Identification and characterization of a new member of the prolactin family, placental lactogen-I variant

This report describes the identification and characterization of a new member of the placental prolactin (PRL) family, termed placental lactogen-I variant (PL-Iv). PL-Iv was isolated from medium conditioned by late gestation placental explants. Rat PL-Iv was found to be closely related to rat PL-I. Amino-terminal sequence analysis indicated that PL-Iv shared approximately 88% sequence identity with the amino terminus of PL-I. PL-Iv proteins cross-reacted with antiserum to recombinant mouse PL-I and PL-Iv mRNA hybridized with a PL-I cDNA. Multiple PL-I and PL-Iv species were present in placental cytosol. Despite the structural similarities between PL-I and PL-Iv, distinct differences were also evident. Antibodies generated to the amino-terminal 19 amino acids of PL-Iv specifically recognized PL-Iv, while failing to recognize PL-I. Secreted PL-Iv had an affinity for concanavalin A, whereas secreted PL-I lacked affinity for the lectin. PL-I was predominantly secreted as a 36-40-kDa species and PL-Iv was predominantly secreted as a 33-kDa species. Furthermore, PL-I and PL-Iv were synthesized at different times during gestation and by different cell types. PL-I was synthesized by trophoblast giant cells during the first half of gestation, while PL-Iv was predominantly synthesized by spongiotrophoblast cells during the later stages of gestation. PL-Iv was shown to stimulate the proliferation of rat Nb2 lymphoma cells, an in vitro measure of lactogenic activity. In summary, PL-Iv shares structural similarities with PL-I; however, it shows other structural differences in addition to unique cell- and temporal-specific patterns of expression in the rat chorioallantoic placenta.     

Progressive expression of trophoblast-specific genes during formation of mouse trophoblast giant cells in vitro

The expression of a battery of trophoblast-specific mRNAs was studied during trophectoderm development in vivo and in vitro to assess the use of these mRNAs as markers of trophoblast differentiation and to examine lineage relationships between various trophectoderm derivatives. In situ hybridization of sectioned day 6.5–18.5 mouse embryos localized mRNAs for mouse placental lactogens I and II and mouse proliferin (PLF) to trophoblast giant cells and proliferin-related protein mRNA to the spongiotrophoblast and giant cell layers. A fifth marker, cDNA 4311, was found only in spongiotrophoblast. Day 3.5 blastocyst outgrowths and day 7.5 diploid extraembryonic ectoderm (EX) and ectoplacental cone (EPC) were then cultured to produce polyploid giant cells in vitro. Cultures were processed for in situ hybridization after 2, 4, or 6 days. EX and EPC both formed secondary giant cells, which expressed all markers in the same sequence as was observed in vivo, and primary giant cells in blastocyst outgrowths expressed the early giant cell markers PLF and PL-I on days 4 and 6 of culture. EPC progressed through the sequence 2 days ahead of EX, indicating commitment of EPC to giant cell formation. These results suggest that EX, EPC, and primary and secondary giant cells all share in a common pathway of differentiation and that the highly ordered sequence of gene expression characteristic of this pathway occurs similarly in vivo and in vitro. © 1993 Wiley-Liss, Inc.     

Changes in the temporal and spatial expression of H beta 58 during formation and maturation of the chorioallantoic placenta in the Rat

Cloning and sequencing of a cDNA amplified by RNA fingerprinting at the implantation site of pregnant rats revealed 80% similarity with H beta 58, previously shown to be essential for formation of the chorioallantoic placenta in the mouse. H beta 58 mRNA was detected in the endometrium of hormonally sensitized rats stimulated to undergo decidualization and in the contralateral uterine horns lacking a decidual stimulus, indicating that uterine expression of H beta 58 mRNA did not require decidualization or the presence of a blastocyst. Immunodetection in the early postimplantation uterus (Days 6-8 of pregnancy) showed H beta 58 localized in the luminal and glandular epithelia and some stromal cells. Decidual cells at Day 6 of pregnancy expressed H beta 58, and by Day 9 of pregnancy, the protein localized throughout the maternal decidua. The temporal and spatial distribution of H beta 58 in the developing chorioallantoic placenta was assessed at Days 10, 12, and 14 of pregnancy. Immunoreactive H beta 58 localized to erythroid cells within the developing fetal vasculature of the chorioallantoic primordia at Day 10 of pregnancy. By Day 12, the fetal vasculature extended into the placental labyrinth, and the erythroid stem cells continued to strongly express H beta 58. At Day 14 of pregnancy, immunoreactivity became evident in the trophoblast giant cells and syncytiotrophoblast of the fetal placenta. As the chorioallantoic placenta matured (Day 18), H beta 58 mRNA was 3.6-fold higher in the labyrinth compared with the junctional region. Stable cell lines (HRP/LRP) isolated from the rat labyrinthine placenta expressed H beta 58 mRNA and protein. The expression pattern of H beta maternal and fetal placental tissues and its early expression in fetal erythroid stem cells during formation and maturation of the chorioallantoic placenta suggest that H beta 58 plays key roles in the regulatory networks that control hematopoietic development and placentation.     

Expression of steroidogenic acute regulatory protein mRNA in rat placenta during mid-late pregnancy

The production of a steroid hormone in the placenta is essential for maintaining the pregnancy and developing the fetus during gestation. In various steroidogenic tissues (including gonads and adrenal cortex), the steroidogenic-acute-regulatory protein (StAR) acutely transfers cholesterol from the outer to the inner mitochondrial membrane for rapid steroidogenesis. Although steroid hormones were synthesized in the rat placenta, the developmental expression of StAR has been poorly understood in the rat placenta during mid-late pregnancy. Therefore, the aim of the present study was to establish the expression and localization of StAR mRNA in the rat placenta during mid-late pregnancy using Northern blots and in situ hybridization. The Northern blot analysis showed that the StAR mRNA expression significantly changed as the gestation day (GD) progressed. The placental expression of StAR mRNA increased between GD 11 and 13, and then slightly decreased until term. In situ hybridization showed a strong StAR expression in giant trophoblast cells on GD 11 and 13, and a moderate expression in trophoblast and stroma cells within the villi of the labyrinth zone throughout the pregnancy. In this study, we reveal for the first time the existence of StAR mRNA in steroidogenic cells of the placenta during mid-late pregnancy. In conclusion, our results suggest that StAR may regulate steroidogenesis in the rat placenta to maintain the pregnancy and developing the fetus.     

Ontogeny of adenosine deaminase in the mouse decidua and placenta: immunolocalization and embryo transfer studies

This study has determined the cellular site of adenosine deaminase (ADA) expression in the mouse during development from Days 5 through 13 (day vaginal plug was found = Day 0) of gestation. Developmental expression of ADA progressed in two overlapping phases defined genetically (maternal vs. embryonal) and according to region (decidual vs. placental). In the first phase, ADA enzyme activity increased almost 200-fold in the antimesometrial region (decidua capsularis + giant trophoblast cells) from Days 6 through 9 of gestation but remained low in the mesometrial region. Immunohistochemical staining revealed a major localization of ADA to the secondary decidua. In the second phase, ADA activity increased several-fold in the placenta (labyrinth + basal zones) from Days 9 through 13 of gestation but remained low in the embryo proper. Immunohistochemical staining revealed a major localization of ADA to secondary giant cells, spongiotrophoblast, and labyrinthine trophoblast. Regression of decidua capsularis and growth of the spongiotrophoblast population accounted for an antimesometrial to placental shift in both ADA enzyme activity and a 40-kDa immunoreactive protein band. To verify a shift from maternal to fetal expression, studies were performed with two strains of mice (ICR, Eday) homozygous for a different ADA isozyme (ADA-A, ADA-B). Blastocysts homozygous for Adab were transferred to the uterus of pseudopregnant female recipients homozygous for Adaa. The isozymic pattern in chimeric embryo-decidual units analyzed at Days 7, 9, 11, and 13 revealed a predominance of maternal-encoded enzyme at Days 7 through 11 of gestation and a shift to fetal-encoded enzyme by Day 13. Thus, maternal expression of ADA in the antimesometrial decidua may play a role during establishment of the embryo in the uterine environment, whereas fetal expression of ADA in the trophoblast might be important to placentation.     

Trophoblast giant cell release of placental lactogens: temporal and regional characteristics

Placental lactogen (PL) production by rat trophoblast giant cells was studied using in vitro methods. The influence of trophoblast giant cell location within the conceptus and day of trophoblast giant cell isolation on the type of PL released in vitro were investigated. The effect of trophoblast giant cell location on the amount of PL, progesterone, and testosterone released in vitro was also evaluated. Trophoblast giant cells release two types of PLs in vitro; a high-molecular-weight lactogen, PL-1, and a low-molecular-weight lactogen, PL-2. The type of PL released by trophoblast giant cells was not influenced by their location within the conceptus at the time of dissection. Location did influence the amount of hormone produced by trophoblast giant cells. Mural trophoblast giant cells were more active in the production of PL, progesterone, and testosterone. The type of PL released by trophoblast giant cells is highly dependent upon the day of gestation the cells are removed for study. Trophoblast giant cells isolated on Day 10 of gestation release predominantly PL-1, while those cells isolated 24 hr later (Day 11 of gestation) release predominantly PL-2. The switch from PL-1 to PL-2 production that occurs in vivo does not occur under the in vitro conditions employed in this report.     

Assessment of expression of the receptor for colony-stimulating factor-1 (fms) in bovine trophoblast.

Bovine trophoblast was employed to assess the questions of whether the receptor for CSF-1 is expressed by noninvasive trophoblast and whether expression changes with differentiation within placentomes. Bovine placental poly(A) mRNA contained sequences cross-reactive with cDNA probes to c-fms and v-fms. Using a monoclonal antibody to v-fms, immunohistochemistry of postattachment bovine trophoblast showed expression of an fms-like protein between Day 29 and term. Expression occurred in both the intercotyledonary and cotyledonary trophoblast. Reactivity that was fms-like was also demonstrated on preattachment conceptuses flushed at Days 14 and 7 of gestation and on Day 7 embryos derived from in vitro oocyte maturation and fertilization. Unexpectedly, in the second half of pregnancy some cells, including binucleate cells, showed nuclear rather than cytoplasmic reactivity to the antibody. These data indicate expression of an fms-like protein in bovine placenta that does not correlate with properties of trophoblast cell invasiveness or major morphological differentiation. The data do support a universal role for this protein during mammalian placental development.     

Transgenic major histocompatibility complex class I antigen expressed in mouse trophoblast affects maternal immature B cells

We have produced transgenic mice using the mouse placental lactogen type II promoter to force and restrict the expression of the mouse major histocompatibility complex (MHC) class I molecule, H-2K(b), to the placenta. We show that the transgenic MHC antigen H-2K(b) is expressed exclusively in trophoblast giant cells from Day 10.5 until the end of gestation. This expression affects neither the fetal development nor the maternal tolerance to the fetus in histoincompatible mothers. We have used the 3.83 B cell receptor (BcR) transgenic mouse line to follow the fate of H-2K(b)-specific maternal B cells in mothers bearing H-2K(b)-positive placentas. Our results suggest that transgenic H-2K(b) molecules on trophoblast giant cells are recognized by 3.83 BcR-transgenic B cells in the bone marrow of pregnant females. This antigen recognition triggers the deletion of a bone marrow B cell subpopulation, including immature and transitional B cells. Their percentage decreases during the second half of gestation and is down to 8% on Day 17.5, compared to 22% in the (3.83 Tg female x Fvb) control group. This deletion might contribute to the process of maternal tolerance of the conceptus.     

Immunocytochemical localization of relaxin in the golden hamster (Mesocricetus auratus) during the last half of gestation

The objective of this study was to determine the tissue source of relaxin in pregnant hamsters by immunocytochemical techniques. Ovarian, uterine, and placental tissues were recovered from hamsters on Days 8, 10, 12, 14, and 15 of gestation and processed for light microscopy. Relaxin immunoreactivity was localized in tissue sections by the avidin-biotin-peroxidase technique using antiserum to porcine relaxin. On Day 8 of gestation, relaxin immunoreactivity was localized in primary giant trophoblast cells (GTC-1s) adjacent to the uterine decidua. On Day 10, relaxin immunoreactivity was localized in GTC-1s, secondary giant trophoblast cells (GTC-2s) adjacent to the ectoplacental cone, and endometrial granulocytes in the wall of sheathed arteries. On Day 12, relaxin immunoreactivity was observed primarily in GTC-2s interspersed among cells of the placental trophospongium but not in cells of the placental labyrinth. The intensity of staining and number of relaxin immunoreactive GTCs increased between Days 12 and 14 but was decreased by Day 15 PM. Relaxin was not localized in uterine glands or corpora lutea. These observations suggest that the placenta is the tissue source of relaxin in pregnant hamsters.     

Expression of carbonic anhydrase IV in mouse placenta

Carbonic anhydrase (CA) facilitates acid-base transport in several tissues. Acidosis upregulates membrane-bound SDS-resistant hydratase activity in various tissues and CA IV mRNA in rabbit kidney. This study was designed to assess whether the expression of membrane-bound CA IV isozyme in mouse placenta is regulated developmentally and by maternal ammonium chloride loading at the end of pregnancy. For this purpose we used Northern blot analysis, Western blots of microsomal membranes, and immunocytochemistry. The expression of CA IV mRNA on Northern blots tripled from day 11 to day 15 and then remained stable until the end of pregnancy. Expression of CA IV immunoreactive protein on Western blot tripled from day 11 to day 15 and decreased almost to baseline by day 19. Strong staining for CA IV was detected by immunocytochemistry in labyrinthine trophoblast, in the endodermal layer of the yolk sac (both intra- and extraplacental) and in the uterine epithelium. Weak staining was observed in most fetal endothelial cells at 11 days but not later in gestation. Maternal acidosis did not upregulate the expression of CA IV mRNA or CA IV immunoreactive protein. Thus CA IV expression in mouse placenta is developmentally regulated. Maternal acidosis during the last quarter of pregnancy does not upregulate CA IV mRNA or CA IV immunoreactive protein.     

Measurement of plasma and tissue relaxin concentrations in the pregnant hamster and fetus using a homologous radioimmunoassay

A homologous hamster relaxin RIA was developed to evaluate plasma and tissue concentrations of relaxin in the latter half of pregnancy in this species. Relaxin protein and mRNA were localized using antibodies developed to synthetic hamster relaxin and gene-specific molecular probes, respectively. Molecular weight and isoelectric point of the synthetic and native hormones were identical by electrophoretic methods, and synthetic hamster relaxin was active in the mouse interpubic ligament bioassay. Synthetic hormone was used as tracer and standard with rabbit antiserum to the synthetic hormone in the RIA. Relaxin was assayed in blood samples recovered from the retro-orbital plexus on Days 6, 8, 10, 12, 14, 15, and 16 of gestation and on Days 1 and 5 postpartum. Relaxin was first detected on Day 8 of gestation (3.7 +/- 0.6 ng/ml), increased to reach a maximum in the evening of Day 15 (826.0 +/- 124.0 ng/ml), and decreased by Day 16 (day of parturition). Relaxin concentrations were assayed in aqueous extracts of implantation sites (Days 6, 8, and 10) and chorioallantoic placentae (Days 12, 14, and 15). Concentrations were low on Day 6 (0.02 +/- 0.001 microg/g tissue), increased to Day 15 (6.96 +/- 0.86 microg/g tissue), and subsequently declined by the evening of Day 15. Relaxin protein and mRNA were localized to primary and secondary giant trophoblast cells in the chorioallantoic placental trophospongium. However, relaxin protein was not localized in ovaries of pregnant animals or oviductal tissues of cycling animals. Significant quantities of relaxin were detected in the serum of fetal hamsters recovered on Day 15.     

Localization and subcellular distribution of prolyl oligopeptidase in the mouse placenta

Prolyl oligopeptidase (POP) is a serine endopeptidase which selectively digests a -Pro-X- peptide bond. Our previous study showed that POP mRNA was strongly expressed in the spongiotrophoblast of the mouse placenta at E17.5, suggesting its importance in development. To gain more insight into POP’s role during gestation, we investigated its expression using different developmental stages of placenta. As a result of in situ hybridization, we found that localization of POP mRNA changed at E12.5. POP mRNA was strongly expressed in the spongiotrophoblast and labyrinth at E10.5 and E11.5 but thereafter only in the spongiotrophoblast. Immunohistochemistry revealed that POP was present in the parietal trophoblast giant cell, the spongiotrophoblast cell, and the labyrinth at E11.5 but the strong expression in the labyrinth was maintained only in the canal-associated and sinusoidal trophoblast giant cells at E16.5 and E18.5. To determine subcellular distribution of the POP protein, we fractionated the placental extract into cytoplasmic, membrane, and nuclear subfractions. By Western blot analysis, POP was detected in the cytoplasmic and membrane fractions but not in the nuclear fraction at E11.5 and E16.5. Interestingly, the cytoplasmic POP exhibited higher enzymatic activity than the membrane-associated type. These data suggest that the cytoplasmic and membrane-associated POP have distinct roles in different types of placental cells.     

Immunohistochemical analysis of protein gene product 9.5, a ubiquitin carboxyl-terminal hydrolase, during placental and embryonic development in the mouse.

Protein gene product 9.5 (PGP9.5) is expressed at high level in the neural and neuroendocrine systems. We investigated the localization and degree of expression of PGP9.5 in the developing mouse placenta and embryo at 6.5, 10.5 and 14 days of gestation using an immunohistochemical technique. At 6.5 days of gestation PGP9.5 was detected at various levels in decidual and primary trophoblast giant cells in the placenta, and in embryonic ectodermal cells in the embryo. At 10.5 and 14 days of gestation PGP9.5 was expressed at moderate to strong levels in neurons in the embryo, but rarely in the placenta. These findings suggest that the protein may play a significant role in implantation and placental development, and differentiation of embryonic ectoderm.     

Identification of three prolactin-related hormones as markers of invasive trophoblasts in the rat

An expressed-sequence tag database search has identified three rat cDNA clones in the prolactin/growth hormone family, including a homologue of mouse proliferin-related protein (PRP). The encoded proteins of the two novel clones, designated prolactin-like proteins L (PLP-L) and M (PLP-M), are predicted to be synthesized as precursors of 229 and 227 amino acids, modified by N-linked glycosylation, and secreted as mature glycoproteins of 199 and 200 residues, respectively. Murine homologues to PLP-L and PLP-M were also identified. The open reading frame of rat PRP encodes a precursor protein of 245 amino acids and predicts a secreted 215-amino acid glycoprotein with 81% identity to mouse PRP. All three rat mRNAs are expressed in the placenta, and expression is not detected in other tissues. PLP-L mRNA expression is observed from Days 11-20, with highest levels at Day 13; highest levels of PLP-M are observed from Day 11 until parturition, with peak levels also on Day 13; and highest levels of PRP are also observed from Day 11 until term, with maximal expression on Day 17. All three genes are most highly expressed in invasive trophoblast cells lining the central placental vessel. The identification of molecular markers for endovascular trophoblasts serves to highlight the invasive nature of rodent placentation and may prove useful for future studies of placental function.     

金属蛋白酶-2(MMP-2)和金属蛋白酶抑制因子(TIMP-1,-3)mRNA在小鼠胎盘中的表达

本实验利用原位杂交对小鼠妊娠不同时期胎盘中MMP－2,TIMP－2,－3mRNA的表达进行了研究。结果表明;MMP－2主要在具有很强的侵润能力的海绵滋养层细胞中表达,到妊娠13．5天时,MMP－2的表达明显降低,说明此时的滋养层细胞基本上失去侵润能力。TMIP－1和TMIP－3在滋养层细胞和蜕膜细胞中都有表达,这两种抑制因子的协同表达,一方面能够调控滋养层细胞侵入子宫内膜的深度,另一方面,滋养层细胞自身既表达MMP－2又表达TIMPs,可能对其自身有保护作用,使得MMP的水解功能局限于子宫蜕膜的特定区域。在妊娠10．5天,滋养层巨细胞同时表达TIMP－1,－3mRNA,这可能与其功能的转换是一致的;因为此时小鼠滋养层巨细胞体积最大,且不再增殖,同时其功能屯从侵入型向内分泌型转换。所以,MMPs和TIMPs在小鼠滋养层细胞和子宫蜕膜中的协同表达表明其在着床过程中可能发挥重要作用。     

Mammary gland differentiation in hypophysectomized, pregnant mice treated with corticosterone and thyroxine.

Swiss Webster mice were hypophysectomized or sham-operated on Day 11 of pregnancy. The animals were fitted s.c. with osmotic minipumps containing either corticosterone (B) dissolved in Molecusol (Pharmatec, Alachua, FL) or the vehicle alone immediately after they were hypophysectomized. Animals in some of the experimental groups also received thyroxine (T4) in their drinking water. The mice were killed on Day 18 of gestation, and mammary tissue was homogenized and extracted for assessment of DNA, RNA, alpha-lactalbumin, and alpha-casein. Serum was assayed for placental lactogen-I (PL-I), and placental lactogen-II (PL-II), B, and T4. The concentration of PL-II in serum was elevated in the hypophysectomized mice, whereas the PL-I concentration did not differ among experimental groups. Hypophysectomy decreased both T4 and B concentrations in serum, and administration of these hormones restored their serum concentrations to normal or, in some cases, somewhat higher than normal levels. Hypophysectomy reduced the total RNA content and RNA/DNA ratio of the mammary gland, but treatment with B alone or with B and T4 restored RNA levels to those of sham-operated animals. T4 alone was ineffective in restoring RNA levels. Sham-operated animals that received hormonal treatment (B and T4) had the highest levels of RNA in the mammary tissue. Hypophysectomized animals had reduced content and concentration of alpha-lactalbumin in the mammary gland as compared to all other experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)