Placental lactogen-I Research Articles

DNA methylation contributes to the tissue-specific expression of the rPL-Iv gene

To understand the tissue-specific expression of the rat placental lactogen-I variant (rPL-Iv) gene, we investigated the methylation pattern of the 5′-flanking region of this gene in various rat tissues. We report that the 5′-flanking region of the rPL-Iv gene was hypomethylated in placenta that expressed the gene and hypermethylated in those tissues that did not express the gene. Moreover, the intron region of the rPL-Iv gene was hypomethylated in the placenta, but hypermethylated in the liver, kidney and pituitary. Although there are 5 CpG sites and the density of CpG dinucleotide is lower within 2 kb of the rPL-Iv 5′-flanking region, the methylated promoter reporter gene produced strong repression in the transcriptional activity of the gene. In addition, the 5′-flanking and intron regions of the rPL-Iv gene were hypomethylated on day 12 of gestation, and the methylation pattern in the placenta remained unchanged from mid-pregnancy until term. The entire genomic region of the rPL-Iv gene might be hypermethylated in tissues other than the placenta, within which its methylated status repress expression of the placenta-specific rPL-Iv gene. Interestingly, the methylation status of the intron region of the rPL-Iv in proliferating Rcho-1 cells was changed to the unmethylated status on day 8 and 12 of differentiation of Rcho-1 cells. These results demonstrate that demethylation in the rPL-Iv upstream region was induced at an early stage of placental development, and once the 5′-flanking region of the rPL-Iv had been demethylated, its status on the rPL-Iv genomic region was continued during pregnancy. Taken together, these results suggest that DNA methylation is responsible for the silencing of tissue-specific genes in non-expressing cells, while defined combinations of trophoblast factors dictate the expression of unmethylated rPL-Iv gene in placenta trophoblast cells.

Identification of trophoblast-specific binding sites for GATA-2 that are essential for rat placental lactogen-I gene expression

We identified a 3.4-kb 5'-flanking region of the rPL-I gene and examined its promoter activity using rat trophoblast Rcho-1 cells. A regulatory element between base pairs (bp) -2,487 and -2,310 in the 5'-flanking region was essential for maximum promoter activity of the rPL-I gene. This regulatory element was further characterized between bp -2,443 to -2,415 and -2,374 to -2,345. Electrophoretic mobility shift analysis showed that the interaction of nuclear extract proteins from differentiated Rcho-1 cells was inhibited by competition with a GATA-like sequence in the promoter, but not by a mutated GATA sequence. Moreover, the promoter activity of 2487 eLuc containing two novel GATA sites was significantly elevated by co-transfection of a GATA-2 expression vector in proliferating Rcho-1 cells. Our results demonstrate that GATA-2 is involved in multiple promoter regions to activate the specific expression of the rPL-I gene in placental tissue.

Interleukin‐11 signaling is required for the differentiation of natural killer cells at the maternal–fetal interface

Interleukin-11 (IL-11) is a multifunctional hematopoietic growth factor that has been implicated in the control of reproduction. Studies on IL-11 receptor-alpha (IL-11R alpha)-deficient mice showed that female mice are infertile due to defective decidualization. In this report, we evaluated the development of decidual cells, immune cells, and the vasculature associated with the implantation site of IL-11R alpha-deficient mice; with the aim of better understanding the nature of the fertility defect. Messenger RNAs for decidual differentiation, such as decidual prolactin-related protein and prolactin-like protein-J are expressed in the IL-11R alpha mutant. However, the number of decidual cells expressing these genes is decreased in the mutant compared with the wild-type control. Although, trophoblast cells differentiate and express placental lactogen-I in the IL-11R alpha-deficient uterine environment, they fail to progress and expand in number. Defects in the organization of the decidual vasculature were also apparent in the IL-11R alpha mutant uterus. The most dramatic effect of IL-11 signaling was on the hematopoietic environment of the uterine decidua. Differentiated/perforin-expressing uterine natural killer (NK) cells were virtually absent from implantation sites of IL-11R alpha mutant mice. NK cell precursors were capable of homing to the IL-11R alpha-deficient uterus and a known regulator of NK cell differentiation; IL-15 was expressed in the IL-11R alpha mutant uterus. Splenic NK cells from IL-11R alpha mutant mice were also able to respond to IL-15 in vitro. Thus, the defect in NK precursor cell maturation was not intrinsic to the NK precursor cells but was dependent upon the tissue environment. In summary, IL-11 signaling is required for decidual-specific maturation of NK cells.

Effect of high concentrations of glucose on differentiation of rat trophoblast cells in vitro.

Previous studies have shown that diabetic placentas are characterized by structural and biochemical anomalies, including defects in the differentiation of trophoblasts. In this study, the Rcho-1 cell line was used to investigate the impact of high glucose concentrations on different markers of differentiation of rat trophoblast cells in giant cells (endoreduplication, invasive phenotype and endocrine phenotype). Rcho-1 cells were incubated for 12 days in medium supplemented with different concentrations of glucose and 10% horse serum to optimize differentiation. The cells were examined for the proportion of nuclei showing signs of apoptosis. The effect of high glucose was investigated on the endoreduplication process, on invasive phenotype (secretion of gelatinase B) and on endocrine phenotype (expression of placental lactogen I (PL-I) and II (PL-II) and progesterone secretion). Apoptosis was not induced by high glucose in Rcho-1. The number of cells was higher in the cultures exposed to high glucose (p<0.05) and their nuclei contained more DNA compared with control cells (p<0.001), while their nuclear size was smaller (p<0.001). Gelatinase B secretion increased during differentiation but no difference was found when gelatinase B secretion from trophoblasts exposed to high glucose was compared with the control cells. Rcho-1 cell cultures showed an increase in PL-I and PL-II mRNA expressions during differentiation and which was not affected by high glucose. Progesterone secretion increased during differentiation in control cultures. However, this increase was abolished when trophoblasts were cultured in high glucose. Our data suggest that high glucose influences the endoreduplication process and the steroidogenesis during differentiation of rattrophoblasts.

The mouse prolactin gene family locus.

In the mouse, there is a large family of paralogous genes closely related to PRL. The objective of this report was to investigate the organization of the mouse PRL gene family locus. PRL family genes reside on chromosome 13 of the mouse genome. The PRL gene family members were localized to a series of overlapping bacterial artificial chromosome clones and arranged based on structural relationships. Additionally, several new members of the PRL gene family were identified. Placental lactogen I (PL-I) was found to be encoded by three closely related (>98% exon sequence identity) contiguous genes (termed: PL-Ialpha, PL-Ibeta, and PL-Igamma). Two previously unidentified mouse orthologs for members of the rat PRL family, PRL-like protein-I (PLP-I) and PLP-K were discovered, as were two new members of the PLP-C subfamily, PLP-Cgamma and PLP-Cdelta, and two new entirely unique members of the PRL family, PLP-N and PLP-O. Amino acid sequences predicted from the latter two genes most closely resembled proliferin-related protein. Each of the nine newly discovered genes is expressed in trophoblast cells of the mouse placenta in a gestationally specific pattern. In summary, elucidation of the mouse PRL gene family locus provides new insights into the expansion of the mouse PRL family and new tools for studying the genetics and biology of its members.

Characterization of the Expression of the Retinoid-related, Testis-associated Receptor (RTR) in Trophoblasts

Previous studies have provided evidence indicating that the nuclear orphan receptor RTR plays an important role during embryonic development and in spermatogenesis. In this study, we examine the expression of RTR in murine placenta and several human placental choriocarcinoma cell lines. Northern blot analysis showed high expression of RTR mRNA in placental tissue. In contrast to murine testis, which contains 7.4 and 2.3kb transcripts, placental tissue expressed only the larger transcript. Examination of RTR expression in murine placental tissue by immunohistochemistry demonstrated the presence of RTR protein in the nuclei of giant trophoblasts and spongiotrophoblasts. RTR mRNA was also expressed in rat choriocarcinoma Rcho-1 cells and in the human placental choriocarcinoma cell lines BeWo, JAR, and JEG-3. In trophoblasts, RTR was co-expressed with the estrogen-related receptors ERRα and ERRβ. Giant trophoblast differentiation in Rcho-1 cells, characterized by induction of placental lactogen I (PL-I), was accompanied by a steady decrease in the expression of RTR mRNA and down-regulation of ERRβ expression while levels of ERRα mRNA did not change significantly. RTR was able to inhibit ERRα-mediated transactivation through the consensus RTR-response element (RTRE) likely by competing with ERRα for binding to the RTRE. These results suggest the possibility of cross-talk between RTR and ERRα receptor signalling pathways in trophoblasts.

PAL31, a Nuclear Protein Required for Progression to the S Phase

PAL31 is a nuclear protein expressed by various cell types. In the present study, the expression and function of PAL31 were examined in the cytokine-regulated growth of T and B cell lines. Treatment of the cells with mitogens [ovine PRL, recombinant rat placental lactogen-I (PL-I) and human IL-3] caused a dose-dependent increase in the expression of PAL31 mRNA in the PRL-dependent cell line Nb2, and IL-3 dependent cell line BaF3. A time-course study on synchronized Nb2 cells revealed that the expression of PAL31 is specific to the late G1 and S phases. Immunocytological studies revealed that PAL31 accumulates in the nuclei at the S phase. Furthermore, the antisense oligonucleotide for PAL31 severely inhibited the proliferation of Nb2 cells by inhibiting cells progressing to the S phase. Thus, PAL31 is a nuclear protein associated with cell cycle progression.

Placental lactogen-I gene activation in differentiating trophoblast cells: extrinsic and intrinsic regulation involving mitogen-activated protein kinase signaling pathways.

Trophoblast giant cells are one of the primary endocrine cell types of the rodent placenta. Placental lactogen-I (PL-I) is the initial prolactin (PRL) family member expressed as trophoblast giant cells differentiate. In this report, we use the Rcho-1 trophoblast cell line as a model for studying the regulation of PL-I gene expression during trophoblast giant cell differentiation. Evidence is provided for trophoblast cell expression of epidermal growth factor receptor (EGFR), ErbB2, fibroblast growth factor receptor 1 (FGFR1), transforming growth factor-alpha, and heparin-binding EGF. EGF and FGF-2 stimulated PL-I mRNA and protein accumulation and PL-I promoter activity in a concentration-dependent manner. These latter growth factor actions on PL-I promoter activities were specifically inhibited by cotransfection with dominant negative constructs for EGFR and FGFRs respectively. Utilization of the mitogen-activated protein kinase (MAPK) pathway by EGF and FGF-2 in trophoblast cells was demonstrated by growth factor stimulation of a Gal4 DNA binding/Elk1 transactivational domain fusion construct, and more specifically by activation of extracellular signal regulated kinase and p38 MAPK. PL-I gene activation was also sensitive to disruption of MAPK and activation protein-1 (AP-1) signaling pathways. In conclusion, autocrine/paracrine pathways involving EGFR and FGFR1, MAPK and AP-1 are shown to participate in the regulation of the PL-I gene in differentiating trophoblast cells.

Influence of rat placental lactogen-I on the development of whole rat embryos in culture.

Rat placental lactogen-I (rPL-I), the first prolactin-like hormone expressed in the placenta during pregnancy in the rat, is known to influence maternal functions. In the present study, we have investigated the effects of rPL-I on the growth and development of cultured whole rat embryos. Rat embryos, with or without ectoplacental cone (EPC) attached, were explanted at day 9 of gestation. After 48 h of culture, the embryos, enclosed by the yolk sacs, were assessed by the presence of visible heart contractions ('heart beats'), crown-rump length (CRL) and yolk sac diameter (YSD). When intact embryos with EPC were cultured, the concentrations of rPL-I and rPL-II (products of EPC) in the medium were 850+/-841 and 92+/-181 ng/ml respectively (means+/-s.e.m.). In embryo cultures with the EPC removed, rPL-I levels decreased to</=10 ng/ml, and only 70% of the embryos were viable, with visible heart beats. In the viable embryos, both CRL and embryonic DNA synthesis were reduced compared with controls, and the addition of rPL-I (1 microg/ml) did not prevent this reduction. YSD and yolk sac DNA synthesis were also reduced compared with control embryos, and the addition of rPL-I significantly prevented this decrease by 45%. In embryos cultured without EPC in the presence of neutralizing rabbit anti-rat prolactin serum (anti-rPRL), embryonic and yolk sac DNA synthesis were reduced by 35% compared with embryos exposed to normal rabbit serum. Addition of rPL-I significantly increased (P<0.05) embryonic and yolk sac growth. Thus the effects of rPL-I on embryo growth could only be seen in the absence of prolactin. The addition of human prolactin in the presence of anti-rPRL also resulted in significant increases (P<0.05) in embryonic DNA synthesis and CRL. These results suggest that rPL-I may substitute for prolactin to influence the growth of the rat embryo.

Placental lactogen-I (PL-I) target tissues identified with an alkaline phosphatase-PL-I fusion protein.

The rat placenta expresses a family of genes related to prolactin (PRL). Target tissues and physiological roles for many members of the PRL family have yet to be determined. In this investigation we evaluated the use of an alkaline phosphatase (AP) tag for monitoring the behavior of a prototypical member of the PRL family, placental lactogen-I (PL-I). A probe was generated consisting of a fusion protein of human placental AP and rat PL-I (AP-PL-I). The AP-PL-I construct was stably expressed in 293 human fetal kidney cells, as was the unmodified AP vector that served as a control. AP activity was monitored with a colorimetric assay in conditioned medium from transfected cells. Immunoreactivity and PRL-like biological activities of the AP-PL-I fusion protein were demonstrated by immunoblotting and the Nb2 lymphoma cell proliferation assay, respectively. AP-PL-I specifically bound to tissue sections known to express the PRL receptor, including the ovary, liver, and choroid plexus. Binding of AP-PL-I to tissues was specific and could be competed with ovine PRL. The results indicate that AP is an effective tag for monitoring the behavior of PL-I and suggest that this labeling system may also be useful for monitoring the actions of other members of the PRL family.

Interaction of mouse placental lactogens and androgens in regulating progesterone release in cultured mouse luteal cells.

Pituitary hormones are essential for the maintenance of the corpus luteum in the pregnant mouse during the first half of gestation. Thereafter, hormones from the placenta take over the luteotropic role of the pituitary hormones. Mouse placental lactogen-I (mPL-I) and mPL-II, two PRL-like hormones produced in the placenta, are probably necessary for the maintenance of the corpus luteum in the latter half of pregnancy. A culture system of luteal cells from pregnant mice was developed to investigate the role of hormones from the placenta that may be important for the function of the corpus luteum. Mice were killed on days 10, 14, and 18 of pregnancy, and the corpora lutea were excised from the ovaries and digested in 0.1% collagenase, 0.002% DNase for 1 h. The resulting luteal cell suspension was plated onto 96-well plates coated with fibronectin (1 x 10(5) cells/well) and cultured for 1-3 days. Medium was changed daily. The cells were treated with various concentrations and combinations of mPL-I, mPL-II, mouse PRL, androstenedione, dihydrotestosterone, 17beta-estradiol (E2), testosterone, hydroxyflutamide, cycloheximide, actinomycin D, and fadrozole to study the effects of these different treatments on progesterone (P4) production. The three lactogens (mPL-I, mPL-II, and mouse PRL) all stimulated the release of P4 from the luteal cells. The potency of the lactogens was similar and did not depend on the stage of pregnancy at which the luteal tissue was obtained. However, the responsiveness of the cells to all hormone-stimulated P4 release was gradually reduced the later in pregnancy the tissue was collected. Androgens also stimulated the release of P4 from the luteal cells, and when administered together, the lactogens and the androgens acted synergistically to stimulate P4 release. The androgens acted directly but not through conversion to E2, as determined by the findings that 1) the effects of the androgens could not be reproduced by E2 administration, 2) nonaromatizable androgen dihydrotestosterone was as effective as aromatizable androgens, and 3) aromatase inhibitor did not prevent the action of the androgens to stimulate the P4 release. The effect of the androgens on the P4 release was rapid, occurring within 15 min of hormone administration. It was not prevented by inhibitors of protein and RNA synthesis, and the intracellular androgen receptor antagonist hydroxyflutamide did not affect the androgen action. Therefore, the androgen effects were not mediated through the intracellular androgen receptor and de novo protein synthesis was not needed for androgen-stimulated P4 release.

Rat placental lactogen-I variant (rPL-Iv), product of an unique gene, is biologically different from rPL-I

Rat placental lactogen-I variant (rPL-Iv), product of an unique gene, is biologically different from rPL-I

Rcho-1 trophoblast cell placental lactogens: complementary deoxyribonucleic acids, heterologous expression, and biological activities.

In this report, we have investigated placental lactogens (placental lactogen-I, PL-I; PL-I variant, PL-Iv; PL-II) expressed by differentiated Rcho-1 trophoblast cells. A complementary DNA (cDNA) library to differentiated Rcho-1 trophoblast cells was constructed and screened with probes to detect PL-I and PL-II. Sequence analysis of three independent Rcho-1 PL-I cDNAs indicated that they significantly differed from the previously reported PL-I sequence but more closely resembled a related cDNA referred to as PL-I mosaic (PL-Im). Upon further analysis, Rcho-1 PL-I/PL-Im transcripts could be detected in Rcho-1 trophoblast cells and normal developing placental tissue; however, the previously reported PL-I transcript could not be identified from the same sources. Given these results, we examined the original PL-I cDNA by PCR and nucleotide sequence analyses. The sequence differed from the original report and was found to be identical to the Rcho-1 PL-I and PL-Im cDNA clones. Thus, PL-I, Rcho-1 PL-I, and PL-Im are equivalent and should be referred to as PL-I. The PL-I gene was localized to chromosome 17 of the rat genome, similar to other PRL family members. Rcho-1 PL-II cDNAs were identical to the published PL-II sequence. PL-Iv cDNAs were isolated from differentiated Rcho-1 cells via an RT-PCR strategy and found to be identical to previously isolated PL-Iv cDNAs. Rcho-1 PL-I and PL-II cDNAs were subcloned into the pcDNA3 expression vector and recombinant protein produced in HRP-1 cells. Both recombinant Rcho-1 PL-I and PL-II proteins significantly stimulated the proliferation of lactogen-dependent rat Nb2 lymphoma cells and mouse mammary epithelial cells. In summary, we show that the Rcho-1 PL-I corresponds to PL-Im and Rcho-1 PL-Iv and PL-II are identical to their previously described placental counterparts. Additionally, both recombinant Rcho-1 PL-I and PL-II proteins are biologically active.

Regulation of mouse placental lactogen secretion by G proteins before midpregnancy.

To determine whether G proteins are involved in the regulation of mouse placental lactogen-I (mPL-I) and/or mPL-II secretion before midpregnancy, mouse placental tissue from day 7 of pregnancy was dispersed with collagenase, cells were fractionated on a percoll gradient, and the purified trophoblast cells were cultured in a serum-free medium with cholera toxin (CTX) or pertussis toxin (PTX) which modulate the activities of distinct G proteins for 5 days. CTX inhibited both mPL-I and mPL-II secretion, but PTX inhibited mPL-I secretion and stimulated mPL-II secretion in a time- and dose-dependent manner. Addition of both CTX and PTX additionally inhibited mPL-I secretion but did not affect mPL-II secretion. 8-Bromo cAMP, which increases intracellular cAMP accumulation, inhibited both mPL-I and mPL-II secretion similarly to CTX. In contrast, H8, an inhibitor of cAMP-dependent protein kinase A, stimulated both mPL-I and mPL-II secretion. Addition of PTX and H8 synergistically stimulated mPL-II secretion. These findings suggest that G proteins play important roles in regulation of mPL-I and mPL-II secretion before midpregnancy.

Identification, Characterization, and Expression of a New Prolactin-Like Molecule in the Hamster Placenta1

In the hamster, serum total lactogenic activity increases during the latter half of gestation (Days 8-16). On Days 10 and 12 a substantial amount of lactogenic activity cannot be attributed to prolactin (PRL) and hamster placental lactogen-II (haPL-II); therefore, the presence of a molecule similar to placental lactogen-I (PL-I), as found in the rat and mouse at midpregnancy, has been hypothesized for the hamster. The objectives of this study were to identify PRL-like molecules synthesized by the hamster placenta and to determine the temporal and cellular synthesis of identified molecules. Oligonucleotides (20-23 bp) corresponding to regions of nucleotide homology between mouse PL-I (mPL-I) and rat PL-I (rPL-I) along with midgestation hamster placental RNA were used in 3' rapid amplification of cDNA ends (RACE) methodology to generate PRL-like cDNA. A 444-bp cDNA fragment that had nucleotide sequence similarity with members of the prolactin-growth hormone (PRL-GH) gene family was generated. This cDNA fragment was utilized to screen a Day 16 hamster placental bacteriophage cDNA library, and a clone containing the entire coding region was identified and sequenced. The molecule had 77% nucleotide sequence homology with mouse proliferin-related protein (mPRP) and somewhat less homology (approximately 60%) with hamster, rat, and mouse PRL or placental lactogens (PL). The derived amino acid sequence of the identified molecule contained a 15-residue signal sequence and a 219-residue peptide with a calculated molecular weight of 25477. The peptide shared 58% amino acid sequence identity with mPRP. Placental expression of the PRL-like molecule during the latter half of gestation was evaluated by Northern and slot-blot analyses using the 444-bp cDNA fragment as a hybridization probe. A 1-kb transcript was detected on Days 9-15 with peak expression on Days 10 and 11. Messenger RNA for the PRL-like molecule was localized to cytotrophoblast but not giant trophoblast cells of the placental trophospongium region. In addition, specific immunostaining using an antibody to mPRP was confined to cytotrophoblast cells.

Lipopolysaccharides selectively inhibit mouse placental lactogen-II secretion through stimulation of interleukin-1 alpha (IL-1 alpha) and IL-6 production.

To determine whether lipopolysaccharides (LPS) regulate mouse placental lactogen-I (mPL-I), mPL-II, and mouse GHRF (mGHRF) secretion, mouse placental tissue from days 7, 9, and 12 of pregnancy was dispersed with collagenase and the purified trophoblast cells were cultured in a serum-free medium with or without LPS for 5 days. LPS significantly inhibited mPL-II secretion by cells from days 9 and 12 of pregnancy. However, LPS did not affect mPL-II secretion by cells from day 7 of pregnancy, mPL-I secretion by cells from days 7 and 9 of pregnancy, or mGHRF secretion by cells from day 12 of pregnancy. The inhibitory effect of LPS on mPL-II secretion by cells from day 12 of pregnancy was dose-dependent. Steady-state levels of mPL-II mRNA were significantly reduced by incubation of placental cells from day 12 of pregnancy with LPS. The inhibitory effect of LPS on mPL-II secretion was abolished by the addition of antibodies to IL-1 alpha and IL-6. These findings suggest that LPS selectively inhibit mPL-II secretion, at least partly through increases in IL-1 and IL-6 production, after midpregnancy.

Regulation of placental lactogen-I in differentiating trophoblast cells

Regulation of placental lactogen-I in differentiating trophoblast cells

Rat placental lactogen-I abolishes nocturnal prolactin surges in the pregnant rat

The twice-daily surges of prolactin (PRL) present during the first half of pregnancy abruptly terminate at midpregnancy concurrent with the appearance of high levels of placental lactogen-I (PL-I) in the blood. This study addressed the role PL-I and other pituitary or placental hormones have in terminating PRL surges in pregnant rats. Implantation of rat PL-I (rPL-I) or ovine PRL into the arcuate-median eminence area of the hypothalamus of day 7 pregnant rats totally eliminated nocturnal PRL surges on days 8 and 9. To assess the specificity of the inhibitory effects of hormones from the PRL-growth hormone (GH) family, rat growth hormone (rGH), human growth hormone (hGH), and rat prolactin-like protein-A (PLP-A) were tested. Only the lactogenic hormone, hGH, had any effect. Since lactogenic hormones may inhibit PRL by stimulation of dopamine synthesis and release into the hypophysial portal blood vessels leading to the anterior pituitary, the effect of these hormones on tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine activity, was determined. In pregnant rats, both ovine prolactin (oPRL) and hGH significantly increased (64%) TH activity, whereas rPL-I was less effective. In ovariectomized, bromocriptine-treated rats, both rPL-I and oPRL increased TH activity 207 and 151%, respectively. This supports the concept that termination of PRL surges at midpregnancy are owing to secretion of placental lactogens (PLs) from the placenta. However, the mechanism for the inhibition cannot be entirely attributed to an increase in tuberoinfundibular dopaminergic neuronal activity.

Transforming growth factor-alpha regulates subpopulation of giant cells which secrete mouse placental lactogen-I (mPL-I) and/or mPL-II at midpregnancy.

The aim of this study was to determine whether transforming growth factor-alpha (TGF-alpha) regulates expression of mouse placental lactogen-I (mPL-I) and mPL-II at midpregnancy in vitro. Treatment of placental cells from day 9 of pregnancy with TGF-alpha resulted in stimulation of mPL-I secretion and inhibition of mPL-II secretion in a time- and dose-dependent manner without changing the amount of newly synthesized trichloroacetic acid precipitable proteins and cell viability. Moreover, TGF-alpha increased the intra-cellular mPL-I concentration and immuno-precipitable newly synthesized mPL-I concentration both in the medium and cells. TGF-alpha increased the number of cells containing only mPL-I and cells containing both mPL-I and mPL-II, but decreased that of the cells containing only mPL-II assessed by double immunocytochemistry. TGF-alpha increased the number of cells releasing only mPL-I but decreased the number of cells releasing only mPL-II, however TGF-alpha did not affect the number of cells releasing both mPL-I and mPL-II assessed by sequential reverse hemolytic plaque assay (RHPA). TGF-alpha decreased the expression of mPL-II mRNA, but did not change the expression of mPL-I mRNA. In situ hybridization for epidermal growth factor receptor (EGF-R) after RHPA for mPL-I indicated that giant cells releasing mPL-I express EGF-R mRNA. These findings suggest that TGF-alpha regulates the subpopulation of giant cells which produces and releases mPL-I and mPL-II and it results in an increase of mPL-I secretion and decrease of mPL-II secretion, and suggests possible post-transcriptional stimulation of mPL-I secretion and transcriptional inhibition of mPL-II secretion by TGF-alpha. Giant cells releasing mPL-I express EGF-R mRNA, suggesting direct regulation of giant cell differentiation by EGF and TGF-alpha at midpregnancy.

Endocrine communication between conceptus and mother: placental lactogen stimulation of maternal behavior.

The possible role of the conceptus in stimulating the onset of maternal behavior through its secretion of placental lactogens and their passage into the brain was investigated in female rats. In the first study, significant mitogenic activity in the Nb2 lymphoma cell bioassay was detected in cerebrospinal fluid (CSF) samples collected by push-pull perfusion from rats on days 12-21 of pregnancy, coincident with the establishment of placental function. In contrast, mitogenic activity was absent from CSF in lactating and gonadectomized, virgin females. In a second study the mitogenic activity in day 12 pregnant samples was neutralized 71% with antibodies to rat placental lactogen-I (rPL-I) and > 90% with a combination of antibodies to rPL-I plus rPL-II. In contrast, activity on day 21 of pregnancy, 1 day prepartum, was reduced by antibodies to rPL-II (> 85%), but not by antibodies to rPL-I, indicating that the predominant lactogen in the CSF prepartum is rPL-II. The behavioral actions of placental secretions were assessed in the third experiment by infusing recombinant rPL-I and purified rPL-II directly into the medial preoptic area of the brain of steroid-primed, nulliparous rats. Latencies to respond maternally to foster young were significantly reduced in rPL-I- and rPL-II-treated rats (2- to 3-day latencies) when compared with latencies in control females (5- to 6-day latencies). Thus, the conceptus through its secretion of rPLs which apparently gain access to the CSF helps to prime the pregnant female's brain to respond maternally at the end of gestation. This endocrine communication between the developing conceptus and pregnant female appears to be an important part of the biological system which helps to establish successful maternal care.