The Mechanism of Oocyte Activation Influences the Cell Cycle-Related Genes Expression During Bovine Preimplantation Development

The first cleavage divisions and preimplantation embryonic development are supported by mRNA and proteins synthesized and stored during oogenesis. Thus, mRNA molecules of maternal origin decrease and embryonic development becomes gradually dependent on expression of genetic information derived from the embryonic genome. However, it is still unclear what the role of the sperm cell is during this phase and whether the absence of the sperm cell during the artificial oocyte activation affects subsequent embryonic development. The objective of this study was to determine, in bovine embryos, changes in cell cycle-associated transcript levels (cyclin A, cyclin B, cyclin E, CDC2, CDK2, and CDK4) after oocyte activation in the presence or absence of the sperm cell. To evaluate that, in vitro-produced (IVP) and parthenogenetically activated (PA) embryos (2-4 cells (2-4C), 8-16 cells (8-16C) and blastocysts) were evaluated by real-time PCR. There was no difference in cleavage and blastocyst rates between IVP and PA groups. Transcript level was higher in oocytes than in IVP and PA embryos. Cleaved PA embryos showed higher expression of cyclin A, cyclin B, cyclin E, and CDK2 and lower expression of CDC2 when compared with that from the IVP group. At the time of activation, all transcripts were expressed less in PA than in IVP embryos, whereas at the blastocyst stage, almost all genes were expressed at a higher level in the PA group. These results suggest that in both groups there is an initial consumption of these transcripts in the early stages of embryonic development. Furthermore, 8-16C embryos seem to synthesize more cell cycle-related genes than 2-4C embryos. However, in PA embryos, activation of the cell cycle genes seems to occur after the 8- to 16-cell stage, suggesting a failure in the activation process.

The use of parthenotegenetic and IVF bovine blastocysts as a model for the creation of human embryonic stem cells under defined conditions

Clinical application of human embryonic stem cells will be possible, when cell lines are created under xeno-free and defined conditions. We aimed to establish methodologies for parthenogenetic activation, culture to blastocyst and mechanical isolation of the inner cell mass (ICM) using bovine oocytes, as a model for derivation and proliferation of human embryonic stem cells under defined xeno-free culture conditions. Cumulus-oocyte-complexes were in vitro matured and activated using Ca(2+)Ionophore and 6-DMAP or in vitro fertilized (IVF). Parthenotes and biparental embryos were cultured to blastocysts, when their ICM was mechanically isolated and placed onto a substrate of fibronectin in StemProA (R) medium. After attachment, primary colonies were left to proliferate and stained for pluripotency markers, alkaline phosphatase and Oct-4. Parthenogenesis and fertilization presented significantly different success rates (91 and 79 %, respectively) and blastocyst formation (40 and 43 %, respectively). ICMs from parthenogenetic and IVF embryos formed primary and expanded colonies at similar rates (39 % and 33 %, respectively). Six out of eight parthenogenetic colonies tested positive for alkaline phosphatase. Three colonies were analyzed for Oct-4 and they all tested positive for this pluripotency marker. Our data show that Ca2+ Ionophore, and 6-DMAP are efficient in creating large numbers of blastocysts to be employed as a model for human oocyte activation and embryo development. After mechanical isolation, parthenogetic derived ICMs showed a good rate of derivation in fibronectin and Stem-Pro forming primary and expanded colonies of putative embryonic stem cells. This methodology may be a good strategy for parthenogenetic activation of discarded human oocytes and derivation in defined conditions for future therapeutic interventions.

Nuclear Transfer with Apoptotic Bovine Fibroblasts: Can Programmed Cell Death Be Reprogrammed?

Cell death by apoptosis is considered to be irreversible. However, reports have indicated that its reversibility is possible if the cells have not yet reached the "point of no return.'' In order to add new information about this topic, we used cells at different moments of apoptotic process as nuclear donors in somatic cell nuclear transfer (SCNT) in order to test if programmed cell death can be reversed. Adult bovine fibroblasts were treated with 10 mu M of staurosporine (STP) for 3 h and analyzed for phosphatidylserine externalization (Annexin assay) and presence of active caspase-9. Annexin-positive (Anx +) and Caspase-9-positive (Casp-9 +) cells were isolated by FACS and immediately transferred into enucleated in vitro matured bovine oocytes. After STP treatment, 89.9% of cells were Anx + (4.6% in control cells; p < 0.01) and 24.9% were Casp-9 + (2.4% in control cells; p < 0.01). Fusion and cleavage were not affected by the use apoptotic cells (p > 0.05). Also, the use of Anx + cells did not affect blastocyst production compared to control (26.4% vs. 22.9%, respectively; p > 0.05). However, blastocyst formation was affected by the use of Casp-9 + cells (12.3%; p < 0.05). These findings contribute to the idea of that apoptosis is reversible only at early stages. Additionally, we hypothesize that the "point of no return'' for apoptosis may be located around activation of Caspase-9.

Heparin and penicillamine–hypotaurine–epinephrine (PHE) solution during bovine in vitro fertilization procedures impair the quality of spermatozoa but improve normal oocyte fecundation and early embryonic development

The presence of heparin and a mixture of penicillamine, hypotaurine, and epinephrine (PHE) solution in the in vitro fertilization (IVF) media seem to be a prerequisite when bovine spermatozoa are capacitated in vitro, in order to stimulate sperm motility and acrosome reaction. The present study was designed to determine the effect of the addition of heparin and PHE during IVF on the quality and penetrability of spermatozoa into bovine oocytes and on subsequent embryo development. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes and mitochondrial function, was diminished (P<0.05) in the presence of heparin and PHE. Oocyte penetration and normal pronuclear formation rates, as well as the percentage of zygotes presenting more than two pronuclei, was higher (P<0.05) in the presence of heparin and PHE. No differences were observed in cleavage rates between treatment and control (P>0.05). However, the developmental rate to the blastocyst stage was increased in the presence of heparin and PHE (P>0.05). The quality of embryos that reached the blastocyst stage was evaluated by counting the inner cell mass (ICM) and trophectoderm (TE) cell numbers and total number of cells; the percentage of ICM and TE cells was unaffected (P>0.05) in the presence of heparin and PHE (P<0.05). In conclusion, this study demonstrated that while the supplementation of IVF media with heparin and PHE solution impairs spermatozoa quality, it plays an important role in sperm capacitation, improving pronuclear formation, and early embryonic development

Photobiological effect of low-level laser irradiation in bovine embryo production system

The objectives of this study were to evaluate the effect of low-level laser irradiation (LLLI) on bovine oocyte and granulosa cells metabolism during in vitro maturation (IVM) and further embryo development. Cumulus-oocytes complexes (COCs) were subjected (experimental group) or not (control group) to irradiation with LLLI in a 633-nm wavelength and 1 J/cm2 fluency. The COCs were evaluated after 30 min, 8, 16, and 24 h of IVM. Cumulus cells were evaluated for cell cycle status, mitochondrial activity, and viability (flow cytometry). Oocytes were assessed for meiotic progression status (nuclear staining), cell cycle genes content [real-time polymerase chain reaction (PCR)], and signal transduction status (western blot). The COCs were also in vitro fertilized, and the cleavage and blastocyst rates were assessed. Comparisons among groups were statistically performed with 5% significance level. For cumulus cells, a significant increase in mitochondrial membrane potential and the number of cells progressing through the cycle could be observed. Significant increases on cyclin B and cyclin-dependent kinase (CDK4) levels were also observed. Concerning the oocytes, a significantly higher amount of total mitogen-activated protein kinase was found after 8 h of irradiation, followed by a decrease in all cell cycle genes transcripts, exception made for the CDK4. However, no differences were observed in meiotic progression or embryo production. In conclusion, LLLI is an efficient tool to modulate the granulosa cells and oocyte metabolism

Gewebeprotektive und antiinflammatorische Wirkung von humanem Choriogonadotropin in Mausmodellen mit autoimmunem Leberschaden

Während der Schwangerschaft kommt es häufig zu einer spontanen Verbesserung von klinischen Symptomen der autoimmunen Hepatitis und anderen Th1-vermittelten Autoimmunerkrankungen. Die Gründe hierfür sind bis heute noch nicht vollständig aufgeklärt. Eines der wichtigsten Hormone in der Schwangerschaft ist das humane Choriogonadotropin (hCG), welches schon in der frühen Schwangerschaft eine entscheidende Rolle spielt. Es sorgt für die Stimulation des Corpus luteums, wodurch es zur Ausschüttung von Progesteron kommt und somit die Einnistung der Blastozyte gewährleistet und die Abstoßung des Embryos verhindert wird. In dieser Arbeit wurden Effekt und Signalweg von hCG in primären murinen und humanen Hepatozyten sowie in Mausmodellen mit T-Zell-abhängigem Leberschaden untersucht. hCG führte sowohl bei akuten als auch bei chronischen Leberschäden zu einer drastischen Senkung der Aspartat-Aminotransferase, einem Indikator für Lebererkrankungen. Die Histologie der Leber hCG-behandelter Tiere wies außerdem signifikant weniger apoptotische Zellen und eine deutliche Reduktion infiltrierender CD4+ T-Zellen auf. Die Analyse des hCG-Signalweges zeigte, dass hCG die Langlebigkeitsproteine Foxo3a und Sirt1 reguliert. Die Aktivierung des PI3-Kinase/Akt-Signalweges durch hCG führte zu einem Transport des Transkriptionsfaktors Foxo3a aus dem Zellkern, wodurch die proapoptotischen Zielgene Bim und Puma nicht mehr transkribiert werden können. Eine zusätzliche Hemmung von Foxo3a erfolgte durch die Aktivierung der Deacetylase Sirt1, indem diese phosphoryliert wird und in den Zellkern transloziert. In weiteren Untersuchungen wurde der immunsuppressive Effekt von hCG näher betrachtet. Dabei stellte sich heraus, dass hCG effektiv die proteolytische Aktivität der Caspase-3 in Hepatozyten hemmt, wodurch die Ausschüttung der biologisch aktiven Form von Interleukin-16, einem chemotaktischen Faktor für CD4+ Zellen, herabgesetzt wird. Dadurch wird die Leber erfolgreich vor der Infiltration durch autoaggressive CD4+ Zellen geschützt. IL-16 spielt bei vielen inflammatorischen Krankheiten eine Rolle, was auch in dieser Arbeit durch den Nachweis hoher IL-16-Konzentrationen in Seren von Patienten mit autoimmuner Hepatitis bestätigt werden konnte. Die in dieser Studie beschriebene Wirkung von hCG und die Tatsache, dass hCG ein bereits bewährtes und auf Nebenwirkungen getestetes Medikament bei Infertilität ist, macht es zu einem idealen Kandidaten für immunsuppressive Therapieansätze bei akuten und chronisch entzündlichen Lebererkrankungen.

[Implantation: physiology, pathology and therapeutic options in disorders of implantation]

So far, implantation is a poorly understood process, which involves several paradoxical cell-biological mechanisms. First, 50% of the embryo is paternal and immunologically foreign material, and second, both the endometrium and embryo are covered by epithelial tissue to prevent cellular fusion. The adhesion and invasion of the blastocyst require an accurate coordination of embryonic and endometrial physiology and the modulation of maternal immune tolerance. Endometrial function plays an important role in assisted reproduction. Pathologies such as fibroids, hydrosalpinges, endometriosis and the polycystic ovary syndrome have a significant negative impact on implantation but can be treated in most cases. Therapeutic strategies to improve endometrial and embryonic function in recurrent implantation disorders are however still controversially discussed.

Assessment of BoviPureTM for the In Vitro Production of Bovine Embryos

The objective of this study was to examine the potential utility of a commercially available sperm separation and purification product for the in vitro production of bovine embryos. Bovine oocytes were purchased from a commercial supplier, and matured oocytes were randomly allocated to one of two treatments. Oocytes were co-incubated with frozen-thawed semen washed twice with BoviPureTM (BoviPure group) or with modified Brackett-Oliphant medium (control group). After a 6-hour insemination period, oocytes were cultured in vitro for 8 days. Cleavage rate of embryos was determined 48 hours post-insemination, and blastocyst formation rate was assessed on day 8 of culture. The experiment was replicated three times, and data were analyzed using chi-square analysis. Washing of sperm in BoviPureTM had no effect (P>.05) on either cleavage rate (77.2%) or blastocyst development (21.6%) when compared with controls (71.9% and 17.1%, respectively). These results indicate that, under conditions of our study, the washing of sperm with BoviPureTM did not significantly enhance the ability to produce bovine embryos in vitro.

Expression and hormonal regulation of Muc-1 at the apical surface of mouse uterine epithelial cells and its role in modulating embryo implantation

Previous studies from our lab have established that large molecular weight mucin glycoproteins are major apically-disposed components of mouse uterine epithelial cells in vitro (Valdizan et al., (1992) J. Cell. Physiol. 151:451-465). The present studies demonstrate that Muc-1 represents one of the apically-disposed mucin glycoproteins of mouse uterine epithelia, and that Muc-1 protein and mRNA expression are regulated in the peri-implantation stage mouse uterus by ovarian steroids. Muc-1 expression is high in the proestrous and estrous stages, and decreases during diestrous. Both Muc-1 protein and mRNA levels decline to barely detectable levels by day 4 of pregnancy, i.e., prior to the time of blastocyst attachment. In contrast, Muc-1 expression in the cervix and vagina is maintained during this same period. Delayed implantation was established in pregnant mice by ovariectomy and maintained by administration of exogenous progesterone. Initiation of implantation was triggered by coinjection of progesterone maintained mice with a nidatory dose of 17$\beta$-estradiol. Muc-1 levels in the uterine epithelia of progesterone maintained mice declined to similar low levels as observed on day 4 of normal pregnancy. Coinjection of estradiol did not alter Muc-1 expression suggesting that down-regulation of Muc-1 is a progesterone dominated event. This was confirmed in ovariectomized, non-pregnant mice which displayed stimulation of Muc-1 expression following 6 hr of estradiol injection. Estradiol stimulated Muc-1 expression was inhibited by the pure antiestrogen, ICI 164,384. While progesterone alone had no effect on Muc-1 expression, it antagonized estradiol action in this regard. Injection of pregnant mice with the antiprogestin, RU 486, a known implantation inhibitor, on day 3 of pregnancy restored high level expression of Muc-1 mRNA on day 4, indicating that down-regulation of Muc-1 is progesterone receptor-mediated. Muc-1 appears to function as an anti-adhesive molecule at the apical cell surface of mouse uterine epithelial cells. Treatment of polarized cultures of mouse uterine epithelial cells with O-sialoglycoprotein endopeptidase reduced mucin expression in vitro, by about 50%, and converted polarized uterine epithelia to a functionally receptive state. Similarly, ablation of Muc-1 in Muc-1 null mice resulted in polarized uterine epithelia that were functionally receptive as compared to their wild-type counterparts in vitro. Collectively, these data indicate that Muc-1 and other mucins function as anti-adhesive molecules and that reduction or removal of these molecules is a prerequisite for the generation of a receptive uterine state. ^

A study of the biology of perlecan

Extracellular matrix (ECM) is a component of a variety of organisms that provides both structural support and influence upon the cells it surrounds. The importance of the ECM is becoming more apparent as matrix defects are linked to human disease. In this study, the large, extracellular matrix heparan sulfate proteoglycan, perlecan (Pln) is examined in two systems. First, the role of Pln in the interaction between a blastocyst and uterine epithelial cells is investigated. In mice, blastocyst attachment and implantation occurs at approximately d 4.5 post coitus. In addition, a delayed implantation model has been used to distinguish between the response of the blastocyst to that of hatching and of becoming attachment competent. ^ The second series of experiments described in this study focuses on the process of chondrogenesis in mice. Pln, commonly expressed with other basement membrane (BM) proteins, was found to be expressed in cartilaginous tissue without other BM proteins. This unusual expression pattern led to further study and the development of an in vitro chondrogenesis assay using the mouse embryonic fibroblast cell line, C3H/10T1/2. When cultured on Pln in vitro, these cells form aggregates and express the cartilage proteins, collagen type II and aggrecan. In examining the participation of the heparan sulfate (HS) chains in this process, the proteoglycan was enzymatically digested to remove the HS chains before the initiation of 10T1/2 cell culture. After digestion, the ability of Pln to stimulate aggregate formation was greatly diminished. Thus, the HS chains participate in the cell induction process. To determine which domain of Pln might be responsible for this activity, recombinant fragments of Pin were used in the cell culture assay. Of all recombinant protein fragments tested, only the domain including the HS chains, domain 1, was able to initiate the morphological change exhibited by the 10T1/2 cells. Similar to native Pln, when HS chains were removed from domain I, chondrogenic activity was abolished. A variant of domain I carrying both HS and chondroitin sulfate (CS) chains retained activity when only HS chains were removed. When both HS and CS chains were removed, then activity was lost. ^ The ability to rapidly stimulate differentiation of 10T1/2 cells in vitro may lead to better control of chondrogenesis in vitro and in vivo, providing better understanding and manipulation of the chondrogenic process. This greater understanding may have benefits for study of cartilage and bone diseases and subsequent treatment options. (Abstract shortened by UMI.)^

The Differential Requirement of Albumin and Sodium Citrate on the Development of In Vitro Produced Bovine Embryos

In vitro culture for bovine embryos is largely not optimal. Our study was to determine the components necessary for early embryo development. In experiment 1, IVF embryos were cultured for two days in CR1aa medium containing sodium citrate and BSA from two sources (Sigma vs. ICPbio), subsequently for additional five days with cumulus monolayer in 10% FBS CR1aa. We found that supplementation with both Sigma-BSA and sodium citrate significantly increased total blastocyst (BL) development compared with the ICPbio-BSA groups (37% vs. 19-21%), and enhanced the total number of high quality (C1 BL, IETS standard) blastocysts (26% vs. 11-17%) (P < 0.05). In experiment 2 with serum free and/or somatic free culture, we found that CR1aa culture can support a comparable embryo development with a supplement of Sigma BSA. The addition of sodium citrate did not increase blastocyst development in either the Sigma-BSA or the ICPbio-BSA groups. An inferior blastocyst development occurring in ICPbio-BSA culture (1-3%) could be rescued by culture in CRlaa supplemented with 10% FBS (29%), more importantly, by culture in CR1aa with a replacement of Sigma BSA (24%) (P <0.05). C1 blastocysts rescued by FBS and Sigma BSA in ICPbio-BSA culture possessed indistinguishable morphology to embryos developed in a Sigma-BSA, FBS and somatic co-culture system, showing similar cell number/blastocyst (129-180, P > 0.05). Our study found a beneficial effect of sodium citrate and BSA on the in vitro development of bovine IVF embryos during co-culture. We also determined that differential embryotrophic factor(s) contained in BSA and serum, probably not sodium citrate, is necessary for promoting competent morula and blastocyst development in cattle.

Differential Cytoplast Requirement for Embryonic and Somatic Cell Nuclear Transfer in Cattle

Effective activation of a recipient oocyte and its compatibility with the nuclear donor are critical to the successful nuclear reprogramming during nuclear transfer. We designed a series of experiments using various activation methods to determine the optimum activation efficiency of bovine oocytes. We then performed nuclear transfer (NT) of embryonic and somatic cells into cytoplasts presumably at G1/S phase (with prior activation) or at metaphase II (MII, without prior activation). Oocytes at 24 hr of maturation in vitro were activated with various combinations of calcium ionophore A23187 (A187) (5 microM, 5 min), electric pulse (EP), ethanol (7%, 7 min), cycloheximide (CHX) (10 micro g/ml, 6 hr), and then cultured in cytochalasin D (CD) for a total of 18 hr. Through a series of experiments (Exp. 1-4), an improved activation protocol (A187/EP/CHX/CD) was identified and used for comparison of NT efficiency of embryonic versus somatic donor cells (Exp. 5). When embryonic cells from morula and blastocysts (BL) were used as nuclear donors, a significantly higher rate of blastocyst development from cloned embryos was obtained with G1/S phase cytoplasts than with MII-phase cytoplasts (36 vs. 11%, P < 0.05). In contrast, when skin fibroblasts were used as donor cells, the use of an MII cytoplast (vs. G1/S phase) was imperative for blastocyst development (30 vs. 6%, P < 0.05). Differential staining showed that parthenogenetic, embryonic, and somatic cloned BL contained 26, 29, and 33% presumptive inner cell mass (ICM) cells, respectively, which is similar to that of frozen-thawed in vivo embryos at a comparable developmental stage (23%). These data indicate that embryonic and somatic nuclei require different recipient cytoplast environment for remodeling/ reprogramming, and this is likely due to the different cell cycle stage and profiles of molecular differentiation of the transferred donor nuclei.

Maternal ingestion of radon-222 in drinking water and the absorbed radiation dose to the embryo

Maternal ingestion of high concentrations of radon-222 (Rn-222) in drinking during pregnancy may pose a significant radiation hazard to the developing embryo. The effects of ionizing radiation to the embryo and fetus have been the subject of research, analyses, and the development of a number of radiation dosimetric models for a variety of radionuclides. Currently, essentially all of the biokinetic and dosimetric models that have been developed by national and international radiation protection agencies and organizations recommend calculating the dose to the mother's uterus as a surrogate for estimating the dose to the embryo. Heretofore, the traditional radiation dosimetry models have neither considered the embryo a distinct and rapidly developing entity, the fact that it is implanted in the endometrial layer of the uterus, nor the physiological interchanges that take place between maternal and embryonic cells following the implantation of the blastocyst in the endometrium. The purpose of this research was to propose a new approach and mathematical model for calculating the absorbed radiation dose to the embryo by utilizing a semiclassical treatment of alpha particle decay and subsequent scattering of energy deposition in uterine and embryonic tissue. The new approach and model were compared and contrasted with the currently recommended biokinetic and dosimetric models for estimating the radiation dose to the embryo. The results obtained in this research demonstrate that the estimated absorbed dose for an embryo implanted in the endometrial layer of the uterus during the fifth week of embryonic development is greater than the estimated absorbed dose for an embryo implanted in the uterine muscle on the last day of the eighth week of gestation. This research provides compelling evidence that the recommended methodologies and dosimetric models of the Nuclear Regulatory Commission and International Commission on Radiological Protection employed for calculating the radiation dose to the embryo from maternal intakes of radionuclides, including maternal ingestion of Rn-222 in drinking water would result in an underestimation of dose. ^

RAG2 is regulated differentially in B and T cells by elements 5′ of the promoter

To study RAG2 gene regulation in vivo, we developed a blastocyst complementation method in which RAG2-deficient embryonic stem cells were transfected with genomic clones containing RAG2 and then assessed for their ability to generate lymphocytes. A RAG2 genomic clone that contained only the RAG2 promoter sequences rescued V(D)J recombination in RAG2-deficient pro-B cell lines, but did not rescue development of RAG2-deficient lymphocytes in vivo. However, inclusion of varying lengths of sequences 5′ of the RAG2 promoter generated constructs capable of rescuing only in vivo B cell development, as well as other constructs that rescued both B and T cell development. In particular, the 2-kb 5′ region starting just upstream of the RAG2 promoter, as well as the region from 2–7 kb 5′, could independently drive B cell development, but not efficient T cell development. Deletion of the 2-kb 5′ region from the murine germ line demonstrated that this region was not required for RAG expression sufficient to generate normal B or T cell numbers, implying redundancy among 5′ elements. We conclude that RAG2 expression in vivo requires elements beyond the core promoter, that such elements contribute to differential regulation in the B vs. T lineages, and that sequences sufficient to direct B cell expression are located in the promoter-proximal 5′ region.

Production of identical sextuplet mice by transferring metaphase nuclei from four-cell embryos

Mouse clones were produced by serial nuclear transfer commencing with the transfer of four-cell nuclei at metaphase into unfertilized ooplasts. The donor four-cell-stage nuclei were synchronized in metaphase with nocodazole. The oocytes receiving a four-cell nucleus at metaphase formed two nuclei after artificial activation and inhibition of cytokinesis with cytochalasin B. To obtain embryos with diploid sets of chromosomes, nuclei from each reconstructed embryo were transferred individually into separate enucleated fertilized one-cell embryos, thus doubling the number of identical embryos. This procedure produced a high frequency of development of reconstructed embryos to the blastocyst stage. Of 11 sets of identical embryos produced by serial nuclear transplantation, 83% developed into blastocysts, including three sets of identical septuplet blastocysts. After transfer to recipient mice, a total of 25 (57%) live young were obtained, which included one set of identical sextuplet and two sets of identical quadruplet mice.