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1.  Turning a Spermatogenic Wave into a Tsunami: Synchronizing Murine Spermatogenesis Using WIN 18,4461 
Biology of Reproduction  2013;88(2):40.
The BDADs (bis-[dichloroacetyl]-diamines) are compounds that can inhibit spermatogenesis via blocking the metabolism of vitamin A. We utilized one specific BDAD, WIN 18,446, to manipulate the endogenous production of retinoic acid (RA) in the testis to further investigate the action of this compound on mammalian sperm production. Transient treatment of adult male mice with WIN 18,446 blocked spermatogonial differentiation and induced significant changes in the cycle of the seminiferous epithelium. WIN 18,446 treatment of neonatal mice also blocked spermatogonial differentiation and, followed by injection of RA, induced synchronous spermatogenesis in adulthood. The net result was pulsatile, rather than normal continuous, release of sperm from the seminiferous epithelium. This study describes a novel technique that can enrich for specific germ cell populations within the testis, representing a valuable new tool for studying spermatogenesis.
The treatment of mice with WIN 18,446 suppresses retinoic acid-mediated differentiation of spermatogonia and synchronizes spermatogenesis.
PMCID: PMC3589231  PMID: 23284139
contraception; retinoids; spermatogenesis; testis; WIN 18,446
2.  Hormonal Coordination of Natriuretic Peptide Type C and Natriuretic Peptide Receptor 3 Expression in Mouse Granulosa Cells1 
Biology of Reproduction  2012;88(2):42.
Natriuretic peptide type C (NPPC) and its receptor natriuretic peptide receptor 2 (NPR2) regulate cGMP in ovarian follicles and participate in maintaining oocyte meiotic arrest. We investigated the regulation of Nppc expression in mouse granulosa cells in vivo and in vitro. In mural granulosa cells (MGCs) in vivo, eCG caused an increase in Nppc mRNA, and subsequent human chorionic gonadotropin (hCG) treatment caused a decrease. A culture system was established for MGCs isolated from follicles not stimulated with equine chorionic gonadotropin to further define the mechanisms controlling Nppc expression. In this system, expression of Nppc mRNA was increased by estradiol (E2), with augmentation by follicle-stimulating hormone (FSH), but FSH or luteinizing hormone (LH) alone had no effect. Thus, estrogens are important for regulating Nppc expression, probably by feedback mechanisms enhancing the action of gonadotropins. In MGCs treated with E2 plus FSH in vitro, subsequent treatment with EGF, but not LH, decreased Nppc mRNA. MGCs express higher levels of both Nppc and Lhcgr mRNAs than cumulus cells. Oocyte-derived paracrine factors suppressed cumulus cell Lhcgr but not Nppc expression. Thus, higher Nppc expression by MGCs is not the result of oocyte suppression of expression in cumulus cells. Another possible regulator of the LH-induced NPPC decrease is NPR3, an NPPC clearance receptor. Human chorionic gonadotropin increased Npr3 expression in vivo and LH increased Npr3 mRNA in cultured MGCs, independently of EGF receptor activation. Interestingly, despite the increase in Npr3 mRNA, the hCG-induced decrease in ovarian NPPC occurred normally in an Npr3 mutant (lgj), thus NPR3 probably does not participate in regulation of ovarian NPPC levels or oocyte development.
Expression of natriuretic peptide type C (NPPC) in mouse granulosa cells is regulated by estrogen, gonadotropins, and EGF, but ovarian NPPC peptide levels are unaffected by mutation of the NPR3 clearance receptor.
PMCID: PMC3589232  PMID: 23255339
epidermal growth factor; estradiol/estradiol receptor; estrogens; follicle-stimulating hormone; gonadotropins; granulosa cells; luteinizing hormone; natriuretic peptide receptor 3; natriuretic peptide type C
3.  Induced Endometriosis in Nonhuman Primates1 
Biology of Reproduction  2013;88(2):43.
In this month's issue of Biology of Reproduction, Afshar et al. report on the effects of endometriosis on gene expression in the baboon endometrium.
PMCID: PMC3589233  PMID: 23303683
4.  Changes in Eutopic Endometrial Gene Expression During the Progression of Experimental Endometriosis in the Baboon, Papio Anubis1 
Biology of Reproduction  2013;88(2):44.
Endometriosis is associated with aberrant gene expression in the eutopic endometrium of women with disease. To determine if the development of endometriotic lesions directly impacts eutopic endometrial gene expression, we sequentially analyzed the eutopic endometrium across the time course of disease progression in a baboon model of induced disease. Endometriosis was induced in baboons (n = 4) by intraperitoneal inoculation of autologous menstrual endometrium. Eutopic endometria were collected during the midsecretory phase (Days 9–11 postovulation) at 1, 3, 6–7, 10–12, and 15–16 mo after disease induction and compared with tissue from disease-free baboons. RNA was hybridized to Human Genome U133 Plus 2.0 Arrays, and data were extracted using Gene-Chip Operating Software. Subsequently, both Gene Set Enrichment Analysis and Ingenuity Pathways Analysis were used to find biological states that have a statistically significant enrichment concomitant with pairwise comparison of human endometriosis arrays. Within 1 mo of induction of the disease, 4331 genes were differentially expressed (P < 0.05). Hierarchical clustering revealed self-segregation into two groups—a) 1, 3, and 10–12 mo and b) 6–7 and 15–16 mo—together with controls. Clustering analysis at each stage of disease validated dysregulation of several signaling pathways, including Nodal-like receptor, EGF, ERK/MAPK, and PI3/AKT. Sequential analysis of the same animals during disease progression demonstrated an early disease insult and a transitory dominance of an estrogenic phenotype; however, as the disease progressed, a progesterone-resistant phenotype became evident. Furthermore, we demonstrate a 38.6% differential gene expression overlap with endometrial samples in the midsecretory phase from women with endometriosis, concomitant with similar dysregulation in human disease candidate genes Fos, Nodal, Suclg2, and Kras, among others. Molecular changes in the eutopic endometrium, associated with endometriosis, are directly impacted by endometriotic lesions, providing strong evidence that it is the disease rather than inherent defective endometrium that results in aberrant gene expression in the eutopic endometrium. Furthermore, this baboon model provides a powerful means whereby the early events associated with the pathology of disease and the resulting infertility may be elucidated.
Molecular changes in the eutopic endometrium associated with endometriosis are directly affected by endometriotic lesions, providing evidence that it is the disease, rather than inherently defective endometrium, that causes aberrant gene expression in the eutopic endometrium.
PMCID: PMC3589234  PMID: 23284138
baboon; endometriosis; endometrium; primate; transcriptome
5.  Locus-Specific DNA Methylation Reprogramming During Early Porcine Embryogenesis1 
Biology of Reproduction  2013;88(2):48.
During early mammalian embryogenesis, there is a wave of DNA demethylation postfertilization and de novo methylation around implantation. The paternal genome undergoes active DNA demethylation, whereas the maternal genome is passively demethylated after fertilization in most mammals except for sheep and rabbits. However, the emerging genome-wide DNA methylation landscape has revealed a regulatory and locus-specific DNA methylation reprogramming pattern in mammalian preimplantation embryos. Here we optimized a bisulfite sequencing protocol to draw base-resolution DNA methylation profiles of several selected genes in gametes, early embryos, and somatic tissue. We observed locus-specific DNA methylation reprogramming in early porcine embryos. First, some pluripotency genes (POU5F1 and NANOG) followed a typical wave of DNA demethylation and remethylation, whereas CpG-rich regions of SOX2 and CDX2 loci were hypomethylated throughout development. Second, a differentially methylated region of an imprint control region in the IGF2/H19 locus exhibited differential DNA methylation which was maintained in porcine early embryos. Third, a centromeric repeat element retained a moderate DNA methylation level in gametes, early embryos, and somatic tissue. The diverse DNA methylation reprogramming during early embryogenesis is thought to be possibly associated with the multiple functions of DNA methylation in transcriptional regulation, genome stability and genomic imprinting. The latest technology such as oxidative bisulfite sequencing to identify 5-hydroxymethylcytosine will further clarify the DNA methylation reprogramming during porcine embryonic development.
DNA methylation reprogramming in early porcine embryos is locus-specific and associated with multiple functions of DNA methylation in transcriptional regulation, genomic imprinting, and genome stability.
PMCID: PMC3589235  PMID: 23303676
CDX2; DNA methylation; NANOG; porcine preimplantation embryos; POU5F1; reprogramming; SOX2
6.  DMRT1 Owner's Manual: Synchronized Installation Required to Operate 1 
Biology of Reproduction  2013;88(2):50.
The study by Agbor et al. in this issue of Biology of Reproduction provides a novel approach to determine the functions of Sertoli cell and germ cell-expressed Dmrt1.
PMCID: PMC3589236  PMID: 23349232
7.  A Wt1-Dmrt1 Transgene Restores DMRT1 to Sertoli Cells of Dmrt1−/− Testes: A Novel Model of DMRT1-Deficient Germ Cells1 
Biology of Reproduction  2012;88(2):51.
DMRT1 is an evolutionarily conserved transcriptional factor expressed only in the postnatal testis, where it is produced in Sertoli cells and germ cells. While deletion of Dmrt1 in mice demonstrated it is required for postnatal testis development and fertility, much is still unknown about its temporal- and cell-specific functions. This study characterized a novel mouse model of DMRT1-deficient germ cells that was generated by breeding Dmrt1-null (Dmrt1−/−) mice with Wt1-Dmrt1 transgenic (Dmrt1+/−;tg) mice, which express a rat Dmrt1 cDNA in gonadal supporting cells by directing it from the Wilms tumor 1 locus in a yeast artificial chromosome transgene. Like Dmrt1−/− mice, male Dmrt1−/− transgenic mice (Dmrt1−/−;tg) were infertile, while female mice were fertile. Immunohistochemistry and Western blot analysis showed transgenic DMRT1 expressed in supporting cells of the newborn gonads of both sex and in Sertoli cells of the testis afterbirth. Sertoli cells were evaluated by electron microscopy, revealing that maturation of Dmrt1−/−;tg Sertoli cells was incomplete. Morphological analysis of testes from 42-day-old mice showed that, compared to Dmrt1−/− mice, Dmrt1−/−;tg mice have improved seminiferous tubule structure, with lumens present in many. Immunohistochemistry of the polarity markers ESPIN and NECTIN-2 showed that DMRT1 in Sertoli cells is required for NECTIN-2 expression and influences organization of ectoplasmic specializations. Further functional analyses of the transgene on a Dmrt1−/− background showed that it did not rescue the decrease in Dmrt1−/− testis size, but when expressed on a wild-type background, exogenous DMRT1 prevented the normal age-related decline in testis size and enhanced sperm progressive motility. The studies suggest that DMRT1 in Sertoli cells regulates tubule morphology, spermatogenesis, and sperm function via its effects on Sertoli cell maturation and polarity. Furthermore, expression and function of transgenic DMRT1 in Sertoli cells establishes a novel mouse model of DMRT1-deficient germ cells generated by breeding Dmrt1-null mice with Wt1-Dmrt1 transgenic mice (rescue; Dmrt1−/−;tg).
A novel mouse model of DMRT1-deficient germ cell has been generated by breeding Dmrt1-null mice with Wt1-Dmrt1 transgenic mice (rescue; Dmrt1−/−;tg).
PMCID: PMC3589237  PMID: 23255335
DMRT1; male infertility; Sertoli cells; spermatogenesis; testis
8.  Targeted Genome-Wide Methylation and Gene Expression Analyses Reveal Signaling Pathways Involved in Ovarian Dysfunction after Developmental EDC Exposure in Rats1 
Biology of Reproduction  2013;88(2):52.
Transient exposure to methoxychlor (MXC), an environmental endocrine-disrupting chemical, during fetal and neonatal stages causes ovarian dysfunction in pubertal, adult, and aging animals. Adult animals have reduced number of ovulations and abnormal follicular composition associated with altered gene expression and DNA methylation patterns. To test the hypothesis that the ovarian epigenomic changes induced by MXC are detectable following the exposure period, leading to altered gene expression by adulthood, we conducted a targeted genome-wide methylation study using Nimblegen 3x720K CpG Island Plus RefSeq Promoter Arrays. Control (vehicle), low-dose MXC (20 μg/kg/day), or high-dose MXC (100 mg/kg/day) treatments were administered between Embryonic Day 19 and Postnatal Day (PND) 7. Ovaries were collected at PND 7 immediately after exposure or at adulthood, PND 60. Array hybridizations were conducted with genomic DNA after methylated DNA immunoprecipitation and the array data were analyzed. DNA methylation events were functionally annotated, and candidate loci common to all the treatments or unique to some treatments were identified. Specific loci encoding signaling molecules such as the regulatory subunit p85 of phosphoinositide-3-kinase, insulin-like growth factor-1 receptor, Harvey rat sarcoma viral oncogene, insulin receptor, and forkhead box protein O3 were identified to be hypermethylated in MXC-treated ovaries at PND 7 and/or PND 60. Examination of gene expression changes with TaqMan low-density arrays revealed that nearly 25% of the genes that were assayed were downregulated. These data demonstrate that key molecules in specific signaling pathways such as PTEN signaling, IGF-1 signaling, or rapid estrogen signaling are epigenetically altered in MXC-exposed ovaries, which is associated with ovarian dysfunction and female infertility.
Developmental exposure to the model endocrine-disrupting chemical methoxychlor leads to age-dependent alterations in DNA methylation patterns in multiple signaling molecules, resulting in ovarian dysfunction in adulthood.
PMCID: PMC3589238  PMID: 23303685
development; endocrine-disrupting chemicals; epigenetics; folliculogenesis; functional annotation; gene expression; methylation; ovary
Biology of reproduction  2013;89(6):10.1095/biolreprod.113.113688.
Dynamic control of maternal blood flow to the placenta is critical for healthy pregnancy. In many tissues, microvasculature arteries control flow. The uterine/endometrial vascular bed changes during pregnancy include physiological remodeling of spiral arteries from constricted artery–like structures to dilated vein-like structures between gestation days (gd)8 and12 in mice and weeks 12–16 in humans. These changes occur, in part, due to local environmental changes such as decidualization, recruitment of maternal uterine natural killer cells and invasion of conceptus-derived trophoblasts. No current preparations permit in vivo testing of decidual microvascular reactivity. We report an in vivo intravital fluorescence microscopy model that permits functional study of the entire uterine microvascular bed (uterine, arcuate, radial, basal and spiral arteries) in gravid C57BL/6 mice. Vascular reactivities were measured at gd8 pre-spiral arterial remodeling and gd12 (post-remodeling) to a range of concentrations of adenosine (ADO, 10−8–10−6M), acetylcholine (ACh, 10−7–10−5M), phenylephrine (PHE, 10−7–10−5M) and angiotensin II (AngII, 10−8–10−6M). At baseline, each arterial branch order was significantly more dilated on gd12 than gd8. Each microvascular level responded to each agonist on gd8 and gd12. At gd12, vasodilation to ADO was attenuated in uterine, arcuate and basal arteries while constrictor activity to AII was enhanced in uterine and arcuate arteries. The tendency for increasing vasoconstriction between gd8 to gd12 and the constrictor responses of modified spiral arteries were unexpected findings that may reflect influences of the intact in vivo environment rather than inherent properties of the vessels and be relevant to ongoing human pregnancies.
PMCID: PMC3866111  PMID: 24174571 CAMSID: cams3647
decidua; pregnancy; blood flow; artery; vascular remodeling
10.  FZD1 Regulates Cumulus Expansion Genes and Is Required for Normal Female Fertility in Mice1 
Biology of Reproduction  2012;87(5):104.
WNT4 is required for normal ovarian follicle development and female fertility in mice, but how its signal is transduced remains unknown. Fzd1 encodes a WNT receptor whose expression is markedly induced in both mural granulosa cells and cumulus cells during the preovulatory period, in a manner similar to Wnt4. To study the physiological roles of FZD1 in ovarian physiology and to determine whether it serves as receptor for WNT4, Fzd1-null mice were created by gene targeting. Whereas rare Fzd1−/− females were sterile because of uterine fibrosis and ovarian tubulostromal hyperplasia, most were subfertile, producing ≈1 fewer pup per litter on average relative to controls. Unlike WNT4-deficient mice, ovaries from Fzd1−/− mice had normal weights, numbers of follicles, steroid hormone production, and WNT4 target gene expression levels. Microarray analyses of granulosa cells from periovulatory follicles revealed few genes whose expression was altered in Fzd1−/− mice. However, gene expression analyses of cumulus-oocyte complexes (COCs) revealed a blunted response of both oocyte (Zp3, Dppa3, Nlrp5, and Bmp15) and cumulus (Btc, Ptgs2, Sema3a, Ptx3, Il6, Nts, Alcam, and Cspg2) genes to the ovulatory signal, whereas the expression of these genes was not altered in WNT4-deficient COCs from Wnt4tm1.1Boer/tm1.1Boer;Tg (CYP19A1-cre)1Jri mice. Despite altered gene expression, cumulus expansion appeared normal in Fzd1−/− COCs both in vitro and in vivo. Together, these results indicate that Fzd1 is required for normal female fertility and may act in part to regulate oocyte maturation and cumulus cell function, but it is unlikely to function as the sole ovarian WNT4 receptor.
Frizzled 1 (Fzd1) is induced in periovulatory granulosa and cumulus cells; a null mutation blunts the induction of genes associated with cumulus expansion and results in reduced female fertility.
PMCID: PMC3509776  PMID: 22954793
cumulus cells; fertility; granulosa cells; mouse; ovary
11.  Residency and Activation of Myeloid Cells During Remodeling of the Prepartum Murine Cervix1 
Biology of Reproduction  2012;87(5):106.
Remodeling of the cervix is a critical early component of parturition and resembles an inflammatory process. Infiltration and activation of myeloid immune cells along with production of proinflammatory mediators and proteolytic enzymes are hypothesized to regulate cervical remodeling as pregnancy nears term. The present study standardized an approach to assess resident populations of immune cells and phenotypic markers of functional activities related to the mechanism of extracellular matrix degradation in the cervix in preparation for birth. Analysis of cells from the dispersed cervix of mice that were nonpregnant or pregnant (Days 15 and 18 postbreeding) by multicolor flow cytometry indicated increased total cell numbers with pregnancy as well as increased numbers of macrophages, the predominant myeloid cell, by Day 18, the day before birth. The number of activated macrophages involved in matrix metalloproteinase induction (CD147) and signaling for matrix adhesion (CD169) significantly increased by the day before birth. Expression of the adhesion markers CD54 and CD11b by macrophages decreased in the cervix by Day 18 versus that on Day 15 or in nonpregnant mice. The census of cells that expressed the migration marker CD62L was unaffected by pregnancy. The data suggest that remodeling of the cervix at term in mice is associated with recruitment and selective activation of macrophages that promote extracellular matrix degradation. Indices of immigration and activities by macrophages may thus serve as markers for local immune cell activity that is critical for ripening of the cervix in the final common mechanism for parturition at term.
Remodeling of the preterm cervix involves hyperplasia of cells, as well as increased presence and activities of macrophages that promote inflammation and degradation of the extracellular matrix.
PMCID: PMC3509777  PMID: 22914314
cervical remodeling; extracellular matrix; flow cytometry; immunology; inflammation; macrophage; parturition
12.  Tissue-Restricted Transcription from a Conserved Intragenic CpG Island in the Klf1 Gene in Mice1 
Biology of Reproduction  2012;87(5):108.
Beyond Mendelian inheritance, an understanding of the complexities and consequences of the transfer of nonhereditary information to successive generations is at an early stage. Such epigenetic functionality is exemplified by DNA methylation and, as genome-wide high-throughput methodologies emerge, is increasingly being considered in the context of conserved intragenic and intergenic CpG islands that function as alternate sites of transcription initiation. Here we characterize an intragenic CpG island in exon 2 of the protein-coding mouse Klf1 gene, from which clustered transcription initiation sites yield positive-strand, severely truncated, capped and spliced RNAs. Expression from this CpG island in the testis begins between Postnatal Days 14–20, increases during development, and is temporally correlated with the maturation of secondary spermatocytes as they become the dominant cell population in the seminiferous epithelium. Only full-length KLF1-encoding mRNAs are detected in the hematopoietic tissue, spleen; thus, expression from the exon 2 CpG island is both developmentally regulated and tissue restricted. DNA methylation analysis indicates that spatiotemporal expression from the Klf1 CpG island is not associated with hypermethylation. Finally, our computational analysis from multiple species confirms intragenic transcription initiation and indicates that the KLF1 CpG island is evolutionarily conserved. Currently we have no evidence that these truncated RNAs can be translated via nonconventional mechanisms such as in-frame, conserved non-AUG-dependent Kozak consensus sequences; however, high-quality carboxyl-terminal antibodies will more effectively address this issue.
The truncated noncoding RNAs arising from tissue-specific intragenic transcription start sites from the Klf1 gene are characterized during testis development.
PMCID: PMC3509778  PMID: 22933519
CGI; Cldn11; EKLF; germ cells; somatic cells; testis
13.  Estrogen-Induced Maldevelopment of the Penis Involves Down-Regulation of Myosin Heavy Chain 11 (MYH11) Expression, a Biomarker for Smooth Muscle Cell Differentiation1 
Biology of Reproduction  2012;87(5):109.
Cavernous smooth muscle cells are essential components in penile erection. In this study, we investigated effects of estrogen exposure on biomarkers for smooth muscle cell differentiation in the penis. Neonatal rats received diethylstilbestrol (DES), with or without the estrogen receptor (ESR) antagonist ICI 182,780 (ICI) or the androgen receptor (AR) agonist dihydrotestosterone (DHT), from Postnatal Days 1 to 6. Tissues were collected at 7, 10, or 21 days of age. The smooth muscle cell biomarker MYH11 was studied in depth because microarray data showed it was significantly down-regulated, along with other biomarkers, in DES treatment. Quantitative real time-PCR and Western blot analyses showed 50%–80% reduction (P ≤ 0.05) in Myh11 expression in DES-treated rats compared to that in controls; and ICI and DHT coadministration mitigated the decrease. Temporally, from 7 to 21 days of age, Myh11 expression was onefold increased (P ≥ 0.05) in DES-treated rats versus threefold increased (P ≤ 0.001) in controls, implying the long-lasting inhibitory effect of DES on smooth muscle cell differentiation. Immunohistochemical localization of smooth muscle alpha actin, another biomarker for smooth muscle cell differentiation, showed fewer cavernous smooth muscle cells in DES-treated animals than in controls. Additionally, DES treatment significantly up-regulated Esr1 mRNA expression and suppressed the neonatal testosterone surge by 90%, which was mitigated by ICI coadministration but not by DHT coadministration. Collectively, results provided evidence that DES treatment in neonatal rats inhibited cavernous smooth muscle cell differentiation, as shown by down-regulation of MYH11 expression at the mRNA and protein levels and by reduced immunohistochemical staining of smooth muscle alpha actin. Both the ESR and the AR pathways probably mediate this effect.
Estrogen-induced loss of cavernous smooth muscle cells in the rat penis involves down-regulation of MYH11 expression, mitigated by estrogen receptor antagonist ICI 182,780 and dihydrotestosterone.
PMCID: PMC3509779  PMID: 22976277
development; estrogen; microarray; MYH11; penis
14.  Leptin and the Placental Response to Maternal Food Restriction During Early Pregnancy in Mice1 
Biology of Reproduction  2012;87(5):120.
Several studies have demonstrated that maternal undernutrition or overnutrition during pregnancy can have negative consequences for the health of children born to these pregnancies, but the physiological mechanisms by which this occurs are not completely understood. During periods of food restriction, concentrations of leptin decline, whereas leptin is elevated in obesity, suggesting that it may play a role in the response to altered nutrition during pregnancy. This study compares placental development and global placental gene expression profiles at Day 11.5 in pregnant control mice, mice that were undernourished, and mice that were undernourished but given leptin. Placentas from mothers exposed to food restriction preserved the placental labyrinth zone at the expense of the junctional zone, an effect abrogated in the restricted plus leptin group, which had a significant decrease in the labyrinth zone area compared with controls. Similarly, there were more significant differences in gene expression between placentas from control and restricted plus leptin mothers (1128 differentially expressed genes) than between placentas of control and restricted mothers (281 differentially expressed genes). We conclude that the presence of high concentrations of circulating leptin during food restriction disrupts the normal adaptive response of the placenta to reduced energy availability.
Elevated maternal leptin disrupts the response of the placenta to maternal food restriction during early pregnancy.
PMCID: PMC3509780  PMID: 22993381
developmental origin of adult health and disease (DOHAD); leptin; trophoblast; undernutrition
15.  Imaging of Vascular Development in Early Mouse Decidua and Its Association with Leukocytes and Trophoblasts1 
Biology of Reproduction  2012;87(5):125.
In species with endometrial decidualization and hemochorial placentation (humans, mice, and others), leukocytes localize to early implant sites and contribute to decidual angiogenesis, spiral arterial remodeling, and trophoblast invasion. Relationships between leukocytes, trophoblasts, and the decidual vasculature are not fully defined. Early C57BL/6J implant sites were analyzed by flow cytometry to define leukocyte subsets and by whole-mount immunohistochemistry to visualize relationships between leukocytes, decidual vessels, and trophoblasts. Ptprc+ (CD45+) cells increased in decidua between Gestational Day (GD) 5.5 and GD 9.5. Uterine natural killer (uNK) cells that showed dynamic expression of Cd (CD) 69, an activating receptor, and Klrg1 (KLRG1), an inhibitory receptor, localized mesometrially and were the dominant CD45+ cells between GD 5.5 and GD 7.5. At GD 8.5, immature monocytes that occurred throughout decidua exceeded uNK cells numerically and many leukocytes acquired irregular shapes, and leukocyte-leukocyte conjugates became frequent. Vessels were morphologically heterogeneous and regionally unique. Migrating trophoblasts were first observed at GD 6.5 and, at GD 9.5, breached endothelium, entered vascular lumens, and appeared to occlude some vessels, as described for human spiral arteries. No leukocyte-trophoblast conjugates were detected. Whole-mount staining gave unparalleled decidual vascular detail and cell-specific positional information. Its application across murine models of pregnancy disturbances should significantly advance our understanding of the maternal-fetal interface.
Whole-mount immunohistochemistry of early mouse implant sites defines domain-specific vascular heterogeneity with leukocyte enrichment in the most densely vascularized region as well as in the earliest stages and in the pattern of trophoblast invasion.
PMCID: PMC3509781  PMID: 22954796
angiogenesis; conceptus; decidua; immunology; pregnancy; trophoblast
16.  Prenatal Exposure to Low Doses of Bisphenol A Increases Pituitary Proliferation and Gonadotroph Number in Female Mice Offspring at Birth1 
Biology of Reproduction  2012;87(4):82.
The pituitary gland is composed of hormone-producing cells essential for homeostasis and reproduction. Pituitary cells are sensitive to endocrine feedback in the adult and can have altered hormonal secretion from exposure to the endocrine disruptor bisphenol A (BPA). BPA is a prevalent plasticizer used in food and beverage containers, leading to widespread human exposure. Although prenatal exposure to BPA can impact reproductive function in the adult, the effects of BPA on the developing pituitary are unknown. We hypothesized that prenatal exposure to low doses of BPA impacts gonadotroph cell number or parameters of hormone synthesis. To test this, pregnant mice were administered 0.5 μg/kg/day of BPA, 50 μg/kg/day of BPA, or vehicle beginning on Embryonic Day 10.5. At parturition, pituitaries from female offspring exposed in utero to either dose of BPA had increased proliferation, as assessed by mKi67 mRNA levels and immunohistochemistry. Coincidently, gonadotroph number also increased in treated females. However, we observed a dichotomy between mRNA levels of Lhb and Fshb. Female mice exposed to 0.5 μg/kg/day BPA had increased mRNA levels of gonadotropins and the gonadotropin-receptor hormone (GNRH) receptor (Gnrhr), which mediates GNRH regulation of gonadotropin production and release. In contrast, mice treated with 50 μg/kg/day of BPA had decreased gonadotropin mRNA levels, Gnrhr and Nr5a1, a transcription factor required for gonadotroph differentiation. No other pituitary hormones were altered on the day of birth in response to in utero BPA exposure, and male pituitaries showed no change in the parameters tested. Collectively, these results show that prenatal exposure to BPA affects pituitary gonadotroph development in females.
Developmental exposure to low doses of bisphenol A increases proliferation and alters gonadotroph differentiation in the pituitary.
PMCID: PMC3507543  PMID: 22875908
anterior pituitary; bisphenol A (BPA); development; developmental biology; endocrine disruptors; environmental contaminants and toxicants; follicle-stimulating hormone (FSH); gonadotrophs; gonadotropins; luteinizing hormone (LH); pituitary
17.  Aurora Kinase A Drives MTOC Biogenesis but Does Not Trigger Resumption of Meiosis in Mouse Oocytes Matured In Vivo1 
Biology of Reproduction  2012;87(4):85.
Aurora kinase A (AURKA) is an important mitotic kinase involved in the G2/M transition, centrosome maturation and separation, and spindle formation in somatic cells. We used transgenic models that specifically overexpress in mouse oocytes either wild-type (WT-AURKA) or a catalytically inactive (kinase-dead) (KD-AURKA) AURKA to gain new insights regarding the role of AURKA during oocyte maturation. AURKA activation occurs shortly after hCG administration that initiates maturation in vivo. Although AURKA activity is increased in WT-AURKA oocytes, resumption of meiosis is not observed in the absence of hCG administration. Control oocytes contain one to three microtubule organizing centers (MTOCs; centrosome equivalent) at prophase I. At the time of germinal vesicle breakdown (GVBD), the first visible marker of resumption of meiosis, the MTOC number increases. In WT-AURKA oocytes, the increase in MTOC number occurs prematurely but transiently without GVBD, whereas the increase in MTOC number does not occur in control and KD-AURKA oocytes. AURKA activation is biphasic with the initial activation not requiring CDC25B-CDK1 activity, whereas full activation, which is essential for the increase in MTOCs number, depends on CDK1 activity. AURKA activity also influences spindle length and regulates, independent of its protein kinase activity, the amount of MTOC associated with gamma-tubulin. Both WT-AURKA and KD-AURKA transgenic mice have normal fertility during first 6 mo of life. These results suggest that although AURKA is not a trigger kinase for G2/M transition in mouse oocytes, it regulates MTOC number and spindle length, and, independent of its protein kinase activity, gamma-tubulin recruitment to MTOCs.
Aurora-A protein kinase, activated very early in microtubule organizing centers in mouse oocytes, regulates multiple aspects of their biogenesis and spindle formation but does not trigger resumption of meiosis in vivo.
PMCID: PMC3507544  PMID: 22837479
AURKA; CDC25B; centrosome; γ-tubulin; mouse oocytes; MTOC; resumption of meiosis; spindle formation
18.  Development of Quantitative Microscopy-Based Assays for Evaluating Dynamics of Living Cultures of Mouse Spermatogonial Stem/Progenitor Cells1 
Biology of Reproduction  2012;87(4):90.
Spermatogonial stem cell (SSC) self-renewal and differentiation are required for continuous production of spermatozoa and long-term fertility. Studying SSCs in vivo remains challenging because SSCs are rare cells and definitive molecular markers for their identification are lacking. The development of a method for propagating SSCs in vitro greatly facilitated analysis of SSCs. The cultured cells grow as clusters of a dynamic mixture of “true” stem cells and differentiating progenitor cells. Cells in the stem/progenitor culture system share many properties with spermatogonia in vivo; however, to fully exploit it as a model for spermatogonial development, new assays are needed that account for the dynamic heterogeneity inherent in the culture system. Here, assays were developed for quantifying dynamics of cultures of stem/progenitor cells that expressed histone-green fluorescent protein (GFP). First, we built on published results showing that cluster formation in vitro reliably predicts the relative number of SSCs. The GFP-based in vitro cluster assay allows quantification of SSCs with significantly fewer resources than a transplantation assay. Second, we compared the dynamics of differentiation in two experimental paradigms by imaging over a 17-day time frame. Finally, we performed short-term live imaging and observed cell migration, coordinated cell proliferation, and cell death resembling that of spermatogonia in the testes. The methods that we present provide a foundation for the use of fluorescent reporters in future microscopy-based high-throughput screens by using living spermatogonial stem/progenitor cultures applicable to toxicology, contraceptive discovery, and identification of regulators of self-renewal and differentiation.
Live imaging of cell proliferation and cell death in spermatogonial stem/progenitor cell cultures elucidates properties of cluster formation.
PMCID: PMC3507545  PMID: 22933516
cell death; differentiation; germ cell; green fluorescent protein; lentivirus; live imaging; spermatogenesis; spermatogonial stem cell; undifferentiated spermatogonia
19.  Acute Slices of Mice Testis Seminiferous Tubules Unveil Spontaneous and Synchronous Ca2+ Oscillations in Germ Cell Clusters1 
Biology of Reproduction  2012;87(4):92.
Spermatogenic cell differentiation involves changes in the concentration of cytoplasmic Ca2+ ([Ca2+]i); however, very few studies exist on [Ca2+]i dynamics in these cells. Other tissues display Ca2+ oscillations involving multicellular functional arrangements. These phenomena have been studied in acute slice preparations that preserve tissue architecture and intercellular communications. Here we report the implementation of intracellular Ca2+ imaging in a sliced seminiferous tubule (SST) preparation to visualize [Ca2+]i changes of living germ cells in situ within the SST preparation. Ca2+ imaging revealed that a subpopulation of male germ cells display spontaneous [Ca2+]i fluctuations resulting from Ca2+ entry possibly throughout CaV3 channels. These [Ca2+]i fluctuation patterns are also present in single acutely dissociated germ cells, but they differ from those recorded from germ cells in the SST preparation. Often, spontaneous Ca2+ fluctuations of spermatogenic cells in the SST occur synchronously, so that clusters of cells can display Ca2+ oscillations for at least 10 min. Synchronous Ca2+ oscillations could be mediated by intercellular communication via gap junctions, although intercellular bridges could also be involved. We also observed an increase in [Ca2+]i after testosterone application, suggesting the presence of functional Sertoli cells in the SST. In summary, we believe that the SST preparation is suitable to explore the physiology of spermatogenic cells in their natural environment, within the seminiferous tubules, in particular Ca2+ signaling phenomena, functional cell-cell communication, and multicellular functional arrangements.
Male germ cells display coupled spontaneous Ca2+ oscillations recorded in seminiferous tubules slices; these oscillations are due mainly to Ca2+ entry through CaV channels, and gap junctions participate in this coupling.
PMCID: PMC3507546  PMID: 22914313
calcium; cell coupling; intercellular communication; spermatogenesis; testis
20.  Variants of Stretch-Activated Two-Pore Potassium Channel TREK-1 Associated with Preterm Labor in Humans1 
Biology of Reproduction  2012;87(4):96.
Spontaneous preterm labor (PTL) is a uniquely human problem that results in preterm delivery of an underdeveloped fetus. The underlying cause remains elusive. The cost to societies in human suffering and treasure is enormous. The stretch-activated two pore potassium channel TREK-1 is up-regulated during gestation to term such that it may maintain uterine quiescence by hyperpolarizing the smooth muscle cell membrane. We have hypothesized that the human TREK-1 channel is involved in myometrial relaxation during pregnancy and that splice variants of the TREK-1 channel expressed in preterm myometrium are associated with preterm delivery by interaction with full-length TREK-1. We detected three wild-type human TREK-1 transcript isoforms in nonpregnant and pregnant human myometrium. Using RT-PCR, we identified five unique TREK-1 splice variants in myometrium from women in PTL. These myometrial TREK-1 variants lack either the pore or the transmembrane domains or both. In transiently transfected HEK293T cells, wild-type TREK-1 was predominantly expressed at the plasma membrane. However, individual splice variants were expressed uniformly throughout the cell. Wild-type TREK-1 was localized at the plasma membrane and cytoplasm close to the plasma membrane when coexpressed with each splice variant. Co-immunoprecipitation of FLAG epitope-tagged TREK-1 and six-His epitope-tagged splice variants using Ni bead columns successfully pulled down wild-type TREK-1. These results suggest that each of four TREK-1 splice variants interacts with full-length wild-type TREK-1 and that in vivo, such interactions may contribute to a PTL phenotype.
Splice variants of the TREK-1 stretch-activated K+ channel may be associated with preterm labor.
PMCID: PMC3507547  PMID: 22811574
human; labor; myometrial smooth muscle; pregnancy; potassium channels; preterm labor; splice variants; TREK-1; uterus
21.  Repropedia: A Reproductive Lexicon to Fill the Gap in Reproductive Terminology1  
Biology of Reproduction  2012;87(4):98.
Repropedia ( is a comprehensive, reproductive-focused online lexicon created to increase public literacy in the fields of reproductive health and science.
PMCID: PMC3507548  PMID: 22954794
22.  Characterization of EPPIN's Semenogelin I Binding Site: A Contraceptive Drug Target1 
Biology of Reproduction  2012;87(3):56.
Epididymal protease inhibitor (EPPIN) is found on the surface of spermatozoa and works as a central hub for a sperm surface protein complex (EPPIN protein complex [EPC]) that inhibits sperm motility on the binding of semenogelin I (SEMG1) during ejaculation. Here, we identify EPPIN's amino acids involved in the interactions within the EPC and demonstrate that EPPIN's sequence C102-P133 contains the major binding site for SEMG1. Within the same region, the sequence F117-P133 binds the EPC-associated protein lactotransferrin (LTF). We show that residues Cys102, Tyr107, and Phe117 in the EPPIN C-terminus are required for SEMG1 binding. Additionally, residues Tyr107 and Phe117 are critically involved in the interaction between EPPIN and LTF. Our findings demonstrate that EPPIN is a key player in the protein-protein interactions within the EPC. Target identification is an important step toward the development of a novel male contraceptive, and the functionality of EPPIN's residues Cys102, Tyr107, and Phe117 offers novel opportunities for contraceptive compounds that inhibit sperm motility by targeting this region of the molecule.
Residues Cys102, Tyr107, and Phe117 within EPPIN's Kunitz domain are critical for binding semenogelin I and could be targets for contraceptive drug design.
PMCID: PMC3463412  PMID: 22699487
contraception; EPPIN; semenogelin I; spermatozoa
23.  Localization and Regulation of Murine Esco2 During Male and Female Meiosis1 
Biology of Reproduction  2012;87(3):61.
Meiosis is essential for generation of healthy gametes in both sexes and involves recombination and segregation of homologous chromosomes to produce haploid gametes. The initiation of meiosis in both sexes relies upon retinoic acid (RA) (Griswold MD, Hogarth CA, Bowles J, Koopman P. Initiating Meiosis: The Case for Retinoic Acid. Biol Reprod 2012; 86(35):1–7). Previous studies have demonstrated that the stimulated by retinoic acid gene 8 (Stra8) was required for meiotic progression in both the mouse ovary and postnatal testis. To identify additional candidates that may play a role during meiosis, we used microarray databases to generate lists of transcripts with expression profiles similar to that of Stra8 in the embryonic ovary and postnatal testis. One such gene, establishment of cohesion 1 homolog 2 (Saccharomyces cerevisiae) (Esco2), has been described as a regulator of sister chromatid cohesion during mitosis. This study describes the first in-depth analysis of ESCO2 localization and regulation during meiosis in both males and females. ESCO2 colocalized with the gamma H2A histone family member X (H2AFX) in pachytene spermatocytes, indicating that ESCO2 is a component of the XY body. In pachytene cells of the embryonic ovary, ESCO2 colocalized with H2AFX, which is consistent with the presence of ESCO2 in areas of double-stranded breaks. In addition, the expression of Esco2 was found to be regulated by RA in the postnatal testis. These data indicate that ESCO2 may play a vital role in meiosis in both males and females.
Establishment of cohesion 1 homolog 2 (ESCO) co-localizes with histone H2AFX is consistent with ESCO2 being present in areas of double-stranded breaks.
PMCID: PMC3463413  PMID: 22699483
germ cells; meiosis; ovary; testis
24.  Unique Pattern of ORC2 and MCM7 Localization During DNA Replication Licensing in the Mouse Zygote1 
Biology of Reproduction  2012;87(3):62.
In eukaryotes, DNA synthesis is preceded by licensing of replication origins. We examined the subcellular localization of two licensing proteins, ORC2 and MCM7, in the mouse zygotes and two-cell embryos. In somatic cells ORC2 remains bound to DNA replication origins throughout the cell cycle, while MCM7 is one of the last proteins to bind to the licensing complex. We found that MCM7 but not ORC2 was bound to DNA in metaphase II oocytes and remained associated with the DNA until S-phase. Shortly after fertilization, ORC2 was detectable at the metaphase II spindle poles and then between the separating chromosomes. Neither protein was present in the sperm cell at fertilization. As the sperm head decondensed, MCM7 was bound to DNA, but no ORC2 was seen. By 4 h after fertilization, both pronuclei contained DNA bound ORC2 and MCM7. As expected, during S-phase of the first zygotic cell cycle, MCM7 was released from the DNA, but ORC2 remained bound. During zygotic mitosis, ORC2 again localized first to the spindle poles, then to the area between the separating chromosomes. ORC2 then formed a ring around the developing two-cell nuclei before entering the nucleus. Only soluble MCM7 was present in the G2 pronuclei, but by zygotic metaphase it was bound to DNA, again apparently before ORC2. In G1 of the two-cell stage, both nuclei had salt-resistant ORC2 and MCM7. These data suggest that licensing follows a unique pattern in the early zygote that differs from what has been described for other mammalian cells that have been studied.
The DNA replication licensing protein MCM7 binds to DNA before origin recognition complex, subunit 2 (ORC2), and ORC2 appears to play significant roles in chromatin organization and nuclear structure.
PMCID: PMC3463414  PMID: 22674395
chromatin; DNA replication; early development; fertilization
25.  Bisphenol A Inhibits Follicle Growth and Induces Atresia in Cultured Mouse Antral Follicles Independently of the Genomic Estrogenic Pathway1 
Biology of Reproduction  2012;87(3):63.
Bisphenol A (BPA) is an estrogenic chemical used to manufacture many commonly used plastic and epoxy resin-based products. BPA ubiquitously binds to estrogen receptors throughout the body, including estrogen receptor alpha (ESR1) in the ovary. Few studies have investigated the effects of BPA on ovarian antral follicles. Thus, we tested the hypothesis that BPA alters cell cycle regulators and induces atresia in antral follicles via the genomic estrogenic pathway, inhibiting follicle growth. To test this hypothesis, we isolated antral follicles from 32- to 35-day-old control and Esr1-overexpressing mice and cultured them with vehicle control (dimethylsulfoxide [DMSO]) or BPA (1–100 μg/ml). Additionally, antral follicles were isolated from 32- to 35-day-old FVB mice and cultured with DMSO, BPA (1–100 μg/ml), estradiol (10 nM), ICI 182,780 (ICI; 1 μM), BPA plus ICI, or BPA plus estradiol. Follicles were measured for growth every 24 h for 96–120 h and processed either for analysis of estrogen receptor, cell cycle, and/or atresia factor mRNA expression, or for histological evaluation of atresia. Results indicate that estradiol and ICI do not protect follicles from BPA-induced growth inhibition and that estradiol does not protect follicles from BPA-induced atresia. Furthermore, overexpressing Esr1 does not increase susceptibility of follicles to BPA-induced growth inhibition. Additionally, BPA up-regulates Cdk4, Ccne1, and Trp53 expression, whereas it down-regulates Ccnd2 expression. BPA also up-regulates Bax and Bcl2 expression while inducing atresia in antral follicles. These data indicate that BPA abnormally regulates cell cycle and atresia factors, and this may lead to atresia and inhibited follicle growth independently of the genomic estrogenic pathway.
Bisphenol A inhibits antral follicle growth by aberrant up-regulation cell cycle regulators, inducing the cell cycle inhibitor transformation-related protein 53 and inducing atresia, independently of the genomic estrogenic pathway.
PMCID: PMC3464906  PMID: 22743301
atresia; bisphenol A; cell cycle; follicle growth

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