNA synthesis. A cell line established from the labyrinth region of midgestation rat chorioallantoic placentas was used to examine the actions of various agents. Transferrin was found to stimulate rat placental cell DNA synthesis and cell proliferation. The effects of transferrin on rat placental cell growth paralleled those observed with fetal bovine serum. Rat placental cells were responsive to both rat and human transferrin. Iron-saturated (holo-) transferrin was a more potent stimulator of rat placental cell DNA synthesis than was iron-free (apo-) transferrin. Addition of insulin, epidermal growth factor, or insulin-like growth factor-II to serum-free medium supplemented with rat transferrin did not significantly enhance rat placental cell DNA synthesis beyond that observed with only transferrin. The results demonstrate that a population of cells exists within the rat chorioallantoic placenta that are highly responsive to transferrin.

Cyclic adenosine 3',5'-monophosphate (cAMP) has been implicated in the control of placental function. The present investigation was designed to evaluate the actions of cAMP analogues on the control of rat placental development. Two model systems were used to assess the actions of cAMP in the placenta: 1) a rat placental cell line and 2) rat labyrinth placental explants. Elevation of intracellular cAMP via treatment with cAMP analogues, 3-isobutyl-1-methylxanthine, forskolin, or cholera toxin inhibited placental cell DNA synthesis whereas treatment with an analogue to cyclic guanosine 3',5'-monophosphate was without effect. The inhibitory actions of dibutyryl cAMP on DNA synthesis were at least partially reversible and were not the result of metabolic toxicity. Dibutyryl cAMP had dramatic effects on the organization and morphology of placental cells growing in vitro and diminished the ability of the placental cells to grow following transplantation into allogeneic hosts. Differentiation-associated characteristics of rat placental cells were also affected by cAMP. cAMP analogues stimulated placental cell progesterone release and inhibited placental cell alkaline phosphatase activity. Dibutyryl cAMP had effects on placental labyrinth explants similar to its effects on the placental cell line. Dibutyryl cAMP inhibited explant outgrowth while stimulating explant release of progesterone. In summary, cAMP effectively modulates the growth and differentiation of rat placental cells in vitro.