The newly established bovine endometrial gland cell line (BEGC) forms gland acini in vitro and is only IFNτ-responsive (MAPK42/44 activation) after E2 and P4-pre-incubation.

INTRODUCTION: Uterine glands (UG) are crucial for the establishment of ruminant pregnancy and influenced (orchestrated manner) by estrogen (E2), progesterone (P4) and interferon tau (IFNτ). In the study we established a bovine endometrial glandular cell line (BGEC) and tested its functional reactivity (signaling) to IFNτ. METHODS: BGEC was characterized by light microscopy (LM), epithelial markers (ezrin, CK18) [immunofluorescence (IF)/immunohistochemistry (IHC)] and ultrastructure (TEM/SEM) (apical microvilli). In vitro formation of gland acini and transepithelial-electric-resistance (TEER) measurements (EVOM) were done. The expression of mRNA-transcripts (RT-PCR) of steroid receptors (PR, PGRMC1/2, ESR1/2) and the IFNτ-system (IFNAR1/2, IRF1, 2, 9) was checked. BEGC was stimulated with IFNτ (10 ng/ml;1000 ng/ml) (15 min) after steroid pre-treatment [10 pg/ml E2 (two days)/20 ng/ml P4 (two days)]. Activation of MAPK42/44;STAT1 was evaluated (densitometrical Western Blot). RESULTS: BGEC cells expressed epithelial markers and possessed apical microvilli. High TEER-values could be measured (2320-2620 ohm/cm2). The assembled BEGC acini (25 days) were similar to UG in vivo (markers/ultrastructure). All transcripts (steroid receptors/IFNτ-system) could be detected in BEGC (mRNA). MAPK42/44 were significantly activated after E2/P4 pre-treatment and IFNτ stimulation (10 ng/ml) (p < 0.05), whilst 1000 ng/ml IFNτ did not activate MAPK42/44. Neither a STAT1 (by IFNτ) nor an activation (MAPK42/44;STAT1) by IFNτ-only was observed. DISCUSSION: BGEC retains its epithelial phenotype in culture and forms gland acini in vitro thereby confirming its glandular character. Cells were only reactive to (low) IFNτ concentrations when pre-treated with steroids thereby closely resembling implantation physiology in vivo. BEGC can be used as a bovine implantation model to study embryo-maternal communication during early pregnancy in cattle.

A Comparative Study of Growth Kinetics, In Vitro Differentiation Potential and Molecular Characterization of Fetal Adnexa Derived Caprine Mesenchymal Stem Cells.

The present study was conducted with an objective of isolation, in vitro expansion, growth kinetics, molecular characterization and in vitro differentiation of fetal adnexa derived caprine mesenchymal stem cells. Mid-gestation gravid caprine uteri (2-3 months) were collected from abattoir to derive mesenchymal stem cells (MSCs) from fetal adnexa {amniotic fluid (cAF), amniotic sac (cAS), Wharton's jelly (cWJ) and cord blood (cCB)} and expanded in vitro. These cultured MSCs were used at the 3rd passage (P3) to study growth kinetics, localization as well as molecular expression of specific surface antigens, pluripotency markers and mesenchymal tri-lineage differentiation. In comparison to cAF and cAS MSCs, cWJ and cCB MSCs showed significantly (P<0.05) higher clonogenic potency, faster growth rate and low population doubling (PDT) time. All the four types of MSCs were positive for alkaline phosphatase (AP) and differentiated into chondrogenic, osteogenic, and adipogenic lineages. These stem cells expressed MSC surface antigens (CD73, CD90 and CD105) and pluripotency markers (Oct4, Sox2, Nanog, KLF, cMyc, FoxD3) but did not express CD34, a hematopoietic stem cell marker (HSC) as confirmed by RT-PCR, immunocytochemistry and flow cytometric analysis. The relative mRNA expression of MSC surface antigens (CD73, CD90 and CD105) was significantly (P<0.05) higher in cWJ MSCs compared to the other cell lines. The mRNA expression of Oct4 was significantly (P<0.05) higher in cWJ, whereas mRNA expression of KLF and cMyc was significantly (P<0.05) higher in cWJ and cAF than that of cAS and cCB. The comparative assessment revealed that cWJ MSCs outperformed MSCs from other sources of fetal adnexa in terms of growth kinetics, relative mRNA expression of surface antigens, pluripotency markers and tri-lineage differentiation potential, hence, these MSCs could be used as a preferred source for regenerative medicine.

Stem cells from foetal adnexa and fluid in domestic animals: an update on their features and clinical application.

Over the past decade, stem cell research has emerged as an area of major interest for its potential in regenerative medicine applications. This is in constant need of new cell sources to conceive regenerative medicine approaches for diseases that are still without therapy. Scientists drew the attention towards alternative sources such as foetal adnexa and fluid, as these sources possess many advantages: first of all, cells can be extracted from discarded foetal material and it is non-invasive and inexpensive for the patient; secondly, abundant stem cells can be obtained; and finally, these stem cell sources are free from ethical considerations. Cells derived from foetal adnexa and fluid preserve some of the characteristics of the primitive embryonic layers from which they originate. Many studies have demonstrated the differentiation potential in vitro and in vivo towards mesenchymal and non-mesenchymal cell types; in addition, the immune-modulatory properties make these cells a good candidate for allo- and xenotransplantation. Naturally occurring diseases in domestic animals can be more ideal as disease model of human genetic and acquired diseases and could help to define the potential therapeutic use efficiency and safety of stem cells therapies. This review offers an update on the state of the art of characterization of domestic animals' MSCs derived from foetal adnexa and fluid and on the latest findings in pre-clinical or clinical setting of the stem cell populations isolated from these sources.

Deciphering the evolutionary history and developmental mechanisms of a complex sexual ornament: the abdominal appendages of Sepsidae (Diptera).

Male abdomen appendages are a novel trait found within Sepsidae (Diptera). Here we demonstrate that they are likely to have evolved once, were lost three times, and then secondarily gained in one lineage. The developmental basis of these appendages was investigated by counting the number of histoblast cells in each abdominal segment in four species: two that represented the initial instance of appendage evolution, one that has secondarily gained appendages, and one species that did not have appendages. Males of all species with appendages have elevated cell counts for the fourth segment, which gives rise to the appendages. In Perochaeta dikowi, which reacquired the trait, the females also have elevated cell count on the fourth segment despite the fact that females do not develop appendages. The species without appendages has similar cell counts in all segments regardless of sex. These results suggest that the basis for appendage development is shared in males across all species, but the sexual dimorphism is regulated differently in P. dikowi.

Isolation and cellular properties of mesenchymal cells derived from the decidua of human term placenta.

The clinical promise of cell-based therapies is generally recognized, and has driven an intense search for good cell sources. In this study, we isolated plastic-adherent cells from human term decidua vera, called decidua-derived-mesenchymal cells (DMCs), and compared their properties with those of bone marrow-derived-mesenchymal stem cells (BM-MSCs). The DMCs strongly expressed the mesenchymal cell marker vimentin, but not cytokeratin 19 or HLA-G, and had a high proliferative potential. That is, they exhibited a typical fibroblast-like morphology for over 30 population doublings. Cells phenotypically identical to the DMCs were identified in the decidua vera, and genotyping confirmed that the DMCs were derived from the maternal components of the fetal adnexa. Flow cytometry analysis showed that the expression pattern of CD antigens on the DMCs was almost identical to that on BM-MSCs, but some DMCs expressed the CD45 antigen, and over 50% of them also expressed anti-fibroblast antigen. In vitro, the DMCs showed good differentiation into chondrocytes and moderate differentiation into adipocytes, but scant evidence of osteogenesis, compared with the BM-MSCs. Gene expression analysis showed that, compared with BM-MSCs, the DMCs expressed higher levels of TWIST2 and RUNX2 (which are associated with early mesenchymal development and/or proliferative capacity), several matrix metalloproteinases (MMP1, 3, 10, and 12), and cytokines (BMP2 and TGFB2), and lower levels of MSX2, interleukin 26, and HGF. Although DMCs did not show the full multipotency of BM-MSCs, their higher proliferative ability indicates that their cultivation would require less maintenance. Furthermore, the use of DMCs avoids the ethical concerns associated with the use of embryonic tissues, because they are derived from the maternal portion of the placenta, which is otherwise discarded. Thus, the unique properties of DMCs give them several advantages for clinical use, making them an interesting and attractive alternative to MSCs for regenerative medicine.

The amount and activity of active matrix metalloproteinase 13 is suppressed by estradiol and progesterone in human pelvic floor fibroblasts.

As a key degrader of fibrillar collagens, matrix metalloproteinase 13 (MMP13), may contribute to the progression of pelvic organ prolapse. Here we aimed to define the regulation of MMP13 by estradiol and progesterone in the vaginal supportive tissues. Fibroblasts cultured from the arcus tendineous fasciae pelvis of three pre- and three postmenopausal women with prolapse were treated with 17-beta-estradiol (E2), progesterone (P4), E2 + P4, or E2 + ICI 182,780 (ICI). Collagenase inhibitor I (CI) and MG-132 were employed to investigate the mechanism of MMP13 degradation into inactive fragments (fragmentation) by hormones. The regulation of MMP13 in vivo was assessed by comparing tissues of ovariectomized (ovx) vs. sham-operated rats. Expression of MMP13 (proenzyme and active and fragment forms) was quantitated by Western immunoblotting, and MMP13 enzymatic activity was measured using a substrate degradation assay. The amount of cellular active MMP13 and MMP13 proteolytic activity decreased in the presence of hormones. The decrease was paralleled by increased proenzyme and fragment forms. MG-132, not CI, suppressed cellular MMP13 fragmentation. Active MMP13 increased in rats following ovx and was suppressed by E2 + P4 supplementation. Active MMP13 is suppressed in vivo and in vitro by estradiol and progesterone, suggesting a protective effect against vaginal supportive tissue deterioration.

Adiponectin mRNA levels in parametrial adipose tissue and serum adiponectin levels are reduced in mice during late pregnancy.

Adiponectin, a fat-derived factor, is downregulated in insulin resistance and obesity; insulin resistance has been demonstrated during late pregnancy in both humans and in rodents. The present study examines the physiological change of adiponectin gene expression as well as the circulating levels of adiponectin during pregnancy. We examined the relative quantity of adiponectin mRNA produced in the adipose tissues of pregnant compared to virgin mice. We also measured serum adiponectin levels and parametrial adipocyte size in mice throughout pregnancy. Adiponectin mRNA was significantly reduced by 74 +/- 8 % and 63 +/- 4 % at days 15 and 18 of pregnancy, respectively, compared to virgin mice. Serum adiponectin concentration decreased on days 15 (30.7 +/- 8.5 microg/ml) and 18 (27.9 +/- 8.7 microg/ml) of pregnancy, and the values were significantly lower than that of virgin mice (56.8 +/- 6.6 microg/ml). Parametrial adipocytes from mice on days 15 and 18 of pregnancy were significantly larger than in virgin mice or during early pregnancy. Fat-cell size was closely correlated to degradation of adiponectin gene expression and serum adiponectin levels. These results suggest that changes of adiponectin expression affect metabolic status in pregnant mice.

Mast cells in the female genital tract.

Mast cells in the human uterus and adnexa have been studied using basic lead acetate fixation and a long toluidine-blue technique to maximise the numbers of cells stained. Counts were performed on measured areas of tissue and the numbers of mast cells related to clinical and pathologic variables. Considerable variation in numbers was found among individual cases at all the sites studied. In the endometrium and myometrium, a drop in the number of mast cells has been demonstrated with advancing age, particularly after menopause. In leiomyomas the highest counts were in the smaller and more cellular lesions. It is concluded that the numbers of mast cells are at least partly related to the degree of cellularity or atrophy of the surrounding tissues. No significant association was found with menorrhagia or with the presence of leiomyomas.