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The concentrations of androgen, progesterone and prolactin-like activity in serum, placentae and media from placental incubations during the second half of pregnancy in the C3H mouse were evaluated. Serum concentrations, placental content and in-vitro placental release of androgen were raised on day 10 of pregnancy. Serum progesterone levels showed minor fluctuations during the second half of gestation, whereas placental content and in-vitro release of progesterone were increased on day 10 of gestation. The serum profile of prolactin-like activity showed a significant mid-pregnancy increase on day 10 which did not correlate with placental content or in-vitro placental release of prolactin-like activity. The placental content and in-vitro release of prolactin-like activity were low during midpregnancy and increased during the latter days of gestation when serum prolactin-like activity was reduced.
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ABSTRACT
Methods for the primary culture of muscle cells from fetal sheep were developed which gave high yields of cells. Myoblasts were grown in vitro, and allowed to fuse to form contractile multinucleate myotubes; these could be maintained in a good condition for at least 2 weeks. Protein turnover in these differentiated cultures was examined for sensitivity to each of four potentially anabolic peptide hormones and growth factors: insulin, insulin-like growth factor I (somatomedin C), epidermal growth factor and growth hormone. Insulin was found to have no effect except at high concentrations (1 μmol/l), compatible with its role as a somatomedin analogue. Insulin-like growth factor I was active at lower levels (1 nmol/l) but the cultures were not as responsive to it as were primary rat muscle cultures or differentiated L6 cells, which were tested in similar experiments. The maximum stimulation of protein synthesis observed with the ruminant system was only 16%. Epidermal growth factor was highly anabolic for primary cultures from sheep muscle, and the cells were very sensitive to it, half-maximal stimulation of protein synthesis being seen with concentrations as low as 20 pmol/l. No effects of bovine growth hormone were seen in the ovine system. However, an inhibition of protein breakdown was found with high concentrations (0·1 μmol/l) in the L6 rat myoblast cell line. It was found that the culture conditions used could affect the observed responses of protein synthesis and degradation, despite withdrawal of serum from the incubation media 22 h before testing.
J. Endocr. (1987) 112, 87–96
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SUMMARY
Effects of progesterone on production of androgen-dependent aggression-eliciting pheromones were investigated. Two groups of anosmic (non-fighting) castrated mice treated with testosterone or with testosterone and progesterone, respectively, were attacked to the same degree by intact, isolated (fighting) mice while control mice (castrated only) were attacked less. The findings support the idea that progesterone may inhibit androgen-induced aggression via a neural and not via a somatic mechanism.
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Differentiation of uterine stromal cells is critical for the establishment of pregnancy. This study had two purposes: (i) to validate the use of the UIII rat uterine stromal cell model for investigating mechanisms underlying decidual cell differentiation, and (ii) to use this cell model to identify a molecular switch for cellular entry into the decidual cell differentiation pathway. Quiescent rat uterine stromal cells were transfected with a 500 bp segment of the decidual prolactin-related protein (dPRP) promoter ligated to a luciferase reporter gene. Cells were incubated in low-serum medium, or in low-serum medium containing progesterone (1 μM), estradiol 17-β (10 nM), cholera toxin (10 ng/ml) and interleukin-11 (10 ng/ml). Protein extracts were collected 48 h later and luciferase was measured in the cellular lysates. Cholera toxin and interleukin-11 stimulated luciferase expression (P< 0.05) and addition of sex steroids further increased (P< 0.05) dPRP promoter activity. Stromal cells did not proliferate (P< 0.05) under differentiation conditions. Deletion analysis of the dPRP promoter revealed maximal luciferase expression between −250 and −500 bp relative to the transcription start site. Comparison of cyclin E/Cdk2 activity between proliferating and differentiating cells showed a 3-fold increase (P< 0.05) at 12 h in differentiating cells. The results suggest that cyclin E/Cdk2 serves as a molecular switch for uterine stromal cell entry into the decidual cell differentiation pathway.
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Abstract
Prolactin-like protein-B (PLP-B) is a member of a family of proteins expressed by the rat placenta and/or decidua with characteristics similar to prolactin (PRL). In this report, we present the heterologous expression and characterization of PLP-B. Recombinant PLP-B heterologously expressed in Chinese hamster ovary cells exhibited similar immunoreactive and electrophoretic characteristics with PLP-B produced by rat placental and decidual tissues. N-terminal sequencing verified the identity and purity of the recombinant PLP-B species and the site of cleavage of the signal peptide from the mature secreted PLP-B species. Polyclonal antibodies were generated to the recombinant PLP-B and used for Western blot and immunocytochemical analyses. Recombinant and native PLP-B migrated as a doublet at 30–31 kDa in SDS-PAGE under reducing conditions. Treatment of recombinant and native PLP-B with N-glycanase accelerated their electrophoretic mobility, indicative of their glycoprotein nature. PLP-B was localized exclusively to decidual cells in the developing deciduum and spongiotrophoblast cells in the placental junctional zone. The level of PLP-B protein expression dramatically declined prior to parturition. Potential PRL-like biological actions of PLP-B were also investigated. PLP-B bound weakly to ovarian and liver PRL receptors and did not stimulate the proliferation of lactogen-dependent Nb2 lymphoma cells. In conclusion, recombinant PLP-B possesses characteristics similar to native decidual and placental PLP-B and may represent a hormone/cytokine that has important modulatory actions during the establishment of pregnancy and the initiation of parturition.
Journal of Endocrinology (1997) 152, 291–302
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ABSTRACT
Rat trophoblast tissue was found to synthesize a number of low molecular weight proteins possessing prolactin-like characteristics. There appear to be at least three proteins that cross-react with antisera to pituitary prolactin. Two of the proteins had a molecular weight of 25 000, similar to ovine pituitary prolactin, and isoelectric points of 6·8 and 7·0. The third immunoreactive protein had a lower molecular weight (23 500), similar in size to human placental lactogen, and a slightly more acidic isoelectric point of 6·75. The molecular weight variants cross-reacted with an antipeptide serum that was generated to a synthetic peptide representing amino acids 150 to 164 of rat placental lactogen-2 (PL-2). Based on this analysis, we consider these proteins to be related to PL-2.Analysis of trophoblast proteins by gel-filtration chromatography resulted in the identification of another trophoblast prolactin. This material eluted earlier than PL-2-related proteins on a gel-filtration column, possessed prolactin-like activity (determined by competition with ovine pituitary prolactin for rabbit mammary gland or rat liver prolactin receptors) but showed limited cross-reactivity with either the antiserum to pituitary prolactin or the antiserum to the PL-2 peptide. We have thus identified multiple low molecular weight trophoblast prolactins, possessing different biochemical and immunological characteristics.
J. Endocr. (1988) 116, 101–106
Developmental Skin Biology Unit, NIAMS, Bethesda, Maryland, USA
Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas, USA
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Developmental Skin Biology Unit, NIAMS, Bethesda, Maryland, USA
Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas, USA
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Developmental Skin Biology Unit, NIAMS, Bethesda, Maryland, USA
Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas, USA
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Developmental Skin Biology Unit, NIAMS, Bethesda, Maryland, USA
Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas, USA
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Developmental Skin Biology Unit, NIAMS, Bethesda, Maryland, USA
Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas, USA
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Developmental Skin Biology Unit, NIAMS, Bethesda, Maryland, USA
Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas, USA
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Developmental Skin Biology Unit, NIAMS, Bethesda, Maryland, USA
Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas, USA
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Developmental Skin Biology Unit, NIAMS, Bethesda, Maryland, USA
Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas, USA
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Developmental Skin Biology Unit, NIAMS, Bethesda, Maryland, USA
Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas, USA
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Distal-less 3 (Dlx3) is a homeobox factor that functions as a placental-specific transcriptional regulator. Dlx3 null mice (−/−) have compromised placental development and do not survive in utero past embryonic day (E) 9.5. The current studies were undertaken to examine the expression of Dlx3 in mouse placenta during gestation, and to determine whether Dlx3 was involved in placental progesterone production. Dlx3 was not detectable at E8.5 but was detected in E9.5 placenta with continuing but diminished expression through E15.5. Dlx3 immuno-localization was restricted to the labyrinth, was nuclear and was found in cytokeratin-positive cells. Previous studies in choriocarcinoma cell lines support the conclusion that Dlx3 is required for expression of 3′-hydroxysteroid dehydrogenase VI (3βHSD VI), an obligate enzyme in the production of progesterone by trophoblast giant cells. In a rat trophoblast stem cell line (Rcho-1), Dlx3 expression was non-detectable in Rcho-1 cells induced to differ-entiate using mitogen withdrawal. In vitro progesterone production in placental cultures and 3βHSD VI mRNA from Dlx3 (+/+), (+/−) and (−/−) mice were equivalent. In situ hybridization for 3βHSD VI revealed mRNA expression restricted to trophoblast giants cells with no detectable expression in the labyrinth suggesting that Dlx3 and 3βHSD VI were not colocalized within the placenta. These studies support the conclusion that Dlx3 protein expression is restricted to the labyrinth region of the murine placenta into late gestation and that Dlx3 does not appear to be expressed in trophoblast giant cells. Further, loss of Dlx3 was not correlated with synthesis of progesterone from E9.5 mouse placentas.
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Abstract
Trophoblast giant cells of the rat placenta express cytochrome P450 17α-hydroxylase (P450c17) and synthesize androgens. The purpose of this study was to investigate androgen production and expression of P450c17 in the Rcho-1 trophoblast cell line. These cells are capable of differentiating along the trophoblast giant cell lineage. Androstenedione production increased approximately 70-fold as Rcho-1 trophoblast cells progressed from the proliferation to the differentiation state. P450c17 enzyme activity and mRNA also showed significant increases associated with trophoblast giant cell differentiation. To study the transcriptional regulation of the P450c17 gene, the activities of a series of P450c17 promoter–luciferase reporter constructs were evaluated following transient transfection into Rcho-1 trophoblast cells. A DNA region located – 98 bp upstream of the P450c17 gene transcriptional start site was the shortest promoter DNA construct consistently possessing activity in Rcho-1 trophoblast cells. Activities of longer constructs (−156 to −1560 bp) in this population of cells were significantly greater than the −98 bp promoter–reporter construct. The − 476 bp P450c17 construct showed maximal promoter activity in transiently transfected Rcho-1 trophoblast cells and was developmentally activated in stably transfected Rcho-1 trophoblast cells. Activation of the cyclic AMP/protein kinase A pathway did not significantly affect P450c17 promoter activity in Rcho-1 trophoblast cells, in contrast to its effects in mouse MA-10 Leydig cells. In summary, Rcho-1 trophoblast cells are capable of endocrine differentiation and are a useful in vitro system for studying the regulation of trophoblast androgen production and P450c17 gene expression.
Journal of Endocrinology (1996) 150, 161–168
Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA
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Waseda Research Institute for Science and Engineering, Waseda University, Tokyo, Japan
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Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA
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Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA
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Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas, USA
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Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA
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Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA
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Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA
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Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA
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Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA
Department of Pediatrics, University of Kansas Medical Center, Kansas City, Kansas, USA
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Mammals share common strategies for regulating reproduction, including a conserved hypothalamic–pituitary–gonadal axis; yet, individual species exhibit differences in reproductive performance. In this report, we describe the discovery of a species-restricted homeostatic control system programming testis growth and function. Prl3c1 is a member of the prolactin gene family and its protein product (PLP-J) was discovered as a uterine cytokine contributing to the establishment of pregnancy. We utilized mouse mutagenesis of Prl3c1 and revealed its involvement in the regulation of the male reproductive axis. The Prl3c1-null male reproductive phenotype was characterized by testiculomegaly and hyperandrogenism. The larger testes in the Prl3c1-null mice were associated with an expansion of the Leydig cell compartment. Prl3c1 locus is a template for two transcripts (Prl3c1 -v1 and Prl3c1-v2) expressed in a tissue-specific pattern. Prl3c1-v1 is expressed in uterine decidua, while Prl3c1-v2 is expressed in Leydig cells of the testis. 5′RACE, chromatin immunoprecipitation and DNA methylation analyses were used to define cell-specific promoter usage and alternative transcript expression. We examined the Prl3c1 locus in five murid rodents and showed that the testicular transcript and encoded protein are the result of a recent retrotransposition event at the Mus musculus Prl3c1 locus. Prl3c1-v1 encodes PLP-J V1 and Prl3c1-v2 encodes PLP-J V2. Each protein exhibits distinct intracellular targeting and actions. PLP-J V2 possesses Leydig cell-static actions consistent with the Prl3c1-null testicular phenotype. Analysis of the biology of the Prl3c1 gene has provided insight into a previously unappreciated homeostatic setpoint control system programming testicular growth and function.
