Joseph C. Chen

Joseph C. ChenSiemens Clinical Laboratory · R&D

33.09
· Phd, MS
  • About
    67
    Research items
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    Research Experience
    Dec 2010
    PostDoc Position
    University of California, San Francisco · Center for Reproductive Sciences
    San Francisco, United States
    Sep 2005 - Sep 2010
    Graduate Student
    Rutgers, The State University of New Jersey · Department of Animal Sciences
    New Brunswick, United States
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    Derar Refaat Derar
    Irmgard Classen-Linke
    Julie Lee
    Carol A Bagnell
    Robbie L Mcleod
    Kai Wang
    Martyna M Małopolska
    Maria Pinzon-Ortiz
    Igor Lvovych Popovych
    Masuma Khatun
    Following (19)
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    Derar Refaat Derar
    Irmgard Classen-Linke
    Barbara Shacklett
    Terhi Piltonen
    Julie Lee
    Guy A Higgins
    Lynn A Hyde
    Carol A Bagnell
    David W Erikson
    Chiara Bellio
    Research
    Research Items (67)
    Objective: To develop a protocol for cryopreservation and recovery of human endometrial epithelial cells (eECs) retaining molecular and functional characteristics of endometrial epithelium in vivo. Design: In vitro study using human endometrial cells. Setting: University research laboratory. Patient(s): Endometrial biopsies were obtained from premenopausal women undergoing benign gynecologic procedures. Intervention(s): Primary eECs were cryopreserved in 1% fetal bovine serum/10% dimethylsulfoxide in Defined Keratinocyte Serum-Free Medium (KSFM). Recovered cells were observed for endometrial stromal fibroblast (eSF) contamination and subsequently evaluated for morphology, gene expression, and functional characteristics of freshly cultured eECs and in vivo endometrial epithelium. Main outcome measure(s): Analysis of eEC morphology and the absence of eSF contamination; evaluation of epithelial-specific gene and protein expression; assessment of epithelial polarity. Result(s): Endometrial epithelial cells recovered after cryopreservation (n = 5) displayed epithelial morphology and expressed E-cadherin (CDH1), occludin (OCLN), claudin1 (CLDN1), and keratin18 (KRT18). Compared with eSF, recovered eECs displayed increased (P<.05) expression of epithelial-specific genes AREG, CDH1, DEFB4A, MMP7, and WNT7A, while exhibiting low-to-undetectable (P<.05) stromal-specific genes COL6A3, HOXA11, MMP2, PDGFRB, and WNT5A. Recovered eECs secreted levels of cytokines and growth factors similarly to freshly cultured eECs. Recovered eECs could form a polarized monolayer with high transepithelial electrical resistance (TER) and impermeability to small molecules, and expressed apical/basolateral localization of CDH1 and apical localization of OCLN. Conclusion(s): We have developed a protocol for cryopreservation of eECs in which recovered cells after thawing demonstrate morphologic, transcriptomic, and functional characteristics of human endometrial epithelium in vivo.
    To determine the effects of coculturing endometrial epithelial cells (eEC) with paired endometrial stromal fibroblasts (eSF) on cell-specific gene expression and cytokine secretion patterns. In vitro study. University research laboratory. Endometrial biopsies were obtained from premenopausal women. Polarized eEC and subject-paired eSF were cultured for 12.5 hours alone (monoculture) or combined in a two-chamber coculture system without cell-cell contact. Cells and conditioned media were analyzed for global gene expression and cytokine secretion, respectively. Purified, endometrial tissue-derived eEC and eSF isolated by fluorescent activated cell sorting (FACS) were used as noncultured controls. Cell-specific global gene expression profiling and analysis of secreted cytokines in eEC/eSF cocultures and respective monocultures. Transepithelial resistance, diffusible tracer exclusion, expression of tight junction proteins, and apical/basolateral vectorial secretion confirmed eEC structural and functional polarization. Distinct transcriptomes of eEC and eSF were consistent with their respective lineages and their endometrial origin. Coculture of eEC with eSF resulted in altered cell-specific gene expression and cytokine secretion. This coculture model provides evidence that interactions between endometrial functionally polarized epithelium and stromal fibroblasts affect cell-specific gene expression and cytokine secretion underscoring their relevance when modeling endometrium in vitro.
    The neonatal porcine cervix is sensitive to hormones, including relaxin (RLX), from birth. Whether nursing is required to establish the cervical developmental program or to determine cervical developmental trajectory is unknown. The objective of study 1 was to determine effects of age and nursing on expression of molecular markers and mediators of porcine cervical growth and remodeling from birth to postnatal day (PND) 2 and to document effects of RLX treatment during this period on expression of targeted gene products in nursed vs. replacer-fed gilts. Study 2 was conducted to determine effects of age at first nursing and duration of nursing from birth on expression of targeted transcripts or proteins at PND 14. Nursing supported cervical estrogen receptor-α, vascular endothelial growth factor, matrix metalloproteinase (MMP)9, and antiapoptotic B-cell lymphoma-2 protein expression on PND 2. These proteins were undetectable in replacer-fed gilts. Returning replacer-fed gilts to nursing after PND 2 did not restore cervical expression of these proteins by PND 14. RLX increased (P < 0.05) cervical estrogen receptor-α, vascular endothelial growth factor, and B-cell lymphoma-2 protein in nursed gilts, MMP2 protein in nursed and replacer-fed gilts, and decreased (P < 0.05) pro-MMP9 protein in nursed gilts, and RXFP1 mRNA levels in nursed and replacer-fed gilts at PND 2. Replacer feeding for 2 wk from birth increased (P < 0.05) RXFP1 mRNA levels on PND 14. Results support the lactocrine hypothesis for maternal programming of neonatal tissues. Nursing from birth is required to establish the neonatal cervical developmental program and to maintain cervical developmental trajectory to PND 14.
    Accumulation of amyloid beta-peptide (Abeta) is considered a key step in the etiology of Alzheimer's disease. Abeta is produced by sequential cleavage of the amyloid precursor protein by beta- and gamma-secretase enzymes. Consequently, inhibition of gamma-secretase provides a promising therapeutic approach to treat Alzheimer's disease. Preclinically, several gamma-secretase inhibitors have been shown to reduce plasma and brain Abeta, although they also produce mechanism-based side effects, including thymus atrophy and intestinal goblet cell hyperplasia. The present studies sought to establish an efficient screen for determining the therapeutic window of gamma-secretase inhibitors and to test various means of maximizing this window. Six-day oral administration of the gamma-secretase inhibitor N(2)-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N(1)-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-l-alaninamide (LY411,575) reduced cortical Abeta(40) in young (preplaque) transgenic CRND8 mice (ED(50) approximately 0.6 mg/kg) and produced significant thymus atrophy and intestinal goblet cell hyperplasia at higher doses (>3 mg/kg). The therapeutic window was similar after oral and subcutaneous administration and in young and aged CRND8 mice. Both the thymus and intestinal side effects were reversible after a 2-week washout period. Three-week treatment with 1 mg/kg LY411,575 reduced cortical Abeta(40) by 69% without inducing intestinal effects, although a previously unreported change in coat color was observed. These studies demonstrate that the 3- to 5-fold therapeutic window for LY411,575 can be exploited to obtain reduction in Abeta levels without induction of intestinal side effects, that intermittent treatment could be used to mitigate side effects, and that a 6-day dosing paradigm can be used to rapidly determine the therapeutic window of novel gamma-secretase inhibitors.
    Human endometrium undergoes extensive regeneration on a cyclic basis in premenopausal women and likely occurs through the contribution of stem/progenitor cells. Menopause results in the permanent cessation of menstrual cycles and is preceded by perimenopause, a period of several years in which endocrine and biological changes occur and is a period of risk for endometrial proliferative disorders. The objectives of this study were to identify mesenchymal stem cells (eMSC) and stromal fibroblasts (eSF) in endometrium of perimenopausal women and perform expression profile analysis of perimenopausal eMSC and eSF to gain insight into the biology of stem/progenitor and lineage cell populations during the transition to menopause. Endometrial tissue was collected from perimenopausal and premenopausal women (n = 9 each). Microarray analysis was performed on FACS-isolated eSF and eMSC, and data were validated by quantitative RT-PCR. Principal component analysis showed that cells clustered into three distinct groups in 3-dimensional space: perimenopausal eMSC and premenopausal eMSC clustered together, while perimenopausal eSF and premenopausal eSF formed two discrete clusters separate from eMSC. Hierarchical clustering revealed a branching pattern consistent with PCA results, indicating that eMSC from premenopausal and perimenopausal women exhibit similar transcriptomic signatures. Pathway analysis revealed dysregulation of cytoskeleton, proliferation, and survival pathways in perimenopausal vs. premenopausal eSF. These data demonstrate that cell populations have altered gene expression in perimenopausal vs. premenopausal endometrium and that perimenopausal eSF had altered pathway activation when compared to premenopausal eSF. This study provides insight into aging endometrium with relevance to function in reproductively older women.
    Objective Intrinsic inflammatory characteristics play a pivotal role in stem cell recruitment and homing through migration where the subsequent change in niche has been shown to alter these characteristics. The bone marrow mesenchymal stem cells (bmMSCs) have been demonstrated to migrate to the endometrium contributing to the stem cell reservoir and regeneration of endometrial tissue. Thus, the aim of the present study was to compare the inflammation-driven migration and cytokine secretion profile of human bmMSCs to endometrial mesenchymal stem cells (eMSCs) and endometrial fibroblasts (eSFs). Materials and methods The bmMSCs were isolated from bone marrow aspirates through culturing, whereas eMSCs and eSFs were FACS-isolated. All cell types were tested for their surface marker, proliferation profiles and migration properties towards serum and inflammatory attractants. The cytokine/chemokine secretion profile of 35 targets was analysed in each cell type at basal level along with lipopolysaccharide (LPS)-induced state. Results Both stem cell types, bmMSCs and eMSCs, presented with similar stem cell surface marker profiles as well as possessed high proliferation and migration potential compared to eSFs. In multiplex assays, the secretion of 16 cytokine targets was detected and LPS stimulation expanded the cytokine secretion pattern by triggering the secretion of several targets. The bmMSCs exhibited higher cytokine secretion of vascular endothelial growth factor (VEGF)-A, stromal cell-derived factor-1 alpha (SDF)-1α, interleukin-1 receptor antagonist (IL-1RA), IL-6, interferon-gamma inducible protein (IP)-10, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)1α and RANTES compared to eMSCs and/or eSFs after stimulation with LPS. The basal IL-8 secretion was higher in both endometrial cell types compared to bmMSCs. Conclusion Our results highlight that similar to bmMSCs, the eMSCs possess high migration activity while the differentiation process towards stromal fibroblasts seemed to result in loss of stem cell surface markers, minimal migration activity and a subtler cytokine profile likely contributing to normal endometrial function.
    Understanding early events of HIV transmission within mucosal tissues is vital for developing effective prevention strategies. Here, we report that primary stromal fibroblasts isolated from endometrium, cervix, foreskin, male urethra, and intestines significantly increase HIV infection of CD4+ T cells–by up to 37-fold for R5-tropic HIV and 100-fold for X4-tropic HIV–without themselves becoming infected. Fibroblasts were more efficient than dendritic cells at trans-infection and mediate this response in the absence of the DC-SIGN and Siglec-1 receptors. In comparison, mucosal epithelial cells secrete antivirals and inhibit HIV infection. These data suggest that breaches in the epithelium allow external or luminal HIV to escape an antiviral environment to access the infection-favorable environment of the stromal fibroblasts, and suggest that resident fibroblasts have a central, but previously unrecognized, role in HIV acquisition at mucosal sites. Inhibiting fibroblast-mediated enhancement of HIV infection should be considered as a novel prevention strategy.
    Kinetics of HIV infection of T cells in the absence and presence of eSF. PHA/IL2-activated PBMCs cultured with or without eSF were infected with NLENG1I (A) or BaLGFP (B) and monitored for infection levels at the indicated timepoints. Results are gated on live, singlet CD3+CD8- cells. The proportions of HIV-infected cells are displayed on the left, while the absolute numbers of HIV-infected cells are displayed on the right. *p<0.05, **p<0.01, and ****p<0.0001 in group-wise comparisons (one-way analysis of variance with a Bonferroni posttest). n.s.: non-significant. Numbers underneath each colored bar correspond to fold-enhancement over the “No eSF” condition for each timepoint. (EPS)
    Characterization of HIV attachment to eSF and DCs. (A) Expression of DC and activation markers on iDCs and mDCs. Cells were analyzed by flow cytometry for the DC markers CD40 and CD1a, or the DC maturation marker CD83. (B) Attachment of HIV to eSF and DCs. Equal number of the indicated cells were incubated with virus for 2 h at 37°C, washed 4x, and then assessed by p24 ELISA for levels of bound virus. (C) eSF or DCs were assessed by flow cytometry for cell-surface expression of the trans-infection receptors DC-SIGN or Siglec-1. iDCs served as a positive control for DC-SIGN staining, while mDCs served as a positive control for Siglec-1 staining. (D) Mannan does not inhibit trans-infection to CD4+ T cells. iDCs, RAJI-DC-SIGN, or eSF were pre-treated with mannan (50 μg/ml), pulsed with NLENG1I, and incubated with PHA/IL2-activated PBMCs, and infection levels were monitored 3 d later and reported as percentage inhibition relative to the corresponding cell type not treated with mannan. ****p<0.0001 relative to no mannan control in a group-wise comparison (one-way analysis of variance with a Bonferroni posttest) for each cell type. (E) PHA/IL2-activated PBMCs cultured with mock- or heparinase-treated eSF were infected with NLENG1I and then monitored for infection levels 3 d later. The panel on the left shows the levels of cell-surface HSPG in heparinase-treated eSF, gated on live, singlet cells. The panel on the right shows the ability of mock- and heparinase-treated eSF to enhance HIV infection of T cells, and is gated on live, singlet CD3+CD8- cells. (EPS)
    eSF-mediated enhancement of HIV infection of CD4+ T cells is most potent at low viral inocula. (A) PHA/IL2-activated PBMCs cultured with or without eSF were infected with the indicated concentrations of NLENG1I and monitored by flow cytometry for infection levels 3 d later. Numbers in green correspond to fold-enhancement of HIV infection. (B) Enhancement of viral infection rates can reach up to ~100-fold when baseline level of infection in the absence of eSF is minimal. PHA/IL2-activated PBMCs cultured with or without eSF from 3 different donors were monitored for infection levels 3 d later. Results are gated on live, singlet CD3+CD8- cells. (EPS)
    eSF enhance the numbers of HIV-infected CD4+ T cells in culture. (A) PHA/IL2-activated PBMCs from 2 donors were infected with NLENG1I in the absence or presence of eSF and monitored for absolute numbers of HIV-infected cells 3 d later by flow cytometry. Results correspond to live, singlet CD3+CD8-GFP+ cells. (B) PHA/IL2- or CD3/CD28-activated PBMCs were infected with an HIV reporter virus encoding Firefly luciferase (NL4-3.Luc) with or without eSF, and monitored for infection levels 3 d later by luminescence. **p<0.01, ***p<0.001 (by 2-tailed t test). (EPS)
    Comparison of HIV infection enhancement by eSF and 10-fold excess DCs. PHA/IL2-activated PBMCs were infected with replication-competent NLENG1I (A) or replication-incompetent NL4-3.env*GFP (B) in isolation, or in the presence of eSF, an equal number of DCs, or 10x the amount of DCs. Infection levels were monitored 3 d later by flow cytometry. Results are gated on live, singlet CD3+CD8- cells. *p<0.05, ***p<0.001, and ****p<0.0001 relative to No co-culture control in a group-wise comparison (one-way analysis of variance with a Bonferroni posttest). (EPS)
    Fibroblasts do not proliferate over the course of co-culture. eSF were seeded in 96-well flat-bottom tissue culture plates and cultured until confluency, at which time cells were removed by trypsin/EDTA and counted with a hemocytometer. A separate set of wells were counted three days after cells achieved confluency. (EPS)
    eSF exhibit fibroblast morphology and express PDGFRB. (A) Phase-contrast image showing a confluent monolayer of primary eSF. Scale bar corresponds to 250 μm. (B) Flow cytometric assessment of cell-surface expression of the eSF marker PDGFRB. Gated on live, singlet events. (EPS)
    eSF enhance HIV infection of naturally permissive tissue-derived cells. CD19-depleted cells isolated from human tonsils from 2 donors were infected with NLENG1I, in the absence or presence of eSF. Infection levels were monitored 3 d later by flow cytometry. Results are gated on live, singlet CD3+CD8- cells, and are representative of four experiments with different eSF donors. **p<0.01 (by 2-tailed t test). (EPS)
    eSF-mediated enhancement of R5-tropic HIV infection T cells occurs under low vial inoculum conditions, and with purified CD4+ T cells activated by either PHA/IL2 or anti-CD3/CD28. (A) PHA/IL2-activated PBMCs cultured with or without eSF were infected with limiting amounts of BaLGFP and monitored by flow cytometry for infection levels 3 d later. Results are gated on live, singlet CD3+CD8- cells. (B) PBMC-derived CD4+ T cells were isolated to >96% purity for experiments described in Panels C and D. (C) The purified CD4+ T cells shown in panel B were activated with PHA/IL2 or anti-CD3/CD28 and then infected with BaLGFP. Infection levels were monitored 3 d later by flow cytometry. Results are gated on live, singlet CD3+CD8- cells. ***p<0.001, ****p<0.0001 relative to no co-culture in a group-wise comparison (one-way analysis of variance with a Bonferroni posttest). (D) As in panel C, except that the T/F strain THROGFP was used instead of BaLGFP. (EPS)
    Stromal fibroblasts isolated from endometrium, ectocervix, and endocervix of the same subject enhance HIV infection of CD4+ T cells to similar extents. PHA/IL2-activated PBMCs were infected with NLENG1I in isolation, or in the presence of eSF, cSF, or cnSF. Infection levels were monitored 3 d later by flow cytometry. Results are gated on live, singlet CD3+CD8- cells. **p<0.01 and ***p<0.001 in a group-wise comparison (one-way analysis of variance with a Bonferroni posttest) of all samples. (EPS)
    Expression of anti-HIV factors is highly enriched in eEC compared to eSF. Shown are the top 40 genes more highly expressed in eEC than eSF as assessed by Genespring. Highlighted genes encode secreted proteins with known anti-HIV activity. (PPTX)
    eSF do not activate CD4+ T cells and eSF-mediated enhancement occurs with autologous T cells. (A) Unstimulated or PHA/IL2-activated CD4+ T cells were cultured in the absence or presence of eSF for 3 d, and then monitored for cell-surface levels of the activation markers CD25 (left) and HLA-DR (right). The activation profiles of T cells in the absence (black) and presence (red) of eSF largely overlay. PHA/IL2 upregulates both CD25 and HLA-DR expression, as expected, but this level is not further increased with eSF co-culture. Results are gated on live, singlet CD3+CD8- cells. (B) Unstimulated (No Stim) or activated (PHA or PHA/IL2) PBMCs from four donors were co-cultured with autologous or heterologous eSF and infected with NLENG1I. Infection levels were monitored 3 d later by flow cytometry. Right: No Stim data are shown on a smaller scale to demonstrate the low level of eSF-mediated enhancement of infection of unstimulated CD4+ T cells. (EPS)
    Effect of HeLa and T ESCs cell lines on HIV infection of CD4+ T cells. (A) Unstimulated or PHA/IL2-activated PBMCs cultured with or without eSF or HeLa were infected with NLENG1I and monitored for infection levels 3 d later. Results are gated on live, singlet CD3+CD8- cells. (B) Activated PBMCs cultured with or without T ESCs or eSF were infected with NLENG1I and monitored for infection levels 3 d later. Results are gated on live, singlet CD3+CD8- cells, and representative FACS plots of triplicate conditions are shown. ***p<0.001 and ****p<0.0001 relative to No co-culture control in a group-wise comparison (one-way analysis of variance with a Bonferroni posttest). Results are representative of one of two experiments conducted. (EPS)
    eSF-mediated enhancement of HIV infection is not due to productive infection of eSF. (A) PBMCs co-cultured with eSF were inoculated with NLENG1I (left) or BaLGFP (right) and both cell types were assessed for productive infection after 3 d. (B) Unstimulated or activated PBMCs cultured with or without eSF were infected with replication-incompetent NL4-3.env*GFP, and monitored for infection levels 3 d later by flow cytometry. Results are gated on live, singlet CD3+CD8- cells. (C) PHA/IL2-activated PBMCs cultured with or without eSF from 3 different donors were infected with replication-incompetent Nef-deficient R5-tropic HIV and monitored for infection levels 3 d later by flow cytometry. Results are gated on live, singlet CD3+CD8- cells. ****p<0.0001 relative to No co-culture control in a group-wise comparison (one-way analysis of variance with a Bonferroni posttest). Left: Results of experimental triplicate measurements. Right: Representative flow cytometric plots. (EPS)
    eSF enhance fusion of HIV to CD4+ T cells. PBMCs infected with BlaM-encoding X4-tropic NL4-3 (A) or R5-tropic BaL (B) with or without eSF were monitored for viral fusion levels. Top: Representative flow cytometric plots. Bottom: Results of experimental triplicate measurements. **p<0.01, ***p<0.001 (by 2-tailed t test). Shown are one of two representative experiments with different eSF donors. (EPS)
    Effect of eSF on cell-surface levels of HIV-1 receptor, HIV-1 co-receptors, and adhesion molecules on T cells. PHA/IL2-activated T cell were co-cultured for 3 d with eSF, and then assessed for cell-surface levels of CD4 or the co-receptors CXCR4 and CCR5 (A), or the adhesion molecules CD11a (α-chain component of LFA-1), CD18 (β-chain component of LFA-1) and CD54 (ICAM-1) (B). Unstained samples correspond to Fluorescence Minus One (FMO) controls. Results are gated on live, singlet CD3+CD8- cells, and are representative of one of two experiments using different sets of eSF and PBMC donors. (EPS)
    Gating strategy used for determining percentage of HIV-infected CD4+ T cells. PBMCs were gated on live cells through use of an amine-reactive dye (Zombie Aqua), followed by gating on singlets. CD4+ T cells were identified by gating on CD3+CD8- cells. Productively infected cells are in large part CD4- because of HIV-mediated CD4 downregulation. (EPS)
    Human endometrium undergoes cyclic regeneration involving stem/progenitor cells, but the role of resident endometrial mesenchymal stem cells (eMSC) as progenitors of endometrial stromal fibroblasts (eSF) has not been definitively demonstrated. In endometriosis, eSF display progesterone (P4)-resistance with impaired decidualization in vivo and in vitro. To investigate eMSC as precursors of eSF and whether endometriosis P4-resistance is inherited from eMSC, we analyzed transcriptomes of eutopic endometrium eMSC and eSF isolated by fluorescence activated cell sorting (FACS) from endometriosis (eMSCendo, eSFendo) and controls (eMSCcontrol, eSFcontrol) and their derived primary cultures. Differentially expressed lineage-associated genes (LG) of FACS-isolated eMSC and eSF were largely conserved in endometriosis. In culture, eSFcontrolmaintained in vitro expression of a subset of eSF LG and decidualized in vitro with P4 eMSCcontrolcultures differentiated in vitro to eSF lineage, down-regulating eMSC LG and up-regulating eSF LG, showing minimal transcriptome differences vs. eSFcontrolcultures and decidualizing in vitro. Cultured eSFendodisplayed less in vitro LG stability and did not decidualize in vitro. eMSCendodifferentiated in vitro to eSF lineage but showed more differentially expressed genes vs. eSFendocultures, and did not decidualize in vitro, demonstrating P4-resistance inherited from eMSCendo Compared to controls, cultures from tissue-derived eSFendouniquely had a proinflammatory phenotype not present in eMSCendodifferentiated to eSF in vitro, suggesting divergent niche effects for in vivo vs. in vitro lineage differentiation. These findings substantiate eMSC as progenitors of eSF and reveal eSF in endometriosis have P4-resistance inherited from eMSC and a proinflammatory phenotype acquired within the endometrial niche.
    Question - How can I determine the type of collagenase is being used for endometrial tissue dissociation?
    Answer
    Hi there!  Please check out this bioprotocol for details on how to dissociate endometrial biopsies into single cell suspensions, preserving viability for endometrial stromal cells, epithelial cells, leukocytes, and stem cells. 
    Also check out this paper for cryopreservation of endometrial epithelial cells for epithelial cell culture models.
    Good luck!
    Joe
    Purification and culture of endometrial epithelial cells (eEC) and stromal fibroblasts (eSF) from endometrial biopsies allows for downstream cell-specific in vitro studies. The utility of this protocol is the ease with which cells are purified without contamination from unwanted cell types, and the ability to use patient-paired eEC and eSF in experiments. These methods have been previously published, but here the protocol has been updated for maximum efficiency.
    Do endometrial stromal fibroblasts (eSF) in women with polycystic ovary syndrome (PCOS) (eSFpcos) exhibit altered estrogen and/or progesterone (P4) responses, which may explain some of the adverse reproductive outcomes and endometrial pathologies in these women? In vitro, eSF from women with PCOS exhibit an aberrant decidualization response and concomitant changes in pro-inflammatory cytokine, chemokine and matrix metalloproteinase (MMP) release and immune cell chemoattraction. In vivo these aberrations may result in suboptimal implantation and predisposition to endometrial cancer. The endometrium in women with PCOS has several abnormalities including progesterone (P4) resistance at the gene expression level, likely contributing to subfertility, pregnancy complications and increased endometrial cancer risk in PCOS women. Prospective, university-based, case-control, in vitro study. Cultures of eSFPCOS (n = 12, Rotterdam and NIH criteria) and eSFControl (Ctrl) (n = 6, regular cycle length, no signs of hyperandrogenism) were treated with vehicle, estradiol (E2, 10 nM) or E2P4 (10 nM/1 μM) for 14 days. Progesterone receptor (PGR) mRNA was assessed with quantitative real-time PCR (qRT-PCR) and eSF decidualization was confirmed by insulin-like growth factor-binding protein-1 (IGFBP-1) transcript and protein expression. Fractalkine (CX3CL1), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL) 6, 8 and 11, macrophage chemoattractant protein (MCP) 1 and 3, CCL5 (RANTES) and MMPs (MMP1, 2, 3, 7, 9, 10 and 12) were measured in conditioned media by Luminex multiplex assays, and chemotactic activity of the conditioned media was tested in a migration assay using CD14+ monocyte and CD4+ T-cell migration assay. Effects of IL-6 (0.02, 0.2, 2 or 20 ng/ml) or IL-8 (0.04, 0.4, 4, or 40 ng/ml) or combination (0.2 ng/ml IL-6 and 4.0 ng/ml IL-8) on 14-d decidualization were also tested. ANOVA with pre-planned contrasts was used for statistical analysis. Hormonal challenge with E2P4 to induce decidualization revealed two distinct subsets of eSFPCOS. Eight eSFPCOS (dPCOS) and all eSFCtrl (dCtrl) cultures showed a normal decidualization response to E2P4 as determined by morphology and IGFBP-1 secretion. However, 4 eSFPCOS cultures showed blunted decidualization (ndPCOS) in morphological assessment and low IGFBP-1 levels even though all three groups exhibited normal estrogen-mediated increase in PGR expression. Interestingly dPCOS had decreased IL-6 and GM-SCF secretion compared with dCtrl, whereas the ndPCOS cultures showed increased IL-6 and 8, MCP1, RANTES and GM-CSF secretion at base-line and/or in response to E2 or E2P4 compared with dCtrl and/or dPCOS. Furthermore, even though PGR expression was similar in all three groups, P4 inhibition of MMP secretion was attenuated in ndPCOS resulting in higher MMP2 and 3 levels. The conditioned media from ndPCOS had increased chemoattractic activity compared with dCtrl and dPCOS media. Exogenously added IL-6 and/or 8 did not inhibit decidualization in eSFCtrl indicating that high levels of these cytokines in ndPCOS samples were not likely a cause for the aberrant decidualization. This is an in vitro study with a small sample size, utilizing stromal cell cultures from proliferative and secretory phase endometrium. The effect of PCOS on endometrial epithelium, another major histoarchitectural cell compartment of the endometrium, was not evaluated and should be considered in future studies. Furthermore, results obtained should also be confirmed in a larger data set and with mid/late secretory phase in vivo samples and models. The alterations seen in ndPCOS may contribute to endometrial dysfunction, subfertility and pregnancy complications in PCOS women. The results emphasize the importance of understanding immune responses related to the implantation process and normal endometrial homeostasis in women with PCOS. Sigrid Juselius Foundation, Academy of Finland, Finnish Medical Foundation, Orion-Farmos Research Foundation (to T.T.P.), the NIH Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) U54HD 055764-07 Specialized Cooperative Centers Program in Reproduction and Infertility Research (to L.C.G.), the NICHD the Ruth L. Kirschstein National Research Service Awards grant 1F32HD074423-03 (to J.C.C.). The authors have no competing interests. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
    Epidemiological studies indicate that progestin-containing contraceptives increase susceptibility to HIV, although the underlying mechanisms involving the upper female reproductive tract are undefined. To determine the effects of depot medroxyprogesterone acetate (DMPA) and the levonorgestrel intrauterine system (LNG-IUS) on gene expression and physiology of human endometrial and cervical transformation zone (TZ), microarray analyses were performed on whole tissue biopsies. In endometrium, activated pathways included leukocyte chemotaxis, attachment, and inflammation in DMPA and LNG-IUS users, and individual genes included pattern recognition receptors, complement components, and other immune mediators. In cervical TZ, progestin treatment altered expression of tissue remodeling and viability but not immune function genes. Together, these results indicate that progestins influence expression of immune-related genes in endometrium relevant to local recruitment of HIV target cells with potential to increase susceptibility and underscore the importance of the upper reproductive tract when assessing the safety of contraceptive products. © The Author(s) 2015.
    Intravaginal anti-HIV microbicides could provide women with a self-controlled means for HIV prevention, but results from clinical trials have been largely disappointing. We postulated that unrecognized effects of intravaginal gels on the upper female reproductive tract might contribute to the lower-than-expected efficacy of HIV microbicides. Our objective was to study the effects of intravaginal gels on the immune microenvironment of the cervix and uterus. In this randomized crossover study, 27 healthy female volunteers used a nightly application of intravaginal nonoxynol-9 (N9) gel as a "failed" microbicide or the universal placebo gel (UPG) as a "safe" gel (intervention cycles), or nothing (control cycle) from the end of menses to the mid-luteal phase. At a specific time-point following ovulation, all participants underwent sample collection for measurements of T-cell phenotypes, gene expression, and cytokine/chemokine protein concentrations from 3 anatomic sites above the vagina: the cervical transformation zone, the endocervix and the endometrium. We used hierarchical statistical models to estimate mean (95% CI) intervention effects, for N9 and UPG relative to control. Exposure to N9 gel and UPG generated a common "harm signal" that included transcriptional up-regulation of inflammatory genes chemokine (C-C motif) ligand 20 (macrophage inflammatory factor-3alpha) and interleukin 8 in the cervix, decreased protein concentrations of secretory leukocyte protease inhibitor, and transcriptional up-regulation of inflammatory mediators glycodelin-A and osteopontin in the endometrium. These results need to be replicated with a larger sample, but underscore the need to consider the effects of microbicide agents and gel excipients on the upper female reproductive tract in studies of vaginal microbicides.
    The major metabolite of the estrogenic pesticide methoxychlor (MXC) HPTE is a stronger ESR1 agonist than MXC and acts also as an ESR2 antagonist. In granulosa cells (GCs), FSH stimulates estradiol via the second messenger cAMP. HPTE inhibits estradiol biosynthesis, and this effect is greater in FSH-treated GCs than in cAMP-treated GCs. Therefore; we examined the effect of MXC/HPTE on FSH-stimulated cAMP production in cultured GCs. To test involvement of ESR-signaling, we used the ESR1 and ESR2 antagonist ICI 182,780, ESR2 selective antagonist PHTPP, and ESR2 selective agonist DPN. ESR1 and ESR2 mRNA and protein levels were quantified. Both HPTE and MXC inhibited the FSH-induced cAMP production. ICI 182,780 and PHTPP mimicked the inhibitory action of HPTE. MXC/HPTE reduced FSH-stimulated Esr2 mRNA and protein to basal levels. MXC/HPTE also inhibited FSH-stimulated Esr1. The greater inhibition on FSH-stimulated GCs is likely due to reduced cAMP level that involves ESR-signaling, through ESR2. Copyright © 2014 Elsevier Inc. All rights reserved.
    Question - Does anyone have experience in preparing strongly adherent cells (U2OS) for FACS without using acute dissociation treatments such as trypsin?
    Answer
    I agree with Elsenoor from above.  I use Accutase for both epithelial preps as well as macrophage preps.  I think it works really well while preserving viability.  Also, Accutase does not require serum deactivation (although I always wash the prep with relevant media, KSFM for epithelial cells and RPMI with 10% serum for macrophages).
    There is also Accumax, which is effective at room temperature and I have found that it also works well although I do not really know the differences between the two besides that Accutase has some phenol red.
    The synthesis of a series of iminoheterocycles and their structure-activity relationships (SAR) as inhibitors of the aspartyl protease BACE1 will be detailed. An effort to access the S3 subsite directly from the S1 subsite initially yielded compounds with sub-micromolar potency. A subset of compounds from this effort unexpectedly occupied a different binding site and displayed excellent BACE1 affinities. Select compounds from this subset acutely lowered Aβ40 levels upon subcutaneous and oral administration to rats
    Intravaginal anti-HIV microbicides could provide women with a self-controlled means for HIV prevention, but results from clinical trials have been largely disappointing. We postulated that unrecognized effects of intravaginal gels on the upper female reproductive tract might contribute to the lower-than-expected efficacy of HIV microbicides. Our objective was to study the effects of intravaginal gels on the immune microenvironment of the cervix and uterus. In this randomized crossover study, 27 healthy female volunteers used a nightly application of intravaginal nonoxynol-9 (N9) gel as a "failed" microbicide or the universal placebo gel (UPG) as a "safe" gel (intervention cycles), or nothing (control cycle) from the end of menses to the mid-luteal phase. At a specific time-point following ovulation, all participants underwent sample collection for measurements of T-cell phenotypes, gene expression, and cytokine/chemokine protein concentrations from 3 anatomic sites above the vagina: the cervical transformation zone, the endocervix and the endometrium. We used hierarchical statistical models to estimate mean (95% CI) intervention effects, for N9 and UPG relative to control. Exposure to N9 gel and UPG generated a common "harm signal" that included transcriptional up-regulation of inflammatory genes chemokine (C-C motif) ligand 20 (macrophage inflammatory factor-3alpha) and interleukin 8 in the cervix, decreased protein concentrations of secretory leukocyte protease inhibitor, and transcriptional up-regulation of inflammatory mediators glycodelin-A and osteopontin in the endometrium. These results need to be replicated with a larger sample, but underscore the need to consider the effects of microbicide agents and gel excipients on the upper female reproductive tract in studies of vaginal microbicides.
    Purpose: Decidualization comprises specific biochemical and morphological changes in uterine endometrium essential for establishment of pregnancy. This process is abnormal in women with endometriosis, a disorder in which endometrial-like tissue is present outside the uterus. The aim of this study was to restore cAMP-induced decidualization marker expression in endometrial stromal fibroblasts from women with endometriosis by using chemical inhibitors to PI3K/AKT/mammalian target of rapamycin (mTOR), mitogen-activated protein kinase (MAPK) and epidermal growth factor receptor (EGFR) signaling pathways in vitro.
    Question - Comparison of RT-PCR vs Western blot for tissue protein expression?
    Answer
    Late to the party but my congratulations as well. I think the fact that protein expression does not always correlate with mRNA expression is an important issue that you should not back down from (and good to see you did not!).
    Emerging studies suggest that certain protein families (like cytokines) do not always correlate with mRNA expression (see: Comparison of mRNA and protein measures of cytokines following vaccination with HPV-16 L1 virus like particles).
    Various papers hypothesize the mechanisms for this. For example, in a JBC article (see: Gene Array and Protein Expression Profiles Suggest Post-transcriptional Regulation during CD8+ T Cell Differentiation) the authors speculate that post-transcriptional modification can account for their data, in which 50% of the proteins showed discordant mRNA expression.
    Others suspect that micro-RNA regulation of transcripts actually suppresses protein expression, while stabilizing the transcript, causing detection at the transcript level but…
    How does seminal plasma (SP) affect the transcriptome of human primary endometrial epithelial cells (eEC) and stromal fibroblasts (eSF)? Exposure of eEC and eSF to SP in vitro increases expression of genes and secreted proteins associated with cellular migration, proliferation, viability and inhibition of cell death. Studies in both humans and animals suggest that SP can access and induce physiological changes in the upper female reproductive tract (FRT), which may participate in promoting reproductive success. This is a cross sectional study involving control samples versus treatment. SP (pooled from twenty donors) was first tested for dose- and time-dependent cytotoxic effects on eEC and eSF (n = 4). As exposure of eEC or eSF to 1% SP for 6 h proved to be non-toxic, a second set of eEC/eSF samples (n = 4) was treated under these conditions for transcriptome, protein and functional analysis. With a third set of samples (n = 3), we further compared the transcriptional response of the cells to SP versus fresh semen. eEC and eSF were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. RNA was isolated and processed for microarray studies to analyze global transcriptomic changes. Secreted factors in conditioned media from SP-treated cells were analyzed by Luminex and for the ability to stimulate migration of CD14+ monocytes and CD4+ T cells. Pathway identifications were determined using the Z-scoring system in Ingenuity Pathways Analysis (Z scores ≥|1.5|). SP induced transcriptomic changes (P < 0.05) associated with promoting leukocyte and endothelial cell recruitment, and proliferation of eEC and eSF. Cell viability pathways were induced, while those associated with cell death were suppressed (P < 0.05). SP and fresh semen induced similar sets of pathways, suggesting that SP can model the signaling effects of semen in the endometrium. SP also induced secretion of pro-inflammatory and pro-chemotactic cytokines, as well as pro-angiogenic and proliferative growth factors (P < 0.05) in both eEC and eSF. Finally, functional assays revealed that conditioned media from SP-treated eEC and eSF significantly increased (P < 0.05) chemotaxis of CD14+ monocytes and CD4+ T cells. This study is limited to in vitro analyses of the effects of SP on endometrial cells. In addition, the measured response to SP was conducted in the absence of the ovarian hormones estradiol and progesterone, as well as epithelial-stromal paracrine signaling. While this study focused on establishing the baseline cellular response of endometrial cells to SP, future work should assess how hormone signaling in the presence of appropriate paracrine interactions affects SP-induced genes in these cells. The results of this study support previous findings that SP and semen contain bioactive factors capable of eliciting chemotactic responses in the uterus, which can lead to recruitment of leukocytes to the endometrium. Future directions will explore if similar changes in gene expression do indeed occur after coitus in vivo, and how the signaling cascades initiated by SP in the endometrium can affect reproductive success, female reproductive health and susceptibility to sexually transmitted diseases. The gene list provided by the transcriptome analysis reported here should prove a valuable resource for understanding the response of the upper FRT to SP exposure. This project was supported by NIH AI083050-04 (W.C.G./L.C.G.); NIH U54HD 055764 (L.C.G.); NIH 1F32HD074423-02 (J.C.C.); DOD W81XWH-11-1-0562 (W.C.G.); NIH 5K12-DK083021-04, NIH 1K99AI104262-01A1, The UCSF Hellman Award (N.R.R.). The authors have nothing to disclose.
    Question - Can anyone help with a qRTPCR problem: good melt curve but not band in an agarose gel?
    Answer
    Agree with Jose and Muhammad, the NTC is very high, and some low-expression genes can actually be measured past 30Ct. The shape and quality of your amp curve determines what is a viable measurement.
    Agree with Paula, use EtBr gel (there are safe, battery-powered gels with precast gels and docing bases that you can use). You should see equal bands across the line, since the gel is picking up your endpoint product, NOT the product at the dynamic stage of amplification.
    Usually the issue can be resolved by:
    1. Redesigning primers as suggested, are you using a program with a dedicated ability to calculate the various penalties of your primers? (potential for dimers, CG content, etc.)
    2. Make sure there's no contaminants, increase your DNAse time, run your water just to be sure (we had a problem with contaminated water with nucleic acids in it). Hide your water! Water is not a product for sharing. Your dissociation curve highly suggests that multiple reagents may be contaminated.
    Question - Can anyone help with RNA integrity: TAE gel electrophoresis?
    Answer
    I agree with EL above. When working with endometrial biopsies, I had to polytron on ice, and i always washed the polytron in cold water, NaOH, and TRI reagent.
    The freeze thaw comment, I would highly recommend against freeze-thawing tissues. Not only are proteases and RNAses released, but your increasing your chance of external contamination every time. To be honest, I have never heard of freeze-thawing tissues.
    Lastly, its highly recommended that you utilize a fresh, cell derived RNA as positive control, otherwise, as obvious as the degradation is, there's no reference point, even with your ladder. If you cannot obtain perfect 18/28S bands with cell-derived RNA, there is an important technical problem in your protocol.
    In Vitrogen makes a great kit called the E-gel. You do not have to do too much prepping, it comes with a precast gel that you load into a battery kit. just load total RNA without having to heat denature, and the disposal is also much safer. Its cheap, check it…
    Context:Endometrium in polycystic ovary syndrome (PCOS) presents altered gene expression indicating progesterone resistance and predisposing to reduced endometrial receptivity and endometrial cancer.Objective:We hypothesized that an altered endocrine/metabolic environment in PCOS may result in an endometrial "disease phenotype" affecting the gene expression of different endometrial cell populations, including stem cells and their differentiated progeny.Design and setting:A prospective study conducted at an academic medical center.Patients and Main Outcome Measures:Proliferative phase endometrium was obtained from 6 overweight/obese PCOS (NIH criteria) and 6 overweight/obese controls. Microarray analysis was performed on fluorescence-activated cell sorting (FACS)-isolated endometrial epithelial cells (eEP), endothelial cells (eEN), stromal fibroblasts (eSF) and mesenchymal stem cells (eMSC). Gene expression data were validated using microfluidic Q-RT-PCR and immunohistochemistry (IHC).Results:The comparison between eEPPCOS and eEPCtrl showed dysregulation of inflammatory genes and genes with oncogenic potential (CCL2, IL-6, ORM1, TNAIFP6, SFRP4, SPARC). eSFPCOS and eSFCtrl showedupregulation of inflammatory genes (C4A/B, CCL2, ICAM1, TNFAIP3). Similarly, in eMSCPCOS vs. eMSCCtrl the most upregulated genes were related to inflammation and cancer (IL-8, ICAM1, SPRR3, LCN2). IHC scoring showed increased expression of CCL2 in eEPPCOS and eSFPCOS compared to eEPCtrl and eSFCtrl and IL-6 in eEPPCOS compared to eEPCtrl.Conclusions:Isolated endometrial cell populations in women with PCOS showed altered gene expression revealing inflammation and pro-oncogenic changes, independent of BMI, especially in eEPPCOS and eMSCPCOS, compared to controls. The study reveals an endometrial "disease phenotype" in women with PCOS with potential negative effects on endometrial function and long-term health.
    Substantial evidence implicates β-amyloid (Aβ) peptides in the etiology of Alzheimer's disease (AD). Aβ is produced by the proteolytic cleavage of the amyloid precursor protein by β- and γ-secretase suggesting that γ-secretase inhibition may provide therapeutic benefit for AD. Although many γ-secretase inhibitors have been shown to be potent at lowering Aβ, some have also been shown to have side effects following repeated administration. All of these side effects can be attributed to altered Notch signaling, another γ-secretase substrate. Here we describe the in vivo characterization of the novel γ-secretase inhibitor SCH 697466 in rodents. Although SCH 697466 was effective at lowering Aβ, Notch-related side effects in the intestine and thymus were observed following subchronic administration at doses that provided sustained and complete lowering of Aβ. However, additional studies revealed that both partial but sustained lowering of Aβand complete but less sustained lowering of Aβ were successful approaches for managing Notch-related side effects. Further, changes in several Notch-related biomarkers paralleled the side effect observations. Taken together, these studies demonstrated that, by carefully varying the extent and duration of Aβ lowering by γ-secretase inhibitors, it is possible to obtain robust and sustained lowering of Aβ without evidence of Notch-related side effects.
    The first two weeks of neonatal life constitute a critical period for estrogen receptor-alpha (ESR1)-dependent uterine adenogenesis in the pig. A relaxin receptor (RXFP1)-mediated, lactocrine-driven mechanism was proposed to explain how nursing could regulate endometrial ESR1 and related gene expression events associated with adenogenesis in the porcine neonate during this period. To determine effects of nursing on endometrial morphogenesis and cell compartment-specific gene expression, gilts (n = 6-8/group) were assigned at birth to be either: A) nursed ad libitum for 48 h; B) gavage-fed milk-replacer for 48 h; C) nursed ad libitum to PND 14; or D) gavage-fed milk-replacer for 48 h followed by ad libitum nursing to PND 14. Uteri were collected on PND 2 or PND 14. Endometrial histoarchitecture and both ESR1 and proliferating cell nuclear antigen (PCNA) labeling indices (LI) were evaluated. Laser microdissection was used to capture epithelium and stroma to evaluate treatment effects on cell compartment-specific ESR1, VEGFA and RXFP1 expression. Imposition of a lactocrine-null state by milk replacer feeding for 48 h from birth retarded endometrial development and adenogenesis. Effects of replacer feeding, evident by PND 2, were marked by PND 14 when endometrial thickness, glandularity and gland depth were reduced. Consistently, in lactocrine-null gilts, PCNA LI was reduced in glandular epithelium (GE) and stroma on PND 14, when epithelial ESR1 expression and ESR1 LI in GE were reduced and stromal VEGFA and RXFP1 expression increased. Results establish that lactocrine signaling effects morphogenetic changes in developing uterine tissues that may determine reproductive capacity later in life.
    Lactocrine signaling is defined as transmission of bioactive factors from mother to offspring as a consequence of nursing. Lactocrine transmission of signaling molecules may be an evolutionarily-conserved process through which bioactive factors necessary for support of neonatal development are delivered postnatally. Dependence on maternal resources for development in eutherian mammals extends into neonatal life for at least that period of time when nutrition is obtained solely from first milk (i.e., colostrum). Data for the pig (Sus scrofa domesticus) provide evidence of lactocrine mediated effects on development of the female reproductive tract and other somatic tissues. Porcine uterine gland development, an estrogen receptor-alpha (ESR1)-dependent process, begins within 2 d of birth (postnatal day = PND 0). A lactocrine-driven, ESR1-mediated process was proposed as a regulatory mechanism governing onset of uterine gland development and endometrial maturation in the neonatal pig. Gilts maintained in a lactocrine-null state for 2 d from birth by milk-replacer feeding displayed altered patterns of endometrial gene expression and retarded uterine gland development by PND 14. In lactocrine-null gilts, inhibition of endometrial and cervical ESR1 and vascular endothelial growth factor (VEGFA) expression observed on PND 2 persisted to PND 14, even after gilts were returned to nursing on PND 2. Collectively, data support a role for lactocrine signaling in regulation of critical neonatal developmental events. Maternal lactocrine programming of postnatal development may help to insure healthy developmental outcomes. A systems biology approach will be required to define and understand mechanistic dynamics of lactocrine signaling events that may ultimately connect genotype to phenotype and establish the parameters of reproductive potential.
    The lactocrine hypothesis describes a mechanism whereby milk-borne bioactive factors are delivered to offspring via nursing and affect development of neonatal tissues. In support of this hypothesis, data for the uterus indicate that, in comparison with gilts fed a porcine milk replacer for two days from birth (postnatal day = PND 0), nursing is essential to support normal uterine protein expression of developmental markers at PND 2 including estrogen receptor-alpha (ESR1) and vascular endothelial growth factor (VEGFA). The extent to which nursing and, therefore, lactocrine signaling is required to support porcine neonatal testicular ESR1 expression and other markers of testicular development is unknown. However, the importance of testicular ESR1 expression for subsequent testicular development and spermatogenesis is clear from studies in ESR1-null mice. Therefore, objectives of this study were to determine the effects of lactocrine signaling on testicular development as marked by expression of ESR1 and VEGFA, as well as markers of Sertoli cell number (GATA-4), and Leydig cell steroidogenesis (P450scc). At birth, male pigs were randomly assigned (n=6-7/group) to nurse ad libitum for 48 h or to be pan-fed porcine milk replacer for 48 h. Testes were collected on PND 2 (50 h). In addition, for comparison, testes were obtained from a third group of boars that were euthanized at birth, before nursing. Protein expression for ESR1, VEGFA, GATA-4, and P450scc was evaluated by immunoblot using actin as a loading control and quantified by densitometry. Results showed that nursing from birth was required for testicular ESR1 and VEGFA expression. These proteins were undetectable in testicular tissue obtained at birth or after feeding replacer for 48 h. In contrast, GATA-4 protein was detected in testicular tissue from newborn pigs as well as those fed replacer for 48 h, but was higher (P<0.05) in nursed pigs on PND 2. No effects of age or nursing were detected for testicular P450scc protein expression. Results indicate that nursing is required to support normal patterns of testicular ESR1 and VEGFA expression. Increased testicular GATA-4 protein expression observed in nursed boars on PND 2 suggests that nursing supports testicular development via increased Sertoli cell number. In contrast, nursing did not affect expression of P450scc, the rate-limiting enzyme required for Leydig cell steroidogenesis. This work extends the scope of the lactocrine hypothesis by showing that milk-borne bioactive factors support protein expression patterns in reproductive tissues of males as well as females. Results reinforce the importance of lactocrine signaling in support of reproductive tissue development in neonatal pigs. [Support: USDA-NRICGP 2007-35203-18098 and NSF-EPS-0814103]
    The lactocrine hypothesis describes a mechanism whereby milk-borne bioactive factors are delivered to offspring via nursing and affect development of neonatal tissues. In support of this hypothesis, data for the uterus indicate that, in comparison with gilts fed a porcine milk replacer for two days from birth (postnatal day = PND 0), nursing is essential for support of normal uterine estrogen receptor-α (ESR1), vascular endothelial growth factor (VEGF) A, and matrix metalloproteinase (MMP) 9 protein expression at PND 2. Depressed expression of these markers and mediators of uterine growth and remodeling was not restored when gilts fed milk replacer through PND 2 were switched to nursing until PND 14. These results suggest that a window of opportunity for lactocrine signaling exists within the first two days of life; however, the critical time period for such signaling within the first 48 h from birth is unknown. Here, objectives were to determine the effects of: (1) age at first nursing (0 h, 30 min, 12 h); and (2) duration of nursing (30 min, 12 h, 48 h) on uterine ESR1, VEGFA and MMP9 expression. Gilts (n=5-6/group) were randomly assigned at birth to one of six treatment groups: (1) nursed for 30 min from birth and gavage-fed milk replacer (30ml/kg BW/2 h) for 47.5 h; (2) nursed for 12 h from birth and gavage-fed milk replacer for 36 h; (3) nursed ad libitum for 48 h; (4) gavage-fed milk replacer for 30 min from birth and nursed for 47.5 h; (5) gavage-fed milk replacer for 12 h from birth and nursed for 36 h; or (6) gavage-fed milk replacer for 48 h. Uteri were collected on PND 2 (50 h). ESR1, VEGFA, and MMP9 protein expression was evaluated by immunoblotting using actin as a loading control and quantified by densitometry. Immunoreactive bands at 51 kDa for ESR1, 46 kDa for VEGFA, and 84 and 92 kDa for MMP 9 and pro-MMP9 were detected consistently in uterine tissues from gilts allowed to nurse beginning at 0 h or 30 min of age. However, as observed in gilts fed replacer for 48 h from birth, targeted proteins were undetectable in uterine tissues on PND 2 when gilts were nursed starting at 12 h after birth. Nursing for as little as 30 min from birth was sufficient to induce uterine ESR1, VEGFA and MMP9 expression at PND 2. Increasing the duration of nursing from 30 min to 12 h increased expression of targeted proteins to levels similar to those seen in gilts nursed for 48 h. Results indicate that age at first nursing is important for lactocrine signaling. Nursing initiated at 0 h or 30 min of age is sufficient to induce a normal developmental program as reflected by uterine ESR1, VEGFA, and MMP9 expression on PND 2. However, when a lactocrine-null period is imposed from birth to 12 h of age, resumption of nursing through PND 2 failed to rescue the normal uterine developmental program. Thus, the window for lactocrine signaling important for neonatal uterine development is open within 12 h of birth. Results also show that increased nursing duration correlates positively with levels of uterine ESR1, VEGFA, and MMP9 expression. This study illustrates the importance of age at first nursing and duration of nursing in support of the porcine uterine developmental program and defines the critical period for lactocrine signaling more precisely within two days of birth. [Support: USDA-NRICGP 2007-35203-18098 and NSF-EPS-0814103] (poster)
    Transmission of human immunodeficiency virus (HIV) among women occurs primarily through heterosexual intercourse. Nonoxynol-9 (N9), a non-ionic detergent spermicide in vaginal gel formulations that has in vitro HIV antiviral activity, paradoxically, in clinical trials for prevention of male to female HIV transmission, increases HIV infection in women. N9 in part compromises the vaginal epithelium and increases inflammatory responses with recruitment of HIV target immune cells in the lower female reproductive tract (FRT). We hypothesize that N9 also affects the upper FRT and stimulates local inflammation in the endometrium through epithelial-stromal interactions, contributing to recruitment of target immune cells and increased susceptibility to HIV infection. The objective of this study was to use a human epithelial/stromal co-culture system to evaluate N9 effects on cytokine levels in the endometrial microenvironment. Epithelial and stromal fibroblast cells were isolated from endometrial tissue from two women of reproductive age by enzymatic dissociation, size fractionation, and selective attachment. To model the endometrial microenvironment, polarized confluent epithelial cultures established on Matrigel-coated inserts were co-cultured with patient-paired stromal fibroblasts such that cells in the stromal compartment were not exposed to agents added to the apical epithelial chamber, but basolaterally secreted epithelial products diffuse to the stromal compartment. Epithelial and stromal mono- and co-cultures were exposed to medium (75% DMEM, 25% MCDB-105, 1% FBS, 1 mg/ml BSA) with or without a non-cytotoxic dose of N9 (10 µg/ml) for 13.5 hours. Epithelial integrity was confirmed by apical/basolateral diffusible tracer exclusion. Levels of IL4, IL8, TNFα, and GM-CSF in conditioned media from each cell population/treatment were assessed by Luminex assays. Epithelial apical secretion of IL4 (78 ± 19 pg/ml) was unaffected by co-culture with stromal fibroblasts but was reduced by 58-69% with N9 treatment in mono- or co-culture. Levels of IL8, TNFα, and GM-CSF in the epithelial apical compartment were detectable but unaffected by N9 treatment or co-culture with stromal fibroblasts. Epithelial basolateral levels of IL8 (2101 ± 835 pg/ml), TNFα (22 ± 13 pg/ml), and GM-CSF (39 ± 17 pg/ml) were increased 2-, 4-, and 8-fold respectively with apical exposure to N9. Stromal fibroblast conditioned medium had undetectable IL4 and low to undetectable TNFα and GM-CSF levels. Stromal secretion of these three cytokines was unaffected by N9 treatment or by co-culture with either untreated or N9-treated epithelial cells. Stromal IL8 secretion (149 ± 15 pg/ml) was increased 2.8-fold by direct exposure to N9. IL8 levels in the stromal compartment of co-cultures were increase 21-fold with untreated epithelial cells and 26-fold with N9-treated epithelial cells. In conclusion, preliminary data suggest that exposure of human endometrial epithelium to non-cytotoxic levels of N9 has distinct effects on apical/basolateral cytokine secretion. Remarkably, N9 induced increased basolateral secretion of pro-inflamatory cytokines IL8, TNFα, and GM-CSF to the underlying stroma. These changes within the endometrial microenvironment can contribute to increased susceptibility to HIV infection. (NIH AI083050) (platform)
    Colostrum, or first milk, supports porcine neonatal development by providing a conduit for delivery of milk-borne bioactive factors (MbFs) from mother to offspring as proposed in the lactocrine hypothesis. Ingestion of colostrum for 48 h from birth (postnatal day (PND) = 0) supports expression of factors important for cervical development, including estrogen receptor-alpha (ESR1). During this period, changes in the composition of colostrum are likely to affect the array of lactocrine-active MbFs delivered to neonates. The critical period for lactocrine signaling supportive of cervical development within 48 h of birth is undefined. Moreover, whether ingestion of a single dose of early colostrum during this period can rescue an altered, lactocrine-null cervical phenotype disrupted by milk replacer feeding from birth is unknown. Objectives were to determine effects of: 1) age at first nursing (0 h, 30 min, 12 h); 2) duration of nursing from birth (30 min, 12 h, 48 h); and 3) a single feeding of early colostrum (collected at 1 h of lactation), delivered at 8 h or 12 h after birth, on cervical ESR1 protein expression on PND 2. For objectives 1 and 2, gilts (n=4-6/group) were assigned randomly at birth to one of the following groups: 1) nursed ad libitum; 2) gavage-fed milk replacer (30 ml/kg BW/2 h); 3) nursed for 30 min, then switched to milk-replacer; 4) gavage-fed milk replacer 30 min, then switched to nursing; 5) nursed for 12 h, then switched to milk-replacer; and 6) gavage-fed milk-replacer for 12 h, then switched to nursing. Objective 3 gilts (n=4-6/group) were gavage-fed milk replacer from birth, fed a single dose of 1 h lactation colostrum (30 ml/kg BW) at 8 h or 12 h after birth and then returned to milk replacer feeding through 48 h. Cervices were collected on PND 2 (50 h) for all groups. Cervical ESR1 protein was evaluated by immunoblotting using actin as a loading control and quantified by densitometry. Cervical ESR1 protein expression was evident consistently at PND 2 in gilts that began nursing at 0 h or 30 min of age. However, in gilts fed replacer from birth and then switched to nursing at 12 h, cervical ESR1 expression was undetectable at PND 2 and identical to the cervical phenotype of gilts fed replacer for 48 h from birth. Increasing duration of nursing from 30 min to 12 h and 48 h increased cervical ESR1 expression on PND 2. Additionally, cervical ESR1 protein was detected on PND 2 in replacer-fed gilts given a single dose of 1 h lactation colostrum at 8 h or 12 h after birth. Results show that age at first nursing is important for lactocrine signaling supportive of cervical development by PND 2. Switching gilts fed replacer from birth to nursing at 12 h of lactation did not rescue cervical development from the lactocrine-null phenotype. However, nursing for only 30 min from birth was sufficient to trigger cervical ESR1 expression by PND 2. Lactocrine-active MbFs remain to be defined. However, results indicate that colostrum obtained at 1 h of lactation contains MbFs necessary to induce cervical ESR1 expression and that these factors are effective when delivered within 12 h of birth. Results support the lactocrine hypothesis for maternal programming of cervical development during a critical period within 12 h from birth and indicate that lactocrine-active MbFs present in colostrum at 1 h of lactation can initiate the cervical developmental program. [Support: USDA-NRICGP 2007-35203-1809 and NSF-EPS-0814103] (poster)
    A lactocrine mechanism for delivery of maternally derived relaxin (RLX) into the neonatal circulation as a consequence of nursing was proposed for the pig. Immunoreactive RLX was detected in colostrum and in the serum of newborn pigs only if they were allowed to nurse. Milk-borne RLX concentrations are highest during early lactation (9-19  ng/ml), declining to <2  ng/ml by postnatal day 14. Whether milk-borne RLX is bioactive is unknown. Evidence that RLX concentrations in milk are higher than in maternal circulation in several species suggests the mammary gland as a site of local RLX production. It is unknown whether the porcine mammary gland is a source of RLX. Therefore, objectives were to evaluate RLX bioactivity in porcine milk during the first 2 weeks of lactation, identify the form of RLX in porcine milk, and determine whether mammary tissue from early lactation is a source of milk-borne RLX. Milk RLX bioactivity was determined using an in vitro bioassay in which cAMP production by human embryonic kidney (HEK293T) cells transfected with the human RLX receptor (RXFP1) was measured. RLX bioactivity was highest at lactation day (LD) 0, decreasing to undetectable levels by LD 4. Immunoblot analysis of milk proteins revealed an 18  kDa band, indicating proRLX as the primary form of RLX in porcine milk. ProRLX protein and transcripts were detected in porcine mammary tissue on LD 0 and 7. Results support the lactocrine hypothesis by defining the nature and a potential source for bioactive proRLX in porcine colostrum/milk.
    Rheumatoid arthritis (RA) is characterized by joint inflammation and bone destruction. The receptor activator of nuclear factor-kappa B ligand (RANKL)-osteoprotegerin (OPG) system is important for maintaining the balance between bone resorption and formation. Both serum RANKL/OPG protein and synovial tissue RANKL/OPG mRNA ratios are elevated in patients with RA. Studies indicate that hormones of pregnancy, estrogens and relaxin, administered in combination, reduce circulating (TNF)-α and joint inflammation in a rat adjuvant-induced arthritis (AIA) model of RA. The purpose of this study was to investigate whether relaxin (RLX), alone or in combination with estrogens, regulates the bone remodeling markers RANKL and OPG in vivo and in vitro. Results show that in AIA rats, treatment with relaxin, estradiol valerate (EV) or EV in combination with relaxin had no effect on circulating RANKL. However, EV increased systemic OPG and combined treatment with EV and relaxin further increased circulating OPG in comparison to EV alone. Importantly, the RANKL/OPG protein ratio was lower in rats treated with EV or EV+RLX when compared to arthritic controls. Relaxin in combination with EV decreased local RANKL transcripts, increased OPG mRNA and decreased the RANKL/OPG mRNA ratio in joints of arthritic rats when compared to controls. RLX family peptide receptor 1 (RXFP1) gene expression in joints of AIA rats increased in response to EV and EV+RLX. In parathyroid hormone-pretreated murine UMR 106-01 osteoblast cells, 17-β-estradiol (E) and E+RLX increased RXFP1 transcripts, while RLX reduced RANKL mRNA and protein expression. However, in vitamin D-treated primary rat osteoblast cells E+RLX increased OPG protein and reduced the RANKL/OPG protein ratio. These results suggest that modulation of the RANKL-OPG system by estrogens and RLX may contribute to their antiarthritic effects on bone during pregnancy.
    Lactocrine communication of milk-borne bioactive factors (MbFs) from mother to offspring through nursing can affect neonatal development with lasting consequences. Relaxin (RLX), a lactocrine-active peptide found in porcine colostrum, stimulates estrogen receptor-α (ESR1) expression required for uterine development shortly after birth (postnatal day=PND 0). Whether other MbFs or cooperative lactocrine mechanisms affect the neonatal uterine developmental program is unknown. To determine the effects of age, nursing, and exogenous RLX on gene expression associated with uterine development, gilts (n=4-5/group) were assigned to nurse ad libitum or to receive milk replacer, with or without exogenous RLX (20 μg/kg BW i.m./6 h for 48 h), from birth to PND 2 when uteri were collected. Body weight and uterine weight increased (P<0.05) similarly from birth to PND 2 in all gilts. However, colostrum consumption was required for normal uterine ESR1, vascular endothelial growth factor (VEGFA), matrix metalloproteinase 9 (MMP9), and RLX receptor (RXFP1) protein and/or transcript expression on PND 2. Uterine ESR1, VEGFA, and MMP9 protein levels were below (P<0.01) the assay sensitivity in replacer-fed gilts. Supplemental RLX increased (P<0.05) uterine ESR1 protein and mRNA in nursed gilts, as well as VEGFA protein in nursed and VEGFA mRNA in both nursed and replacer-fed gilts. RLX treatment did not affect uterine MMP9 mRNA levels. When compared with replacer-fed gilts on PND 2, uterine RXFP1 mRNA was reduced (P<0.05) in nursed gilts and in RLX-supplemented replacer-fed gilts. These results constitute the first evidence that establishment of the neonatal porcine uterine developmental program requires maternal lactocrine support.
    Matrix metalloproteinases (MMPs) digest the extracellular matrix to facilitate cellular proliferation and tissue growth. The gelatinase MMP9, expressed in the neonatal and prepubertal porcine uterus, has been linked to porcine uterine growth and development. Colostrum (first milk) is rich in bioactive factors that include relaxin and estradiol, both of which can induce uterine MMP9 expression. Lactocrine signaling occurs when bioactive factors are transferred from mother to offspring as a consequence of nursing. Thus, lactocrine-acting factors are likely to affect critical developmental events in neonatal tissues. For example, ingestion of colostrum during the first 48 h of life is necessary to support normal estrogen receptor alpha (ESR1) expression in the neonatal porcine uterus, a requirement for normal uterine development. Whether nursing affects uterine MMP9 expression during this period is unknown. Moreover, effects of nursing after colostrum deprivation for 48 h from birth on the expression of specific markers and mediators of uterine development remain undefined. Here, objectives were to determine effects of: (1) age and nursing on porcine uterine MMP9 expression at birth [postnatal day (PND) 0] and on PND 2 and; (2) the age at first nursing (PND 0 or PND 2) and duration of nursing on expression of porcine uterine MMP9 and ESR1 at PND 14. For objective 1, neonatal gilts (n=4-5/group) were assigned randomly at birth to either nurse ad libitum or to receive hormone-free milk replacer via gavage (50 ml/2 h for 48 h). Uteri were collected on PND 2. In addition, uteri were obtained from gilts at birth (PND 0), prior to their consumption of colostrum. For objective 2, neonatal gilts (n=4-5/group) were: (a) allowed to nurse ad libitum; (b) nursed for 2 d from birth and switched to milk replacer; (c) fed milk replacer ad libitum; or (d) fed milk replacer for 2 d from birth and switched to nursing. Uterine tissues were collected on PND 14. MMP9 and ESR1 proteins were detected by immunoblotting using actin as a loading control. Uterine MMP9 activity was evaluated by gelatin zymography. For objective 1, uterine MMP9 protein was undetectable at birth and induced at PND 2 in nursing, but not replacer-fed animals. For objective 2, uterine MMP9 and ESR1 protein levels were similar at PND 14 in gilts that nursed for either two weeks or for only 48 h from birth. In gilts fed milk replacer for two weeks, uterine MMP9 was reduced (P<0.05) and ESR1 protein was undetectable on PND 14 in comparison to colostrum-fed gilts. Return to nursing after 48 h from birth did not restore uterine MMP9 or ESR1 signals at PND 14 to levels observed for colostrum-fed gilts. Zymographic analyses indicated an increase (P<0.05) in uterine MMP9 gelatinolytic activity in animals that nursed from birth to PND 14 in comparison to gilts that were fed replacer over the same period. Overall, results indicate that normal induction of porcine uterine MMP9 and ESR1 requires ingestion of colostrum during the first 48 h of life. Data support the idea that maternally-driven lactocrine signaling for two days from birth may be essential to establish an optimal developmental program in neonatal uterine tissues. Depriving neonates of such lactocrine-acting factors could alter the developmental trajectory of uterine tissues with negative reproductive consequences in adulthood. (Support USDA-NRI 2003-35203-1357 and 2007-35203-18098; NSF-EPS 0814103) (platform)
    Colostrum (first milk) serves as an important source of bioactive molecules, including antibodies and hormones. Since all neonatal mammals drink milk, such milk-borne bioactive factors (MbFs) are likely to be important for postnatal development. The lactocrine hypothesis states that MbFs delivered into the circulation of nursing infants can influence developmental programming of neonatal tissues. The peptide hormone relaxin (RLX), a prototypical lactocrine-acting factor, is present in the colostrum of several species including pigs and humans. RLX supports events required for development of the neonatal porcine reproductive tract, including expression of estrogen receptor (ER) alpha (ESR1) in the uterus and cervix. Normal ESR1 expression in these tissues is altered in neonatal pigs deprived of colostrum from birth (postnatal day = PND 0). Both ER subtypes, ESR1 and estrogen receptor beta (ESR2), are expressed in human and rodent cardiac tissue. It is not known if the neonatal porcine heart is an estrogen or RLX target tissue, or whether nursing or RLX affect ER or RLX receptor (RXFP1) expression in the heart, as demonstrated for the reproductive tract. Objectives were to determine effects of age, nursing and exogenous RLX treatment on the expression of markers of neonatal porcine cardiac development including ESR1, ESR2 and RXFP1. At birth, gilts were assigned randomly to either nurse ad libitum or to receive hormone-free milk replacer, with or without exogenous RLX treatment (20 µg/kg BW im/6h for 48 h). Left ventricular cardiac tissues were collected from newborn (PND 0) gilts prior to ingestion of colostrum and from treated gilts on PND 2. Immunoblotting was used to assess cardiac ESR1 and ESR2 protein expression, with actin used as a reference for loading. Quantitative RT-PCR was used to quantify cardiac RXFP1 mRNA expression. Cardiac ESR1, undetectable in newborn gilts, was induced in gilts that nursed, but was undetectable in replacer-fed animals at PND 2. Exogenous RLX had no effect on cardiac ESR1 protein expression in nursing gilts, but partially restored (P<0.05) ESR1 expression in replacer-fed gilts at PND 2. Cardiac ESR2 protein was detectable at birth and at PND 2 in both nursed and milk replacer-fed gilts. Exogenous RLX treatment did not affect cardiac ESR2 protein expression on PND 2. Cardiac RXFP1 mRNA levels were higher (P<0.05) on PND 0 than on PND 2 for all treatment groups. Within PND 2 groups, cardiac RXFP1 mRNA was reduced (P<0.05) in animals that nursed or received exogenous RLX in comparison to milk replacer-fed animals. Together, data suggest that factors present in colostrum are necessary for normal expression of neonatal porcine cardiac ESR1. Although exogenous RLX was bioactive in uterine tissues, treatment of milk replacer-fed gilts with RLX alone was not sufficient to restore ESR1 protein expression to levels observed in nursing animals at PND 2. Data support and extend the lactocrine hypothesis by showing that milk-borne factors affect expression patterns for developmentally important genes and proteins in the neonatal heart. Lactocrine signaling may affect neonatal cardiac tissue programming and development in a manner similar to that proposed for reproductive tract tissues. (Support: USDA-NRI 2007-35203-18098; NSF-EPS 0447675) (poster)
    Maternal influence on neonatal reproductive development extends beyond the womb into postnatal life. Bioactive factors present in colostrum (first milk) are delivered to offspring via nursing. The lactocrine hypothesis describes a mechanism whereby such milk-borne factors affect development of neonatal tissues. In support of this hypothesis, data for the uterus and cervix indicate an alteration in estrogen receptor-α (ESR1) protein expression in gilts fed a hormone-free milk replacer via gavage from birth when compared to gilts allowed to nurse through postnatal day (PND) 2. Nursing in pigs encompasses a series of discrete behaviors that include sucking the teat. Whether the absence of one component of nursing behavior in replacer-fed gilts (sucking) influences uterine ESR1 protein expression is unknown. The importance of the ESR1 system on growth and function of male reproductive tissues in animals including the pig is also recognized. However, the extent to which consumption of colostrum affects ESR1 expression in these tissues during early neonatal life is unknown. Objectives were to determine if: (1) sucking behavior associated with ingestion of colostrum during nursing influences uterine ESR1 expression in PND 2 gilts; and (2) nursing in neonatal male pigs influences the expression of ESR1 in the testis, epididymis, and prostate at PND 2. In study 1, gilts (n=4-5/group) were randomly assigned at birth (PND 0) to one of three treatment groups: (1) nursed ad libitum; (2) gavage-fed milk replacer (15ml/kg BW/2h); or (3) bottle-fed milk replacer. In study 2, boars (n=6/group) were assigned randomly at birth to be nursed normally or to be pan-fed milk replacer ad libitum. Uteri (study 1), testes, prostates, and epididymides (caput, corpus and cauda; study 2) were collected on PND 2. ESR1 protein expression was evaluated by immunoblotting using actin as a loading control. In study 1, an immunoreactive 51 kDa band corresponding to ESR1 was detected in uterine tissue extracts from gilts allowed to nurse that was not detectable in tissues from gilts consuming milk replacer, regardless of the mode of delivery (gavage or bottle-fed). In study 2, ESR1 was detected in testis, prostate and all three regions of the epididymis in males allowed to nurse normally, but was not detectable in tissues obtained from males fed replacer from birth. Results indicate that mode of milk replacer ingestion (sucking behavior vs orogastric gavage) is not a factor affecting ESR1 protein expression in the neonatal porcine uterus at PND 2. Thus, it is the absence of milk-borne, lactocrine-acting factors in replacer-fed gilts that accounts for changes in the neonatal uterine developmental program observed in milk replacer-fed animals. Data extend the scope of the lactocrine hypothesis by showing that milk-borne factors affect ESR1 expression patterns in reproductive tract tissues of males as well as females. Overall, this study reinforces the importance of lactocrine signaling to support normal developmental programming of neonatal reproductive tract tissues. (Support: USDA-NRI 2007-35203-18098; NSF-EPS 0447675) (poster)
    The first two weeks of neonatal life in pigs is a critical period for reproductive tract programming. Data for the uterus indicate that disruption of gene expression between birth (postnatal day = PND 0) and PND 14 changes the developmental trajectory of these tissues. Colostrum, or first milk, may play a role in maternal programming of neonatal development. The lactocrine hypothesis was proposed as a mechanism whereby bioactive milk-borne factors delivered to nursing offspring affect tissue development. Compared to nursing animals, expression of markers of cervical development in neonatal gilts fed a hormone-free milk replacer from birth is altered by PND 2. The extent to which this 48 h window of sensitivity to milk-borne factors influences subsequent cervical development is unknown. In addition, whether effects of the absence of nursing on the cervical phenotype at PND 2 can be reversed by returning gilts to nursing has not been investigated. Objectives were to determine effects of age at first nursing (birth or PND 2) and both duration and period of nursing on expression of molecular markers of cervical growth, remodeling and apoptosis at PND 14. Targeted proteins included estrogen receptor-alpha (ESR1) and vascular endothelial growth factor (VEGFA), both mediators of porcine cervical growth, matrix metalloproteinase 2 (MMP2), involved in tissue remodeling, and the anti-apoptotic marker BCL2. Gilts (n = 5-11/group) were assigned randomly to one of four treatment groups at birth, in which they were: 1) allowed to nurse ad libitum; 2) pan-fed a hormone-free milk replacer ad libitum; 3) nursed for 48 h from birth and switched to replacer; or 4) fed replacer for the first 48 h of life and switched to nursing. Cervices were collected on PND 14. Protein expression was evaluated by immunoblotting, using actin as a loading control, and quantified by densitometry. Expression of cervical ESR1 and BCL2 proteins, undetectable at PND 0, was induced in PND 14 gilts nursed from birth, but remained undetectable in gilts fed replacer during the first 48 h of life. VEGFA protein, undetectable at PND 0, was detectable on PND 14 in all groups, but was markedly reduced (p < 0.01) in PND 14 gilts fed replacer from birth. Latent MMP2 was detectable at similar levels at birth and on PND 14 in nursed and replacer-fed gilts. Active MMP2, undetectable at PND 0, was induced in both nursing and replacer-fed gilts by PND 14. There were no differences in cervical ESR1, VEGFA or BCL2 protein levels in gilts allowed to nurse continuously for two weeks or for only 48 h from birth. Data support the lactocrine hypothesis for maternal programming of neonatal development, indicating that milk-borne factors are required to support protein expression patterns important for cervical development in the neonatal gilt. Results indicate that the first 48 h of neonatal life constitutes a potentially critical period for lactocrine signaling in porcine cervical tissues. This idea is supported by the fact that effects of disruption of lactocrine signaling on cervical development between birth and PND 2 persisted to PND 14 even when replacer-fed gilts returned to nursing after PND 2. Thus, in the absence of lactocrine signaling from birth, altered expression patterns for morphoregulatory proteins, as observed here, could affect the cervical developmental trajectory with long-term consequences for reproductive performance and health. (Support: USDA-NRI 2007-35203-18098; NSF-EPS 0447675) (poster)
    Disruption of estrogen-sensitive, estrogen receptor (ER)-dependent events during porcine uterine development between birth (postnatal day=PND 0) and PND 14 affects patterns of uterine morphoregulatory gene expression in the neonate with lasting consequences for reproductive success. Uterine capacity for conceptus support is reduced in pregnant adult gilts exposed to estradiol valerate (EV) for 14 days from birth. Objectives here were to determine effects of EV exposure from birth through PND 13 on neonatal uterine and adult endometrial markers of growth, patterning, and remodeling. Targets included the relaxin receptor (RXFP1), estrogen receptor-alpha (ESR1) and vascular endothelial growth factor (VEGFA), morphoregulatory markers HOXA10 and WNT7A, and the matrix metalloproteinases (MMP)2 and MMP9. Gilts were treated daily with EV (50 microg/kg body weight per day, i.m.) or corn oil vehicle from birth through PND 13. Uteri were obtained from neonates on PND 14 and from adults on pregnancy day 12 (PxD 12). In neonates, EV exposure from birth increased uterine RXFP1 gene expression, and both ESR1 and VEGFA proteins. At PxD 12, endometrial RXFP1 mRNA remained elevated, while ESR1 protein was reduced. Early EV treatment decreased neonatal uterine WNT7A, but increased HOXA10 expression. WNT7A expression was reduced in EV-treated adults. Transient EV exposure increased MMP9 transcripts at PND 14, whereas both latent and active MMP9 activity was increased due to early EV treatment in adults on PxD 12. Results support the hypothesis that transient, estrogen-induced disruption of porcine uterine development from birth alters early programming events that lead to functional consequences in the adult.
    Nutritional and immunological benefits of colostrum (herein referred to as milk) for neonatal growth and survival are well known. However, the role of milk as a conduit for bioactive factors that can affect neonatal development is not well understood. Immunoreactive relaxin (RLX) was detected in porcine milk and in the systemic circulation of newborn pigs only if they were allowed to nurse. In addition, the neonatal cervix is sensitive to trophic effects of RLX by postnatal day (PND) 2. Thus, the lactocrine hypothesis was proposed as a mechanism whereby milk-borne growth factors are delivered to nursing offspring where they can affect target tissues. Whether milk and/or RLX are necessary for neonatal porcine cervical development is unknown. Here, objectives were to determine effects of: 1) age and consumption of milk on expression of molecular markers of cervical growth, remodeling and apoptosis at birth (PND 0; before nursing) and on PND 2; and 2) exogenous RLX on cervical expression of targeted markers in gilts allowed to nurse versus those fed hormone-free milk replacer. Targeted proteins included estrogen receptor-alpha (ESR1) and vascular endothelial growth factor (VEGFA), both mediators of porcine cervical growth, matrix metalloproteinase 2 (MMP2), involved in tissue remodeling, and the anti- and pro-apoptotic markers BCL2 and caspase 3. Gilts (n = 4-7/group) were assigned randomly to one of four treatment groups at birth, in which they were: 1) allowed to nurse ad libitum; 2) fed milk replacer; 3) nursed and given either RLX (20 µg/kg BW, im) or vehicle every 6 h for 48 h; or 4) fed replacer and treated with exogenous RLX/vehicle for the first 48 h of life. Cervices were collected on PND 2, approximately 3 h after the last treatment. Additionally, cervices were collected from a fifth group at birth, before gilts were allowed to nurse. Protein expression was evaluated by immunoblotting and quantified by densitometry. Expression of cervical ESR1, VEGFA and BCL2 proteins, undetectable at PND 0, was induced in gilts allowed to nurse for two days, but remained undetectable in replacer-fed gilts. In contrast, MMP2 expression, also undetectable at PND 0, was induced in both nursing and replacer-fed gilts. RLX increased ESR1, VEGFA and BCL2 expression in nursing (p < 0.05) but not in replacer-fed gilts. MMP2 expression was increased by RLX treatment in both nursing and replacer groups (p < 0.01). Caspase 3 expression was undetectable at both PND 0 and 2, regardless of treatment. Data support the lactocrine hypothesis for maternal programming of neonatal development, showing that milk-borne factors are important for induction of growth and anti-apoptotic proteins in the cervix at PND 2. Data support the idea that lactocrine-acting factors may be cooperative in facilitating the actions of RLX. In the absence of milk, altered expression patterns for morphoregulatory proteins, as observed here, could affect neonatal cervical development with long-term consequences for reproductive performance and health. Data complement earlier results indicating that the neonatal porcine cervix is sensitive to RLX as reflected by increased expression of specific growth, remodeling and anti-apoptotic proteins. Thus, lactocrine-driven programming must be considered a potentially critical component of the maternal continuum of factors affecting female reproductive tract development. (Support: USDA-NRI 2003-35203-1357 and 2007-35203-18098; NSF-EPS 0814103) (poster)
    In the pig, uterine development is initiated prenatally and continues after birth (postnatal day = PND 0). Postnatal development of the porcine uterus involves morphogenetic events that include differentiation and proliferation of endometrial glandular epithelium and temporospatially associated changes in morphoregulatory gene expression patterns. Endometrial development also requires uterine expression of estrogen receptor-alpha (ESR1), evident by PND 2. Relaxin (RLX), a peptide hormone found in porcine colostrum/milk, is uterotrophic in neonatal gilts and stimulates uterine ESR1 expression. Evidence that milk-borne RLX is bioactive supports the idea that such lactocrine-acting factors, delivered from mother to offspring in milk, contribute to the neonatal uterine developmental program. Objectives here were to determine effects of the consumption of colostrum/milk in the presence and absence of exogenous RLX from birth on the expression of molecular markers of endometrial development at PND 2. At birth, neonatal gilts (n=4-5/group) were assigned randomly to either nurse ad libitum (milk) or to receive hormone-free milk-replacer, with or without exogenous RLX treatment (20 µg/kg BW im/6h for 48h). Uteri were collected 3h after the last treatment on PND 2. In addition, uteri were obtained from gilts prior to their consumption of milk at birth. Targeted uterine transcripts, quantified by real-time PCR, included ESR1, the relaxin receptor RXFP1, and the morphoregulatory gene WNT7A. Proteins, quantified by immunoblotting and densitometry, included ESR1, vascular endothelial growth factor (VEGFA), and matrix metalloproteinase 2 (MMP2). Uterine expression of ESR1 mRNA was greater (P<0.05) in milk- than in replacer-fed gilts at PND 2, in which levels were similar to those observed at birth, and increased (P=0.09) with RLX treatment in milk-fed gilts. Detectable at birth, RXFP1 mRNA expression remained at PND 0 levels in replacer-fed gilts on PND 2, but was reduced (P<0.05) at PND 2 in milk-fed gilts and in response to RLX in both groups. WNT7A expression, also detectable at birth, was lower (P<0.05) in milk- than in replacer-fed gilts at PND 2 and unaffected by exogenous RLX. Consistently, uterine ESR1 protein expression, undetectable in newborn gilts, was induced in milk-fed gilts, but remained below assay sensitivity in all replacer-fed gilts at PND 2. Exogenous RLX increased (P<0.01) uterine ESR1 protein expression in milk- but not in replacer-fed gilts at PND 2. Similar expression patterns were observed for VEGFA protein. Uterine MMP2 protein expression, detectable in newborn gilts, was greater (P<0.01) in milk- than in replacer-fed gilts at PND 2 and was further increased (P<0.01) in milk-fed gilts following RLX treatment from birth. Data indicate that factors in milk can regulate uterine expression of ESR1, VEGFA, WNT7A and MMP2, and that both exogenous RLX and/or related milk-borne factors, which could include lactocrine-acting RLX, affect uterine RXFP1 expression in the neonatal pig. Evidence that milk-borne factors affect patterns of morphoregulatory gene expression during a critical period for neonatal uterine development in the pig supports the lactocrine hypothesis for maternal programming of female reproductive tract development. Disruption of this program can have long-term consequences for reproductive performance and health. (Support USDA-NRI 2003-35203-1357 and 2007-35203-18098; NSF-EPS 0814103) (poster)
    The mycotoxin zearalenone (ZEA) is a selective estrogen receptor modulator that can contaminate cereal feeds and lead to reproductive disorders. To determine effects of perinatal ZEA exposure on uterine expression of genes associated with endometrial development in the neonatal gilt, pregnant sows were fed ZEA (1500 microg ZEA/kg of feed/day) or vehicle from 14 days before farrowing through postnatal day (PND) 20-21, when neonatal uterine tissues were collected. At birth, gilts were cross-fostered to generate four ZEA exposure groups (n= 5-6/group): unexposed controls or exposures limited to prenatal, postnatal, or pre- and postnatal (continuous) periods. Results showed that at PND 20-21, uterine Wnt7a, Hoxa10, estrogen receptor alpha, and RXFP2 mRNA levels were decreased in neonates exposed continuously to ZEA (P < 0.05). Uterine RXFP1 transcripts were decreased in postnatally and continuously exposed groups (P < 0.05). Neonatal uterine Wnt4 mRNA levels were unchanged.
    The porcine female reproductive tract undergoes estrogen receptor (ER) alpha-dependent development after birth (postnatal day=PND 0), the course of which can determine adult uterine function. Uterotrophic effects of relaxin (RLX) in the porcine neonate are age specific and may involve ER activation. Here, objectives were to determine effects of RLX and estrogen administered from birth on uterine and cervical growth and expression of ERalpha, vascular endothelial growth factor (VEGF), and the RLX receptor (RXFP1). On PND 0, gilts were treated with the antiestrogen ICI 182 780 (ICI) or vehicle alone and, 2 h later, were given estradiol-17beta (E) or porcine RLX for 2 days. Neither RLX nor E affected uterine wet weight or protein content on PND 2. However, RLX, but not E, increased cervical wet weight and protein content when compared with controls. Pretreatment with ICI did not inhibit RLX-stimulated cervical growth. Uterine and cervical ERalpha increased in response to RLX, but not E. Both RLX and E increased VEGF in the uterus and cervix on PND 2. Pretreatment with ICI increased VEGF in both tissues and increased RLX-induced cervical VEGF. In the uterus E, but not RLX, increased RXFP1 mRNA. In the cervix, E increased RXFP1 gene expression whereas RLX decreased it. Results indicate that the neonatal uterus and cervix are sensitive to E and RLX and that growth responses to RLX in these tissues differ by PND 2. Effects of RLX on uterine and cervical ERalpha and VEGF expression may be important for neonatal reproductive tract development.
    Disruption of estrogen-sensitive, estrogen receptor (ER)–dependent events during development of the porcine uterus between birth (postnatal day = PND 0) and PND 14 affects patterns of uterine morphoregulatory gene expression in the neonate and alters the developmental trajectory of uterine tissues with lasting consequences for reproductive success. Uterine capacity for conceptus support is reduced in pregnant adult gilts exposed to estradiol valerate (EV) for 14 days from birth. Effects of such uterine developmental disruption were reflected dramatically in neonatally EV-exposed adult gilts on pregnancy day 12 (PxD 12), in which a proteomic analysis revealed quantitative changes in relative expression patterns for 300 different endometrial proteins and peptides. Objectives of this study were to determine effects of EV exposure from birth through PND 13 on patterns of change in neonatal uterine and adult endometrial expression of genes implicated in the regulation of critical, estrogensensitive uterine organizational events during early neonatal life. Targeted transcripts included the morphoregulatory genes Hoxa10 and Wnt7a, the relaxin receptor, LGR7, and ER alpha, both recognized to mediate uterine growth and endometrial development in the pig, and the matrix metalloproteinases (MMP)-2 and MMP-9. Gilts (n=4–6/neonatal and adult groups) were treated daily with EV (50μg/kg BW/day, i.m.) or corn oil (CO) vehicle alone from PND 0 to PND 13. Uteri were obtained from CO- and EV-treated neonates on PND 14 and from adult gilts in both groups on PxD 12. Targeted transcripts were quantified using real-time RT-PCR with pig-specific primers and total RNA isolated from individual uteri (neonates) or endometrial samples (adults). In neonates on PND 14, results indicated that EV exposure from birth reduced (p<0.001) levels of Wnt7a mRNA and increased (p<0.05) levels of Hoxa10, LGR7 and MMP-9 mRNA. Treatment with EV did not affect ER alpha or MMP-2 transcript levels on PND 14. In adults on PxD 12, transient neonatal EV exposure reduced (p<0.05) endometrial levels of Wnt7a and MMP-9 mRNA, and increased (p<0.05) levels of LGR7 mRNA. Endometrial Hoxa10, ER alpha and MMP-2 mRNA levels on PxD 12 were unaffected by neonatal EV treatment. These quantitative data complement and extend earlier in situ hybridization analyses indicating that EV exposure from birth affects expression of neonatal uterine epithelial Wnt7a negatively, and stromal Hoxa10 and LGR7 positively, without affecting endometrial ER alpha expression. Data also provide the first evidence that estrogen-sensitive developmental programming of porcine uterine tissues between birth and PND 14 may involve MMP-9. Results support the hypothesis that transient estrogen-induced disruption of porcine uterine development from birth will be reflected by alterations in adult endometrial gene expression patterns during early pregnancy. Persistent dysregulation of endometrial Wnt7a, MMP-9 and LGR7 expression in adult gilts on PxD 12 indicates that EV-exposure from birth not only alters the neonatal uterine developmental program acutely but affects the developmental trajectory of endometrial tissues and, ultimately, endometrial function in adulthood. Further, results indicate that Wnt7a, MMP-9 and LGR7 may be useful as molecular markers of neonatal estrogen exposure in the adult porcine uterus. (Support USDA-NRI 2003-35203-13572 and NSF EPS-0447675) (platform)
    The objective of the present experiments was to study the effects of pulmonary inflammation induced by subacute Sulfur-dioxide (SO(2)) exposure on capsaicin-induced responses in isolated primary vagal sensory neurons and cough. Additionally, we examined the effects of SO(2) exposure on respiratory function and lung histology. All experiments were conducted 24 h after 4 days of subacute SO(2) (1000 ppm, 3 h/day for 4 days) exposure. In in vitro experiments, intracellular Ca(2+) concentrations were measured in single nodose ganglia cells isolated from SO(2) treated and control guinea pigs, using a fluorescence-based methodology. In nodose ganglia cells from SO(2)-exposed animals, intracellular Ca(2+) responses evoked by capsaicin (1 x 10(-7) and 1 x 10(-6) M) were significantly augmented (87% and 59%, respectively) compared to nodose ganglia from control animals. In vivo experiments, cough responses induced by a submaximal dose of aerosolized capsaicin (30 microM) were increased approximately 50% in SO(2) exposed animals compared to control animals. The enhanced cough response produced by SO(2) was inhibited by the corticosteroid, dexamethasone (10 mg/kg, p.o. b.i.d for 4 days and 10 mg/kg, p.o. once on day 5). In separate experiments, guinea pigs exposed to SO(2) displayed a decrease in respiratory frequency and minute ventilation and an increase in enhanced pause (PenH), a surrogate measure for pulmonary obstruction. Associated with the SO(2)-induced increase in cough and changes in respiratory parameters was an increase in BAL neutrophils. BAL neutrophil counts were 5+/-4 and 691+/-141 cells x 10(3)/ml for air and SO(2)-exposed animals, respectively. The neutrophillic inflammation induced by SO(2) was attenuated by dexamethasone treatment. Finally, staining for collagen, smooth muscle and goblet cells showed inflammation, remodeling and goblet cell metaphasia in the SO(2)-exposed animals. Our results demonstrate that SO(2) exposure enhances TRPV1 receptor function at the level of the nodose ganglia. This effect occurs in parallel with an increase sensitivity of the cough response to capsaicin.
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