Maternally encoded stem-loop-binding protein is degraded in 2-cell mouse embryos by the co-ordinated activity of two separately regulated pathways

AbstractOocytes accumulate mRNAs and proteins that direct early embryonic development. Although subsequent development requires the timely degradation of these maternal products, little is known of the underlying mechanisms. The stem-loop-binding protein (SLBP), which regulates the stability and translation of mRNAs encoding histones and is synthesized during S-phase and degraded during G2 in somatic cells, accumulates during oogenesis. Maternal SLBP is required for mouse embryos to develop beyond the 2-cell stage, but must be degraded to allow the cell-cycle-regulated expression of somatic cells to be established. We report that the quantity of maternal SLBP changes little following fertilization until 44–52 h post-hCG, corresponding to mid-/late G2 of the 2-cell stage, when it decreases by 75%. Efficient degradation requires two pathways. The first requires activity of cyclin-dependent kinases (cdk) and embryonic transcription, preferentially targets nuclear SLBP, and likely corresponds to the pathway that degrades SLBP at G2 in somatic cells. The second does not require cdk activity or transcription and becomes active at 44–52 h post-hCG independently of cell-cycle progression to mid-/late G2, but is not solely regulated by the time elapsed since hCG injection. Thus, the co-ordinated activity of two separately regulated pathways eliminates maternally encoded SLBP from early mouse embryos

Analysis of nuclear maturation, DNA damage and repair gene expression of bovine oocyte and cumulus cells submitted to ionizing radiation

Radiotherapy causes destruction of tumor cells, but also threatens the integrity and survival of surrounding normal cells. Then, woman submitted to irradiation for cancer treatment may present permanent ovary damage, resulting in impaired fertility. The objective of this study was to investigate the effects of therapeutic doses of ionizing radiation (IR), used for ovarian cancer treatment in humans, on bovine cumulus-oocyte complexes (COCs) as experimental model. Bovine ovaries were exposed to 0.9 Gy, 1.8 Gy, 3.6 Gy or 18.6 Gy IR, and then COCs were collected and used to evaluate: (a) oocyte nuclear maturation; (b) presence of phosphorylated H2A.X (γH2AX), as an indicator of DNA double-strand breaks (DSBs); and (c) expression of genes involved in DNA repair (TP53BP1, RAD52, ATM, XRCC6 and XRCC5) and apoptosis (BAX). The radiation doses tested in this study had no detrimental effects on nuclear maturation and did not increase γH2AX in the oocytes. However, IR treatment altered the mRNA abundance of RAD52 (RAD52 homolog, DNA repair protein) and BAX (BCL2-associated X protein). We conclude that although IR doses had no apparent effect on oocyte nuclear maturation and DNA damage, molecular pathways involved in DNA repair and apoptosis were affected by IR exposure in cumulus cells

Intravaginal progestagen for estrus and parturition control in sows

O objetivo deste trabalho foi avaliar a utilização de dispositivos intravaginais (DIV) para o controle da reprodução em suínos. Porcas aos 112 dias de gestação receberam injeção de PGF2α (controle, n = 15) ou PGF2α com inserção de DIV contendo acetato de medroxiprogesterona (grupo DIV, n = 14) por 48 horas. As fêmeas iniciaram o parto 27,7±1,6 e 82,3±3,8 horas após aplicação de PGF2α nos grupos controle e tratado, respectivamente. Quanto ao controle do estro, dez porcas receberam DIV por 12 dias, iniciando imediatamente após o desmame, e o estro foi confirmado aos 17,25±0,17 dias após o desmame, em comparação a 4±0,25 dias no grupo controle. Dispositivos intravaginais com progestágeno podem ser utilizados no controle da reprodução em suínos.The objective of this work was to evaluate the use of intravaginal devices (IVD) for the control of reproductive events in swine. Sows at 112 days of pregnancy received an injection of PGF2α (control, n = 15) or PGF2α plus an IVD containing medroxyprogesterone acetate (IVD group, n = 14) for 48 hours. Sows initiated labor 27.7±1.6 and 82.3±3.8 h after PGF2α aplicaction, in control and treated groups, respectively. Regarding control of estrus cycle, ten sows received IVD for 12 days starting immediately after weaning, and estrus was confirmed 17.25±0.17 days after weaning, in comparison to 4.00±0.25 days for the control group. Intravaginal devices with progestagen can be applied for the control of reproduction in swine

Reprogramming of telomerase activity and rebuilding of telomere length in cloned cattle

Nuclear reprogramming requires the removal of epigenetic modifications imposed on the chromatin during cellular differentiation and division. The mammalian oocyte can reverse these alterations to a state of totipotency, allowing the production of viable cloned offspring from somatic cell nuclei. To determine whether nuclear reprogramming is complete in cloned animals, we assessed the telomerase activity and telomere length status in cloned embryos, fetuses, and newborn offspring derived from somatic cell nuclear transfer. In this report, we show that telomerase activity was significantly (P < 0.05) diminished in bovine fibroblast donor cells compared with embryonic stem-like cells, and surprisingly was 16-fold higher in fetal fibroblasts compared with adult fibroblasts (P < 0.05). Cell passaging and culture periods under serum starvation conditions significantly decreased telomerase activity by approximately 30–50% compared with nontreated early passage cells (P < 0.05). Telomere shortening was observed during in vitro culture of bovine fetal fibroblasts and in very late passages of embryonic stem-like cells. Reprogramming of telomerase activity was apparent by the blastocyst stage of postcloning embryonic development, and telomere lengths were longer (15–23 kb) in cloned fetuses and offspring than the relatively short mean terminal restriction fragment lengths (14–18 kb) observed in adult donor cells. Overall, telomere lengths of cloned fetuses and newborn calves (≈20 kb) were not significantly different from those of age-matched control animals (P > 0.05). These results demonstrate that cloned embryos inherit genomic modifications acquired during the donor nuclei's in vivo and in vitro period but are subsequently reversed during development of the cloned animal

Transforming growth factor-beta family members are regulated during induced luteolysis in cattle

The transforming growth factors beta (TGFβ) are local factors produced by ovarian cells which, after binding to their receptors, regulate follicular deviation and ovulation. However, their regulation and function during corpus luteum (CL) regression has been poorly investigated. The present study evaluated the mRNA regulation of some TGFβ family ligands and their receptors in the bovine CL during induced luteolysis in vivo. On day 10 of the estrous cycle, cows received an injection of prostaglandin F2α (PGF) and luteal samples were obtained from separate groups of cows (n= 4-5 cows per time-point) at 0, 2, 12, 24 or 48 h after treatment. Since TGF beta family comprises more than 30 ligands, we focused in some candidates genes such as activin receptors (ACVR-1A, -1B, -2A, -2B) AMH, AMHR2, BMPs (BMP-1, -2, -3, -4, -6 and -7), BMP receptors (BMPR 1A, -1B and -2), inhibin subunits (INH-A, -BA, -BB) and betaglycan (TGFBR3). The mRNA levels of BMP4, BMP6 and INHBA were higher at 2 h after PGF administration (P<0.05) in comparison to 0 h. The relative mRNA abundance of BMP1, BMP2, BMP3, BMP4, BMP6, ACVR1B, INHBA and INHBB was upregulated up to 12 h post PGF (P<0.05). On the other hand, TGFBR3 mRNA that codes for a reservoir of ligands that bind to TGF-beta receptors, was lower at 48 h. In conclusion, findings from this study demonstrated that genes encoding several TGFβ family members are expressed in a time-specific manner after PGF administration

In Vitro Production of Embryos from Prepubertal Holstein Cattle and Mediterranean Water Buffalo: Problems, Progress and Potential

Laparoscopic ovum pick-up (LOPU) coupled with in vitro embryo production (IVEP) in prepubertal cattle and buffalo accelerates genetic gain. This article reviews LOPU-IVEP technology in prepubertal Holstein Cattle and Mediterranean Water Buffalo. The recent expansion of genomic-assisted selection has renewed interest and demand for prepubertal LOPU-IVEP schemes; however, low blastocyst development rates has constrained its widespread implementation. Here, we present an overview of the current state of the technology, limitations that persist and suggest possible solutions to improve its efficiency, with a focus on gonadotropin stimulations strategies to prime oocytes prior to follicular aspiration, and IVEP procedures promoting growth factor metabolism and limiting oxidative and endoplasmic reticulum stress

Effect of the transient pharmacological inhibition of Mapk3/1 pathway on ovulation in mice.

Mitogen-activated protein kinase 3/1 (Mapk3/1) pathway is critical for LH signal transduction during ovulation. However, the mechanisms remain incompletely understood. We hypothesized that Mapk pathway regulates ovulation through transcriptional regulation of ovulatory genes. To test this hypothesis we used immature mice superovulated with equine and human chorionic gonadotropins (eCG and hCG) and PD0325901, to inhibit hCG-induced Mapk3/1 activity. Mice received either the inhibitor PD0325901 (25 μg/g, i.p.) or vehicle at 2h before hCG stimulation. Administration of the inhibitor abolished Mapk3/1 phosphorylation in granulosa cells. While vehicle-treated mice ovulated normally, there were no ovulations in inhibitor-treated mice. First, we analyzed gene expression in granulosa cells at 0h, 1h and 4h post-hCG. There was expected hCG-driven increase in mRNA abundance of many ovulation-related genes including Ptgs2 in vehicle-treated granulosa cells, but not (P<0.05) in inhibitor-treated group. There was also reduced mRNA and protein abundance of the transcription factor, early growth response 1 (Egr1) in inhibitor-treated granulosa cells. We then used GRMO2 cell-line to test if Egr1 is recruited to promoter of Ptgs2 followed by chromatin immunoprecipitation with either Egr1 or control antibody. Enrichment of the promoter regions in immunoprecipitants of Egr1 antibody indicated that Egr1 binds to the Ptgs2 promoter. We then knocked down Egr1 expression in mouse primary granulosa cells using siRNA technology. Treatment with Egr1-siRNA inhibited Egr1 transcript accumulation, which was associated with reduced expression of Ptgs2 when compared to control-siRNA treated granulosa cells. These data demonstrate that transient inhibition of LH-stimulated MAPK3/1 activity abrogates ovulation in mice. We conclude that Mapk3/1 regulates ovulation, at least in part, through Egr1 and its target gene, Ptgs2 in granulosa cells of ovulating follicles in mice