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A chick non-muscle alpha-actinin cDNA probe encoding the EF-hand region of molecule was used to screen a lambda gt10 chick brain cDNA library from 14-day embryos. A partial 2.1-kb alpha-actinin cDNA was isolated (8W cDNA) which encoded a protein identical to chick skeletal-muscle alpha-actinin, except in the C-terminal part of the first EF hand. In the variant, the 22 residues found in the skeletal-muscle isoform were replaced by a stretch of 26 unique residues. Analysis of the structure of the skeletal-muscle alpha-actinin gene showed that the region of divergence was encoded by two exons which are alternatively spliced. Quantitative reverse transcriptase/polymerase chain reaction (RT/PCR) was used to investigate the levels of the alpha-actinin transcripts in various tissues. The skeletal-muscle alpha-actinin variant was expressed at low levels in brain, liver and spleen, but could not be detected in skeletal muscle. Surprisingly, skeletal-muscle alpha-actinin mRNA was also expressed in brain, liver and spleen. The RT/PCR products were authenticated by using diagnostic restriction enzyme sites and by sequencing. The splice variant derived from the skeletal-muscle alpha-actinin gene was also detected in a variety of cDNA libraries from both adult and embryonic tissues by PCR. Although a transcript encoding this alpha-actinin splice variant is expressed in non-muscle tissues, neither of the two EF-hands would be predicted to be functional, making it unlikely to be a typical non-muscle isoform which are calcium-sensitive with respect to binding actin. The two vertebrate non-muscle alpha-actinins sequenced to date also have a spacer of five amino acids between the two EF hands, whereas in the variant, the spacer is just four residues in length. Further analysis will be required before this alpha-actinin isoform, which we refer to as SKv, can be classified as muscle or non-muscle alpha-actinin. We propose a new nomenclature to describe the various alpha-actinin genes and their transcripts.

The cytoskeletal protein vinculin is a component of adherens-type junctions where it is one of a number of interacting proteins thought to link the cytoplasmic domain of adhesion receptors to F-actin. Vinculin has been shown to bind to at least three other cytoskeletal proteins, talin, paxillin and alpha-actinin. In this study, we further characterise the talin-binding domain in vinculin using a series of chick vinculin polypeptides expressed as glutathione-S-transferase fusion proteins in Escherichia coli. Thus 125I-talin bound to a fusion protein spanning residues 1-398, but not to those spanning residues 399-881 or 881-1066 in an SDS-PAGE gel-blot assay. We have previously characterised two chick vinculin cDNAs (2.89 kb cDNA and cVin5) which are identical in the region of overlap except that cVin5 lacks coding sequence for residues 167-207. Interestingly, a fusion protein spanning residues 1-398, but lacking residues 167-207, was unable to bind talin. However, further analysis showed that residues 167-207 are insufficient to support binding, and deletion of as few as 31 N-terminal residues abolished binding activity. The results of the gel-blot assay were essentially confirmed using purified fusion proteins adsorbed to glutathione-agarose beads. The smallest vinculin fusion protein able to bind talin contained residues 1-258. This fusion protein was as effective as whole vinculin in inhibiting the binding of 125I-vinculin to talin-coated microtitre wells. Interestingly, mutations which altered the charge characteristics of the highly conserved residues 178 and 181 abolished binding, whereas conservative substitutions were without effect. However, such mutations did not abolish the ability of mutant polypeptides spanning residues 1-398 to target to cell-matrix junctions in Cos cells. We have investigated the possible origin of the cDNA clone cVin5 by defining the structure of a 5' portion of the chicken vinculin gene, and by analysing vinculin transcripts in a variety of adult tissues and embryonic fibroblasts using reverse transcriptase and polymerase chain reaction. Although residues 167-207 are encoded on a separate exon, we have been unable to identify a tissue where this exon is alternatively spliced.

We have determined the complete sequence of chick brain alpha-actinin (892 amino acids; 107,644 Da). The sequence differs from that of smooth muscle alpha-actinin only in the region of the first EF-hand calcium-binding motif, where 27 residues in brain alpha-actinin are replaced by just 22 residues in the smooth muscle isoform. This probably accounts for the different calcium sensitivities of the two isoforms with respect to actin binding. Analysis of the gene structure showed that this region of sequence divergence is encoded by two separate exons whose incorporation is mutually exclusive. We have determined the proportion of the two transcripts in various tissues and cell lines using poly(A)+ RNA and a quantitative assay based on the polymerase chain reaction. MRC-5 fibroblasts and HeLa cells express mRNAs encoding both isoforms, whereas Namalwa lymphoblastoid cells, which lack actin stress fibers, express only the non-muscle mRNA. Both isoforms of alpha-actinin became incorporated into stress fibers and cell-matrix junctions when full-length chick alpha-actinin cDNAs were expressed in monkey COS cells. The levels of chick alpha-actinin mRNAs were found to be serum-inducible, suggesting that alpha-actinin may be an early response gene.

Central nervous system (CNS) tumor cells possess specific receptors for insulin-like growth factors (IGFs) and respond to the growth-promoting effects of IGFs in cell culture. In the present study, we asked whether CNS tumors also produce IGF-binding proteins (BPs) which may modulate the effects of IGFs on CNS tumor cells. Primary cell cultures were established from 20 CNS tumors. Dot blot analysis with 125I-labeled recombinant human IGF-I revealed IGF-binding activity in serum-free conditioned medium from 5 of 7 meningiomas, 7 of 8 malignant gliomas, and 3 of 5 other CNS tumors. Specific IGF BPs in conditioned medium were characterized further by Western ligand and immunoblotting, affinity labeling, and precipitation with specific antibodies against human IGFBP-1, -2, and -3. All conditioned media tested contained an Mr 35,000 BP which was recognized by antiserum against IGFBP-2 and an Mr 24,000 BP that was not recognized by available antisera. Medium conditioned by meningiomas (and one glioma) also contained Mr 45,000 and 50,000 IGF BPs, similar in size and/or immunological properties to growth hormone-dependent BPs present in normal human serum (IGFBP-3). Ligand blotting also showed that meningiomas produce an Mr 29,000 BP; immunoblotting and immunoprecipitation of affinity-labeled IGF-BP complexes confirmed that this BP is recognized by antiserum against IGFBP-1. Immunohistochemistry with specific monoclonal antibodies demonstrated that IGFBP-1 is abundant in pathological specimens of meningiomas and that lower amounts also are detected in malignant gliomas. We conclude that human CNS tumor cells produce a variety of IGF BPs in cell culture, including several that are similar in size and immunological properties to previously characterized human IGF BPs. Immunohistochemistry with specific monoclonal antibodies against IGFBP-1 confirms that this BP is present in vivo, further supporting the concept that IGF BPs may contribute to the regulation of growth in human CNS tumors.

The occurrence of pregnancy-associated endometrial alpha 1-globulin (alpha 1-PEG), a 29-32 kd insulin-like growth factor binding protein, now termed type 1 or IGF-bp1, has been examined in the human ovary by monoclonal and polyclonal antibody based radioimmunoassay and immunohistological techniques. Follicular fluids aspirated from 51 follicles of 32 women undergoing hyperstimulation involving buserelin or clomiphene-based protocols contained 35.5-276.0 ng/ml (mean 101.0 mg/ml) of immunoreactive IGF-bp1. Mean fluid concentrations were three times the level of IGF-bp1 detected in paired serum samples, available for 21 women. Immunoreactive IGF-bp1 in follicular fluid exhibited similar dose-response curves to purified protein and amniotic fluid and immunoreactive IGF-bp1 coeluted in gel filtration with a peak of [125I]-IGF-1 binding corresponding to the elution profile of purified IGF-bp1. Gel filtration also revealed the presence in follicular fluid of a greater than 100 kd binding protein with a binding capacity equal to IGF-bp1 under the conditions employed. A highly significant correlation (P less than 0.001) was found between follicular fluid progesterone and IGF-bp1 and a correlation of lower significance was found between oestradiol and IGF-bp1 (P less than 0.05). However, only low levels of immunoreactive IGF-bp1 were detected in supernatant media of granulosa cells in culture (range undetectable to 2.3 ng/ml). Employing monoclonal antibody-based immunohistology, immunoreactive IGF-bp1 was consistently associated with luteinized granulosa cells of corpora lutea rather than paraluteal cells and its intensity of reactivity appeared to reflect luteal phase steroid hormone profiles. No consistent reactivity was detected in preovulatory follicles and granulosa cells in culture, although reactivity was associated with primordial oocytes. Immunoreactive IGF-bp1 was detected in six of nine supernatant media of explants of luteal tissue obtained from five corpora lutea, with levels ranging from undetectable to greater than 200 ng/ml. These observations suggest that IGF-bp1 is primarily related to luteinization of the granulosa and the resultant luteal cells, and if produced by the luteal cells, additional exogenous factors are required to induce production by granulosa cells in vitro.

We have previously demonstrated that pregnancy-associated endometrial alpha 2-globulin (alpha 2-PEG), the human glycosylated beta-lactoglobulin homologue (HG-BLG), is quantitatively the major secretory soluble protein product of the secretory endometrium during the latter half of the menstrual cycle and decidua spongiosa of the gestational endometrium during early pregnancy, and is principally localized to the glandular epithelium. In the present study employing monoclonal antibodies in immunohistological techniques, the distribution and localization has been examined in normal and pathological tissues of the adult and first-trimester fetus. No significant staining for alpha 2-PEG was detected in any nonreproduction-associated tissue in the normal adult nor any tissue in the fetus. In the adult, most intense staining was associated with the endometrial glandular epithelium in the uterus or in ectopic sites in patients with endometriosis. During the menstrual cycle and pregnancy, appearance of alpha 2-PEG in endometriosis was strongly linked with its appearance in uterine endometrial tissue, suggesting that endometriotic tissue exhibited competence to respond to the same hormonal milieu required to induce synthesis in the uterine endometrium. Localization to the mucosal epithelium of the Fallopian tube was consistent with synthesis of alpha 2-PEG, albeit at low levels, and staining at this site reflected fluctuations of staining within the uterus. Of the pathological specimens examined, staining was only detected in a proportion of ovarian carcinomas. No staining was detected in the mammary gland, a site of beta-lactoglobulin synthesis, whether obtained during pregnancy or lactation.(ABSTRACT TRUNCATED AT 250 WORDS)

Employing a murine monoclonal antibody to pregnancy-associated endometrial alpha I-globulin, a 32-kDa insulin-like, growth factor-binding protein (IGF-BP) and major product of the decidualized endometrium in the human, reactivity has been detected in the luminal epithelium of the sheep endometrium during the preimplantation period of pregnancy. Staining was associated with supranuclear regions of the cytoplasm. Reactivity was restricted to the pregnant uterus and was distributed throughout the luminal epithelium in both caruncular and intercaruncular regions. The reactivity was absent from endometrial glands in intercaruncular regions. Staining was first detected at day 10, peaked at day 14 of gestation and was absent by day 16. The implications of the induction of the production of this protein by the preimplantation blastocyst in the luminal epithelium is discussed with reference to the potential role of IGF-BP in IGF action in the embryo.

We have previously shown that pregnancy-associated alpha 1-globulin, a small molecular weight (32 kDa) insulin-like growth factor-binding protein (IGF-BP), is quantitatively the major secretory protein product of the decidualized endometrium during human pregnancy and is localized principally in the decidual cell. In the present study, employing monoclonal antibodies in immunohistological techniques, the distribution and localization of IGF-BP has been examined in normal and pathological tissues of the adult and first trimester fetus. In the adult, most intense reactivity was associated with endometrial stroma and their derived decidual cells in the uterus or in ectopic sites in patients with endometriosis. During the menstrual cycle, the appearance of IGF-BP in endometriotic tissue was linked with its appearance in uterine endometrial tissue. The only other adult cells where significant staining was detected was in the luteal cells of the corpus luteum. Production of the protein was not a feature of carcinomas. In the fetus, the protein was localized in lymphoid-myeloid progenitor cells and hepatocytes of the liver and at lower levels in testicular Leydig cells and adenocortical cells. These observations suggest highly specific tissue expression of this protein and support a specialized role for this protein in progenitor cells of the lymphomyeloid system, in certain steroid hormone-producing cells and in the decidual cell in pregnancy.

We have previously demonstrated that pregnancy-associated endometrial alpha 2-globulin (alpha 2-PEG), the human glycosylated beta-lactoglobulin homologue (HG-BLG), is quantitatively the major secretory soluble protein product of the secretory endometrium during the latter half of the menstrual cycle and decidua spongiosa of the gestational endometrium during early pregnancy, and is principally localized to the glandular epithelium. In the present study employing monoclonal antibodies in immunohistological techniques, the distribution and localization has been examined in normal and pathological tissues of the adult and first-trimester fetus. No significant staining for alpha 2-PEG was detected in any non-reproduction-associated tissue in the normal adult nor any tissue in the fetus. In the adult, most intense staining was associated with the endometrial glandular epithelium in the uterus or in ectopic sites in patients with endometriosis. During the menstrual cycle and pregnancy, appearance of alpha 2-PEG in endometriosis was strongly linked with its appearance in uterine endometrial tissue, suggesting that endometriotic tissue exhibited competence to respond to the same hormonal milieu required to induce synthesis in the uterine endometrium. Localization to the mucosal epithelium of the Fallopian tube was consistent with synthesis of alpha 2-PEG, albeit at low levels, and staining at this site reflected fluctuations of staining within the uterus. Of the pathological specimens examined, staining was only detected in a proportion of ovarian carcinomas. No staining was detected in the mammary gland, a site of beta-lactoglobulin synthesis, whether obtained during pregnancy or lactation.(ABSTRACT TRUNCATED AT 250 WORDS)

A pregnancy-associated serum protein of the mouse (alpha 1-PAP) has recently been reported to be also synthesized by the non-pregnant female, but not the untreated male, in response to injury. The role of gonadal hormones in this phenomenon has been investigated. Ovariectomy abolished the ability of injury to induce synthesis of alpha 1-PAP. Although administration to ovariectomized animals of oestradiol alone resulted in the appearance of low concentrations of alpha 1-PAP after injury, injection of either progesterone or oestradiol plus progesterone permitted injury to induce concentrations of alpha 1-PAP similar to those observed in intact injured females. Castrated mice, like intact males, failed to produce alpha 1-PAP in response to injury, but administration of either oestradiol or oestradiol plus progesterone caused injury to induce concentrations of alpha 1-PAP similar to those in injured intact females. Administration of testosterone to intact females abolished the alpha 1-PAP response to injury and administration of oestradiol plus progesterone in intact males failed to induce the same response as in castrated males. These findings suggest that testosterone can inhibit the synthesis of alpha 1-PAP in response to injury in both sexes whereas oestradiol and progesterone exhibit different abilities in males and females to facilitate the induction of alpha 1-PAP synthesis by injury.

We previously reported that the major secretory protein of the endometrium during the first trimester of pregnancy, pregnancy-associated endometrial alpha 2-globulin (alpha 2-PEG), also represented the major secretory protein during the mid- to late luteal phase. Recently, the major secretory endometrial protein during the luteal phase of the menstrual cycle was identified as glycosylated PRL (G-PRL). Although certain properties of alpha 2-PEG resemble G-PRL, in this study G-PRL was demonstrated to be immunochemically distinct from alpha 2-PEG, and deglycosylation of alpha 2-PEG produced a protein unrelated to PRL. The sequence of the 38 N-terminal amnio acid residues of alpha 2-PEG was determined by a gas phase sequenator. A sequence of Met-Asp-Ileu-Pro-Gln-Thr-Lys-Gln-Asp-Leu-Glu-Leu-Pro-Lys-Leu-Ala-G ly-Lys-Trp- His-Ser-Met-Ala-Met-Ala-Thr-Asn-?-Ileu-Ser-Leu-Met-Ala-Thr-Leu-Lys -Ala-Pro was obtained which was unique and unrelated to that of PRL. However, sequence homology between alpha 2-PEG and the major milk whey protein beta-lactoglobulin of the horse was demonstrated. The data indicate that alpha 2-PEG is a unique protein and is a human homolog of the beta-lactoglobulin family.

Bovine type II collagen administered to non-pregnant female DBA/1 mice caused arthritis in 55% of animals with a mean onset time of 70 days following immunization. Of collagen-treated females subsequently becoming syngeneically pregnant before the onset of arthritis, all developed the disease within 10 days of parturition, representing an earlier onset, compared to non-pregnant females, of 41 days. This earlier onset also occurred in females with an allogenic pregnancy, but did not occur in females resorbing their fetuses (only syngeneic pregnancies were examined). In females with arthritis at the time of conception a significant remission was observed during pregnancy followed by exacerbation post-partum. This sequence of remissions during pregnancy and exacerbations post-partum occurred with each pregnancy (parties of up to four studied). The treatment of multiparous females with collagen demonstrated that pregnancy does not provide long-term protection against the development or progression of arthritis, as such females were equally susceptible to post-partum onset of collagen-induced arthritis (CIA) and the remissions and exacerbations described above. The modifying effect of pregnancy on CIA in mice is complex and does not appear to be the result of a single pregnancy-associated phenomenon.

Endometrium from postmenopausal women with endometrial adenocarcinoma was examined immunohistochemically using a monoclonal antibody to pregnancy-associated endometrial alpha 2-globulin (alpha 2-PEG), the major secretory protein of the glandular epithelium during the late luteal phase of the menstrual cycle and early pregnancy. Specimens were obtained at initial diagnostic curettage and at hysterectomy after medroxyprogesterone acetate (MPA) therapy. alpha 2-PEG was not detected in any malignant tissue irrespective of histological differentiation. Non-malignant endometrium obtained in association with malignant tissue was negative for alpha 2-PEG before treatment although after MPA therapy all specimens obtained exhibited marked alpha 2-PEG localization in glands. In four specimens endogenous alkaline phosphatase was observed consistently only in the malignant endometrium. Malignant endometrium does not appear to synthesize alpha 2-PEG nor is its synthesis induced by an oral progestogen, so that it does not represent a useful marker for endometrial carcinoma. Non-malignant endometrium in postmenopausal women appears to be fully capable of alpha 2-PEG production after stimulation with an oral progestogen.

Using an electroimmunoassay, the murine pregnancy-associated protein alpha 1-PAP was detected in the sera of virgin MF1 but not C57BL/10 female mice. During pregnancy, alpha 1-PAP levels rose in both strains, although concentrations were higher in the latter and in both fell towards term. Using the unlabelled peroxidase anti-peroxidase (PAP) staining procedure, alpha 1-PAP was detected within mononuclear leukocytes, the majority resembling macrophages, in the small and large intestinal mucosae, Peyer's patches and hepatocytes of virgin MF1 but not C57BL/10 females. During pregnancy, alpha 1-PAP positive cells were observed in each of these sites and in the decidua and placenta of both strains. Quantitative studies revealed that the incidence of alpha 1-PAP positive cells in the gut and associated lymphoid tissues reflected the circulating levels of the protein. In the placenta, the frequency and intensity of alpha 1-PAP positive staining was also reduced towards parturition. In contrast, hepatocyte staining remained constant throughout gestation in both strains. Our observations suggest that there may be at least two types of alpha 1-PAP synthesis operative and that circulating levels of the protein in female mice are influenced by strain, pregnancy and stage of gestation. These findings are discussed in relation to the cell types involved, their contribution to serum levels and the possible role of alpha 1-PAP in fetal allograft survival.

A murine pregnancy-associated protein (alpha 1-PAP) with alpha 1-electrophoretic mobility and an estimated molecular weight of 150 000 was present in serum from pregnant C57BL/10 mice but could not be detected in serum of mature non-pregnant females and males. During pregnancy alpha 1-PAP was first detected on Day 7, rose to maximum levels between Days 12 and 14, and thereafter declined during the remainder of pregnancy and was undetectable by Day 8 post partum. The protein was also detected in the serum of females, but not males, subjected to an inflammatory stimulus. Examination of the alpha 1-PAP levels during an acute-phase response in females demonstrated that the protein behaved as a typical classical acute-phase reactant, although the levels were only 10% of those observed during Days 12 to 14 of pregnancy. alpha 1-PAP therefore appears to represent a hitherto undescribed female-specific acute phase reactant in the mouse.

We have previously shown that pregnancy-associated endometrial alpha 1-globulin, a small molecular weight insulin-like growth factor-binding protein (IGF-BP), is quantitatively the major secretory protein product of the decidualized endometrium during human pregnancy. In the present study, employing monoclonal antibodies raised against this protein in an immunohistological technique, the cellular localization of the protein has been examined in the decidua and placenta during pregnancy. During the first trimester the protein was principally associated with the decidual cell in the decidualized decidua compacta region of the endometrium with both cytoplasmic and extracellular matrix-associated staining patterns being detected. No extensive staining was observed in the placenta. At term the protein was localized in similar cells in the placental bed and endometrium associated with the amniochorion but not in the placenta. These studies suggest that the decidual cell represents the major source of IGF-BP during pregnancy and have relevance to the origin of amniotic fluid IGF-BP and the paracrine role of the decidual cell in the control of trophoblast growth.

The levels of the acute-phase reactant serum amyloid P-component (SAP) were measured by quantitative rocket immunoelectrophoresis in pregnant and non-pregnant DBA/1 female mice with or without collagen-induced rheumatoid arthritis (CIA). Non-pregnant animals with CIA showed elevated SAP titres related to the severity of the disease. Pregnancy alone also caused increased SAP levels equivalent to those found in animals with established CIA but which were virgin. The clinical remission seen in arthritic animals during pregnancy was not associated with reductions in circulating SAP levels. Increasing parity, however, caused a lowering of SAP levels in animals with or without CIA compared to the primiparous individuals. Pregnancy causes a strain-dependent elevation of serum SAP which is not further elevated by CIA, thus limiting the usefulness of SAP measurements in assessment of disease progression or remission during gestation.

Pregnancy-associated endometrial alpha 1-globulin (alpha 1-PEG), a 29,000 mol wt insulin-like growth factor-binding protein, is the major secretory protein of the human endometrium from the latter half of the first trimester to the end of pregnancy. It is also produced and secreted by nonpregnant endometrial tissue, especially during the late secretory phase of the menstrual cycle. We studied the localization of this protein in the endometrium during different phases of the cycle using immunohistochemistry with monoclonal antibodies. Immunostaining was first observed in the midsecretory phase and was most consistently detected during the late secretory phase, during which it was principally associated with stromal cell populations. These cells were localized initially surrounding spiral arteries and subsequently also in the subluminal epithelial region of the endometrium. These results suggest that alpha 1-PEG synthesis is principally associated with the process of stromal cell differentiation. i.e. predecidualization. However, the presence of alpha 1-PEG was not directly correlated with the presence of mature predecidual cells, suggesting that its synthesis is not an inherent feature of this cell.

The distribution of alpha 2-PEG, a human analogue of beta-lactoglobulin, in endometrium at different phases of the cycle was determined using immunohistochemistry with monoclonal and polyclonal antibodies. In the epithelial cells of glands in the functional zone of the endometrium, alpha 2-PEG was first detectable from Days 19 to 21 during the mid-luteal phase and maximal immunostaining was observed during the end of the late luteal phase. Intense staining in the glandular secretions and weaker staining in surface luminal epithelial cells during this period were observed. A minor population of basal glands contained alpha 2-PEG during the follicular phase. These results suggest that alpha 2-PEG synthesis by the glandular epithelium of the regenerated endometrium is hormonally regulated. Maximal staining occurring during the late luteal phase suggests that regulation may be related to the hormonal requirement for pre-decidualization rather than that required for histologically defined glandular epithelial secretion.

The levels of two murine acute phase proteins, serum amyloid P component (SAP) and haptoglobin, have been measured in the serum of C57BL/10 female mice during syngeneic and allogeneic pregnancy. Both syngeneic and allogeneic pregnancy resulted in alterations in the levels of these proteins as compared to those observed in virgin females. Syngeneic mating resulted in an increase in concentration of both proteins during the final 3 days of pregnancy. During allogeneic pregnancy, SAP levels, after a transient increase on day 4, rose from days 6-8 and, after remaining relatively stable, increased from day 12 to reach maximum levels on day 18 of pregnancy. Levels fell dramatically during the immediate post-partum period. In contrast, although levels of haptoglobin also increased from days 6-8, for the remainder of pregnancy these increased levels remained stable. The implications of these findings are discussed in relation to the mechanisms of regulation of acute phase reactants and the immunological relationship between the mother and fetus.

Pregnancy-associated endometrial alpha 1-globulin (alpha 1-PEG) is quantitatively the major secretory protein product, synthesized and secreted in vitro, of the human decidualized endometrium during pregnancy. This protein has been purified from cytosolic extracts of this tissue and has now been characterized as a 32 kDa somatomedin/insulin-like growth factor (IGF)-binding protein. Immunoreactive alpha 1-PEG isolated from amniotic fluid exhibited identical physiochemical properties and IGF-I-binding characteristics. In cytosolic extracts of pregnancy endometrium, in incubation medium of this tissue and in amniotic fluid, the 32 kDa protein represented the major alpha 1-PEG immunoreactive protein and major IGF-I-binding component. Purified alpha 1-PEG and incubation medium of pregnancy endometrium competed for IGF-I with placental membrane IGF receptors in vitro. The implications of the endometrial source of IGF-I-binding protein are discussed with reference to the origin of the amniotic fluid and serum small Mr IGF-binding protein and to the suggested paracrine effect upon trophoblast proliferation.

Intrauterine instillation of heat-deaggregated glycogen in virgin mice induced a marked but transient luminal infiltration of polymorphonuclear leukocytes (PMNL). Similar injections of glycogen 2 days post coitum resulted in termination of pregnancy in the majority of treated females. Embryos recovered on Day 4 of pregnancy from the glycogen-treated females, before the expected time of implantation, had developed to the cavitated blastocyst stage, and were capable of undergoing characteristic outgrowth when cultured in vitro. A small proportion, however, appeared abnormal and did not develop in vitro. Cavitated blastocysts obtained from control mated females did not undergo outgrowth in vitro in the presence of intact PMNL, homogenates of these cells or a low molecular weight (less than 1500) fraction of the homogenates and after culture appeared similar to the abnormal blastocysts recovered from glycogen-treated uteri. It is proposed that the anti-fertility effect of PMNL infiltrates is due to a low molecular weight component of these cells which acts in utero and is cytotoxic to preimplantation blastocysts before their hatching from the zona pellucida.

A murine pregnancy-associated protein (alpha 1-PAP) was demonstrated by immunoperoxidase (PAP) staining within Ia positive cells in gut-associated lymphoid tissue and intestinal mucosae of normal female MF1 but not C57BL/10 mice. These immunohistochemical findings reflect the differences in serum alpha 1-PAP concentrations between the two strains, being 50-fold higher in the MF1 females. Staining for alpha 1-PAP was also detected within perivascular and periportal hepatocytes of MF1 mice. Using a combined indirect immunoperoxidase (PAP)/direct immunofluorescence procedure, cytoplasmic alpha 1-PAP was demonstrated in a proportion of plasma cells secreting the IgA isotype. In contrast with human pregnancy-associated alpha 2-glycoprotein (alpha 2-PAG) which was observed in the majority of IgA plasma cells within the lamina propria of human gastrointestinal mucosae, on average only 15% of IgA producers in corresponding mouse tissue were positive for alpha 1-PAP. As with the localization of alpha 2-PAG, no other classes of Ig-producing cells stained for alpha 1-PAP. These observations strengthen recent proposals that alpha 1-PAP is a murine analogue of human alpha 2-PAG, a glycoprotein with immunosuppressive properties.

Monoclonal and polyclonal antibodies to pregnancy-associated endometrial alpha 2-globulin (alpha 2-PEG), a glycosylated human beta-lactoglobulin homologue, were used in an immunohistological technique to determine the cellular localization of this protein in the decidua and placental tissues during pregnancy. During the first trimester the protein was principally localized to the glandular epithelium of the decidua spongiosa region of the endometrium with only weak staining associated with glands of the decidualized decidua compacta region. No significant cellular staining was detected in the decidua capsularis. At term in the decidua of the amniochorion and the placental bed weak staining for alpha 2-PEG was only associated with the epithelium of attenuated glands. No significant staining was detected in the placenta during pregnancy. These results suggest that the epithelium of glands associated with non-decidualized stroma represents the primary source of alpha 2-PEG during the first trimester and that a function of the decidua spongiosa in early pregnancy may be related to production of alpha 2-PEG. The decline in production of alpha 2-PEG during pregnancy is suggested to result from involution of the decidua spongiosa and at term the attenuated glands of the decidua represents the source of alpha 2-PEG.

Monoclonal antibodies were raised against pregnancy-associated endometrial alpha 1-globulin (alpha 1-PEG), a 32 KD insulin-like growth factor binding protein (IGF-BP), which represents a major secretory product of the human decidualized endometrium during pregnancy. This class of IGF-BP has been implicated in the modulation of action, inhibitory and stimulatory, of insulin-like growth factors. Immunization with the protein purified from pregnancy endometrium resulted after myeloma fusion in the isolation of six hybridoma clones and the antibodies produced were characterized. The Ka of the antibodies ranged between 4.75 x 10(9) M-1 and 0.7 x 10(8) M-1. In Western blots all monoclonal antibodies reacted with purified protein of molecular weight 32 KD and specifically detected this IGF-BP species in culture medium and cytosolic extracts of pregnancy endometrium and amniotic fluid. The monoclonal antibodies appear to define three epitope-bearing regions as evidenced by their reactivity to polypeptide fragments of the protein. After synthesis and secretion by tissue explants in vitro the protein is susceptible to cleavage into fragments possessing different monoclonal antibody-defined reactivity. Employing immunohistochemical techniques the protein was principally localized to decidual cells in tissue sections of pregnancy endometrium and solely to these cells after enzymic digestion of the tissue. The implications of these results are discussed with respect to potential role of IGF-BP in the action of IGF upon the IGF-1 receptor-bearing populations, including lymphocytes and trophoblast cells, D in the decidua.

A protein exhibiting immunochemical cross-reactivity with the murine alpha 1-pregnancy-associated protein (alpha 1-PAP) has been detected in the sera of female rats. The protein has an alpha 1-electrophoretic mobility, an estimated molecular weight of 150,000 and is readily detectable in the sera of nonpregnant female rats of each strain examined. During pregnancy, the serum concentration increases up to five-fold, reaching maximal levels at the same gestational stage as alpha 1-PAP in the mouse. Immunohistochemical studies revealed a staining pattern for the rat protein similar to that seen for alpha 1-PAP in the mouse, with positive cells being observed in lumbar lymph nodes, the lamina propria of gut mucosa, Peyer's patches, intracellularly in some hepatocytes and, during pregnancy, also in the placenta and metrial gland. On the basis of these immunochemical, physicochemical and immunohistochemical findings, it is proposed that the protein detected in rat sera represents the analogue of the murine alpha 1-PAP, and that the rat strains examined more closely resemble high endogenous alpha 1-PAP-producer mouse strains. It is proposed that alpha 1-PAP, and a number of other rodent pregnancy proteins recently described, may be used in functional studies of human alpha 2-PAG.