Biology of Reproduction (BIOL REPROD)

Publications in this journal

• Article: Changes in Mouse Uterine Transcriptome in Estrus and Proestrus

  Kerri Stanley Yip, Alexander Suvorov, Jeannette Connerney, Nicholas J. Lodato, David J. Waxman
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  ABSTRACT: Changes in the CD-1 mouse uterine transcriptome during proestrus and estrus were investigated to help elucidate mechanisms of uterine tissue remodeling during the estrus cycle and their regulation by estrogen and progesterone in preparation of the uterus for pregnancy. Mice were staged beginning at 6 wk of age and uterine horns were harvested after monitoring two estrus cycles. Microarray analysis of whole uterine horn RNA identified 2,428 genes differentially expressed in estrus compared to proestrus, indicating there is extensive remodeling of mouse uterus during the estrus cycle, affecting ~10% of all protein-coding genes. Many (~50%) of these genes showed the same differential expression in independent analysis of isolated uterine lumenal epithelial cells. Changes in gene expression associated with structural alterations of the uterus include remodeling of the extracellular matrix, changes in cell keratins and adhesion molecules, activation of mitosis, and changes in MHC class II presentation, complement and coagulation cascades, and cytochrome P450 expression. Signaling pathways regulated during the estrus cycle, involving ligand-gated channels, Wnt and hedgehog signaling, and transcription factors with poorly understood roles in reproductive tissues, include several genes and gene networks that have been implicated in pathological states. Many of the molecular pathways and biological functions represented by the genes differentially expressed between proestrus to estrus are also altered during the human menstrual cycle, although not necessarily at the corresponding phases of the cycle. These findings establish a baseline for further studies in the mouse model to dissect mechanisms involved in uterine tissue response to endocrine disruptors and the development of reproductive tract diseases.
  Biology of Reproduction 06/2013;
• Article: FOXC1 Is Enriched in the Mammary Luminal Progenitor Population, but Is Not Necessary for Mouse Mammary Ductal Morphogenesis.

  Bernardo GM, Sizemore ST, Pal B, Booth CN, Seachrist DD, Abdul-Karim FW, Kume T, Keri RA
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  ABSTRACT: Expression of FOXC1, a forkhead box transcription factor, correlates with the human basal-like breast cancer (BLBC) subtype, and functional analyses have revealed its importance for in vitro invasiveness of BLBC cells. Women diagnosed with this breast tumor subtype have a poorer outcome due to the lack of targeted therapies, thus continued investigation of factors driving these tumors is critical to uncover novel therapeutic targets. Several processes that dictate normal mammary morphogenesis parallel cancer progression and enforced expression of FOXC1 can induce a progenitor state in more differentiated mammary epithelial cells. Consequently, evaluating how FOXC1 functions in the normal gland is critical to further understand BLBC biology. While FOXC1 is well known to control normal development of a number of tissues, its role in the mammary gland has not yet been investigated. Herein, we describe FOXC1 expression patterning in the normal breast where it is localized to the basal/myoepithelium, suggesting that FOXC1 would be required for normal development. However, mammary glands lacking Foxc1 have no overt defect in ductal outgrowth, alveologenesis or lineage specification. Of significant interest, we found that expression of FOXC1 is enriched in the normal luminal progenitor population, which is the postulated cell-of-origin of BLBC. These results indicate that FOXC1 is unnecessary for mammary morphogenesis and that its role in BLBC likely involves processes that are unrelated to cell lineage specification.
  Biology of Reproduction 05/2013;
• Article: Aging and Luteinizing Hormone Effects on Reactive Oxygen Species (ROS) Production and DNA Damage in Rat Leydig Cells.

  Beattie MC, Chen H, Fan J, Papadopoulos V, Miller P, Zirkin BR
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  ABSTRACT: We observed previously that after long-term suppression of luteinizing hormone (LH) and thus of Leydig cell steroidogenesis, the re-stimulation of the Leydig cells by LH resulted in significantly higher testosterone production than by age-matched cells from control rats. These studies suggest that stimulation, over time, may elicit harmful effects on the steroidogenic machinery, perhaps through alteration of the intracellular oxidant-to-antioxidant balance. Herein we compared the effects of LH stimulation on stress response genes, intracellular reactive oxygen species (ROS) formation, and ROS-induced damage to ROS-susceptible macromolecules (DNA) in young and aged cells. Microarray analysis indicated that LH stimulation resulted in significant increases in expression of genes associated with stress response and anti-apoptotic pathways. Short-term LH treatment of primary Leydig cells isolated from young rats resulted in transiently increased ROS levels compared to controls. Aged Leydig cells also showed increased ROS soon after LH stimulation. However, in contrast to the young cells, ROS production peaked later and the time to recovery was increased. In both young and aged cells, treatment with LH resulted in increased levels of DNA damage, but significantly more so in the aged cells. DNA damage levels in response to LH and the levels of intracellular ROS were highly correlated. Taken together, these results indicate that LH stimulation causes increased ROS production by young and aged Leydig cells, and that while DNA damage occurs in cells of both ages, there is greater damage in the aged cells.
  Biology of Reproduction 03/2013;
• Article: Molecular Mechanisms and Pathways Involved in Bovine Embryonic Genome Activation and Their Regulation by Alternative In Vivo and In Vitro Culture Conditions.

  Gad A, Hoelker M, Besenfelder U, Havlicek V, Cinar U, Rings F, Held E, Dufort I, Sirard MA, Schellander K, Tesfaye D
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  ABSTRACT: Understanding gene expression patterns in response to altered environmental conditions at different time points of the preimplantation period would improve our knowledge on regulation of embryonic development. Here we aimed to examine the effect of alternative in vivo and in vitro culture conditions at the time of major embryonic genome activation (EGA) on the development and transcriptome profile of bovine blastocysts. Four different blastocyst groups were produced under alternative in vivo and in vitro culture conditions before or after major EGA. Completely in vitro (IVP) and in vivo produced blastocysts were used as controls. We compared gene-expression patterns between each blastocyst group and in vivo blastocyst control group using EmbryoGENE's bovine microarray. The data showed that changing culture conditions from in vivo to in vitro or vice versa, either before or after the time of major EGA, had no effect on the developmental rates, however in vitro conditions during that time critically influenced the transcriptome of the blastocysts produced. The source of oocyte had a critical effect on developmental rates and the ability of the embryo to react to changing culture conditions. Ontological classification highlighted a marked contrast in expression patterns for lipid metabolism and oxidative stress response between blastocysts generated in vivo vs. in vitro, with opposite trends. Molecular mechanisms and pathways that are influenced by altered culture conditions during EGA were defined. These results will help in the development of new strategies to modify culture conditions at this critical stage to enhance the development of competent blastocysts.
  Biology of Reproduction 07/2012;
• Article: Early Expression of Pregnancy-Specific Glycoprotein 22 (PSG22) by Trophoblast Cells Modulates Angiogenesis in Mice.

  Blois S, Tirado-Gonzalez I, Wu J, Barrientos G, Johnson B, Warren J, Freitag N, Klapp B, Irmak S, Ergün S, Dveksler G
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  ABSTRACT: Mouse and human pregnancy-specific glycoproteins (PSG) are known to exert immunomodulatory functions during pregnancy by inducing maternal leukocytes to secrete anti-inflammatory cytokines that promote a tolerogenic decidual microenvironment. Many such anti-inflammatory mediators also function as pro-angiogenic factors, which, along with the reported association of -murine PSG - with the uterine vasculature, suggest that PSG may contribute to the vascular adaptations necessary for successful implantation and placental development. We observed that PSG22 is strongly expressed around the embryonic crypt on gestation day (Gd) 5.5, indicating that trophoblast giant cells are the main source of PSG22 during the early stages of pregnancy. PSG22 treatment up-regulated the secretion of transforming growth factor beta 1 (TGFB1) and vascular endothelial growth factor A (VEGFA) in murine macrophages, uterine dendritic cells and natural killer cells. A possible role of PSGs in uteroplacental angiogenesis is further supported by the finding that incubation of endothelial cells with PSG22 resulted in the formation of tubes in the presence and absence of VEGFA. We determined that PSG22, like human PSG1 and murine PSG17 and 23, binds to the heparan sulfate chains in syndecans. Therefore, our findings indicate that despite the independent evolution and expansion of human and rodent PSG, members in both families have conserved functions, which include their ability to induce anti-inflammatory cytokines and pro-angiogenic factors as well as to induce the formation of capillary structures by endothelial cells. In summary, our results indicate that PSG22, the most abundant PSG expressed during mouse early pregnancy, is likely a major contributor to the establishment of a successful pregnancy.
  Biology of Reproduction 03/2012;
• Article: Effects of Different Temperatures on Testis Structure and Function, with Emphasis on Somatic Cells, in Sexually Mature Nile Tilapias (Oreochromis niloticus)1

  Érika Ramosde Alvarenga
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  ABSTRACT: The Nile tilapia (Oreochromis niloticus) is economically one of the most important freshwater fish and is an excellent model for studies under laboratory conditions. Temperature is considered a very important modulator of reproductive activity in fish, although few studies have specifically addressed the effects of this key factor on morphological and functional aspects of teleost testes. Therefore, our main objectives in the present study were to analyze the effects of different temperatures (20, 25, 30, and 35°C) on testicular somatic and germ cells in sexually mature Nile tilapias. Compared with fish kept at other temperatures, tilapias maintained at 20°C demonstrated increased (P < 0.05) Sertoli cell and Leydig cell proliferation, volume density and frequency of most type B spermatogonia, and germ cell apoptosis. Conversely, tubular fluid secretion was decreased (P < 0.05) in the same animals. Although not significant, type A spermatogonia proliferation followed the pattern established for Sertoli cell and Leydig cell mitotic activity, suggesting that they preferentially would proliferate at lower temperatures. Based on most results found in our study and considering that tilapias are nonseasonal breeders, we suggest a model for temperature action on tilapia testes in which lower temperature (20°C) would favor type A spermatogonial renewal, Sertoli cell and Leydig cell proliferation, and germ cell apoptosis, whereas higher temperatures (30–35°C) would trigger rapid germ cell differentiation. Thus, tilapias could potentially be utilized in studies involving hormones and factors related to Sertoli cell and Leydig cell proliferation and spermatogonial self-renewal or differentiation.
  Biology of Reproduction 01/2012;
• Article: The Bruce Effect in Norway Rats1

  Vera Marashi
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  ABSTRACT: Intrauterine implantation of fertilized ova can be blocked by exposing recently inseminated females with an unfamiliar male. This selective pregnancy failure, designated as the Bruce effect (Bruce, Nature 1959; 184:105), is well studied in laboratory mice and has been confirmed in several other rodent species. However, no clear information exists concerning this phenomenon in the laboratory rat. The present study was conducted to investigate whether or not the Bruce effect exists in the rat. Females of two F1 hybrid strains (ntotal = 354) with different MHC genotypes (F344BNF1, RT1lv1/n, and LEWPVGF1, RT1l/c) were mated with males of their own strain and subsequently exposed during the first 4 days postcoitus either to a male of the other hybrid strain or to an unfamiliar male of the same strain as the stud. The litter rate of each treatment group was determined. As a control, mated females of both strains were reexposed to the stud male to determine baseline litter rates. Female rats of both F1 hybrid strains showed a significantly lower litter rate when exposed to males of a different strain than their stud male, compared to the expected values of birth rates observed in control females (F344BNF1: P = 0.017; LEWPVGF1: P = 0.019). In contrast, there was no difference between expected and observed litter rates in females of both F1 hybrid strains after exposure to an unfamiliar male of the same strain as their stud. Our results demonstrate for the first time that the Bruce effect, well documented in mice, occurs in the Norway rat.
  Biology of Reproduction 09/2011;
• Article: Seasonal Effect on Germinal Vesicle-Stage Bovine Oocytes Is Further Expressed by Alterations in Transcript Levels in the Developing Embryos Associated with Reduced Developmental Competence1

  Mirit Gendelman
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  ABSTRACT: Physiological perturbations of bovine follicle-enclosed oocytes during the lengthy period of follicular development can lead to reduced oocyte developmental competence. It is suggested that heat stress-induced alterations in germinal vesicle (GV)-stage oocytes are further expressed in the transcriptional levels of genes involved in oocyte maturation and early embryonic development. Bovine oocytes were collected during cold (December–April) and hot (May–November) seasons, matured, fertilized, and cultured in vitro. The percentage of fertilized oocytes cleaving to the 2- to 4-cell stage was higher in the cold vs. hot season (89.0% ± 2.63% vs. 75% ± 2.63%, respectively; P < 0.05), as was the percentage of cleaved embryos further developing to blastocysts (26.6% ± 0.9% vs. 10.1% ± 1.8%, respectively; P < 0.05). Total RNA and poly(A) mRNA of oocytes and developing embryos were isolated and subjected to semiquantitative and real-time PCR for MOS, GDF9, and POU5F1 genes. In GV-stage oocytes, their mRNA levels did not differ between the seasons. However, following maturation, mRNA levels were higher in oocytes collected in the cold season (P < 0.05). In 4-cell-stage embryos, GDF9 and POU5F1 showed opposite mRNA patterns between seasons (higher and lower levels, respectively) in the hot season (P < 0.05). In both 8-cell-stage embryos and blastocysts, POU5F1 expression was lower during the hot season (P < 0.05). Exposing the ovarian pool of oocytes to environmental stress appears to impair maternal mRNA storage and/or the mechanism of transcription renewal, in turn affecting embryo gene expression before and after embryonic genome activation. Such impairment might partially explain the carry-over effect of summer heat stress on dairy cow conception rates.
  Biology of Reproduction 09/2011;
• Article: Role of Rho GTPases in Human Trophoblast Migration Induced by IGFBP11

  Jessica Saso, Sarah-Kim Shields, Yufeng Zuo, Chandan Chakraborty
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  ABSTRACT: Insulin-like growth factor binding protein 1 (IGFBP1), the main secretory product of the decidualized endometrium of a pregnant woman, has previously been shown to interact with the alpha5beta1 integrin of extravillous trophoblast (EVT) cell surface to stimulate its migration in an IGF-independent manner. This migration stimulation has also been shown to require activation of extracellular signal regulated kinases 1 and 2 (ERK1/2; mitogen-activated protein kinase [MAPK] 3/1]) and focal adhesion kinase. The present study examined the roles of Rho GTPases RHOA, RHOC, RAC1, and CDC42 as well as RHO kinases ROCK1 and ROCK2 in IGFBP1-mediated migration of an immortalized EVT cell line HTR-8/SVneo. A nonselective RHO kinase inhibitor, Y27632, as well as siRNAs selective for ROCK1 and ROCK2 decreased the migration of these cells in a Transwell migration assay, and this inhibition could not be restored by IGFBP1. Clostridium difficile toxin B, which inhibits all the Rho GTPases, RAC inhibitor NSC23766, RAC1 siRNA, and CDC42 siRNA, decreased their basal migration, but none of these inhibitions except CDC42 siRNA-induced inhibition could be restored by IGFBP1. Clostridium botulinum C3 exoenzyme that inhibits RHOA, RHOB, and RHOC inhibited basal migration but not IGFBP1-induced migration. IGFBP1-induced activation of ERK1/2 (MAPK3/1), which did not require RHO proteins, might function as an alternate pathway for RHO action. However, selective siRNA-mediated downregulation of RHOA inhibited basal, but not IGFBP1-mediated, migration, whereas that of RHOC inhibited both basal and IGFBP1-mediated migration of these EVT cells. Therefore, RHO kinase, RHOC, and RAC1 are essential, but RHOA and CDC42 are not essential, for IGFBP1-induced EVT migration.
  Biology of Reproduction 09/2011;
• Article: Mediators of the JAK/STAT Signaling Pathway in Human Spermatozoa1

  Catherine Lachance
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  ABSTRACT: In their journey to acquire the ability to fertilize the egg, numerous intracellular signaling systems are activated in spermatozoa, leading to an increase in protein tyrosine phosphorylation. Although the JAK/STAT signaling pathway is usually associated with the activation of transcription of specific genes, our laboratory previously demonstrated the presence of the IL6 receptor (IL6R) and the Janus kinase 1 (JAK1) in human spermatozoa, a cell that is mostly transcriptionally inactive. In order to determine the importance of the JAK/STAT signaling pathway, our objectives were to identify and characterize the mediators of this system in human sperm. Cell fractionation and surface biotinylation assays clearly demonstrated that IL6R is expressed at the sperm membrane surface. The kinase JAK1 is enriched in membrane fractions and is activated during human sperm capacitation as suggested by its increase in phosphotyrosine content. Many signal transducer and activator of transcription (STAT) proteins are expressed in human sperm, including STAT1, STAT3, STAT4, STAT5, and STAT6. Among them, only STAT1 and STAT5 were detected in the cytosolic fraction. All the detected STAT proteins were enriched in the cytoskeletal structures. STAT4 was present in the perinuclear theca, whereas JAK1, STAT1, and STAT5 were detected in the fibrous sheath. Indirect immunofluorescence studies showed that JAK1 and STAT1 colocalized in the neck region and that STAT4 is present at the equatorial segment and flagella. The presence of STAT proteins in sperm structural components suggests that their role is different from their well-known transcription factor activity in somatic cells, but further investigations are required to determine their role in sperm function.
  Biology of Reproduction 08/2011;
• Article: Avian SERPINB11 Gene: Characteristics, Tissue-Specific Expression, and Regulation of Expression by Estrogen1

  Whasun Lim, Ji-Hye Kim, Suzie E. Ahn, Wooyoung Jeong, Jinyoung Kim, Fuller W. Bazer, Jae Yong Han, Gwonhwa Song
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  ABSTRACT: Serpins,a group of proteins with similar structural and functional properties, were first identified based on their unique mechanism of action: their inhibition of proteases. While most serpins have inhibitory roles, certain serpins are not involved in canonical proteolytic cascades but perform diverse functions including storage of ovalbumin in egg white, transport of hormones (thyroxine- and cortisol-binding globulin), and suppression of tumors. Of these, serpin peptidase inhibitor, clade B, member 11 (SERPINB11) is not an inhibitor of known proteases in humans and mice, and its function is unknown. In the present study, the SERPINB11 gene was cloned, and its expression profile was analyzed in various tissues from chickens. The chicken SERPINB11 gene has an open reading frame of 1346 nucleotides that encode a protein of 388 amino acids that has moderate homology (38.8%–42.3%) to mammalian SERPINB11 proteins. Importantly, SERPINB11 mRNA is most abundant in the chicken oviduct, specifically luminal and glandular epithelia, but it was not detected in any other chicken tissues of either sex. We then determined effects of diethylstilbestrol (DES; a synthetic nonsteroidal estrogen) on SERPINB11 expression in the chicken oviduct. Treatment of young chicks with DES induced SERPINB11 mRNA and protein only in luminal and glandular epithelial cells of the oviduct. Collectively, these results indicate that the novel estrogen-induced SERPINB11 gene is expressed only in epithelial cells of the chicken oviduct and implicate SERPINB11 in regulation of oviduct development and differentiated functions.
  Biology of Reproduction 08/2011;
• Article: Adrenomedullin 2/Intermedin Regulates HLA-G in Human Trophoblasts1

  Madhu Chauhan, Meena Balakrishnan, Uma Yallampalli, Janice Endsley, Gary D.V. Hankins, Regan Theiler, Chandra Yallampalli
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  ABSTRACT: Adrenomedullin 2 (ADM2), also referred to as intermedin (IMD), is expressed in trophoblast cells in human placenta and enhances the invasion and migration of first-trimester HTR-8SV/neo cells. Further infusion of ADM2 antagonist in pregnant rat causes fetoplacental growth restriction, suggesting a role for ADM2 in maintaining a successful pregnancy. This study was undertaken to assess whether ADM2 protein is present in decidual tissue and colocalized with HLA-G-positive cytotrophoblast cells and natural killer cells; to assess whether ADM2 regulates expression of HLA-G in trophoblast cells; and to identify whether mitogen-activated protein kinase (MAPK) signaling pathway is involved in ADM2-induced trophoblast cell invasion and migration. Using immunohistochemical methods and RT-PCR, this study shows that ADM2 protein is colocalized with HLA-G-expressing cytotrophoblast cells as well as with NCAM1 (CD56) immunoreactivity in human first-trimester decidual tissue, and that ADM2 mRNA is expressed in peripheral blood natural killer cells. Further, ADM2 dose dependently increases the expression of HLA-G antigen in HTR-8SV/neo cells as well as in term placental villi explants, suggesting involvement of ADM2 in the regulation of HLA-G in trophoblast cells. In addition, interference with the activity of RAF and MAPK3/1 by their inhibitors, manumycin and U0126, respectively, reduces ADM2-induced HTR-8SV/neo cell invasion and migration. In summary, this study suggests a potential involvement for ADM2 in regulating HLA-G antigen at the maternal-fetal interface in human pregnancy and facilitating trophoblast invasion and migration via MAPK3/1 phosphorylation.
  Biology of Reproduction 08/2011;
• Article: Beta- and Gamma-Cytoplasmic Actins Are Required for Meiosis in Mouse Oocytes1

  Céline Brockmann, Joachim Huarte, Vera Dugina, Ludivine Challet, Emmanuelle Rey, Béatrice Conne, Adam Swetloff, Serge Nef, Christine Chaponnier, Jean-Dominique Vassalli
  Biology of Reproduction 07/2011;
• Article: Spatiotemporal Expression of Bone Morphogenetic Protein Family Ligands and Receptors in the Zebrafish Ovary: A Potential Paracrine-Signaling Mechanism for Oocyte-Follicle Cell Communication1

  Cheuk Wun Li
  Biology of Reproduction 07/2011;
• Article: Loss of SPEF2 Function in Mice Results in Spermatogenesis Defects and Primary Ciliary Dyskinesia1

  Anu Sironen, Noora Kotaja, Howard Mulhern, Todd A. Wyatt, Joseph H. Sisson, Jacqueline A. Pavlik, Mari Miiluniemi, Mark D. Fleming, Lance Lee
  Biology of Reproduction 06/2011;