Dynamic expression of matrix metalloproteinases (MMP-2, -9 and -14) and the tissue inhibitors of MMPs (TIMP-1, -2 and -3) at the implantation site during tubal pregnancy

Matrix metalloproteinases (MMPs) are responsible for extracellular matrix (ECM) degradation, and their functions are regulated by tissue inhibitors of MMPs (TIMPs). The evidence for the roles of MMPs and TIMPs in implantation and placentation has remained insufficient in humans, especially during the early stages. Tubal pregnancy has some similarities to normal intrauterine pregnancy and therefore may provide a unique model for implantation studies. In the present study, the expression of MMP-2, -9 and -14, and TIMP-1, -2 and -3 at the feto-maternal interface during tubal pregnancy was examined by immunohistochemistry and in situ hybridization. We found that MMP-9 and TIMP-1, -2 and -3 are produced by all types of extravillous cytotrophoblast (EVCT) cells, while MMP-2 and -14 mainly exist in distal column cytotrophoblast (CCT) cells and invasive EVCT cells. Meanwhile, the intensity of MMP-14 and TIMP-1 and -2 increased along the invasive pathway toward maternal interstitium. In addition, MMP-2, -9 and -14 and TIMP-1, -2 and -3 were all detected in the villous CT (VCT) cells. Furthermore, both the mRNA level and immunoreactivity of MMP-9, TIMP-1 and -3 increased, while those of TIMP-2 decreased concurrent with the progression of pregnancy during weeks 3-9. The unique expression pattern of various MMPs and TIMPs at the feto-maternal interface suggests that they may have roles in regulating the controlled invasion of trophoblasts during implantation and placentation. Meanwhile, the study provides a better understanding of the mechanisms involved in cellular events during human pregnancy, especially at the initiation stage of implantation.     

Regulatory roles for mitochondria in the peri-implantation mouse blastocyst: possible origins and developmental significance of differential DeltaPsim

Studies of mitochondria in mouse and human oocytes and preimplantation stage embryos have focused primarily on their metabolic capacity to generate ATP. However, it is becoming increasingly apparent that mitochondria are also regulatory agents in other processes involved in the establishment of developmental competence, including calcium homeostasis and apoptosis. The magnitude of the inner mitochondrial membrane potential, or its polarity (DeltaPsim), is a physiochemical property of mitochondria related to levels of organelle activity, and differences in the magnitude and spatial distribution of high- and low-polarized mitochondria have been suggested to influence oocyte and early embryo competence. Here, we investigated mitochondrial polarity in normal and diapausing peri-implantation-stage mouse blastocysts, and their corresponding outgrowths, for indications of cell-type-specific regulatory functions or activities in which these organelles may be engaged. The results demonstrate that cell-type- and location-specific domains of differential DeltaPsim exist in the peri-implantation blastocyst and remain unchanged during blastocyst outgrowth and during delayed implantation, which for the latter, is accompanied by the suppression of mitochondrial oxidative phosphorylation. Our findings demonstrate that cell-type-specific DeltaPsim in the peri-implantation blastocyst is not an intrinsic property of the corresponding mitochondria but one that can be mediated by the dynamics of intercellular contact. Cells with high- or low-polarized mitochondria are differentially affected by photosensitization, with developmental consequences related to embryo behavior and outgrowth performance. Differences in polarity are discussed with respect to the participation of mitochondria in regulatory and morphogenetic processes in the normal peri-implantation embryo. The persistence of high DeltaPsim in the diapausing embryo is suggested to be associated with the regulation of levels of cytoplasmic free calcium and the ability of the embryo to reactivate development when delayed implantation terminates.     

Testosterone inhibits matrix metalloproteinase-1 production in human endometrial stromal cells in vitro

Androgen receptor (AR) is reported to be expressed in human uterine endometrium, but not much information is available on the role of androgens in human endometrium. The purpose of this study is to investigate the role of androgens in the regulation of matrix metalloproteinase (MMP)-1, which is one of the important MMPs for menstruation and embryo implantation in human endometrium. Human endometrial stromal cells (HESCs) were obtained from human endometrium by enzymatic dissociation method. Purified HESCs were incubated with 17beta-estradiol (E2), testosterone, or E2 + testosterone. Progestins (natural progesterone or medroxyprogesterone acetate) or vehicle (dimethyl sulfoxide) were also added to the media instead of testosterone. Furthermore, hydroxyflutamide (FLU),a specific AR antagonist, was also supplemented to cultured media. The amounts of MMP-1 in cultured media and in HESC lysates were examined by ELISA measurements and western blotting analysis respectively. The expression of ARmRNA in HESCs RNA was analyzed by RT-PCR. Testosterone significantly inhibited MMP-1 in both cultured media and cell lysates in a dose-dependent manner. Progestins also inhibited MMP-1. Furthermore, FLU completely recovered the decrease of MMP-1 induced by testosterone. ARmRNA was detected in all HESCs RNA. The present study demonstrated that the secretion and production of MMP-1 in HESCs in vitro were inhibited by testosterone through androgen receptors in a manner similar to that seen for progesterone. These findings indicate that androgen may play an important role in morphological and functional changes of human endometrium.     

Regulation of proteinases during mouse peri-implantation development: urokinase-type plasminogen activator expression and cross talk with matrix metalloproteinase 9

Trophoblast cells express urokinase-type plasminogen activator (PLAU) and may depend on its activity for endometrial invasion and tissue remodeling during peri-implantation development. However, the developmental regulation, tissue distribution, and function of PLAU are not completely understood. In this study, the expression of PLAU and its regulation by extracellular matrix proteins was examined by RT-PCR, immunocytochemistry, and plasminogen-casein zymography in cultured mouse embryos. There was a progressive increase in Plau mRNA expression in blastocysts cultured on gestation days 4-8. Tissue-type plasminogen activator (55?kDa) and PLAU (a triplet of 40, 37, and 31?kDa) were present in conditioned medium and embryo lysates, and were adsorbed to the culture plate surface. The temporal expression pattern of PLAU, according to semi-quantitative gel zymography, was similar in non-adhering embryos and embryos cultured on fibronectin, laminin, or type IV collagen, although type IV collagen and laminin upregulated Plau mRNA expression. Immunofluorescence revealed PLAU on the surface of the mural trophectoderm and in non-spreading giant trophoblast cells. Exogenous human plasminogen was transformed to plasmin by cultured embryos and activated endogenous matrix metalloproteinase 9 (MMP9). Indeed, the developmental expression profile of MMP9 was similar to that of PLAU. Our data suggest that the intrinsic developmental program predominantly regulates PLAU expression during implantation, and that PLAU could be responsible for activation of MMP9, leading to localized matrix proteolysis as trophoblast invasion commences.     

Quality of porcine blastocysts produced in vitro in the presence or absence of GH

GH receptor (GHR) mRNA is expressed in bovine in vitro produced embryos up to the blastocyst stage and GH improves the quality of bovine embryos by increasing blastocyst cell numbers and reducing the incidence of apoptosis as evaluated by DNA strand-break labelling. Porcine in vitro produced blastocysts have lower cell numbers than in vivo blastocysts and exhibit higher incidences of apoptosis. Therefore we investigated the effects of 100 ng GH/ml NCSU23 medium during in vitro culture of presumptive in vitro fertilized sow zygotes on embryo development and blastocyst quality (defined by diameter, cell number, apoptosis and survival after non-surgical transfer). In vivo produced blastocysts were analysed concurrently as a reference value. GHR was expressed in embryos from the 2-cell to blastocyst stages. GH had no effect on blastocyst development or cell numbers, but increased the mean blastocyst diameter. The incidence of apoptosis, detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), was decreased by GH, but when non-TUNEL-labelled apoptotic fragmented nuclei were included, no difference was seen. GH appeared to slow down the progression of apoptosis though. In vivo produced blastocysts presented no apoptotic nuclei, and contained higher cell numbers and larger diameters. Pregnancy rates on day 11 were similar for all groups, but survival was poorer for in vitro than in vivo produced blastocysts. In this study GH appeared to be beneficial only from the blastocyst stage, but the presence of GHR from early cleavage stages nevertheless indicates a role for GH throughout porcine embryo development and deserves further investigation.     

Culture medium, gas atmosphere and MAPK inhibition affect regulation of RNA-binding protein targets during mouse preimplantation development

During oogenesis, mammalian oocytes accumulate maternal mRNAs that support the embryo until embryonic genome activation. RNA-binding proteins (RBP) may regulate the stability and turnover of maternal and embryonic mRNAs. We hypothesised that varying embryo culture conditions, such as culture medium, oxygen tension and MAPK inhibition, affects regulation of RBPs and their targets during preimplantation development. STAU1, ELAVL1, KHSRP and ZFP36 proteins and mRNAs were detected throughout mouse preimplantation development, whereas Elavl2 mRNA decreased after the two-cell stage. Potential target mRNAs of RBP regulation, Gclc, Slc2a1 and Slc7a1 were detected during mouse preimplantation development. Gclc mRNA was significantly elevated in embryos cultured in Whitten's medium compared with embryos cultured in KSOMaa, and Gclc mRNA was elevated under high-oxygen conditions. Inhibition of the p38 MAPK pathway reduced Slc7a1 mRNA expression while inhibition of ERK increased Slc2a1 mRNA expression. The half-lives of the potential RBP mRNA targets are not regulated in parallel; Slc2a1 mRNA displayed the longest half-life. Our results indicate that mRNAs and proteins encoding five RBPs are present during preimplantation development and more importantly, demonstrate that expression of RBP target mRNAs are regulated by culture medium, gas atmosphere and MAPK pathways.     

Generation of embryonic stem cell lines from mouse blastocysts developed in vivo and in vitro: relation to Oct-4 expression

Embryonic stem (ES) cells are the source of all embryonic germ layer tissues. Oct-4 is essential for their pluripotency. Since in vitro culture may influence Oct-4 expression, we investigated to what extent blastocysts cultured in vitro from the zygote stage are capable of expressing Oct-4 and generating ES cell lines. We compared in vivo with in vitro derived blastocysts from B6D2 mice with regard to Oct-4 expression in inner cell mass (ICM) outgrowths and blastocysts. ES cells were characterized by immunostaining for alkaline phosphatase (ALP), stage-specific embryonic antigen-1 (SSEA-1) and Oct-4. Embryoid bodies were made to evaluate the ES cells' differentiation potential. ICM outgrowths were immunostained for Oct-4 after 6 days in culture. A quantitative real-time PCR assay was performed on individual blastocysts. Of the in vitro derived blastocysts, 17% gave rise to ES cells vs 38% of the in vivo blastocysts. Six-day old outgrowths from in vivo developed blastocysts expressed Oct-4 in 55% of the cases vs 31% of the in vitro derived blastocysts. The amount of Oct-4 mRNA was significantly higher for freshly collected in vivo blastocysts compared to in vitro cultured blastocysts. In vitro cultured mouse blastocysts retain the capacity to express Oct-4 and to generate ES cells, be it to a lower level than in vivo blastocysts.     

Somatic cell nuclear transfer in horses: effect of oocyte morphology, embryo reconstruction method and donor cell type

The objective of the present work was to investigate and clarify the factors affecting the efficiency of somatic cell nuclear transfer (NT) in the horse, including embryo reconstruction, in vitro culture to the blastocyst stage, embryo transfer, pregnancy monitoring and production of offspring. Matured oocytes, with zona pellucida or after zona removal, were fused to cumulus cells, granulosa cells, and fetal and adult fibroblasts, and fused couplets were cultured in vitro. Blastocyst development to Day 8 varied significantly among donor cells (from 1.3% to 16%, P < 0.05). In total, 137 nuclear transfer-embryos were transferred nonsurgically to 58 recipient mares. Pregnancy rate after transfer of NT-embryos derived from adult fibroblasts from three donor animals was 24.3% (9/37 mares transferred corresponding to 9/101 blastocysts transferred), while only 1/18 (5.6%) of NT-blastocysts derived from one fetal cell line gave rise to a pregnancy (corresponding to 1/33 blastocysts transferred). Overall, seven pregnancies were confirmed at 35 days, and two went to term delivering two live foals. One foal died 40 h after birth of acute septicemia while the other foal was healthy and is currently 2 months old. These results indicate that (a) the zona-free method allows high fusion rate and optimal use of equine oocytes, (b) different donor cell cultures have different abilities to support blastocyst development, (c) blastocyst formation rate does not correlate with pregnancy fate and (d) healthy offspring can be obtained by somatic cell nuclear transfer in the horse.     

Recombinant bovine interferon-τ enhances in vitro development of bovine embryos by upregulating expression of connexin 43 and E-cadherin

Interferon-τ (IFNT), produced in ruminants by embryonic trophoblastic cells before implantation, is involved in the maternal recognition of pregnancy. It is a pleiotropic molecule that alters the synthesis of endometrial proteins and inhibits the proliferation of some cells. The present study investigated the effects of recombinant bovine IFNT on the development of early-stage bovine embryos and the molecular mechanism underlying this effect. This study demonstrated that expression of mRNA encoding type I IFN receptor subunits was detectable from d 4 to 8 in in vitro fertilized (IVF) bovine embryos. A considerable number of IVF (n = 1,941) and parthenogenetic activated (n = 1,552) bovine embryos demonstrated that supplementing the culture medium with IFNT (100 ng/mL) produced a greater percentage of blastocysts, and the total cell number within the resulting blastocysts was higher. In addition, IFNT upregulated the expression levels of both mRNA and protein for connexin 43 (GJA1) and E-cadherin (CDH1) and expression levels for granulocyte-macrophage colony-stimulating factor and insulin-like growth factor 2 mRNA but not for their proteins in d 8 embryos. However, IFNT inhibited mRNA expression for leukemia inhibitory factor (LIF), LIF receptor α, and the sodium/potassium-transporting ATPase subunit β-1. We concluded that IFNT promoted the development of bovine embryos by upregulating the expression of GJA1 and CDH1. Thus, supplementing embryo cultures or transfer medium with IFNT may stimulate embryo development and improve embryo transfer efficiency.     

K(+) efflux through two-pore domain K(+) channels is required for mouse embryonic development

Numerous studies have suggested that K(+) channels regulate a wide range of physiological processes in mammalian cells. However, little is known about the specific function of K(+) channels in germ cells. In this study, mouse zygotes were cultured in a medium containing K(+) channel blockers to identify the functional role of K(+) channels in mouse embryonic development. Voltage-dependent K(+) channel blockers, such as tetraethylammonium and BaCl(2), had no effect on embryonic development to the blastocyst stage, whereas K(2P) channel blockers, such as quinine, selective serotonin reuptake inhibitors (fluoxetine, paroxetine, and citalopram), gadolinium trichloride, anandamide, ruthenium red, and zinc chloride, significantly decreased blastocyst formation (P<0.05). RT-PCR data showed that members of the K(2P) channel family, specifically KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9, were expressed in mouse oocytes and embryos. In addition, their mRNA expression levels, except Kcnk3, were up-regulated by above ninefold in morula-stage embryos compared with 2-cell stage embryos (2-cells). Immunocytochemical data showed that KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9 channel proteins were expressed in the membrane of oocytes, 2-cells, and blastocysts. Each siRNA injection targeted at Kcnk2, Kcnk10, Kcnk4, Kcnk3, and Kcnk9 significantly decreased blastocyst formation by ~38% compared with scrambled siRNA injection (P<0.05). The blockade of K(2P) channels acidified the intracellular pH and depolarized the membrane potential. These results suggest that K(2P) channels could improve mouse embryonic development through the modulation of gating by activators.     

Secretion of matrix metalloproteinases 2 and 9 and tissue inhibitor of metalloproteinases into follicular fluid during follicle development in equine ovaries

Extensive tissue remodelling is required in equine ovaries for follicle growth and development and also migration of the follicle to the ovulatory fossa, where ovulation occurs. The mechanisms for these processes are largely unexplored. Matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) are important for control of breakdown of extracellular matrix during tissue remodelling. The aims of this study were to determine the pattern and sites of secretion of the gelatinases MMP-2 and -9 and TIMPs into follicular fluid during follicle development in mare ovaries. The predominant gelatinase detected in follicular fluid was MMP-2, which was present in similar amounts throughout follicular development, as demonstrated by zymography. MMP-9 was also present in follicular fluid and secretion increased significantly (P < 0.05) with development of follicles from < 10 mm to 11-20 mm in diameter. Follicular fluid also contained TIMP-1, TIMP-2, unglycosylated and glycosylated TIMP-3, and TIMP-4, as shown by reverse zymography. The abundance of TIMPs remained largely unchanged during follicle development. MMP-2 and -9 were localized by immunohistochemistry to stromal cells and granulosa and theca cells. TIMP-1, -2, -3 and -4 were present in granulosa and theca cells of the follicle and in stromal cells and also associated with extracellular matrix of the ovarian stromal tissue. The MMPs and TIMPs are likely to be involved in the regulation of the breakdown of extracellular matrix during tissue remodelling for follicle development and migration to the ovulation fossa in mares.     

An improved method for specific-target preamplification PCR analysis of single blastocysts useful for embryo sexing and high-throughput gene expression analysis

Gene expression analysis in preimplantation embryos has been used for answering fundamental questions related to development, prediction of pregnancy outcome, and other topics. Limited amounts of mRNA in preimplantation embryos hinders progress in studying the preimplantation embryo. Here, a method was developed involving direct synthesis and specific-target preamplification (STA) of cDNA for gene expression analysis in single blastocysts. Effective cell lysis and genomic DNA removal steps were incorporated into the method. In addition, conditions for real-time PCR of cDNA generated from these processes were improved. By using this system, reliable embryo sexing results based on expression of sex-chromosome linked genes was demonstrated. Calibration curve analysis of PCR results using the Fluidigm Biomark microfluidic platform (Fluidigm, South San Francisco, CA) was performed to evaluate 96 STA cDNA from single blastocysts. In total, 93.75% of the genes were validated. Robust amplification was detected even when STA cDNA from a single blastocyst was diluted 1,024-fold. Further analysis showed that within-assay variation increased when cycle threshold values exceeded 18. Overall, STA quantitative real-time PCR analysis was shown to be useful for analysis of gene expression of multiple specific targets in single blastocysts.     

母体铅暴露对仔鼠肾脏中MMP-2、MMP-9 mRNA表达的影响

为了探讨母体铅暴露对仔鼠肾脏中MMP-2、MMP-9 m RNA表达的影响。采用自由饮水模式建立铅暴露动物模型,将40只雌性小鼠自妊娠第1 d开始经饮水染铅(1.0 g/L、5.0 g/L和10.0 g/L,对照组饮蒸馏水)至仔鼠出生后21 d,分别测定其血液和肾脏组织中铅的含量,然后取其肾脏组织,通过实时荧光定量PCR(Real-time PCR)的方法检测MMP-2和MMP-9 m RNA的表达情况。21 d仔鼠血液中铅水平均明显高于对照组(p0.05);低、中和高剂量铅暴露组仔鼠肾脏组织中MMP-2 m RNA的表达明显高于对照组(p0.05),铅暴露组仔鼠肾脏组织中MMP-2 m RNA的表达与对照组相比差异不显著(p0.05)。与对照组相比,染毒组仔鼠肾脏内MMP-9m RNA的表达明显高于对照组,且随染毒剂量增加,表达量增加,差异具有统计学意义(p0.05)母体铅暴露使铅在仔鼠体内蓄积,可能通过增强仔鼠肾脏中MMP-2和MMP-9 m RNA的表达进而造成肾脏损伤,引起神经毒性。     

Caspase activity and expression of cell death genes during development of human preimplantation embryos

It has been observed that apoptosis occurs in human blastocysts. In other types of cell, the characteristic morphological changes seen in apoptotic cells are executed by caspases, which are regulated by the BCL-2 family of proteins. This study investigated whether these components of the apoptotic cascade are present throughout human preimplantation development. Developing and arrested two pronucleate embryos at all stages were incubated with a fluorescently tagged caspase inhibitor that binds only to active caspases, fixed, counterstained with 4,6-diamidino-2-phenylindole (DAPI) to assess nuclear morphology and examined using confocal microscopy. Active caspases were detected only after compaction, at the morula and blastocyst stages, and were frequently associated with apoptotic nuclei. Occasional labelling was seen in arrested embryos. Expression of proapoptotic BAX and BAD and anti-apoptotic BCL-2 was examined in single embryos using RT-PCR and immunohistochemistry. BAX and BCL-2 mRNAs were expressed throughout development, whereas BAD mRNA was expressed mainly after compaction. Simultaneous expression of BAX and BCL-2 proteins within individual embryos was confirmed using immunohistochemistry. The onset of caspase activity and BAD expression after compaction correlates with the previously reported appearance of apoptotic nuclei. As in other types of cell, human embryos express common molecular components of the apoptotic cascade, although apoptosis appears to be suppressed before compaction and differentiation.     

Reduced signaling through the hedgehog pathway in the uterine stroma causes deferred implantation and embryonic loss

The role of the hedgehog (HH) signaling pathway in implantation was studied in mice in which the HH signal transducer, smoothened (SMO), was conditionally deleted in the stromal compartment of the uterus, using CRE recombinase expressed through the Amhr2(cre) allele. In Amhr2(cre/+)Smo(null/flox)-mutant mice, Smo mRNA in uterine stroma was reduced 49% compared to that in Amhr2(+/+)Smo(null/flox) control mice, while levels in the luminal epithelium were not different. Litter size was reduced 60% in mutants compared with controls, but ovulation rate and the number of implantation sites on day 7 of pregnancy did not differ. The number of corpora lutea was equivalent to the number of implantation sites, indicating that most ovulations resulted in implanted embryos. However, on days 13 to 15, the rate of embryo resorption was elevated in mutants. In control mice, on day 5, implantation sites were present and blastocysts were well-attached. In contrast, blastocysts were readily flushed from uteri of mutant mice on day 5 and implantation sites were rare. On days 5.5 and 6, implantation sites were present in mutant mice, and by day 6 embryos could not be flushed from the uterus. The weight of implantation sites on day 7 was decreased by 42% in mutant mice, consistent with delayed development. Signaling through SMO in the endometrial stroma is required for optimal timing of implantation, and deferred implantation leads to defective embryo development and subsequent pregnancy loss.     

Endometrial expression of calbindin (CaBP)-d28k but not CaBP-d9k in primates implies evolutionary changes and functional redundancy of calbindins at implantation

The endometrium is hostile to embryo implantation except during the 'window of receptivity'. A change in endometrial gene expression is required for the development of receptivity. Calbindin-d9k (CaBP-d9k) and calbindin-d28k (CaBP-d28k) are proteins possessing EF-hand motifs which have high affinity for Ca2+ ions. Previously, it has been demonstrated that, in mouse endometrium, the expression of both calbindins is highly regulated during implantation and that both proteins play critical but functionally redundant roles at implantation. This study was the first to determine the expression of these two calbindins in the human and rhesus monkey endometrium. Initial RT-PCR analysis demonstrated that CaBP-d28k but not CaBP-d9k mRNA expression is detectable in the endometrium of both species. Western blot analysis confirmed the presence of immuno-reactive CaBP-d28k protein in the primate endometrium. Furthermore, the endometrial expression pattern of CaBP-d28k mRNA and protein was examined by Northern blot analysis and immunohistochemistry respectively in both species across the menstrual cycle and during early pregnancy. Semi-quantitative statistical analysis of the immunohistochemistry results revealed that, in the human, CaBP-d28k protein expression was maximal in luminal and glandular epithelium during the mid-secretory phase, coinciding with the time when the endometrium is receptive to embryo implantation. Expression in rhesus monkey showed a similar trend. These results suggest that, in the primate endometrium, only CaBP-d28k is expressed and that the specific regulation of this calbindin is potentially important for the establishment of uterine receptivity.     

Production of cloned horse foals using roscovitine-treated donor cells and activation with sperm extract and/or ionomycin

We evaluated the effect of different activation treatments on the production of blastocysts and foals by nuclear transfer. Donor cells were prepared using roscovitine treatment, which has previously been associated with increased production of viable offspring. All activation treatments were followed by culture in 6-dimethylaminopurine (6-DMAP) for 4 h. In experiment 1, blastocyst production after activation by injection of sperm extract followed by treatment with ionomycin was significantly higher than that for activation with a serial treatment of ionomycin, 6-DMAP, and ionomycin (12.5 vs 2.8%; P < 0.05) and tended to be higher than that for injection of sperm extract alone (3.4%; P = 0.07). In experiment 2, there were no significant differences in blastocyst development among treatments with ionomycin once or twice, sperm extract then ionomycin, or ionomycin then sperm extract (range 4.6-7.3%). Overall, transfer of 26 blastocysts resulted in 16 pregnancies (62%) and 9 live foals (35% of transferred embryos). Treatment with sperm extract followed by ionomycin produced a live foal rate per embryo transferred of 5/10 (50%). One foal died of pneumonia 48 h post partum and one foal died at 1 week of age after complications during induction of anesthesia; the remaining seven foals are currently 10-14 months of age.     

Basigin expression and hormonal regulation in the rat uterus during the peri-implantation period

Basigin is essential for fertilization and implantation. The aim of this study was to determine the expression and hormonal regulation of the basigin gene in the rat uterus during the peri-implantation period. Basigin mRNA was localized strongly in the luminal epithelium on day 1 of pregnancy and gradually decreased to a basal concentration from day 3 to day 5 of pregnancy. Basigin mRNA and protein were expressed strongly in the implanting blastocyst and primary decidua on day 6 of pregnancy. A similar expression pattern was also induced in the uterus after delayed implantation was terminated by oestrogen treatment and the embryo implanted, whereas expression was not detected during delayed implantation. Basigin expression was not detected on day 6 of pseudopregnancy. Basigin mRNA was expressed strongly in the decidua on days 7 and 8 of pregnancy. Furthermore, both basigin mRNA and protein were induced in the decidua during artificial decidualization. In addition, oestrogen stimulated strong expression of basigin mRNA in the uterine epithelium of ovariectomized rats. These findings indicate that basigin may play a role during implantation and decidualization in rats.     

Fetal bovine serum influences apoptosis and apoptosis-related gene expression in porcine parthenotes developing in vitro

This study was conducted to determine the effects of polyvinyl alcohol (PVA), fetal bovine serum (FBS) and bovine serum albumin (BSA) on blastocoel formation, total cell number, apoptosis and Bcl-xL and Bak gene expression in porcine presumptive diploid parthenotes developing in vitro. The addition of 0.4% BSA to the culture medium enhanced the development of 2-cell or late 4-cell stage parthenotes to the blastocyst stage (P < 0.01) while FBS decreased the incidence of blastocoel formation. FBS also reduced the frequency of blastocysts developed from both 2-cell (P < 0.001) and late 4-cell (P < 0.05) embryos and increased the percentage of blastocysts undergoing apoptosis (P < 0.001). The relative abundance of Bcl-xL mRNA in presumptive diploid parthenotes in the control, PVA- and BSA-supplemented medium was similar to that of in vivo-derived embryos, but was significantly higher than in parthenotes cultured with FBS supplement (P < 0.05). Bak mRNA significantly increased at the blastocyst stage in FBS-supplemented cells (P < 0.01). These results suggest that apoptosis-related gene expression is significantly affected by FBS, and that this may result in alteration of apoptosis and embryo viability of porcine embryos developing in vitro.