Andrew J. Watson

Impact of Bovine Oocyte Maturation Media on Oocyte Transcript Levels, Blastocyst Development, Cell Number, and Apoptosis

Abstract The objectives were 1) to investigate the effects of oocyte maturation in serum-free and amino acid-supplemented defined media on oocyte transcript levels, blastocyst cell number, and apoptosis; 2) to investigate the influence of oocyte maturation culture atmosphere on blastocyst development, total cell number, and apoptosis; and 3) to examine the influence of epidermal growth factor (EGF) during oocyte maturation on blastocyst cell number and apoptosis. The results demonstrate that blastocysts derived from in vitro maturation, fertilization, and embryo culture protocols undergo apoptosis but that apoptotic levels are not greatly influenced by the oocyte maturation environment. Amino acid supplementation of oocyte maturation media was associated with enhanced developmental frequencies, increased blastocyst cell number, and elevated oocyte maternal mRNA levels. Oocyte maturation with supplemented synthetic oviduct fluid medium (cSOFMaa) resulted in blastocyst cell numbers comparable to those observed with Tissue Culture Medium 199 + newborn calf serum. Blastocyst development was reduced following oocyte maturation under a 5% CO2, 7% O2, 88% N2 culture atmosphere. EGF supplementation of oocyte maturation medium resulted in a concentration-dependent increase in blastocyst development but did not influence blastocyst total cell number or apoptosis. Our findings indicate that cSOFMaa medium is an effective base medium for bovine oocyte maturation.

Aquaporin proteins in murine trophectoderm mediate transepithelial water movements during cavitation.

Mammalian blastocyst formation is dependent on establishment of trophectoderm (TE) ion and fluid transport mechanisms. We have examined the expression and function of aquaporin (AQP) water channels during murine preimplantation development. AQP 3, 8, and 9 proteins demonstrated cell margin-associated staining starting at the 8-cell (AQP 9) or compacted morula (AQP 3 and 8) stages. In blastocysts, AQP 3 and 8 were detected in the basolateral membrane domains of the trophectoderm, while AQP3 was also observed in cell margins of all inner cell mass (ICM) cells. In contrast, AQP 9 was predominantly observed within the apical membrane domains of the TE. Murine blastocysts exposed to hyperosmotic culture media (1800 mOsm; 10% glycerol) demonstrated a rapid volume decrease followed by recovery to approximately 80% of initial volume over 5 min. Treatment of blastocysts with p-chloromercuriphenylsulfonic acid (pCMPS, > or =100 microM) for 5 min significantly impaired (P < 0.05) volume recovery, indicating the involvement of AQPs in fluid transport across the TE. Blastocysts exposure to an 1800-mOsm sucrose/KSOMaa solution did not demonstrate volume recovery as observed following treatment with glycerol containing medium, indicating glycerol permeability via AQPs 3 and 9. These findings support the hypothesis that aquaporins mediate trans-trophectodermal water movements during cavitation.

Preimplantation embryo programming: transcription, epigenetics, and culture environment

Preimplantation development directs the formation of an implantation- or attachment-competent embryo so that metabolic interactions with the uterus can occur, pregnancy can be initiated, and fetal development can be sustained. The preimplantation embryo exhibits a form of autonomous development fueled by products provided by the oocyte and also from activation of the embryos genome. Despite this autonomy, the preimplantation embryo is highly influenced by factors in the external environment and in extreme situations, such as those presented by embryo culture or nuclear transfer, the ability of the embryo to adapt to the changing environmental conditions or chromatin to become reprogrammed can exceed its own adaptive capacity, resulting in aberrant embryonic development. Nuclear transfer or embryo culture-induced influences not only affect implantation and establishment of pregnancy but also can extend to fetal and postnatal development and affect susceptibility to disease in later life. It is therefore critical to define the basic program controlling preimplantation development, and also to utilize nuclear transfer and embryo culture models so that we may design healthier environments for preimplantation embryos to thrive in and also minimize the potential for negative consequences during pregnancy and post-gestational life. In addition, it is necessary to couple gene expression analysis with the investigation of gene function so that effects on gene expression can be fully understood. The purpose of this short review is to highlight our knowledge of the mechanisms controlling preimplantation development and report how those mechanisms may be influenced by nuclear transfer and embryo culture.

Temporal patterns of embryonic gene expression and their dependence on oogenetic factors

Successful development of a fertilized egg beyond early cleavage divisions requires the de novo initiation and subsequent regulation of embryonic transcription. The egg provides the specialized environment within which the newly formed zygotic nucleus initiates its developmental program and as a result plays an obligatory role in its regulation. Although the precise timing of the onset of embryonic transcription in mammals varies during early cleavage divisions, several common elements exist. In the present essay we review the current literature on the timing and control of embryonic gene expression in mammals, and discuss recent findings from our laboratory on gene expression patterns in bovine embryos and their relation to other species, and zygotic gene activation (ZGA). Lastly, we discuss the putative role of maternally inherited factors in conferring developmental competence to the blastocyst stage, and a method to identify such factors present in oocytes as mRNA.

Na/K-ATPase β1 Subunit Expression Is Required for Blastocyst Formation and Normal Assembly of Trophectoderm Tight Junction-associated Proteins

Na/K-ATPase plays an important role in mediating blastocyst formation. Despite the expression of multiple Na/K-ATPase α and β isoforms during mouse preimplantation development, only the α1 and β1 isoforms have been localized to the basolateral membrane regions of the trophectoderm. The aim of the present study was to selectively down-regulate the Na/K-ATPase β1 subunit employing microinjection of mouse 1 cell zygotes with small interfering RNA (siRNA) oligos. Experiments comprised of non-injected controls and two groups microinjected with either Stealth™ Na/K-ATPase β1 subunit oligos or nonspecific Stealth™ siRNA as control. Development to the 2-, 4-, 8-, and 16-cell and morula stages did not vary between the three groups. However, only 2.3% of the embryos microinjected with Na/K-ATPase β1 subunit siRNA oligos developed to the blastocyst stage as compared with 73% for control-injected and 91% for non-injected controls. Na/K-ATPase β1 subunit down-regulation was validated by employing reverse transcription-PCR and whole-mount immunofluorescence methods to demonstrate that Na/K-ATPase β1 subunit mRNAs and protein were not detectable in β1 subunit siRNA-microinjected embryos. Aggregation chimera experiments between β1 subunit siRNA-microinjected embryos and controls demonstrated that blockade of blastocyst formation was reversible. The distribution of Na/K-ATPase α1 and tight junction-associated proteins occludin and ZO-1 were compared among the three treatment groups. No differences in protein distribution were observed between control groups; however, all three polypeptides displayed an aberrant distribution in Na/K-ATPase β1 subunit siRNA-microinjected embryos. Our results demonstrate that the β1 subunit of the Na/K-ATPase is required for blastocyst formation and that this subunit is also required to maintain a normal Na/K-ATPase distribution and localization of tight junction-associated polypeptides during preimplantation development.

A null mutation for tissue inhibitor of metalloproteinases-3 (Timp-3) impairs murine bronchiole branching morphogenesis.

Tissue inhibitors of metalloproteinases (TIMPs) regulate extracellular matrix (ECM) degradation by matrix metalloproteinases (MMPs). We have examined the role of TIMP-3 on ECM homeostasis and bronchiole branching morphogenesis during murine embryogenesis. Employing an in vitro organ culture system, we found decreased bronchiolar branching in null lungs when compared with wild type (WT) counterparts after 2 days in culture. When a synthetic inhibitor of MMPs at low dose was added to the culture system, branching was augmented regardless of genotype. Gelatin and in situ zymography revealed that null lungs exhibited enhanced activation of MMPs throughout lung development. We analysed the impact of increased MMP activity on a number of ECM molecules by Western blot analysis, but found that only fibronectin abundance was consistently reduced in the null lungs throughout development. To confirm that our observed defect in culture was not simply a developmental delay in the null lung, we examined null and WT lungs from newborn pups. Here, we found not only a reduced number of bronchioles in the null, but also that the bronchiole tubes were dilated compared with controls and that alveologenesis was attenuated. We propose that the deletion of TIMP-3 disrupts the exquisite TIMP/MMP balance required for proper focal ECM proteolysis, which leads to correct bronchiole branching morphogenesis in the developing mouse lung.

Analysis of variation in relative mRNA abundance for specific gene transcripts in single bovine oocytes and early embryos.

Variation in the abundance of a specific gene transcript was assessed in single bovine oocytes and in vitro–derived blastocysts. Transcripts encoding the Na+,K+‐ATPase α1 subunit were detected by reverse‐transcription polymerase chain reaction (RT‐PCR) and quantified relative to an exogenously supplied rabbit α‐globin mRNA using laser‐induced fluorescence capillary electrophoresis (LIF‐CE). The precision of this relative abundance (RA) calculation was predicted and shown to resolve 2‐fold differences in transcript abundance between individual blastocysts and predicted in oocytes to resolve 3‐fold differences. The RA of the α1 subunit transcript differed by 2‐ to 3‐fold among blastocysts, and 3‐ to 6‐fold among oocytes. Comparison of a general population of oocytes with blastocysts revealed little overlap in RA values between the two groups, with a 8‐ to 14‐fold increase in the mean RA for each group with development observed in two successive experiments (P ≤ 0.05). In contrast, oocytes selected for their developmental competence on the basis of morphologic criteria exhibited only a 1.6‐ to 1.7‐fold developmental increase when the assay was performed on cDNA generated from either embryo pools (n = 6 versus 6) or individuals (n = 7 versus 7), respectively. These results provide the first characterization of the degree of heterogeneity in the abundance of a specific mRNA transcript among individual mammalian oocytes and preimplantation embryos and demonstrate that transcript relative abundance can be correlated with bovine oocyte morphology. Mol. Reprod. Dev. 49:119–130, 1998.

Cyclooxygenase-2 and Prostaglandin E2(PGE2) Receptor Messenger RNAs Are Affected by Bovine Oocyte Maturation Time and Cumulus-Oocyte Complex Quality, and PGE2 Induces Moderate Expansion of the Bovine Cumulus In Vitro

Abstract Expression of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) receptor 2 (EP2) are necessary for rodent cumulus expansion in vivo. Prostaglandin E2 receptor 3 (EP3) has been detected in bovine preovulatory follicles and corpora lutea. The current experiments examined the effect of PGE2 on bovine cumulus expansion in vitro and expression of COX-2, EP1, EP2, EP3, and EP4 mRNAs in bovine cumulus-oocyte complexes (COCs) at 0, 6, 12, 18, and 24 h time points during maturation in vitro. Concentrations of PGE2 above 50 ng/ml resulted in moderate cumulus expansion of bovine COCs, but expansion did not occur in the absence of serum. COX-2 mRNA expression increased in bovine COCs at 6 h and 12 h of maturation, then decreased. EP2 mRNA was detectable by reverse transcription-polymerase chain reaction at all time points. EP3 mRNA expression increased in COCs from 0 to 6 h and remained at this higher level through the culture period. Very low levels of EP4 mRNA expression were detectable, but EP1 was not detected in bovine COCs. Because EP receptor mRNAs and COX-2 mRNA are expressed in bovine COCs, there exists the potential for a prostaglandin autocrine/paracrine regulatory pathway during oocyte maturation. Differential expression of the EP3 mRNA among varying COC classes indicates that this gene product may be a useful marker of oocyte competence. Although the PGE2 pathway is involved in cumulus expansion, serum factors are required to mediate PGE2-induced expansion.

Deletion of the Na/K-ATPase alpha1-subunit gene (Atp1a1) does not prevent cavitation of the preimplantation mouse embryo.

Increases in Na/K-ATPase activity occur concurrently with the onset of cavitation and are associated with increases in Na(+)-pump subunit mRNA and protein expression. We have hypothesized that the alpha1-isozyme of the Na/K-ATPase is required to mediate blastocyst formation. We have tested this hypothesis by characterizing preimplantation development in mice with a targeted disruption of the Na/K-ATPase alpha1-subunit (Atp1a1) using embryos acquired from matings between Atp1a1 heterozygous mice. Mouse embryos homozygous for a null mutation in the Na/K-ATPase alpha1-subunit gene are able to undergo compaction and cavitation. These findings demonstrate that trophectoderm transport mechanisms are maintained in the absence of the predominant isozyme of the Na(+)-pump that has previously been localized to the basolateral membranes of mammalian trophectoderm cells. The presence of multiple isoforms of Na/K-ATPase alpha- and beta-subunits at the time of cavitation suggests that there may be a degree of genetic redundancy amongst isoforms of the catalytic alpha-subunit that allows blastocyst formation to progress in the absence of the alpha1-subunit.

Responsiveness of bovine cumulus-oocyte-complexes (COC) to porcine and recombinant human FSH, and the effect of COC quality on gonadotropin receptor and Cx43 marker gene mRNAs during maturation in vitro

Substantially less development to the blastocyst stage occurs in vitro than in vivo and this may be due to deficiencies in oocyte competence. Although a large proportion of bovine oocytes undergo spontaneous nuclear maturation, less is known about requirements for proper cytoplasmic maturation. Commonly, supraphysiological concentrations of FSH and LH are added to maturation media to improve cumulus expansion, fertilization and embryonic development. Therefore, various concentrations of porcine FSH (pFSH) and recombinant human FSH (rhFSH) were investigated for their effect on bovine cumulus expansion in vitro. Expression of FSHr, LHr and Cx43 mRNAs was determined in cumulus-oocyte complexes to determine whether they would be useful markers of oocyte competence. In serum-free media, only 1000 ng/ml pFSH induced marked cumulus expansion, but the effect of 100 ng/ml pFSH was amplified in the presence of 10% serum. In contrast, cumulus expansion occurred with 1 ng/ml rhFSH in the absence of serum. FSHr mRNA was highest at 0–6 h of maturation, then abundance decreased. Similarly, Cx43 mRNA expression was highest from 0–6 h but decreased by 24 h of maturation. However, the relative abundance of LHr mRNA did not change from 6–24 h of maturation. Decreased levels of FSHr, LHr and Cx43 mRNAs were detected in COCs of poorer quality. In conclusion, expansion of bovine cumulus occurred at low doses of rhFSH in serum-free media. In summary, FSHr, LHr and Cx43 mRNA abundance reflects COC quality and FSHr and Cx43 mRNA expression changes during in vitro maturation; these genes may be useful markers of oocyte developmental competence.