Initiation of a conserved trophectoderm program in human, cow and mouse embryos.

Current understandings of cell specification in early mammalian pre-implantation development are based mainly on mouse studies. The first lineage differentiation event occurs at the morula stage, with outer cells initiating a trophectoderm (TE) placental progenitor program. The inner cell mass arises from inner cells during subsequent developmental stages and comprises precursor cells of the embryo proper and yolk sac1. Recent gene-expression analyses suggest that the mechanisms that regulate early lineage specification in the mouse may differ in other mammals, including human2-5 and cow6. Here we show the evolutionary conservation of a molecular cascade that initiates TE segregation in human, cow and mouse embryos. At the morula stage, outer cells acquire an apical-basal cell polarity, with expression of atypical protein kinase C (aPKC) at the contact-free domain, nuclear expression of Hippo signalling pathway effectors and restricted expression of TE-associated factors such as GATA3, which suggests initiation of a TE program. Furthermore, we demonstrate that inhibition of aPKC by small-molecule pharmacological modulation or Trim-Away protein depletion impairs TE initiation at the morula stage. Our comparative embryology analysis provides insights into early lineage specification and suggests that a similar mechanism initiates a TE program in human, cow and mouse embryos.

Betaine is accumulated via transient choline dehydrogenase activation during mouse oocyte meiotic maturation.

Betaine (N,N,N-trimethylglycine) plays key roles in mouse eggs and preimplantation embryos first in a novel mechanism of cell volume regulation and second as a major methyl donor in blastocysts, but its origin is unknown. Here, we determined that endogenous betaine was present at low levels in germinal vesicle (GV) stage mouse oocytes before ovulation and reached high levels in the mature, ovulated egg. However, no betaine transport into oocytes was detected during meiotic maturation. Because betaine can be synthesized in mammalian cells via choline dehydrogenase (CHDH; EC 1.1.99.1), we assessed whether this enzyme was expressed and active. Chdh transcripts and CHDH protein were expressed in oocytes. No CHDH enzyme activity was detected in GV oocyte lysate, but CHDH became highly active during oocyte meiotic maturation. It was again inactive after fertilization. We then determined whether oocytes synthesized betaine and whether CHDH was required. Isolated maturing oocytes autonomously synthesized betaine in vitro in the presence of choline, whereas this failed to occur in Chdh-/- oocytes, directly demonstrating a requirement for CHDH for betaine accumulation in oocytes. Overall, betaine accumulation is a previously unsuspected physiological process during mouse oocyte meiotic maturation whose underlying mechanism is the transient activation of CHDH.

Telomere length and telomerase activity in bovine pre-implantation embryos in vitro.

Telomeres are specialized structures that cap the ends of chromosomes and help to maintain genomic integrity and stability. Telomeres undergo dynamic changes during embryo development, which also represents an important stage for telomere elongation through telomerase enzyme activity. The objectives of this study were to examine changes in telomere length and telomerase activity from the early oocyte, through to the blastocysts stage of development, and the expression of factors with the potential to directly regulate telomeres. In vitro-produced bovine embryos were lysed and analysed for either relative telomere length, or telomerase activity using quantitative real-time PCR protocols. Our results reveal that relative telomere length is the shortest in the presumptive zygote stage of development and gradually increases to the blastocyst stage. We also demonstrate that differences between the mean telomere lengths throughout these stages are statistically significant (p < 0.05). Telomerase activity in the stages examined appears relatively constant until the blastocyst, where the highest level of activity is detected, leading to a significant difference in telomerase activity across embryonic stages (p < 0.005). Bovine telomerase RNA component (bTERC) expression levels were highest in the blastocyst, TERF1 transcripts showed little change in expression, and TERF2 expression decreased in the blastocysts (p < 0.05). Our results suggest that a complex integration of telomere-related RNA and proteins influences the regulatory mechanisms involved in 'reprogramming' of telomeres during early embryonic stages.

Effects of histone deacetylase inhibitors on the early development of bovine androgenetic embryos.

Histone acetylation is one of the most important posttranslational modifications that contribute to transcriptional initiation and chromatin remodeling. In our previous study, we enhanced sperm chromatin remodeling within the bovine sperm injection-derived androgenentic (SpI-AG) embryos by sperm pretreatment, and thereby improved their early developmental competence. In this study, we found that blastocyst development of SpI-AG embryos could be elevated by the histone deacetylase inhibitor (HDACi). First, we optimized the efficacy of two histone deacetylase inhibitors [trichostatin A (TSA) and Scriptaid (SCR)] in a dose (0, 5, 10, 20, 50, and 100 nM for TSA; 0, 50, 100, 200, 300, and 500 nM for SCR, respectively) and time-dependent (0, 10, 15, 20, and 25 h) manner on the developmental capacity of these embryos. Furthermore, we quantitatively assessed the alterations in histone H3 and H4 overall acetylation levels and blastocyst quality of SpI-AG embryos by immunofluorescence staining. We found a significantly improved morula and blastocyst development rate of SpI-AG embryos at a mild dose of TSA (20 nM) or SCR (200 nM) for 15 h after embryo activation. Furthermore, both HDACi noticeably increased the levels of acetylated histone H3 and H4 in SpI-AG blastocyst embryos, whereas, SCR treatment improved the quality of blastocysts when compared with control group. In conclusion, HDACi is beneficial for early development of bovine SpI-AG embryos and can be used to improve the efficiency of its in vitro production.

Embryos generated from oocytes lacking complex N- and O-glycans have compromised development and implantation.

Female mice generating oocytes lacking complex N- and O-glycans (double mutants (DM)) produce only one small litter before undergoing premature ovarian failure (POF) by 3 months. Here we investigate the basis of the small litter by evaluating ovulation rate and embryo development in DM (Mgat1(F/F)C1galt1(F/F):ZP3Cre) and Control (Mgat1(F/F)C1galt1(F/F)) females. Surprisingly, DM ovulation rate was normal at 6 weeks, but declined dramatically by 9 weeks. In vitro development of zygotes to blastocysts was equivalent to Controls although all embryos from DM females lacked a normal zona pellucida (ZP) and ∼30% lacked a ZP entirely. In contrast, in vivo preimplantation development resulted in less embryos recovered from DM females compared with Controls at 3.5 days post coitum (dpc) (3.2±1.3 vs 7.0±0.6). Furthermore, only 45% of mated DM females contained embryos at 3.5 dpc. Of the preimplantation embryos collected from DM females, approximately half were morulae unlike Controls where the majority were blastocysts, indicating delayed embryo development in DM females. Post-implantation development in DM females was analysed to determine whether delayed preimplantation development affected subsequent development. In DM females at 5.5 dpc, only ∼40% of embryos found at 3.5 dpc had implanted. However, at 6.5 dpc, implantation sites in DM females corresponded to embryo numbers at 3.5 dpc indicating delayed implantation. At 9.5 dpc, the number of decidua corresponded to embryo numbers 6 days earlier indicating that all implanted embryos progress to midgestation. Therefore, a lack of complex N- and O-glycans in oocytes during development impairs early embryo development and viability in vivo leading to delayed implantation and a small litter.

Betaine homocysteine methyltransferase is active in the mouse blastocyst and promotes inner cell mass development.

Methyltransferases are an important group of enzymes with diverse roles that include epigenetic gene regulation. The universal donor of methyl groups for methyltransferases is S-adenosylmethionine (AdoMet), which in most cells is synthesized using methyl groups carried by a derivative of folic acid. Another mechanism for AdoMet synthesis uses betaine as the methyl donor via the enzyme betaine-homocysteine methyltransferase (BHMT, EC 2.1.1.5), but it has been considered to be significant only in liver. Here, we show that mouse preimplantation embryos contain endogenous betaine; Bhmt mRNA is first expressed at the morula stage; BHMT is abundant at the blastocyst stage but not other preimplantation stages, and BHMT activity is similarly detectable in blastocyst homogenates but not those of two-cell or morula stage embryos. Knockdown of BHMT protein levels and reduction of enzyme activity using Bhmt-specific antisense morpholinos or a selective BHMT inhibitor resulted in decreased development of embryos to the blastocyst stage in vitro and a reduction in inner cell mass cell number in blastocysts. The detrimental effects of BHMT knockdown were fully rescued by the immediate methyl-carrying product of BHMT, methionine. A physiological role for betaine and BHMT in blastocyst viability was further indicated by increased fetal resorption following embryo transfer of BHMT knockdown blastocysts versus control. Thus, mouse blastocysts are unusual in being able to generate AdoMet not only by the ubiquitous folate-dependent mechanism but also from betaine metabolized by BHMT, likely a significant pool of methyl groups in blastocysts.

The aryl hydrocarbon receptor agonist 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) alters early embryonic development in a rat IVF exposure model.

Aryl hydrocarbon receptor (AHR) ligands, including 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD), accelerate reproductive senescence and one proposed target is the early embryo. To discriminate between direct effects on the oocyte and early embryo and those mediated by complex ovarian interactions with TCDD, IVF was carried out in the presence of TCDD (10, 100 nM) and the aryl hydrocarbon antagonist CH-223191 (1 µM) combined factorially. TCDD-induced Cyp1a1 mRNA expression was absent in 2-cell embryos; however morulae exhibit dose-dependent Cyp1a1 expression. TCDD induced accumulation of sperm in the perivitelline space and displacement of blastomere nuclei. At 100 nM TCDD, aberrations in cytokinesis and nuclear positioning were observed 2-cell embryos and morula and these effects were reversed in the presence of CH-223191. Our data suggest that acute exposure to TCDD has direct effects on early development in the rat that permit discrimination of AHR-mediated and AHR-independent mechanisms through which environmental toxicants impair mammalian reproduction.

Expression of enzymes involved in the synthesis of prostaglandin E2 in bovine in vitro-produced embryos.

Prostaglandin E2 (PGE2) may play a major role in embryo development and the establishment of pregnancy in cattle. The biosynthesis of PGE2 implies the sequential transformation of arachidonic acid to PGH2 by cyclooxygenases (COXs), then the conversion of PGH2 to PGE2 by prostaglandin E synthases (PGESs). Quantitative RT-PCR was used to examine the expression of COX-1, COX-2, microsomal PGES-1 (mPGES-1), microsomal PGES-2 (mPGES-2) and cytosolic PGES (cPGES) mRNAs in day 7 in vitro-produced (IVP) embryos from oocytes collected by ovum pick-up in Holstein heifers. Transcripts for COX-2 and mPGES-1 were detected in all embryos, whereas transcripts for COX-1 and mPGES-2 were not detected and cPGESs were at the limit of detection in 40% of embryos. Levels of COX-2 and mPGES-1 mRNAs were significantly higher in blastocysts and expanded blastocysts than in morulae and early blastocysts. Furthermore, excellent-quality embryos (grade 1) displayed higher levels of both COX-2 and mPGES-1 than did embryos of good and medium qualities (grades 2-3). Our results suggest that bovine IVP embryos at the morula and blastocyst stages use exclusively the COX-2/mPGES-1 pathway for PGE2 biosynthesis, and that PGE2 is potentially involved in blastocyst expansion and developmental competence.

Targeted disruption of Aurora A causes abnormal mitotic spindle assembly, chromosome misalignment and embryonic lethality.

Aurora A (also known as STK15/BTAK in humans), a putative oncoprotein naturally overexpressed in many human cancers, is a member of the conserved Aurora protein serine/threonine kinase family that is implicated in the regulation of G(2)-M phases of the cell cycle. In vitro studies utilizing antibody microinjection, siRNA silencing and small molecule inhibitors have indicated that Aurora A functions in early as well as late stages of mitosis. However, due to limitations in specificity of the techniques, exact functional roles of the kinase remain to be clearly elucidated. In order to identify the physiological functions in vivo, we have generated Aurora A null mouse embryos, which show severe defects at 3.5 d.p.c. (days post-coitus) morula/blastocyst stage and lethality before 8.5 d.p.c. Null embryos at 3.5 d.p.c. reveal growth retardation with cells in mitotic disarray manifesting disorganized spindle, misaligned and lagging chromosomes as well as micronucleated cells. These findings provide the first unequivocal genetic evidence for an essential physiological role of Aurora A in normal mitotic spindle assembly, chromosome alignment segregation and maintenance of viability in mammalian embryos.

Intracellular signal transduction in mouse oocytes and irradiated early embryos.

In order to determine the effect of X-irradiation on intracellular signal transduction in mouse oocytes and embryos, JNK, ERK and p38 kinase activities were measured by the state of phosphorylation of their respective substrates (c-Jun, Elk-1 and ATF-2, respectively) in two mouse strains differing in radiation sensitivity, namely C57BL and BALB/c. In a first step, control oocytes and embryos were compared for their respective kinase activities at various stages of oocyte maturation (germinal vesicle and metaphases of 1st and 2nd meiosis stages) and early embryonic development (1-, 2-, 4-, 8- and 16-cell, morula and blastula stages). Levels of p38, ERK or JNK kinase activities were shown to vary with the stage of oocyte maturation and embryo development. In a second step, 1- and 2-cell embryos were X-irradiated with 2.5 Gy during the S-phase of the 1st or the 2nd cell-cycle, respectively. There were no significant differences in p38, ERK and JNK kinase activities between control and irradiated embryos, whatever the stage or mouse strain was considered. In conclusion, p38, ERK and JNK kinase activities were shown to vary during oocyte maturation and early embryonic development. Apparently, X-irradiation did not affect these kinase activities at the 1- and 2-cell stages in either mouse strains regardless of their difference in radiation sensitivity.

Temporal detection of caspase-3 and -7 in bovine in vitro produced embryos of different developmental capacity.

Embryo quality is most frequently evaluated at the blastocyst stage, although quality parameters further back along the developmental axis, such as early developmental kinetics or oocyte quality, can be equally valuable. Despite the fact that previous studies in bovine have linked oocyte diameter and early developmental kinetics with blastocyst formation and viability, their relation with the incidence of apoptosis during embryo development remains relatively unexplored. Therefore, we related non-invasive parameters of oocyte and embryo quality, such as embryo kinetics, embryo morphology, and oocyte diameter, to the incidence of apoptosis throughout embryo development using fluorescent detection of active caspase-3 and -7. First, bovine in vitro embryos were selected according to developmental kinetics and morphology at four set times during culture and subjected to fluorescent detection of active caspase-3 and -7. Caspase activity was significantly higher in slow developing embryos in comparison with fast cleavers (P < 0.05), but was not related to embryo morphology. Second, bovine oocytes were divided into three groups on the basis of oocyte diameter and the resulting embryos were used for staining at the same four set times. Caspase activity was significantly higher in embryos derived from growing oocytes compared with those of fully grown oocytes at 45, 80, and 117 hours post-insemination (hpi; P < 0.05), but not at 168 hpi.

Expression profile of protein kinase C isozymes in preimplantation mouse development.

In the preimplantation mouse embryo, the protein kinase C (PKC) family has been implicated in regulation of egg activation, progression of meiotic and mitotic cell cycles, embryo compaction, and blastulation, but the involvement of the individual isozymes is largely unknown. Here, using semiquantitative immunocytochemistry and confocal microscopy we analyze the relative amount and subcellular distribution of ten isozymes of PKC (alpha, betaI, betaII, gamma, delta, epsilon, eta, theta, zeta, iota/lambda) and a PKC-anchoring protein, receptor for activated C-kinase 1 (RACK1). Our results show that all of these isoforms of PKC are present between the two-cell and blastocyst stages of mouse preimplantation development, and that each has a distinct, dynamic pattern and level of expression. The data suggest that different complements of the isozymes are involved in various steps of preimplantation development, and will serve as a framework for further functional studies of the individual isozymes. In particular, there was a transient increase in the nuclear concentration of several isozymes at the early four-cell stage, suggesting that some of the PKC isozymes might be involved in regulation of nuclear organization and function in the early mouse embryo.

Nitric oxide in blastocyst implantation in the rhesus monkey.

Successful blastocyst implantation depends on the interaction between cells of maternal endometrium and conceptus, as well as adequate blood supply to the site of blastocyst implantation. Nitric oxide (NO) generally plays a significant role in the local regulation of vascular physiology in a variety of mammalian tissue systems, however, its role in blastocyst implantation and placentation in the primate is not known. The aim of the present study was to examine: (i) NADH-diaphorase activity and expression of three isoforms of nitric oxide synthase (NOS), namely endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS) in pre-implantation stage monkey embryos, morula (n = 4) and blastocyst (n = 10), as well as, in different compartments of conceptus and maternal endometrium at primary implantation sites during lacunar (n = 6) and villous (n = 9) stages of placentation in the rhesus monkey, and (ii) the potential anti-nidatory effect of vaginal administration of NOS inhibitor during the peri-implantation period of conception cycles in rhesus monkeys. Pre-implantation stage blastocysts exhibited marked NADPH-diaphorase activity along with immunopositive iNOS mainly in the inner cell mass. During the lacunar stage, marked eNOS expression was observed in cytotrophoblast cells lining the embryonic cavity. However, cytotrophoblast cells lining villi, forming columns, and constituting anchoring villi expressed all the three isoforms of NOS in villous placenta stage tissue. During the lacunar stage, eNOS and iNOS protein expressions were observed in epithelial and decidual cells of endometrium. As gestation advanced, mRNAs for all three isoforms of NOS were observed to increase in epithelial and decidual cells, however, with no marked change in protein expression. Vaginal administration of a NOS inhibitor (N(G)-nitro-l-arginine methyl ester, L-NAME, 4, 6, and 8 mg/kg body weight or aminoguanidine, AG, 4 mg/kg body weight) during days 6 to 12 after ovulation resulted in pregnancy failure in a higher number of animals (L-NAME: 8 confirmed pregnancies in 25 animals; AG: 2 confirmed pregnancies in 8 animals) compared with control animals (5 pregnancies in 7 animals). It appears that NO may play an important role in the establishment of pregnancy in the rhesus monkey.

Heparanase improves mouse embryo implantation.

OBJECTIVE: To improve mouse embryonic implantation by recombinant heparanase supplementation. Heparanase, an endoglycosidase-degrading heparan sulfate proteoglycan, may have a role in embryonic implantation because of its enzymatic, angiogenic, and adhesive properties. Increasing endometrial receptivity could improve one of the most difficult pathologies in human fertility. DESIGN: Comparison between mouse blastocysts obtained after 24-hour incubation of morulae with or without heparanase. SETTING: Experimental laboratory in a medical center. ANIMAL(S): Mice. INTERVENTION(S): Morulae were flushed from CB6F1 female mice and incubated for 24 hours at 37 degrees C in M16 medium supplemented with 0.1 mg/mL heparanase (n = 203), with albumin (n = 60), or with medium alone (n = 258). MAIN OUTCOME MEASURE(S): Blastocysts were evaluated by heparanase immunostaining (n = 10), activity assay (n = 283), and transfer to foster mice uterine horns (n = 228). The number of implantation sites was compared. RESULT(S): Immunostaining demonstrated that heparanase is constitutively expressed in mouse morulae and blastocyts. Heparanase supplementation resulted in increased staining and enzymatic activity in blastocyts. Implantation rates for the heparanase, M16 medium, and albumin groups, were 36.9%, 17.8%, and 20%, respectively (P<.01). CONCLUSION(S): Heparanase was found to be constitutively expressed by blastocyst-stage embryos. Moreover, the amount of heparanase was markedly increased by incubation of morulae with recombinant heparanase, evaluated by immunostaining and enzymatic activity. Heparanase supplementation resulted in approximately a twofold increase in embryo implantation rate in vivo. Taken together, these data suggest that heparanase is actively involved in embryo implantation.

Early embryo-endometrial signaling modulates the regulation of matrix metalloproteinase-3.

OBJECTIVE: To examine matrix metalloproteinase-3 (MMP-3) expression in human stromal cell culture after P stimulation and the effect of conditioned medium from human embryo-epithelial cells coculture on its expression and activity. DESIGN: Metabolic and endocrine studies on human tissue. SETTING: In vitro fertilization (i.v.f.) unit and endocrine research unit. PATIENT(S): Infertile patients undergoing endometrial tissue sampling for dating at the luteal phase before i.v.f. INTERVENTION(S): Endometrial sampling and collection of human embryos culture media. MAIN OUTCOME MEASURE(S): Expression and activity of secreted MMP-3 by P-induced stromal cells, and in stromal cells exposed to conditioned medium from embryo-epithelial cell coculture. RESULT(S): Expression and activity of MMP-3 in human stromal cells decrease after P induction. Following incubation of these stromal-derived decidual cells with conditioned medium from embryo-epithelial cell coculture, MMP-3 expression and activity increased in a statistically significant manner. CONCLUSION(S): Progesterone inhibition of MMP-3 expression and its support of endometrial integrity were prevented by local expression of MMP-3 in response to embryonic signaling.

Essential role for poly (ADP-ribosyl)ation in mouse preimplantation development.

BACKGROUND: Poly (ADP-ribosyl)ation is a covalent modification of many nuclear proteins. It has a strong chromatin modifying potential involved in DNA repair, transcription and replication. Its role during preimplantation development is unknown. RESULTS: We have observed strong but transient synthesis of poly ADP-ribose polymers on decondensing chromosomes of fertilized and parthenogenetically activated mouse oocytes. Inhibition of this transient upregulation with a specific enzyme inhibitor, 3-aminobenzamide, has long-term effects on the postimplantation development of the embryos. In addition, inhibition of poly (ADP-ribosyl)ation at the 4-8 cell stage selectively blocks morula compaction. CONCLUSION: These observations suggest that poly (ADP-ribosyl)ation is involved in the epigenetic chromatin remodeling in the zygote.

Abnormal regulation of DNA methyltransferase expression in cloned mouse embryos.

Cloning by somatic cell nuclear transfer is inefficient. This is evident in the significant attrition in the number of surviving cloned offspring at virtually all stages of embryonic and fetal development. We find that cloned preimplantation mouse embryos aberrantly express the somatic form of the Dnmt1 DNA (cytosine-5) methyltransferase, the expression of which is normally prevented by a posttranscriptional mechanism. Additionally, the maternal oocyte-derived Dnmt1o isoform undergoes little or none of its expected translocation to embryonic nuclei at the eight-cell stage. Such defects in the regulation of Dnmt1s and Dnmt1o expression and cytoplasmic-nuclear trafficking may prevent clones from completing essential early developmental events. Furthermore, aberrant Dnmt1 localization and expression may contribute to the defects in DNA methylation and the developmental abnormalities seen in cloned mammals.

Inhibitor profiles of alkaline phosphatases in bovine preattachment embryos and adult tissues.

The alkaline phosphatases are a small family of isozymes. Bovine preattachment embryos transcribe mRNA for two tissue-specific alkaline phosphatases (TSAP2 and TSAP3) beginning at the 4- and 8-cell stages. Whereas no mRNA has been detected in oocytes, there is maternally inherited alkaline phosphatase activity. It is not known which isozyme(s) is responsible for the maternal activity or when TSAP2 and TSAP3 form functional protein. No antibodies are available that recognize the relevant bovine alkaline phosphatases. Therefore, sensitivity to heat and chemical inhibition was used to separate the different isozymes. By screening tissues, it was determined that the bovine tissue-nonspecific alkaline phosphatase (TNAP) is inactivated by low temperatures (65C) and low concentrations of levamisole (<1 mM), whereas bovine tissue-specific isozymes require higher temperatures (90C) and levamisole concentrations (>5 mM). Inhibition by L-homoarginine and L-phenylalanine was less informative. Cumulus cells transcribe two isozymes and the pattern of inhibition suggested heterodimer formation. Inhibition of alkaline phosphatase in bovine embryos before the 8-cell stage indicated the presence of only TNAP. At the 16-cell stage the pattern was consistent with TNAP plus TSAP2 or -3 activity, and in morulae and blastocysts the pattern indicated that the maternal TNAP is fully supplanted by TSAP2 or TSAP3.

A novel murine tryptase involved in blastocyst hatching and outgrowth.

Before implantation the blastocyst is maintained within a proteinaceous coat, the zona pellucida, which prevents polyspermy and ectopic pregnancy. An extracellular trypsin-like activity, which is necessary for hatching from the zona pellucida in vitro, is localized to the abembryonic pole of the blastocyst. Upon hatching, the extracellular matrix-degrading proteinases urokinase plasminogen activator (uPA) and matrix metalloproteinase 9 (MMP-9) are thought to promote blastocyst invasion. However, gene disruption experiments have demonstrated that uPA and MMP-9 are dispensable and, thus, that other key enzymes are involved in implantation. In this study, a novel implantation serine proteinase (ISP1) gene, which is distantly related to haematopoietic tryptases and represents a novel branch of the S1 proteinase family, was cloned. ISP1 is expressed throughout morulae and blastocysts during hatching and outgrowth. Abrogation of ISP1 mRNA accumulation using antisense oligodeoxynucleotides disrupts blastocyst hatching and outgrowth in vitro. The results of this study indicate that the ISP1 gene probably encodes the long sought after 'hatching enzyme' that is localized to the abembryonic pole during hatching in vitro. ISP1 is the earliest embryo-specific proteinase to be expressed in implantation and may play a critical role in connecting embryo hatching to the establishment of implantation competence at the abembryonic pole of the blastocyst.