PGF2alpha induced differential expression of genes involved in turnover of extracellular matrix in rat decidual cells

In the rat, the decidual tissue is an important component for maternal recognition of pregnancy. Decidualization can be induced by either the implantation of the blastocyst or by artificial stimuli. The process of decidua formation or decidualization, is characterized by growth and differentiation of endometrial stromal cells. Prostaglandin F2alpha (PGF2α) has been shown to be involved in inhibition of implantation, alteration of embryo development, induction of luteal regression, and the mediation of pregnancy loss induced by microorganism infections. In order to establish a direct role for PGF2α in decidual function, we have evaluated its effects on the expression of an extensive array of genes using primary decidual cell culture. Upon treatment with PGF2α sixty genes were significantly down-regulated whereas only six genes were up-regulated (from a total of 1176 genes studied). Interestingly, the majority of the genes inhibited by PGF2α are either directly or indirectly involved in the turnover of the extracellular matrix (ECM). Genes such as gelatinase A (MMP2), cathepsin L, tissue inhibitor metalloproteinases 2 (TIMP2) and 3 (TIMP3), plasminogen activator inhibitor1 (PAI1), tissue type plasminogen activator (tPA), urokinase plasminogen activator (tPA), endothelin 1, calponin, carboxypeptidase D and calponin acidic were down regulated. The opposite effect was observed for prostromelysin 53 kDa (proMMP3), plasma proteinase I alpha and alpha 1 antiproteinase, all of which were significantly up-regulated by PGF2α. The results strongly suggest that the abortificient role of elevated levels of PGF2α after implantation is due, in large part, to inhibition of genes involved in the normal turnover of the extracellular matrix necessary for decidual formation

The involvement of apoptotic regulators during in vitro decidualization

Objectives: The uterus responds to an implanting blastocyst by undergoing extensive tissue modification leading to decidualization. This modification includes differentiation and apoptosis of epithelial as well as stromal cell compartments. It is generally accepted that the decidual cell regression pattern is similar to the pattern of initial differentiation, suggesting that decidual cell death is the end point of timed differentiation. However, the molecular mechanisms controlling these events are not understood clearly. Therefore, we aimed to investigate the involvement of apoptotic factors using an in vitro cell culture system. Design: In order to assess the role of apoptotic factors during decidualization, we used a decidual cell line (GG-AD) that had been transformed with a temperature-sensitive SV-40 mutant. At the non-permissive temperature (39°C), these cells showed the characteristics of differentiated decidual cells. They dedifferentiated into stromal cells when the temperature was shifted back to 33°C. Methods: We performed Northern blot analysis for bax, bcl-XL and bcl-2 at both temperatures. The onset of apoptosis was examined by Annexin V staining. The expression of p53 protein was also determined by Western blot. Results: We found an increase in the expression of bax when GG-AD cells were grown at 39°C. We also showed apoptosis with Annexin V staining at 39°C. The p53 protein expression was also similar to that of the animal models, suggesting that the programmed cell death of the decidual cells occurred in a p53-independent manner. Conclusions: These data indicate that a parallelism exists between the increased expression of pro-apoptotic genes and decidual cell death, similar to animal models. Therefore, an in vitro model of GG-AD cells can be used to assess directly the relationship between apoptotic regulators and decidualization and could be used to study the mechanism of decidual cell regression

Prolactin signaling through the short isoform of the mouse prolactin receptor regulates DNA binding of specific transcription factors, often with opposite effects in different reproductive issues

<p>Abstract</p> <p>Background</p> <p>It has been well established that prolactin (PRL) signals through the long form of its receptor (PRL-RL) and activates the Jak/Stat pathway for transcription of PRL target genes. However, signaling pathways mediated through the short PRL-R isoform (PRL-RS) remains controversial. Our recent finding that PRL signaling through PRL-RS represses two transcription factors critical for follicular development lead us to examine other putative PRL/PRL-RS target transcription factors in the decidua and ovary, two well-known target tissues of PRL action in reproduction.</p> <p>Methods</p> <p>In this investigation we used mice expressing PRL-RS on a PRL-R knockout background and a combo protein/DNA array to study the transcription factors regulated by PRL through PRL-RS only.</p> <p>Results</p> <p>We show that PRL activation of the PRL-RS receptor either stimulates or inhibits the DNA binding activity of a substantial number of transcription factors in the decidua as well as ovary. We found few transcription factors to be similarly regulated in both tissues, while most transcription factors are oppositely regulated by PRL in the decidua and ovary. In addition, some transcription factors are regulated by PRL only in the ovary or only in the decidua. Several of these transcription factors are involved in physiological pathways known to be regulated by PRL while others are novel.</p> <p>Conclusion</p> <p>Our results clearly indicate that PRL does signal through PRL-RS in the decidua as well as the ovary, independently of PRL-RL, and activates/represses transcription factors in a tissue specific manner. This is the first report showing PRL/PRL-RS regulation of specific transcription factors. Many of these transcription factors were not previously known to be PRL targets, suggesting novel physiological roles for this hormone.</p

Molecular weight-dependent activity of aminated poly(&#945;)glutamates as siRNA nanocarriers

RNA interference (RNAi) can contribute immensely to the area of personalized medicine by its ability to target any gene of interest. Nevertheless, its clinical use is limited by lack of efficient delivery systems. Polymer therapeutics can address many of the challenges encountered by the systemic delivery of RNAi, but suffer from inherent drawbacks such as polydispersity and batch to batch heterogeneity. These characteristics may have far-reaching consequences when dealing with therapeutic applications, as both the activity and the toxicity may be dependent on the length of the polymer chain. To investigate the consequences of polymers&rsquo; heterogeneity, we have synthesized two batches of aminated poly(&alpha;)glutamate polymers (PGAamine), differing in their degree of polymerization, but not in the monomer units or their conjugation. Isothermal titration calorimetry study was conducted to define the binding affinity of these polymers with siRNA. Molecular dynamics simulation revealed that Short PGAamine:siRNA polyplexes exposed a higher amount of amine moieties to the surroundings compared to Long PGAamine. This resulted in a higher zeta potential, leading to faster degradation and diminished gene silencing. Altogether, our study highlights the importance of an adequate physico-chemical characterization to elucidate the structure&ndash;function-activity relationship, for further development of tailor-designed RNAi delivery vehicles

Generation of mice expressing only the long form of the prolactin receptor reveals that both isoforms of the receptor are required for normal ovarian function

Prolactin (PRL), a pleiotropic hormone essential for maintenance of corpus luteum (CL) function and pregnancy, transduces its signal through two types of receptors, a short form (PRLR-S) and a long form (PRLR-L). Both types of receptors are expressed in the CL, yet their individual roles are not well defined. We have shown previously that female transgenic mice expressing only PRLR-S display total infertility characterized by defective follicular development and early degeneration of CL, suggesting that expression of PRLR-L is a prerequisite for normal follicular development and maintenance of CL. To determine whether PRLR-L alone is the sole receptor required to maintain normal CL formation, differentiation, and progesterone secretion, we generated two transgenic mice which express only PRLR-L, either ubiquitously (Tg-RL) or in a CL-specific manner (CL-RL). To generate CL-specific expression, we used the HSD17B7 promoter. We found both transgenic mice models cycled normally, displayed no apparent defect in follicular development, and had normal ovulation rates. The STAT5 signaling pathway, considered essential for luteinization and progesterone production, was activated by PRL in both transgenic mice models. However, soon after mating, Tg-RL and CL-RL mice showed early regression of CL, lack of progesterone production, and implantation failure that rendered them totally infertile. Embryo transfer studies demonstrated no embryo abnormalities, and supplementation with progesterone rescued implantation failure in these mice. Close observation revealed lack of luteinization and reduced expression of proteins involved in progesterone biosynthesis despite normal levels of LHCGR (LHR), ESR1 (ER-alpha), CEBPB (C/EBP-beta) and CDKN1B (p27), proteins essential for luteinization. However, we found VEGFA, a key regulator of angiogenesis and vascularization, to be dramatically reduced in both Tg-RL and CL-RL mice. We also found collagen IV, a marker for the basal lamina of endothelial cells, aberrantly expressed and a discordant organization of endothelial cells in CL. Although luteinization did not occur in vivo, granulosa cells isolated from these mice luteinized in culture. Taken together, these results suggest that a vascularization defect in the CL may be responsible for lack of luteinization, progesterone production, and infertility in mice expressing only PRLR-L. This investigation therefore demonstrates that in contrast to earlier presumptions that PRLR-L alone is able to support normal CL formation and function, both isoforms of the PRL receptor are required in the CL for normal female fertility.Fil: Le, Jamie A.. University of Illinois; Estados UnidosFil: Wilson, Heather M.. University of Illinois; Estados UnidosFil: Shehu, Aurora. University of Illinois; Estados UnidosFil: Mao, Jifang. University of Illinois; Estados UnidosFil: Devi, Y. Sangeeta. University of Illinois; Estados UnidosFil: Halperin, Julia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Aguilar, Tetley. University of Illinois; Estados UnidosFil: Seibold, Anita. University of Illinois; Estados UnidosFil: Maizels, Evelyn. University of Illinois; Estados UnidosFil: Gibori, Geula. University of Illinois; Estados Unido

Can luteal regression be reversed?

The corpus luteum is an endocrine gland whose limited lifespan is hormonally programmed. This debate article summarizes findings of our research group that challenge the principle that the end of function of the corpus luteum or luteal regression, once triggered, cannot be reversed. Overturning luteal regression by pharmacological manipulations may be of critical significance in designing strategies to improve fertility efficacy

Prolactin signaling and Stat5: going their own separate ways?

Miyoshi et al. compared the role of the prolactin receptor (PrlR) and its downstream mediator, the signal transducer and activator of transcription 5 (Stat5), in mammary epithelial cells in vivo by studying PrlR(-/-) and Stat5ab(-/-) mouse mammary epithelial transplants during pregnancy. At first glance, the two mutant epithelia appear to have similar defects in the differentiation of the alveolar epithelium. However, a closer examination by Miyoshi et al. revealed defects in the epithelial architecture of the smallest ducts of Stat5ab(-/-) transplants not apparent in the PrlR(-/-) transplants, suggesting that Stat5 is more than a simple mediator of PrlR action