Spherical Blastocysts Research Articles

Single-cell transcriptomic characterization of sheep conceptus elongation and implantation

Bidirectional communication between the developing conceptus and endometrium is essential for pregnancy recognition and establishment in ruminants. We dissect the transcriptomic dynamics of sheep conceptus and corresponding endometrium at pre- and peri-implantation stages using single-cell RNA sequencing. Spherical blastocysts contain five cell types, with 68.62% trophectoderm cells. Strikingly, elongated conceptuses differentiate into 17 cell types, indicating dramatic cell fate specifications. Cell-type-specific gene expression delineates the features of distinctive trophectoderm lineages and indicates that the transition from polar trophectoderm to trophoblast increases interferon-tau expression and likely drives elongation initiation. We identify 13 endometrium-derived cell types and elucidate their molecular responses to conceptus development. Integrated analyses uncover multiple paired transcripts mediating the dialogues between extraembryonic membrane and endometrium, including IGF2-IGF1R, FGF19-FGFR1, NPY-NPY1R, PROS1-AXL, and ADGRE5-CD55. These data provide insight into the molecular regulation of conceptus elongation and represent a valuable resource for functional investigations of pre- and peri-implantation ruminant development.

78 Effects of invitro production on the epigenome and transcriptome of bovine embryos determined through a multi-omics data integration approach

In cattle, ovarian superovulation followed by invivo embryo collection and transfer (MOET), and the invitro production (IVP) of embryos are used all over the world to improve animal genetics. Application of MOET has resulted in the production of billions of healthy animals during the past 40 years, and IVP has evolved and given rise to significant numbers of calves during the past 10 years. Nevertheless, the use of MOET and IVP can affect the embryo epigenome, and therefore its transcriptome, before and after elongation, as shown by different studies. The integration of publicly available epigenome-transcriptome datasets generated by these studies could lead to a robust characterisation of the impacts of the application of MOET and IVP. The goal of this study was to integrate all publicly available data about MOET and IVP embryos to determine temporally differentially methylated regions (DMRs) and differentially expressed genes (DEGs) from blastocyst to elongation between IVP and MOET embryos. Datasets were downloaded from the Gene Expression Omnibus (GEO) database. Accession numbers were (1) for epigenomics: GSE69173, GSE97517, and GSE101895, plus one provided dataset from O’Doherty et al. (2018 BMC Genomics, 19, 438; https://doi.org/10.1186/s12864-018-4818-3), all hybridized to the EDMA platform GPL18384; (2) for transcriptomics: GSE12327, GSE21030, GSE24596, GSE24936, GSE27817, and GSE40101, all hybridized to the Affymetrix platform GPL2112. Both types of data were analysed with the limma package for R software, and functional enrichment analysis was done with the DAVID database. For DMRs, comparisons between IVP and MOET were made from spherical blastocysts (n=16 per group) on Day 7, to embryos on Day 15, specifically in the trophectoderm (TE) or embryonic disc (ED) regions (n=4 per region and per group). For DEGs, comparisons between IVP and MOET were made from spherical blastocysts (n=9 per group) to elongated blastocysts on Day 13 and embryos undergoing gastrulation on Day 16 (n=6 per group). Considering a P-value <0.05 and fold-change >2, there were 16 672 (TE) and 26 264 (ED) DMRs and 2236 DEGs that temporally differed between IVP and MOET. Most of the identified DMRs were found in intronic regions (around 36%) rather than exonic regions (8%). However, DMRs that were more methylated at IVP compared with MOET contained exons encoding for genes that enriched the Wnt signalling Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway in the ED, and focal adhesion and ECM-receptor interaction KEGG pathways (P<0.05) in the TE. Accordingly, DEGs with lower expression in elongated embryos (Day 13 and Day 16) at IVP as opposed to MOET were mainly associated with these three pathways. In conclusion, this multi-omics analysis demonstrates that even when embryos are produced under different conditions and experiments, the main changes imposed by IVP affected genes involved in embryonic development and adhesion to the endometrium, which could explain the lower survival rates at IVP compared with MOET.

Functional role of arginine during the peri-implantation period of pregnancy. II. Consequences of loss of function of nitric oxide synthase NOS3 mRNA in ovine conceptus trophectoderm.

Nitric oxide (NO) is a gaseous molecule that regulates angiogenesis and vasodilation via activation of the cGMP pathway. However, functional roles of NO during embryonic development from spherical blastocysts to elongated filamentous conceptuses (embryo and extraembryonic membrane) during the peri-implantation period of pregnancy have not been elucidated in vivo. In order to assess roles of NO production in survival and development of the ovine conceptus, we conducted an in vivo morpholino antisense oligonucleotide (MAO)-mediated knockdown trial of nitric oxide synthase-3 (NOS3) mRNA, the major isoform of NO synthase, in ovine conceptus trophectoderm (Tr). Translational knockdown of NOS3 mRNA results in small, thin, and underdeveloped conceptuses, but normal production of interferon-tau, the pregnancy recognition signal in sheep. MAO-NOS3 knockdown in conceptuses decreased the abundance of NOS3 (72%, P < 0.05) and the arginine transporter SLC7A1 proteins in conceptus Tr. Furthermore, the amounts of ornithine and polyamines were less (P < 0.01) in uterine fluid, whereas the amounts of arginine (58%, P < 0.01), citrulline (68%, P < 0.05), ornithine (68%, P < 0.001), glutamine (78%, P < 0.001), glutamate (68%, P < 0.05), and polyamines (P < 0.01) were less in conceptuses, which likely accounts for the failure of MAO-NOS3 conceptuses to develop normally. For MAO-NOS3 conceptuses, there were no compensatory increases in the expression levels of either nitric oxide synthase-1 (NOS1) or nitric oxide synthase-2 (NOS2) or in expression of enzymes for synthesis of polyamines (ornithine decarboxylase, arginine decarboxylase, agmatinase) from arginine or ornithine with which to rescue development of MAO-NOS3 conceptuses. Thus, the adverse effect of MAO-NOS3 to reduce NO generation and the transport of arginine and ornithine into conceptuses is central to an explanation for failure of normal development of MAO-NOS3, compared to control conceptuses. The study, for the first time, created an NO-deficient mammalian conceptus model in vivo and provided new insights into the orchestrated events of conceptus development during the peri-implantation period of pregnancy. Our data suggest that NOS3 is the key enzyme for NO production by conceptus Tr and that this protein also regulates the availability of arginine in conceptus tissues for synthesis of polyamines that are essential for conceptus survival and development.

Transcriptome Changes at the Initiation of Elongation in the Bovine Conceptus1

The majority of embryonic loss in cattle occurs before maternal recognition of pregnancy, at around Day 16 postconception. The origin of the embryo can have a significant impact on the dynamics of embryo mortality. The aim of this study was to examine the temporal changes in transcriptional profile as the embryo develops from a spherical blastocyst on Day 7 to an ovoid conceptus at the initiation of elongation on Day 13 and to highlight differences in these temporal gene expression dynamics between in vivo- and in vitro-derived blastocysts that may be associated with embryonic survival/mortality using the bovine Affymetrix microarray. All embryos were produced either in vitro by in vitro fertilization or in vivo by superovulation. A proportion of Day 7 blastocysts were snap frozen, and the remainder were transferred (n = 10 per recipient) to synchronized heifers, recovered on Day 13, and snap frozen individually. Three pools of Day 7 blastocysts (n = 25 per pool) and Day 13 conceptuses (n = 5 per pool) were used for microarray analysis. In Day 7 blastocysts, 50 genes were found to be differentially expressed (P < 0.05), of which 19 were up-regulated and 31 down-regulated in the in vivo compared to in vitro embryos. In Day 13 conceptuses, 288 genes were found to be differentially expressed (P < 0.05), of which 133 were up-regulated and 155 down-regulated in the in vivo compared to in vitro embryos. The comparison between Day 7 and Day 13 embryos revealed significant temporal changes in transcript profile with 1806 and 909 transcripts differentially expressed in the in vitro- and in vivo-derived embryos, respectively. Across the three array comparisons between Day 7 and Day 13 embryos, 444 genes were consistently exclusively present in the in vivo embryos, whereas 1341 were exclusively present in the in vitro embryos. Regardless of the origin of the embryo, 465 differentially expressed genes between Day 7 and 13 were common to both in vivo- and in vitro-derived embryos; these genes are likely critical for the transition between the blastocyst (Day 7) and ovoid conceptus (Day 13) stages of embryo development. In order to validate the microarray findings, differences in the expression of six genes (CYP51A1, FADS1, TDGF1, HABP2, APOA2, and SLC12A2) were confirmed by quantitative real-time PCR on in vivo- and in vitro-derived embryos on Day 7 and Day 13 using independent samples from those used for the microarray. Subsequent mapping of these differentially expressed genes into relevant functional groups and pathways identified important pathways involved in conceptus elongation in cattle. In conclusion, this analysis has identified genes and pathways crucial for the transition from a spherical blastocyst to an ovoid conceptus as well as those uniquely associated with a greater likelihood of embryonic survival (those unique to in vivo embryos) or loss (those unique to in vitro embryos).

TopBP1 Deficiency Causes an Early Embryonic Lethality and Induces Cellular Senescence in Primary Cells

TopBP1 plays important roles in chromosome replication, DNA damage response, and other cellular regulatory functions in vertebrates. Although the roles of TopBP1 have been studied mostly in cancer cell lines, its physiological function remains unclear in mice and untransformed cells. We generated conditional knock-out mice in which exons 5 and 6 of the TopBP1 gene are flanked by loxP sequences. Although TopBP1-deficient embryos developed to the blastocyst stage, no homozygous mutant embryos were recovered at E8.5 or beyond, and completely resorbed embryos were frequent at E7.5, indicating that mutant embryos tend to die at the peri-implantation stage. This finding indicated that TopBP1 is essential for cell proliferation during early embryogenesis. Ablation of TopBP1 in TopBP1(flox/flox) mouse embryonic fibroblasts and 3T3 cells using Cre recombinase-expressing retrovirus arrests cell cycle progression at the G(1), S, and G(2)/M phases. The TopBP1-ablated mouse cells exhibit phosphorylation of H2AX and Chk2, indicating that the cells contain DNA breaks. The TopBP1-ablated mouse cells enter cellular senescence. Although RNA interference-mediated knockdown of TopBP1 induced cellular senescence in human primary cells, it induced apoptosis in cancer cells. Therefore, TopBP1 deficiency in untransformed mouse and human primary cells induces cellular senescence rather than apoptosis. These results indicate that TopBP1 is essential for cell proliferation and maintenance of chromosomal integrity.

200 IN VITRO EMBRYO PRODUCTION IS ASSOCIATED WITH DISTINCT ALTERATIONS IN THE TRANSCRIPTOME BETWEEN THE BLASTOCYST STAGE AND THE INITIATION OF ELONGATION IN CATTLE

The majority of embryonic loss in cattle occurs before maternal recognition of pregnancy around Day 16 postconception. The origin of the embryo can have a significant impact on the dynamics of embryo mortality. The aim of the present study was to compare transcript profiles between Day 7 (spherical blastocyst) and Day 13 (ovoid conceptus) bovine embryos derived in vitro or in vivo using the bovine Affymetrix microarray. Subsequent mapping of these differentially expressed genes (DEG) into relevant functional groups and pathways using ingenuity pathway analysis would identify the most important pathways involved in conceptus elongation in cattle. All embryos were produced either in vitro or in vivo by superovulation. A proportion of Day 7 blastocysts were snap frozen and the remainder were transferred (n = 10 per recipient) to synchronized heifers, recovered on Day 13, and snap frozen individually. Three pools of Day 7 blastocysts (n = 25 per pool) and Day 13 conceptuses (n = 5 per pool) were used for microarray analysis. A total of 909 and 1806 transcripts were differentially expressed between Day 7 and 13 in in vivo- and in vitro-derived embryos, respectively, of which a core of 465 transcripts was common to both groups. These 465 genes, likely to be crucial for the transition from blastocyst to the initiation of elongation, were associated with 5 canonical pathways, including interleukin (IL)-6 and IL-10 signalling, the endoplasmic reticulum stress pathway, and nitrogen metabolism. The top networks identified genes associated with 1) cellular development, lipid metabolism, and small molecular biochemistry; 2) cell-to-cell signaling; and 3) amino acid metabolism, including transcripts such as MYC, SLC25A12, HSPH1, LXN, ALDH18A1, PMP22, PEG3, CDH2, and Hsp70, which were up-regulated in the conceptus on Day 13. The top canonical pathways among the 444 DEG unique to in vivo embryos were 1) glycerolipid metabolism, 2) steroid biosynthesis, and 3) acute phase response signalling. One of the interaction networks identified was associated with cardiovascular system development and function, gene expression, and organismal development, including transcripts of proteins such as laminins and claudins, which are important for cell communication and morphogenesis during embryonic development. Among the 1341 DEG unique to in vitro-produced embryos, the top pathways were 1) glycerophospholipid metabolism, 2) IL-6 signalling, and 3) endothelial-1 signalling, whereas one of the interaction networks identified was associated with hematological disease, organismal injury, and abnormalities and cellular development, including POU5F1, a transcription factor known to bind to DNA and activate or repress transcription of several genes important for early embryonic development. In conclusion, this analysis has identified genes and pathways crucial to the transition from a spherical blastocyst to an ovoid conceptus as well as those uniquely associated with a greater likelihood of embryonic survival (those unique to in vivo embryos) or loss (those unique to in vitro embryos).

Three‐dimensional localisation of NANOG, OCT4, and E‐cadherin in porcine pre‐ and peri‐implantation embryos

The expression patterns of NANOG and OCT4 have previously been reported to differ markedly between mammalian species indicating distinct species-specific roles during development. We investigate the three-dimensional expression pattern of NANOG and OCT4 in porcine pre- and peri-implantation embryos. The expression of NANOG differed remarkably from that reported in other species. NANOG was not detected in the inner cell mass of hatched porcine blastocysts, but later appeared in the epiblast and hypoblast of spherical blastocysts where Rauber's layer had disintegrated. In pre-gastrulating, filamentous embryos NANOG was localised to nuclei in a minor portion of the epiblast cells in which E-CADHERIN seemed to be up-regulated and OCT4 down-regulated. Later NANOG was restricted to the potential PGCs. OCT4 was detected in inner cell mass, epiblast, and mesoderm, and we found that OCT4 expression, in contrast to earlier speculations, at least in hatched blastocysts, resembles the expression pattern in the mouse embryo.

Production of Retinol-binding Protein by Caprine Conceptus during the Time Period of Maternal Recognition of Pregnancy

The purpose of the study were to characterize the proteins secreted by elongating caprine conceptus, to identify a group of low molecular weight proteins as retinol-binding protein (RBP), to identify RBP cell-specific localization in conceptus tissue, and to demonstrate that the conceptuses secreted continuously RBP during the time period maternal recognition of pregnancy. Caprine conceptuses were removed from the uterus between days 16 and 22 of pregnancy, the time period maternal recognition of pregnancy. Isolated conceptuses were cultured in a modified minimum essential medium in the presence of radiolabeled amino acids. Proteins synthesized and secreted into medium were analyzed by fluorography of two-dimensional polyacrylamide gel electrophoresis and fluorography. At least five proteins showed consistently a grouping of spots with characteristic location on two-dimensional gels. A major low molecular weight protein consisted of two major isoforms (pI 5.3-6.0) of similar molecular mass (21 kDa) was identified as RBP by using antiserum against RBP. Presence of RBP in conceptus culture medium and uterine flushings between days 16 and 22 of pregnancy were determined by immunoprecipitation and Western blotting using anti-RBP serum. In immunocytochemical study, strong immunostaining for RBP was localized in trophectoderm and endoderm of conceptus. These results clearly demonstrated that the caprine conceptus was active in protein synthesis as early as day 16 of pregnancy. Secretion of RBP by caprine conceptuses (days 16-22) coincident with the rapid transformation of the conceptus from a spherical blastocyst to a filamentous structure. Production of RBP by the elongating conceptuses may be indicative of an important role for conceptus RBP in the transport, availability and metabolism of retinol during maternal recognition of pregnancy.

Secretion and Immunolocalization of Retinol-Binding Protein in Bovine Conceptuses during Periattachment Periods of Early Pregnancy.

The purpose of the study was to determine and compare the secretion of RBP by bovine spherical, elongating and filamentous conceptuses, and to identify the cellular location of RBP in developing conceptuses by immmunocytochemistry. Bovine conceptuses were removed from the uterus between days 13 and 22 of pregnancy. Events of early bovine embryonic development were observed. The conceptuses underwent a transformation from a spherical to a filamentous morphology during the periattachment period of placentation. Isolated conceptuses were cultured in a modified minimum essential medium in the presence of radiolabeled amino acids. Presence of retinol-binding protein (RBP) in culture medium was determined by immunoprecipitation using bovine placental anti-RBP serum. Presence of immunoreactive RBP in detectable quantities in spherical blastocyst (day 13) culture medium was evident. Increased amounts of RBP were clearly detected in cultures on days 14 and 15, the time of elongating conceptuses. RBP was abundant in cultures on day 22, when the conceptuses were filamentous. Cellular sources of RBP in day 15 and 22 conceptuses were determined by immunocytochemistry with anti-RBP serum. Strong immunoreactive RBP was localized in trophectoderm of day 15 conceptuses, and in epithelial cells lining the chorion and allantois of day 22 conceptuses. RBP originating from the conceptus may serve to transport retinol locally from the uterus to embryonic tissues. Secretion of RBP by spherical blastocysts (day 13) suggests that retinol is needed for initiating the early events of embryonic development. Increased secretion of RBP on days 14 and 15, the time of elongating conceptuses, was coincident with the timing of rapid trophoblast hyperplasia and remodeling. Greater secretion of RBP by the filamentous conceptus (day 22) coincided with the timing of differentiation of the chorion and allantois as well as growth of the embryo proper. Production of RBP by the developing conceptuses may be indicative of an important role for conceptus RBP in the transport, availability and metabolism of retinol during early pregnancy.

Glucose transporter GLUT3: Ontogeny, targeting, and role in the mouse blastocyst

The first differentiative event in mammalian development is segregation of the inner cell mass and trophectoderm (TE) lineages. The epithelial TE cells pump fluid into the spherical blastocyst to form the blastocyst cavity. This activity is fuelled by glucose supplied through facilitative glucose transporters. However, the reported kinetic characteristics of blastocyst glucose transport are inconsistent with those of the previously identified transporters and suggested the presence of a high-affinity glucose carrier. We identified and localized the primary transporter in TE cells. It is glucose transporter GLUT3, previously described in the mouse as neuron-specific. In the differentiating embryo, GLUT3 is targeted to the apical membranes of the polarized cells of the compacted morula and then to the apical membranes of TE cells where it has access to maternal glucose. In contrast, GLUT1 was restricted to basolateral membranes of the outer TE cells in both compacted morulae and blastocysts. Using antisense oligodeoxynucleotides to specifically block protein expression, we confirmed that GLUT3 and not GLUT1 is the functional transporter for maternal glucose on the apical TE. More importantly, however, GLUT3 expression is required for blastocyst formation and hence this primary differentiation in mammalian development. This requirement is independent of its function as a glucose transporter because blastocysts will form in the absence of glucose. Thus the vectorial expression of GLUT3 into the apical membrane domains of the outer cells of the morula, which in turn form the TE cells of the blastocyst, is required for blastocyst formation.

Isolation and identification of porcine embryonic basic protein as a fragment of pregnancy-associated glycoprotein-2

Between days 11 and 12 of gestation, the porcine conceptus undergoes a metamorphosis from a spherical blastocyst to an elongate thread-like form. During this process, the conceptus secretes a variety of products. One of these products is a protein previously referred to as porcine embryonic basic protein (BP). This protein has been shown to be a major secreted product between days 13 and 18. In this study, we report a simple two-step procedure to isolate BP from day 15 porcine conceptus conditioned medium, utilizing ion-exchange chromatography and reverse-phase HPLC. Purified BP was subjected to Edman degradation amino-terminal sequencing and a 25 amino acid residue sequence was obtained. Comparing the N-terminal sequence of BP to sequences in the GenBank database determined that BP shared amino acid homology with porcine pregnancy-associated glycoprotein-2 (PAG-2). The region of identity corresponded to an internal site of PAG-2, suggesting BP was a proteolytic fragment of PAG-2. The purified protein was confirmed to be BP by Western blot using a previously characterized anti-BP antiserum. Also, the BP was immunolocalized within the trophectoderm of day 11 blastocysts. Staining intensity was diminished in spherical blastocysts compared to elongated blastocysts. Although the function of PAG-2 and its cleavage product BP are unknown, the large quantity produced by the porcine conceptus and its sequence conservation across species may indicate a necessary role in early pregnancy.

The effect of excess rumen degradable protein during the pre, peri or post-ovulatory period on conception rate and conceptus interferon secretion in naturally cycling ewes

Low conception rates in high producing dairy cows have been linked to the feeding of excessive quantities of rumen degradable protein (Elrod &amp; Butler, 1993). Similarly, a recent study in non-lactating ewes, superovulated during seasonal anoestrous, has shown that high rumen degradable protein intakes throughout the preovulatory and early pregnancy period have an adverse effect on embryo survival (Bishonga et al., 1994). The present study aimed to identify the window of oocyte/embryo development which is sensitive to variations in the degradability of maternal protein intake in naturally cycling ewes expressing their normal ovulation rate. Nutritional treatments were applied at three crucial developmental time points spanning (1) preovulatory oocyte maturation (2) fertilisation and early cell division in the oviduct and (3) entry of the embryo into the uterus, hatching and the transition from a spherical blastocyst to an elongating conceptus.

The effect of excess rumen degradable protein during the pre, peri or post-ovulatory period on conception rate and conceptus interferon secretion in naturally cycling ewes

Low conception rates in high producing dairy cows have been linked to the feeding of excessive quantities of rumen degradable protein (Elrod &amp; Butler, 1993). Similarly, a recent study in non-lactating ewes, superovulated during seasonal anoestrous, has shown that high rumen degradable protein intakes throughout the preovulatory and early pregnancy period have an adverse effect on embryo survival (Bishonga et al., 1994). The present study aimed to identify the window of oocyte/embryo development which is sensitive to variations in the degradability of maternal protein intake in naturally cycling ewes expressing their normal ovulation rate. Nutritional treatments were applied at three crucial developmental time points spanning (1) preovulatory oocyte maturation (2) fertilisation and early cell division in the oviduct and (3) entry of the embryo into the uterus, hatching and the transition from a spherical blastocyst to an elongating conceptus.

The influence of endometrium on morphology and oestrogen release of day-11 and day-13 pig blastocysts in vitro

Day-11 spherical and Day-13 filamentous blastocysts were cultured in vitro without supplementation of oestrogen precursors for 2 consecutive periods of 24 h, with or without endometrial tissue. Spherical Day-11 blastocysts flushed from one uterine horn (5.5 +/- 0.8 blastocysts; 5.3 +/- 0.4 mm in diameter; mean +/- s.d.) did not show gross morphological changes during the culture period. Free and conjugated oestrone (E1) and oestradiol-17 beta (E2) were released during the first and second 24 h culture period and was maximal during the first 24 h. In the presence of endometrial tissue the release of oestrogens from the blastocysts was reduced to 30-50%. Clewed Day-13 filamentous blastocysts, recovered after flushing, formed trophospheres and also in these cultures both free and conjugated E1 and E2 were released. In this case the addition of endometrial tissue did not affect the release of oestrogens from the blastocysts. Single Day-13 filamentous blastocysts cultured without endometrial tissue (n = 33, from 4 gilts), developed trophospheres and all blastocysts released E1 and E2. During the first 24 h on average per blastocyst 2.6 ng E1 (range 1.6-4.0) and 9.7 ng E2 (range 4.3-15.3) were released. In all cultures of spherical and filamentous blastocysts E2 release exceeded E1 release.These data show that Day 11-13 blastocysts are able to release both free and conjugated E1 and E2 during in vitro culture from endogenous precursors. The addition of endometrial tissue reduced the release of oestrogens from Day-11 but not Day-13 blastocysts. Whether this is due to an altered oestrogen release of the blastocysts in the presence of endometrium on Day-11 or due, to an increased uptake of oestrogens by the endometrium on Day-11 remains to be investigated.

Effect of time of mating relative to ovulation on morphological diversity of swine blastocysts.

The effect of time of mating relative to ovulation on the amount of embryonic diversity was investigated in 37 mature crossbred gilts. Ovulation was induced by injecting proestrous gilts with 1000 IU of hCG. Gilts were naturally mated once at 24-32 (control), 41, or 43 h after hCG injection (n = 10, 9, and 18, respectively). Blastocysts were collected surgically 288 h post-hCG from gilts mated at 24-32 h post-hCG, from those mated at 41 h post-hCG, and from 8 of those mated at 43 h post-hCG. Collection of blastocysts was performed at 295 h in 10 gilts mated at 43 h post-hCG. Blastocysts within the ranges 1.0-4.0, 4.5-7.0, 7.5-10.0, 10.5-15.0, and 15.5-40.0 mm and those > 40.0 mm were classified morphologically as small, medium, and large spheres and as ovoidal, tubular, and filamentous, respectively. Blastocysts were incubated for 6 h to quantify secretion of estradiol-17 beta (estradiol). Morphological diversity (standard deviation of size) did not differ among blastocysts of treatment groups. Delaying the time of mating reduced (p < 0.07) the subsequent size and estradiol secretion of blastocysts. Waiting until 295 h after hCG to evaluate blastocysts of gilts mated at 43 h resulted in blastocysts size and estradiol secretion similar to that of conception of controls. Estradiol secretion by medium and large spherical blastocysts of gilts mated at 43 h post-hCG and evaluated at 288 h was lower (p < 0.05) than that by blastocysts of the same classifications from controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Aromatase activity in individual day-11 pig blastocysts.

Blastocysts were flushed from both uterine horns of 10 gilts on Day 11 of pregnancy. In these gilts 15.1 +/- 2.3 (mean +/- s.d.) corpora lutea were present and 12.7 +/- 3.1 spherical blastocysts were recovered. In all the gilts variation in blastocyst diameter was observed. Aromatase activity was measurable in 118 of 121 examined blastocysts and varied from 0.005 to 19.330 pmol [1 beta-3H]androstenedione converted into 3H2O in 20 min (mean 1.31). This variation in aromatase activity reflected a difference between and within gilts. Of the total variation between all blastocysts, 67% was due to differences between gilts. A positive exponential relationship existed between blastocyst diameter and aromatase activity, but this relationship was different between gilts (P less than 0.0001). The observed variation in aromatase activity may be caused by differences in germ layer differentiation of the blastocysts.

Nuclear Lamin Antigens are Developmentally Regulated during Porcine and Bovine Embryogenesis1

The nuclear lamins, proteins that reside on the inner face of the nuclear envelope, are thought to provide attachment sites for anchoring the chromatin to the nuclear envelope, thus facilitating the overall organization of the nucleus. The composition of the nuclear lamin proteins changes during differentiation and development in a variety of mammalian and nonmammalian tissues. Bovine and porcine oocytes and early embryos were prepared for immunocytochemical detection of nuclear lamins using three different antibodies (recognizing lamin B, lamins A/B/C, or lamins A/C). In both species, germinal vesicle nuclei and early cleavage stage nuclei react positively with the antibodies. However, on nuclei of bovine embryos, the A/C epitope was not detectable at the 16-cell stage, compact morula, spherical blastocyst, or the chorionic cell nuclei of a Day 35 conceptus, but was detectable on both amniotic and embryonic ectodermal cell nuclei of a Day 35 conceptus. All three antibodies reacted with nuclei from two bovine tissue culture cell lines (bovine embryonic cells and Madin-Darby bovine kidney cells) and one porcine kidney cell line. Nuclei in porcine embryos followed a similar pattern, except the loss of the A/C epitope occurred at the 8-cell stage and the epitope was absent from compact morula and spherical blastocyst stage nuclei. All interphase nuclei in both species reacted with both anti-lamin A/B/C and anti-lamin B antibodies, whereas metaphase chromosomes did not react with any of the lamin antibodies tested. The change in recognizing the lamin epitope occurred one cell cycle after the expected transition from maternal control to zygotic control of development. Nuclear transplantation showed that 16-cell stage porcine nuclei, which are lamin A/C negative, acquired the A/C epitope after transfer to an enucleated metaphase II oocyte. These results suggest that the A/C epitope is developmentally regulated.

Purification, characterization, and immunocytochemical localization of the major basic protein of pig blastocysts.

The major basic protein (BP) synthesized and secreted by elongating pig blastocysts was purified from medium of Day 14-17 conceptus cultures. Sequential ion-exchange and gel-filtration chromatographies resulted in isolation of BP as a single polypeptide of Mr = 43,100 or 42,800 under denaturing or native conditions, respectively. BP was found to be a glycoprotein by incorporation of [3H] glucosamine and susceptibility to N-glycopeptidase F. Two BP polypeptides were produced by N-glycopeptidase F (Mr = 39,800 and 36,300). Antiserum to BP immunoprecipitated radiolabeled BP from blastocyst culture medium. BP was not detected in medium from 1-2 mm diameter spherical (Day 10) blastocysts but was found in medium from 3-5 mm spherical (Day 10) and filamentous (less than 50 cm, Day 12) conceptuses, suggesting that BP synthesis and secretion began at the initiation of trophoblast expansion. With immunocytochemical procedures, BP was located in the apical cytoplasm of trophectoderm cells of Day 11 expanding (5-7 and 10-20 mm) blastocysts. These results suggest that trophoblast epithelium secrete BP apically toward the uterine lumen and that BP may play a role in maternal-fetal interactions during the peri-implantation period.

The pig blastocyst: its ultrastructure and the uptake of protein macromolecules.

Between days 8 and 11 of pregnancy spherical blastocysts from 0.3 to 10 mm in diameter were flushed from the uterine horns of Dutch Landrace pigs. A description of their ultrastructure is given, and the uptake of horseradish peroxidase and ferritin is demonstrated. The ultrastructure of the trophoblast was similar at all ages studied. The trophoblast which has many apical microvilli is able to take up and digest the macromolecules which were offered in the in vitro incubation medium. The hypoblast consists of flattened cells. In blastocysts 2 mm and larger, compact cells bearing microvilli are found below the embryoblast. Cell organelles indicating protein synthesis are found within hypoblast cells of such blastocysts. In the embryoblast, local concentrations of cell organelles are visible, indicating that differentiation has started. After the disappearance of Rauber's layer, which takes place when the blastocyst reaches a diameter of about 2 mm, superficial embryoblast cells develop short microvilli. The cells do not absorb ferritin or peroxidase but are dependent on the trophoblast for their food requirements. All cell layers in the blastocyst contain mitochondria that have characteristics of those found in steroid-producing cells. The significance of the uptake and digestion of macromolecules by trophoblast cells, the synthesis of protein by hypoblast cells and the possible synthesis of steroids is discussed with respect to the relationship between the cell layers of the blastocyst and in the context of concepto-maternal relationships.

Establishment of pregnancy in the pig: II. Cellular remodeling of the porcine blastocyst during elongation on day 12 of pregnancy.

The morphology of pig blastocysts changes dramatically just preceding initial attachment of the trophoblast to the uterine epitheliuin. Blastocysts undergo a rapid transition from spherical to tubular and elongated filamentous forms between Days 10 to 12 of pregnancy. The present study investigated rates of blastocyst development and possible mechanism(s) involved with changes in blastocyst morphology. Estimates of blastocyst growth were determined by removing the uterine horns from each animal at different intervals. Estimates of blastocyst growth were 0.25 mm/h for development of spherical blastocysts from 4 to 9 mm in diameter; however, blastocysts elongated at a rate of 30 to 45 mm/h after reaching a diameter of 10 mm. Although elongation from 10 to 150 mm occurred within a few hours, an increase in cellular hyperpiasia (mitosis) was not detected. Although blastocyst content of DNA aiıd RNA did increase between Days 10 and 16 of pregnancy (P<0.01), total DNA was not different between spherical (9 to 10 mm), tubular (11 to 50 mm) and early filamentous (>100 mm) blastocysts (Day 12). The mitotic index of tubular blastocysts was reduced (P<0.01) compared to that for spherical blastocysts, which also suggested that cellular hyperplasia is not involved in initial elongation of pig blastocysts. Morphological changes in cellular organization of both trophectoderm and extraembryonic endoderm were observed when blastocysts developed to 10 mm in diameter. At this stage, a dense band of cells (elongation zone) composed of both endoderm and trophectoderm was visualized extending from the inner cell mass to the tip of the ovoid blastocyst. Scanning and transmission electron microscopy indicated ultrastructural changes in endodermal and trophectodermal cells associated with formation of the elongation zone. Blastocyst elongation occurs rapidly after formation of the elongation zone through alterations in microfilaments and junctional complexes of trophectoderm cells and formation of filapodia by endodermal cells.