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M. J. Soares

Summary. The junctional and labyrinth regions of the rat chorioallantoic placenta during the second half of gestation showed different patterns of development with regard to DNA, protein, placental lactogen and alkaline phosphatase content. DNA and protein measurements indicated that growth of the labyrinth region was more rapid and persisted for longer during gestation than did growth in the junctional zone. At midpregnancy the junctional zone was the main source of placental lactogen whereas by late pregnancy both regions contributed considerable amounts. On Day 20 of gestation the labyrinth region contained significantly more placental lactogen than did the junctional zone. Alkaline phosphatase activity was predominant in the labyrinth zone throughout the second half of gestation. The results indicate that the chorioallantoic placenta is composed of two functionally distinct regions.

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M. J. Soares and S. R. Glasser

Summary. Cells from the labyrinth region of the developing rat chorioallantoic placenta were able to differentiate in vitro into cells capable of producing placental lactogen. Progesterone selectively inhibited placental lactogen production by labyrinth cell cultures undergoing differentiation but had no apparent effect on lactogen production by mature trophoblast giant cells. The measurement of placental lactogen production is a useful method for monitoring the functional differentiation of rat trophoblast cells in vitro.

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T. Yamamoto, B. M. Chapman and M. J. Soares

Trophoblast giant cells are the steroidogenic cells of the rat placenta. In this study, the role of protein kinase C signalling pathways in the control of DNA synthesis and differentiation-dependent progesterone biosynthesis by trophoblast cells were investigated. Rcho-1 trophoblast cells, derived from a rat choriocarcinoma, can be experimentally manipulated to proliferate or differentiate and provide a useful model for studying trophoblast giant cell endocrine differentiation. The role of protein kinase C signal transduction was examined through the treatment of Rcho-1 trophoblast cells with isoquinolinesulfonamide derivatives (H7, a protein kinase C inhibitor; HA1004, a control compound), chelytherine (a protein kinase C inhibitor), and phorbol esters (protein kinase C activators). Treatment with H7 significantly attenuated DNA synthesis in proliferating and differentiating trophoblast cells and accelerated the acquisition of progesterone biosynthetic capabilities by trophoblast cells. Treatment with HA1004, the related but functionally distinct isoquinolone, did not significantly affect trophoblast DNA synthesis or proliferation and only weakly increased progesterone accumulation. Chelytherine significantly inhibited trophoblast cell proliferation but failed to influence trophoblast progesterone production significantly. The phorbol ester, 12-O-tetradecanoylphorbol acetate, did not significantly influence progesterone accumulation. H7 did not significantly influence the concentration of either P450scc or the mRNA encoding it in Rcho-1 trophoblast cells, or the transcriptional activity of the P450scc gene. The results indicate that signalling pathways sensitive to protein kinase C are involved in the control of trophoblast cell proliferation. Differentiation-dependent production of progesterone is sensitive to H7 but appears to be independent of protein kinase C and occurs at a stage other than P450scc expression.

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S M Khorshed Alam, Toshihiro Konno and Michael J Soares

Prolactin family 8, subfamily a, member 2 (PRL8A2; also called decidual prolactin-related protein; dPRP) is a member of the expanded prolactin family. PRL8A2 is expressed in the uterine decidua and contributes to pregnancy-dependent adaptations to hypoxia. The purpose of this study was to identify gene targets for PRL8A2 action within the uteroplacental compartment. Affymetrix DNA microarray analysis was performed for RNA samples from WT and Prl8a2 null tissues. Validation of the DNA microarray was performed using quantitative RT-PCR. Nine genes were confirmed with decreased expression in Prl8a2 null tissues (e.g. Klk7, Rimklb, Arhgef6, Calm4, Sprr2h, Prl4a1, Ccl27, Lipg, and Htra3). These include potential decidual, endothelial and trophoblast cell targets positively regulated by PRL8A2. A significant upregulation of Derl3, Herpud1, Creld2, Hsp90b1, Ddit3 and Hspa5 was identified in Prl8a2 null tissues, reflecting an increased endoplasmic reticulum (ER) stress response. ER stress genes were prominently expressed in the uterine decidua. We propose that PRL8A2 is a mediator of progesterone-dependent modulation of intrauterine responses to physiological stressors.

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A. Amador, H. G. Klemcke, A. Bartke, M. J. Soares, T. M. Siler-Khodr and F. Talamantes

Summary. Adult male hamsters were given transplants of ½, 1, 2, 3 or 4 pituitaries under the kidney capsule and were killed 4 weeks later. Pituitary transplants produced a significant, dose-related increase in plasma prolactin levels, no changes in plasma LH and an increase in plasma FSH. Concentration of LH/hCG receptors in the testes was significantly increased in animals with 2 or 3 transplants and concentration of testicular prolactin receptors was significantly increased in those given 2 transplants. The apparent stimulatory effects of ½, 1 or 4 transplants on testicular LH/hCG and prolactin binding were not statistically significant. Some of the animals were injected with 0·3 i.u. hCG/g body weight 24 h before being killed. This produced a significant reduction in the levels of prolactin receptors and an apparent reduction in the levels of LH/hCG receptors in the testes. Elevation of plasma testosterone concentrations in response to hCG was significantly greater in animals given 3 or 4 pituitary transplants than in the remaining groups. These results provide further evidence that prolactin increases the number of LH/hCG and prolactin receptors in the hamster testis and suggest that changing the number of ectopic pituitary transplants may result in biphasic effects on the testis, with 2 or 3 transplants being maximally stimulatory.

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M. H. Stallings, K. S. Matt, A. Amador, A. Bartke, T. M. Siler-Khodr, M. J. Soares and F. Talamantes

Summary. During prepubertal development in the golden hamster, there are major age-related changes in the number of testicular LH/hCG receptors. Between 22 and 35 days of age, there was > 10-fold increase in testicular LH/hCG receptors, followed by a decrease at Day 37. Concomitant with, but preceding slightly, the changes in receptors, were increases in plasma LH and FSH and most noticeably prolactin concentrations, between Days 10 and 20 of age. Inhibition of the increases in plasma levels of prolactin by daily injections of bromocriptine, between 14 and 31 days of age, resulted in suppressed testicular and seminal vesicle weights, and decreased content and concentration of testicular LH/hCG receptors. Similarly, the premature increase in plasma prolactin concentrations in prepubertal hamsters between 6 and 20 days of age, by means of ectopic pituitary transplants, resulted in increased testicular and seminal vesicle weights, as well as an increase in the concentration of testicular LH/hCG receptors. These results strongly suggest that increases in plasma prolactin values during development are important in enhancement of the development of testicular LH/hCG receptors.

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Kazuhiko Imakawa, Pramod Dhakal, Kaiyu Kubota, Kazuya Kusama, Damayanti Chakraborty, M A Karim Rumi and Michael J Soares

Trophoblast stem (TS) cells possess the capacity to differentiate along a multi-lineage pathway yielding several specialized cell types. The regulatory network controlling trophoblast cell differentiation is poorly understood. Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain, 2 (CITED2) has been implicated in the regulation of placentation; however, we know little about how CITED2 acts to influence trophoblast cells. Rat Rcho-1 TS cells can be manipulated to proliferate or differentiate into specialized trophoblast lineages and are an excellent model for investigating trophoblast differentiation. CITED2 transcript and protein showed a robust induction during Rcho-1 TS cell differentiation. We used an shRNA knockdown approach to disrupt CITED2 expression in order to investigate its involvement in trophoblast cell differentiation. RNA-sequencing was used to examine the impact of CITED2 on trophoblast cell differentiation. CITED2 disruption affected the differentiating trophoblast cell transcriptome. CITED2 possessed a prominent role in the regulation of cell differentiation with links to several signal transduction pathways and to hypoxia-regulated and coagulation processes. In summary, our findings indicate that CITED2 contributes to the regulation of trophoblast cell differentiation.

Reproduction (2016) 151 1–8