Abstract:
BACKGROUND:The objectives of this study were to develop an easy and rapid method for measuring gene expression in a small number of cells by real-time PCR without RNA extraction and purification, and to use this method to determine more precisely IGF-I gene expression in the cumulus cells surrounding oocytes. METHODS:First, after small numbers of cumulus cells were lysed in cell lysis buffer, they were digested with various concentrations of DNase I for different periods at 37 degrees C to determine the optimal conditions for digestion of genomic DNA in the lysate. Since nonspecific amplification was liable to occur when the non-purified RT product of the cell lysate was used for real-time PCR with the given primers, the optimal conditions for Mg2+ and annealing temperature were well investigated. Further, to create the same conditions as in the actual sample reaction for measurement by real-time PCR, RT-minus product was added to the reaction mixture of the standard curve, and then the amplification efficiency was assessed. Next, IGF-I gene expression in cumulus cells collected from cumulus oocyte complexes (COCs) every 4 h during maturation was determined using the developed method. RESULTS:The optimal conditions for measuring gene expression using the cell lysate from a small number of cells were as follows: incubation of the cell lysate with 0.16 U/microL DNase I with 10 U/microL for 30 min, an Mg concentration of 1.5 mM for amplification of target gene by real-time PCR using RT-product of the cell lysate. When the RT-minus products added to the reaction mixture for the standard curve, which was prepared for purified 18SrRNA plasmid, the PCR efficiency was similar between the sample and the standard. The IGF-I gene expression in the cumulus cells was elevated up through the first 8 h of the culture and then declined gradually by the end of maturation, with the maximal gene expression (778-fold) seen at 8 h. CONCLUSION:It can be concluded that the method developed here, in which equivalent to cumulus cells collected from 0.03-0.075 COCs were employed per reaction, permits rapid and easy determination of target gene expression in a limited number of cells using real-time PCR without RNA extraction.
journal_name
Reprod Biol Endocrinoljournal_title
Reproductive biology and endocrinology : RB&Eauthors
Kumamoto K,Wang H,Yamashiro H,Terada Tdoi
10.1186/1477-7827-3-59keywords:
subject
Has Abstractpub_date
2005-10-27 00:00:00pages
59issn
1477-7827pii
1477-7827-3-59journal_volume
3pub_type
杂志文章abstract::Human spermatozoa generate low levels of reactive oxygen species in order to stimulate key events, such as tyrosine phosphorylation, associated with sperm capacitation. However, if the generation of these potentially pernicious oxygen metabolites becomes elevated for any reason, spermatozoa possess a limited capacity ...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章,评审
doi:10.1186/1477-7827-3-67
更新日期:2005-11-29 00:00:00
abstract:BACKGROUND:Nucleotide-binding oligomerization domain (NACHT), leucine rich repeat (LRR) and pyrin domain (PYD) 7 containing protein, NLRP7, is a member of the NLR family which serves as innate immune sensors. Mutations and genetic variants of NLRP7 have been found in women with infertility associated conditions, such a...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章
doi:10.1186/s12958-017-0286-x
更新日期:2017-08-15 00:00:00
abstract:BACKGROUND:Infertile men have higher levels of semen reactive oxygen species (ROS) than fertile men. High levels of semen ROS can cause sperm dysfunction, sperm DNA damage and reduced male reproductive potential. This study investigated the effects of supplementation with N-acetyl-cysteine (NAC) on the sperm quality, c...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章,随机对照试验
doi:10.1186/s12958-019-0468-9
更新日期:2019-02-16 00:00:00
abstract:BACKGROUND:The fibrinolytic system and its inhibitors play a number of roles, apart from their function in blood haemostasis and thrombosis, namely in ovarian folliculogenesis and in ovulation. Plasminogen is converted to active plasmin at the time of follicular rupture through a decrease in plasminogen activator inhib...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章
doi:10.1186/s12958-019-0472-0
更新日期:2019-03-16 00:00:00
abstract:BACKGROUND:Polo-like kinase 1 (Plk1), as a characteristic regulator in meiosis, organizes multiple biological events of cell division. Although Plk1 has been implicated in various functions in somatic cell mitotic processes, considerably less is known regarding its function during the transition from metaphase I (MI) t...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章
doi:10.1186/s12958-017-0289-7
更新日期:2017-08-29 00:00:00
abstract::Polycystic ovary syndrome (PCOS) is a common endocrine disorder, affecting 9-18% of women in reproductive age that causes hyperandrogenism and infertility due to dysfunctional follicular maturation and anovulation. The etiology of PCOS is still poorly known, and information from experimental animal models may help imp...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章,评审
doi:10.1186/s12958-017-0231-z
更新日期:2017-02-10 00:00:00
abstract:BACKGROUND:A decrease in HOXA11 expression in eutopic mid-secretory endometrium has been found in women with endometriosis-associated infertility. METHODS:Using Real-time quantitative PCR (RQ-PCR) and western blotting analysis we studied the HOXA11 transcript and protein levels in mid-luteal eutopic endometrium from e...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章
doi:10.1186/1477-7827-10-1
更新日期:2012-01-10 00:00:00
abstract:BACKGROUND:To assess the amount of variability in ovarian follicular growth rate and maximum follicular diameter related to different centers, women and cycles of the same women in a multicenter observational study of follicular growth. METHODS:Secondary analysis of a prospective cohort study from eight centers in Eur...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章,多中心研究
doi:10.1186/1477-7827-6-61
更新日期:2008-12-10 00:00:00
abstract:BACKGROUND:We characterized the spectrum and etiology of hypogonadism in a cohort of Prader-Willi syndrome (PWS) adolescents and adults. METHODS:Reproductive hormonal profiles and physical examination were performed on 19 males and 16 females ages 16-34 years with PWS. Gonadotropins, sex-steroids, inhibin B (INB) and ...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章
doi:10.1186/1477-7827-10-39
更新日期:2012-05-06 00:00:00
abstract::Cloning mammals by nuclear transfer is a powerful technique that is quickly advancing the development of genetically defined animal models. However, the overall efficiency of nuclear transfer is still very low and several hurdles remain before the power of this technique will be fully harnessed. Among these hurdles in...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章,评审
doi:10.1186/1477-7827-1-84
更新日期:2003-11-07 00:00:00
abstract::Human embryonic stem cells (hESC) have emerged as attractive candidates for cell-based therapies that are capable of restoring lost cell and tissue function. These unique cells are able to self-renew indefinitely and have the capacity to differentiate in to all three germ layers (ectoderm, endoderm and mesoderm). Harn...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 历史文章,杂志文章,评审
doi:10.1186/s12958-015-0005-4
更新日期:2015-02-22 00:00:00
abstract:BACKGROUND:To examine the effects of IVF, ICSI and FET, as well as in vitro culture, on the safety of offspring, this study was conducted from the perspective of genetic imprinting to investigate whether assisted reproductive technology would influence the parental and maternal imprinting genes. METHODS:Eighteen foetu...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章
doi:10.1186/s12958-018-0344-z
更新日期:2018-07-05 00:00:00
abstract:BACKGROUND:There is limited literature investigating the effects of body mass index (BMI) and androgen level on in vitro fertilization (IVF) outcomes with a gonadotropin-releasing hormone (GnRH)-antagonist protocol in polycystic ovary syndrome (PCOS). Androgen-related variation in the effect of body mass index (BMI) on...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章
doi:10.1186/s12958-018-0438-7
更新日期:2018-11-19 00:00:00
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journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章,随机对照试验
doi:10.1186/1477-7827-9-130
更新日期:2011-09-29 00:00:00
abstract:BACKGROUND:Decidualization is a prerequisite for successful implantation and the establishment of pregnancy. A critical role of impaired decidualization in subfertility has been established. In human endometrial stromal cells (hESCs), Krüppel-like factor 12 (KLF12) and Nur77 are novel regulators of decidualization. We ...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章
doi:10.1186/s12958-017-0243-8
更新日期:2017-03-31 00:00:00
abstract::The prolonged lockdown of health services providing high-complexity fertility treatments -as currently recommended by many reproductive medicine entities- is detrimental for society as a whole, and infertility patients in particular. Globally, approximately 0.3% of all infants born every year are conceived using assis...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 信件
doi:10.1186/s12958-020-00605-z
更新日期:2020-05-13 00:00:00
abstract:BACKGROUND:Women with Polycystic Ovary Syndrome (PCOS) present a heterogeneous reproductive and metabolic profile with an increased lifetime risk of Type 2 Diabetes (T2D). Early biomarkers of these metabolic disturbances in PCOS women have not been identified. The abundance of circulating insulin gene promotor cell-fre...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章
doi:10.1186/s12958-019-0478-7
更新日期:2019-04-05 00:00:00
abstract:BACKGROUND:Di(2-ethylhexyl) phthalate (DEHP) exposure reduces embryo implantations, increases embryonic loss, and decreases fetal body weights. However, whether it is associated with the alteration of luteal function remains unknown. Thus, our aim in this study was to explore the effect and mechanism of DEHP on luteal ...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章
doi:10.1186/s12958-015-0013-4
更新日期:2015-03-03 00:00:00
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journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章
doi:10.1186/1477-7827-1-10
更新日期:2003-02-06 00:00:00
abstract:BACKGROUND:L-carnitine (LC), and its acetylated form, acetyl L-carnitine (ALC), have immense functional capabilities to regulate the oxidative and metabolic status of the female reproductive system. The vulnerability of this system to free radicals demand for advanced strategies to combat them. For this purpose, the 'q...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章,评审
doi:10.1186/s12958-018-0323-4
更新日期:2018-01-26 00:00:00
abstract:BACKGROUND:We found previously that the expression of SET gene was up-regulated in polycystic ovaries. Evidences suggested that SET protein was essential for regulating both the promoter activity of CYP17A1 and the biological activity of P450c17. In this study, we explored whether SET regulated androgen production in p...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章
doi:10.1186/1477-7827-11-9
更新日期:2013-02-19 00:00:00
abstract:BACKGROUND:The aim of this randomized controlled trial was to compare the efficacy of extended letrozole regimen with clomiphene citrate in women with unexplained infertility undergoing superovulation and intrauterine insemination (IUI). METHODS:Two hundred and fourteen patients with unexplained infertility were rando...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章,多中心研究,随机对照试验
doi:10.1186/1477-7827-9-84
更新日期:2011-06-21 00:00:00
abstract:BACKGROUND:Testicular microliths are calcifications found within the seminiferous tubules. In humans, testicular microlithiasis (TM) has an unknown etiology but may be significantly associated with testicular germ cell tumors. Factors inducing microlith development may also, therefore, act as susceptibility factors for...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章
doi:10.1186/1477-7827-7-88
更新日期:2009-08-27 00:00:00
abstract:BACKGROUND:Nomegestrol acetate (NOMAC), a synthetic progestogen derived from 19-nor-progesterone, recently completed clinical trials for use with 17beta-estradiol in a new monophasic combined oral contraceptive. In this review, published as well as previously unpublished preclinical studies that detail the effects of N...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章,评审
doi:10.1186/1477-7827-10-85
更新日期:2012-10-08 00:00:00
abstract:BACKGROUND:Endocannabinoids (ECs) are important contributors to implantation and decidualization and are suppressed in early pregnancy. Elevated levels of anandamide (AEA), the endogenous ligand for the CB1 and CB2 receptors (R), interfere with receptivity of the blastocyst. Ang-(1-7) is down-regulated in the implantat...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章
doi:10.1186/1477-7827-13-5
更新日期:2015-01-17 00:00:00
abstract:BACKGROUND:Relaxin is detected in seminal plasma of many species and its association with sperm motility may be beneficial in some aspects of assisted reproduction. Here, we immunolocalized relaxin receptors and investigated the effects of exogenous relaxin on motility characteristics, viability, and cAMP content of bo...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章
doi:10.1186/s12958-015-0021-4
更新日期:2015-03-31 00:00:00
abstract:BACKGROUND:Spontaneous abortion is one of the most common complications in early pregnancy. A preventive test to identify women who will experience a miscarriage, even before first symptoms occur, is not established. Activation of maternal immunological tolerance seems to be essential for early fetal development and va...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章
doi:10.1186/s12958-018-0411-5
更新日期:2018-09-28 00:00:00
abstract::The aim of this study was to analyse the multigenerational effects of para-nonylphenol (NP) and resveratrol (RES) on the body weight, organ weight and reproductive fitness of outbred CD-1 mice. The data indicate that in male mice, NP had an effect on the weight of selected reproductive organs and the kidneys in the pa...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章
doi:10.1186/1477-7827-1-30
更新日期:2003-03-24 00:00:00
abstract::Macaques are menstruating nonhuman primates that provide important animal models for studies of hormonal regulation in the uterus. In women and macaques the decline of progesterone (P) at the end of the cycle triggers endometrial expression of a variety of matrix metalloproteinase (MMP) enzymes that participate in tis...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章,评审
doi:10.1186/1477-7827-4-S1-S6
更新日期:2006-01-01 00:00:00
abstract:BACKGROUND:Septate uterus, one of the most common forms of congenital uterine malformations, negatively affects female reproductive health. METHODS:In a retrospective cohort study, we evaluated the reproductive outcome after hysteroscopic septoplasty in 64 women with septate uterus and primary or secondary infertility...
journal_title:Reproductive biology and endocrinology : RB&E
pub_type: 杂志文章,评审
doi:10.1186/1477-7827-8-52
更新日期:2010-05-21 00:00:00