Sheep Oviduct Research Articles

Expression profiles of oviductal mRNAs and lncRNAs in the follicular phase and luteal phase of sheep (Ovis aries) with 2 fecundity gene (FecB) genotypes.

FecB (also known as BMPR1B) is a crucial gene in sheep reproduction, which has a mutation (A746G) that was found to increase the ovulation rate and litter size. The FecB mutation is associated with reproductive endocrinology, such mutation can control external estrous characteristics and affect follicle-stimulating hormone during the estrous cycle. Previous researches showed that the FecB mutation can regulate the transcriptomic profiles in the reproductive-related tissues including hypothalamus, pituitary, and ovary during the estrous cycle of small-tailed Han (STH) sheep. However, little research has been reported on the correlation between FecB mutation and the estrous cycle in STH sheep oviduct. To investigate the coding and noncoding transcriptomic profiles involved in the estrous cycle and FecB in the sheep oviduct, RNA sequencing was performed to analyze the transcriptomic profiles of mRNAs and long noncoding RNAs (lncRNAs) in the oviduct during the estrous cycle of STH sheep with mutant (FecBBB) and wild-type (FecB++) genotypes. In total, 21,863 lncRNAs and 43,674 mRNAs were screened, the results showed that mRNAs had significantly higher expression levels than the lncRNAs, and the expression levels of these screened transcripts were lower in the follicular phase than they were in the luteal phase. Among them, the oviductal glycoprotein gene (OVGP1) had the highest expression level. In the comparison between the follicular and luteal phases, 57 differentially expressed (DE) lncRNAs and 637 DE mRNAs were detected, including FSTL5 mRNA and LNC_016628 lncRNA. In the comparison between the FecBBB and FecB++ genotypes, 26 DE lncRNAs and 421 DE mRNAs were detected, including EEF1D mRNA and LNC_006270 lncRNA. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional enrichment analysis indicated that the DE mRNAs were enriched mainly in terms related to reproduction such as the tight junction, SAGA complex, ATP-binding cassette, nestin, and Hippo signaling pathway. The interaction network between DE lncRNAs and DE mRNAs indicated that LNC_018420 may be the key regulator in sheep oviduct. Together, our results can provide novel insights into the oviductal transcriptomic function against a FecB mutation background in sheep reproduction.

Expression of dihydrotestosterone synthases and androgen receptor in sheep oviduct ampulla and its regulation by estradiol and progesterone

Oviduct ampulla plays an important role in steroid hormone-regulated sperm-oocyte binding in female animals. Although studies have shown that androgen receptor are expressed in many species oviduct, the interaction among androgen receptor (AR), estradiol (E2) and progesterone (P4) in the sheep oviduct have rarely been reported. In this study, we evaluated the localization of two isoforms of dihydrotestosterone (DHT) sythetase enzymes 5α-reductase (5α‐red1, 5α‐red2) and AR in sheep oviduct ampulla by immunohistochemistry and immunofluorescence. Results showed that they were all distributed in oviduct epithelium layer. In epithelial cells, 5α‐red1, 5α‐red2 were expressed in cytoplast and nuclear, but AR were stained in nuclear. We also investigated their expression pattern in the sheep oviduct ampulla at different development stages of follicles (Large follicles stage; hemorrhagium, luteum and albicans of corpus stage) by molecular experiments. We found that 5α‐red1, 5α‐red2 and AR mRNA abundance and protein were expressed highest in corpus albicans stage and lowest in corpus hemorrhagium stage. In vitro, when sheep oviduct ampulla epithelial cells (SOAECs) were cultured and treated with different concentrations of E2/P4 (10−9–10−6 M), we found that E2 inhibited the expression of AR mRNA and protein, while P4 promoted this expression. In addition, when the SOAECs were treated with E2 (10−8 M) and/or its non-selective inhibitor ICI182780 (10−7 M) as well as with P4 (10−6 M) and/or its non-specific inhibitor RU486 (10−5 M), we found that E2 and P4 inhibited and promoted the expression of AR mRNA and proteins, respectively, via their nuclear receptor pathways. This study provides a basic insight for the further research of oviduct epithelium physiological function closely related to androgen.

S100A8 expression in oviduct mucosal epithelial cells is regulated by estrogen and affects mucosal immune homeostasis.

Chronic inflammation can cause oviduct mucosal damage and immune dysfunction, leading to infertility, early pregnancy loss, ectopic pregnancy, tumors, and a decrease in reproductive capacities in female animals. Estrogen can suppress immune responses in different tissues and oviducts, and regulate the oviduct immune balance; however, the underlying mechanisms remain unclear. The objective of this study was to explore the mechanism of estrogen-regulated oviduct mucosal immunity and discover new estrogen targets for regulating oviduct mucosal immune homeostasis. Sheep oviduct epithelial cells (SOECs) were treated with 17-β estradiol (E2). Transcriptome sequencing and analysis showed differentially expressed S100 calcium-binding protein A (S100A) genes that may participate in the oviduct mucosa immunoregulation of estrogen. Quantitative polymerase chain reaction and immunocytochemistry analysis showed that S100A8 expression changed dynamically in E2-treated SOECs and peaked after 7 h of treatment. Estrogen nuclear receptors and G protein-coupled membrane receptors promoted E2-dependent S100A8 upregulation. The S100A8 gene was disrupted using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 method. Levels of inflammatory factors interleukin (IL)-1β and IL-4 were significantly upregulated in S100A8-knockdown SOECs, whereas those of the anti-inflammatory factor IL-10 was downregulated. Following S100A8 knockdown in SOECs treated with E2 for 7 h, IL-10 levels increased significantly. Estrogen affected oviduct mucosa immune function and dynamically regulated S100A8 in SOECs. S100A8 knockdown caused an excessive immune response, indicating that S100A8 is beneficial for maintaining immune homeostasis in the oviduct mucosa. Moreover, estrogen can compensate for the effect of S100A8 knockdown by upregulating IL-10.

Analysis of Expression Profiles of CircRNA and MiRNA in Oviduct during the Follicular and Luteal Phases of Sheep with Two Fecundity (FecB Gene) Genotypes.

Simple SummaryThe oviduct is an important part of the female reproductive organs, but few people pay attention to its role in the reproductive process. In this study, we perform RNA-Seq to analyze the expression of circRNA and miRNA in the oviduct of sheep (FecBBB and FecB++) during follicular and luteal phases. Enrichment analysis showed that the host genes of differentially expressed (DE) circRNAs were mainly enriched in the Rap1 signaling pathway, PI3K–Akt signaling pathway and neuroactive ligand–receptor interactions. Enrichment analysis showed that the target genes of DE miRNAs were mainly enriched in insulin secretion, the cAMP signaling pathway, the cGMP–PKG signaling pathway, the Rap1 signaling pathway, the TGF-β signaling pathway and other pathways related to reproduction. Our study, although not establishing direct causal relationships of the circRNA and miRNA changes, provides data for further exploring the mechanism of sheep fecundity.CircRNA and miRNA, as classes of non-coding RNA, have been found to play pivotal roles in sheep reproduction. There are many reports of circRNA and miRNA in the ovary and uterus, but few in the oviduct. In this study, RNA-Seq was performed to analyze the expression profile of circRNA and miRNA in the oviduct during the follicular phase and luteal phase of sheep with FecBBB and FecB++ genotypes. The results showed that a total of 3223 circRNAs and 148 miRNAs were identified. A total of 15 DE circRNAs and 40 DE miRNAs were found in the comparison between the follicular phase and luteal phase, and 1 DE circRNA and 18 DE miRNAs were found in the comparison between the FecBBB genotype and FecB++ genotype. GO and KEGG analyses showed that the host genes of DE circRNAs were mainly enriched in the Rap1 signaling pathway, PI3K–Akt signaling pathway and neuroactive ligand–receptor interactions. Novel_circ_0004065, novel_circ_0005109, novel_circ_0012086, novel_circ_0014274 and novel_circ_0001794 were found to be possibly involved in the oviductal reproduction process. GO and KEGG analyses showed that the target genes of DE miRNAs were mainly enriched in insulin secretion, the cAMP signaling pathway, the cGMP–PKG signaling pathway, the Rap1 signaling pathway and the TGF-β signaling pathway, and the target genes LPAR1, LPAR2, FGF18, TACR3, BMP6, SMAD4, INHBB, SKP1 and TGFBR2 were found to be associated with the reproductive process. Miranda software was used to identify 27 miRNAs that may bind to 13 DE circRNAs, including miR-22-3p (target to novel_circ_0004065), miR-127, miR-136 (target to novel_circ_0000417), miR-27a (target to novel_circ_0014274) and oar-miR-181a (target to novel_circ_ 0017815). The results of this study will help to elucidate the regulatory mechanisms of circRNAs and miRNAs in sheep reproduction. Our study, although not establishing direct causal relationships of the circRNA and miRNA changes, enriches the sheep circRNA and miRNA database and provides a basis for further studies on sheep reproduction.

17β-estradiol protects sheep oviduct epithelial cells against lipopolysaccharide-induced inflammation in vitro

Estrogen has known anti-inflammatory effects, but the mechanism whereby 17β-estradiol (E2) protects oviduct sheep epithelial cells from inflammation remains unknown. In this study, we detected the E2 synthetase and E2 nuclear and membrane receptors in sheep oviducts, primarily in epithelial cells. Using lipopolysaccharide (LPS)-stimulated sheep oviduct epithelial cells as an in vitro inflammation model, we demonstrated that E2 attenuates the expression of inflammatory factors in a concentration-response manner. E2 also inhibited the LPS-stimulated phosphorylation of p38 MAPK and NF-κB p65 but did not reduce the phosphorylation of JNK and ERK 1/2. This attenuation was partially antagonized by an intracellular estrogen antagonist that was involved in genomic regulation and enhanced by a G protein-coupled estrogen receptor agonist that was involved in nongenomic cellular modulation. These results suggest that E2 has an inhibitory effect on LPS-induced oviduct epithelial cell inflammation in sheep, which is mediated by the downstream regulatory effects of estrogen receptors.

The expression of melatonin receptors MT1 and MT2 is regulated by E2 in sheep oviduct

Some of the functions of melatonin in mammals are exerted through its membrane receptors (MRs) and studies have shown that estradiol (E2) might play an important role in regulating the expression of these proteins in female reproductive organs. However, no reports have reported the expression of MRs in the sheep oviduct or whether they are regulated by E2. Thus, herein, we detected the localization of MT1 and MT2 in the sheep oviduct. Moreover, we also investigated the expression pattern of these markers in the ovulating and non-ovulating side of the oviduct in the sheep ampulla and isthmus. Immunohistochemistry analyses revealed that both MT1 and MT2 are mainly expressed on oviduct epithelial cells. Both real-time polymerase chain reaction (qPCR) and western blot analyses showed that MT1 and MT2 genes and proteins are highly expressed on the non-ovulating side of the oviduct ampulla, but not the ovulating side. However, regarding the oviduct isthmus, there were no significant differences between the ovulating and non-ovulating sides. In vitro, 10 ng/ml and 1 µg/ml of E2, as well as 1 µg/ml of E2 combined with 0.1 µg/ml, 1 µg/ml, and 10 µg/ml of ICI182780 (a non-selective estrogenreceptor antagonist), were used to treat oviduct epithelial cells. We found that E2 inhibited the expression of MT1 and MT2 in cultured oviduct cells. Moreover, the inhibitory effect was suppressed by ICI182780. In conclusion, it was demonstrated that MRs are present in the sheep oviduct, and that E2, via the ER pathway, regulates their expression in the oviduct.

Role of the oviduct and oviduct-derived products in ruminant embryo development

The fact that embryos can be obtained in vitro undermines the role of the oviduct. However, it has been demonstrated that when in vitro produced bovine zygotes are cultured in the oviduct of sheep, cattle or mice the embryo quality is improved compared to the embryos produced in vitro. Thus the oviduct is not simply a passive organ required only for transporting the embryo to the uterus but also provides a suitable microenvironment for the early embryo. The study of physiological mechanisms and interactions between the embryo and the oviductal environment is essential to understand the correct processes of early embryo developmental. This knowledge can be used to improve current in vitro procedures providing high quality embryos capable of continued development and implantation, and resulting in viable births.

Role of the Oviduct in Early Embryo Development

This review highlights the role of the oviduct in early embryo development, which has to fulfil many aligned and well-tuned tasks during early embryogenesis. The oviductal lining is subjected to dynamic changes to timely accomplish gamete transport, fertilization and embryo development and to deliver a competent and healthy conceptus to the endometrium which can implant and develop to term. Although knowledge about the role of the oviduct is limited, we know that embryos are very sensitive to the environment in which they develop. The success of in vitro embryo production techniques demonstrates that it is possible to bypass the oviduct during early development and, to a certain extent, replicate the conditions in vitro. However, comparative studies show that embryos developed in vivo are superior to their in vitro produced counterparts, underlining our relatively poor knowledge of the biology of the oviduct. Oviduct activity is orchestrated by various factors, depending on cyclic dynamics, which crucially affect the success of tubal transfer and/or (re-)collection of embryos in embryo transfer studies. This paper reviews data which demonstrate that in vivo culture of embryos in the bovine oviduct is a useful tool for the assessment of embryos developed under various conditions (e.g. superovulation vs single ovulation, lactating dairy cows vs non-lactating cows). It is concluded that more work in the field of early embryo development within the oviduct would contribute to improved ART protocols leading to healthy pregnancies and offspring.

Angiogenesis in Day-30 Bovine Pregnancies Derived from Nuclear Transfer

Impaired placental angiogenesis during early pregnancy may result in placental defects that adversely affect development of nuclear-transfer (NT) embryos later in pregnancy. These experiments were designed to quantify and compare development of placental microvasculature and expression of genes associated with angiogenesis, including members of the VEGF and angiopoietin (Ang) families, in maternal and embryonic placental tissues of day 30 bovine concepti derived from NT or in vitro fertilization (IVF) followed by in vivo development to the blastocyst stage in the sheep oviduct. Microvascular volume density (MVD) within the caruncular tissues, as determined using Periodic Acid-Schiff's staining as well as immunohistochemical staining for von Willebrand's factor, was not different between NT- and IVF- derived pregnancies. Expression of genes implicated in angiogenic mechanisms, including VEGF-A and -C, placental growth factor (PlGF), VEGF receptors (Flt-1, Flk-1, and Flt-4), angiopoietin-1 (Ang1), Ang2, Tie1, Tie2, and hypoxia-inducible factor (HIF), were determined. In chorio-allantoic membranes, levels of PlGF transcripts were significantly lower in NT- than IVF-derived tissues (p<0.05), whereas HIF-1alpha transcription in chorio-allantoic membranes of cloned concepti was higher at p<0.10. Caruncular expression of HIF-1alpha and Ang1 also was increased in NT-derived pregnancies at p <or= 0.10. Immunohistochemical staining of caruncular tissues for VEGF-A and the Flt-1 receptor revealed few differences in protein expression between NT- and IVF-derived pregnancies. These results indicate that expression of most angiogenic factors at day 30 of gestation is not altered as a result of the NT procedure; however, given reports of impaired placental vascular development in NT-derived bovine embryos, perturbations in angiogenesis may occur subsequently during early placental development and throughout gestation. Elevated expression of the HIF-1alpha gene in maternal and chorio-allantoic tissues of cloned concepti may suggest a generalized hypoxic condition in early placental tissues of NT-derived concepti, which could adversely affect subsequent development of the placenta.

In Vitro Fertilization and Development of OPU Derived Goat and Sheep Oocytes

The recent upgrade in IVP technology seen in cattle can be adapted to embryo production in small ruminants to overcome limitations exhibited by surgical procedures on preserving the reproductive potential of donors and the efficiency of embryo production. The aim of the present study was to assess the current procedures used in cattle for the production of IVP embryos in goats and sheep based on laparoscopic-aided ovum pick-up (LOPU) supplied oocytes. Sexually matured goat and sheep donors were treated during the breeding season with FSH and subjected to laparoscopic-guided follicular puncture under general anaesthesia. The collected cumulus-oocyte complexes were matured in medium 199 and fertilized by frozen-thawed spermatozoa using Talp medium supplemented with heparin and oestrus-sheep serum. Cleaved ova were either cultured in sheep in vitro fertilization medium plus amino acids or transferred to sheep oviducts. Blastocyst rate, hatching rate and development rate up to term were used as markers of embryo function. The results obtained for goat and sheep involving 30 and 35 donors respectively (10 and 9 LOPU sessions) were 81.2% and 85.2% of oocyte collection rate; 88.3% and 98.6% oocyte incubation rate; 85.6% and 76.0% fertilization rate; 82.4% and 93.4% of cleavage rate; 50.0% and 61.5% IVP blastocyst rate; 42.1% and 45.5% blastocyst rate in oviducts; 73.0% and 66.7% embryo survival up to term, respectively. The results are comparable to those obtained in small ruminants and in bovines suggesting that requirements for embryo production and development are similar.

Birth of Cloned Pigs from Zona-Free Nuclear Transfer Blastocysts DevelopedIn Vitrobefore Transfer

The objective of this work was to obtain cloned pig offspring by uterine transfer of blastocysts produced by zona-free manipulation. We started by defining the most suitable culture media for growing pig nuclear transfer embryos produced by zona-free micromanipulation comparing NCSU-23aa with Synthetic Oviduct Fluid (SOFaa) and with in vivo culture in the sheep oviduct. We found that parthenogenetic development to day 7 blastocyst in NCSU-23aa and sheep oviduct was significantly superior as compared to SOFaa (61.8%, 64% and 42.4 respectively) although blastocyst cell number was higher in the latter. Interestingly, when we compared the two media for the culture of nuclear transfer (NT) embryos derived from 3 different donor cell lines, we observed lower rates of development with NCSU-23aa (from 24.5% to 32.4%) while with SOFaa the development was significantly higher for two donor cell lines as compared to the third (44.4%, 48.9% and 20.6% respectively). A total of 244 blastocysts grown in SOFaa were transferred in four synchronized sows on day 5 or 6 of development. Two recipients farrowed 6 and 8 piglets corresponding to an efficiency of development to term of 8% and 16% of the transferred embryos respectively. Eleven pigs are now 10 month of age and those that have reached puberty have been proven to be fertile. Finally, this is the first report on the production of cloned pigs derived from the transfer of NT embryos at the blastocyst stage.

Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer

Development of assisted reproductive technologies in horses has been relatively slow compared to other domestic species, namely ruminants and pigs. The scarce availability of abattoir ovaries and the lack of interest from horse breeders and breed associations have been the main reasons for this delay. Progressively though, the technology of oocyte maturation in vitro has been established followed by the application of ICSI to achieve fertilization in vitro. Embryo culture was initially performed in vivo, in the mare oviduct or in the surrogate sheep oviduct, to achieve the highest embryo development, in the range of 18–36% of the fertilised oocytes. Subsequently, the parallel improvement of in vitro oocyte maturation conditions and embryo culture media has permitted high rates of embryo development from in vitro matured and in vitro cultured ICSI embryos, ranging from 5 to 10% in the early studies to up to 38% in the latest ones. From 2003, with the birth of the first cloned equids, the technology of somatic cell nuclear transfer has also become established due to improvement of the basic steps of embryo production in vitro, including cryopreservation. Pregnancy and foaling rates are still estimated based on a small number of in vitro produced equine embryos transferred to recipients. The largest set of data on non-surgical embryo transfer of in vitro produced embryos, from ICSI of both abattoir and in vitro-matured Ovum Pick Up (OPU) oocytes, and from somatic cell nuclear transfer, has been obtained in our laboratory. The data demonstrate that equine embryos produced by OPU and then cryopreserved can achieve up to 69% pregnancy rate with a foaling rate of 83%. These percentages are reduced to 11 and 23%, respectively, for cloned embryos. In conclusion, extensive evidence exists that in vitro matured equine oocytes can efficiently develop into viable embryos and offspring.

Sex steroid receptor expression in the oviduct and uterus of sheep with estrus synchronized with progestagen or prostaglandin analogues

The objective of this study was to investigate differences in the expression of estrogen receptor-α (ERα), progesterone receptor (PR) and the proliferative indexes (Ki-67), in the uterus and oviduct of sheep with estrus synchronized either by prostaglandin analogues (Group PA, n = 27) or by treatment with progestagens (Group P, n = 29) on days 4 and 7 (day 0 = estrus), when the embryos were collected. Immunohistochemical methods were used to quantify ERα, PR and Ki-67 in six superficial and deep compartments in the uterus and oviduct. The expression of ERα was significantly ( P < 0.01) lower in progestagen treated ewes than in prostaglandin analogues treated group in the luminal epithelium, superficial glands and superficial stroma in the uterus on day 4. The expression of PR was significantly lower in progesterone treated ewes than in the PA Group in the superficial gland ( P < 0.05) in both days studied. The lowest expression of PR was observed in the luminal caruncular epithelium and superficial glands in both treatments, obtaining the lowest levels on day 4 ( P < 0.05). There were significant differences between days 4 and 7 in the Ki-67 immunostaining in the luminal epithelium ( P < 0.01) and superficial glands ( P < 0.05). A higher cell proliferation was observed in the uterine epithelium ( P < 0.05) on day 4 in the animals treated with progestagens. Results indicate that sheep with synchronization of estrus with progestagens showed a reduction of ERα and PR protein expression in most of oviductal and uterine cells.

Measures of Maternal-Fetal Interaction in Day-30 Bovine Pregnancies Derived from Nuclear Transfer

Embryonic mortality and abnormal placental morphology have been reported by most researchers studying nuclear transfer (NT), and it now is accepted that placental anomalies and poor development of cloned embryos are related. As early as day 50 of gestation, cloned bovine concepti exhibit poor structural organization of the developing placentomes. These experiments were designed to identify alterations in maternal-fetal interactions during establishment of the placentas of NT-derived embryos at day 30 of gestation. Bovine NT embryos were produced using cultured fibroblast cells from a single Hereford donor cow, and control embryos were derived from in vitro fertilization (IVF). Following in vivo culture in ligated sheep oviducts, day-8 blastocysts were transferred to synchronized recipient heifers. Tissues recovered from viable day-30 pregnancies were analyzed by real-time RT-PCR, immunohistochemistry, and quantitative histological techniques. Immunoperoxidase staining of caruncular tissue from NT- and IVF-derived pregnancies revealed no significant differences in expression of the extracellular matrix proteins, collagen type IV and laminin, or the receptor subunits, integrins alpha1 and alpha3, suggesting that altered expression of these proteins at day 30 of gestation is not a primary cause of abnormal placentome structure in cloned concepti. Percentage of binucleate cells (BNC) within the trophoblast also was similar in NT- and IVF-derived pregnancies; however, expression of the BNC-specific placental lactogen (PL) transcript was elevated in NT-derived concepti (p < 0.05). These results indicate that regulation of PL transcription was altered in cloned day-30 placental tissues, suggesting the presence of irregular fetal-maternal signaling patterns that might undermine continued development of NT-derived concepti.

Gametes Alter the Oviductal Secretory Proteome

The mammalian oviduct provides an optimal environment for the maturation of gametes, fertilization, and early embryonic development. Secretory cells lining the lumen of the mammalian oviduct synthesize and secrete proteins that have been shown to interact with and influence the activities of gametes and embryos. We hypothesized that the presence of gametes in the oviduct alters the oviductal secretory proteomic profile. We used a combination of two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry to identify oviductal protein secretions that were altered in response to the presence of gametes in the oviduct. The oviductal response to spermatozoa was different from its response to oocytes as verified by Western blotting. The presence of spermatozoa or oocytes in the oviduct altered the secretion of specific proteins. Most of these proteins are known to have an influence on gamete maturation, viability, and function, and there is evidence to suggest these proteins may prepare the oviductal environment for arrival of the zygote. Our findings suggest the presence of a gamete recognition system within the oviduct capable of distinguishing between spermatozoa and oocytes.

173 EFFECT OF CULTURE SYSTEM FOR IVM-IVF PIG EMBRYOS ON THE ICMS ABILITY TO PRODUCE OUTGROWTHS FOR EMBRYONIC STEM CELL DERIVATION

Attempts to derive true embryonic stem cells in large farm animals rely on the supply of good quality embryos. In these species, including the pig, pre-implantation-stage embryos can be produced by in vitro techniques from slaughterhouse ovaries. The objective of this study was to evaluate the ability of the inner cell masses (ICMs) of pig embryos, produced in vitro by different methods, to provide viable initial outgrowths of ICM cells that could be subsequently subcultured and expanded. Porcine oocytes were recovered from slaughtered donors and matured in vitro for 40–44 h in DMEM-F12 supplemented with 10% FCS, 0.05 IU LH and FSH (Menogon, Ferring, Milan, Italy), 0.3 mM cystine, 0.5 mM cysteamine, 50 ng/mL long-EGF, 100 ng/mL long-IGF1, 5 ng/mL bFGF (Sigma-Aldrich, Milan, Italy) in 5% CO2 at 38.5°C. Boar frozen-thawed semen was separated on a percoll gradient and diluted in TALP medium with PHE (penicillamine, hypotaurine, epinefrine) to a concentration ranging from 0.05 to 0.1 million sperm per mL. Oocytes were partially decumulated, co-incubated with sperm for 24 h, and finally denuded and cultured in microdrops of mSOFaa or NCSU. After cleavage, approximately half of the cleaved embryos were surgically transferred into the sheep oviduct for 4 days of in vivo culture and the remaining embryos were left in vitro in the two media. On Day +6 in vivo-cultured embryos were recovered from the sheep oviduct. Blastocyst formation and quality were comparatively evaluated in the three culture groups. Quality specifically referred to the morphology/size of the ICM according to the following criteria: ICM A (large/prominent), ICM B (flat), and ICM C (non-visible). All embryos with a visible inner cell mass were subjected to microdissection with needles to recover the ICMs that were then plated on feeder-layers of mitomycin-treated STO fibroblasts. Attachment and outgrowth was evaluated 48–72 h post-plating. Results are presented in Table 1. Our data indicate that in vivo culture of pig embryos in the sheep oviduct greatly enhance both blastocyst development and ICM quality. As a consequence the efficiency of outgrowth formation, following plating for ES cell derivation, was significantly higher with ICMs derived from IVM-IVF pig embryos cultured in vivo as compared to their in vitro-cultured counterparts. Within the two culture media tested for in vitro culture, SOF and NCSU, the rate of blastocyst formation was similar but the quality of SOF-cultured embryos is higher. In conclusion, embryo/ICM quality represents a fundamental requirement for the derivation of ES cell lines, and in vivo culture in the sheep oviduct provides the most efficient source of high quality IVM-IVF pig embryos. Table 1. Blastocyst development and ICM quality of in vitro-produced pig embryos This work was supported by the Istituto Superiore di Sanità, Programma Nazionale Cellule Staminali, Rome, Italy, grant No. CS 11.

42ANGIOGENESIS IN CLONED AND IVF-DERIVED BOVINE PREGNANCIES AT DAY 30 OF GESTATION

New and expanded microvascular networks in maternal and embryonic tissue are vital to development of a functional placenta. Typically a large proportion of bovine pregnancies derived from somatic cell nuclear transfer (NT) are lost between Days 25 and 45 of gestation. Several laboratories have noted aberrant placental development of NT embryos and fetuses; one factor that may contribute to these defects is faulty maternal or embryonic angiogenesis during placentation. The aim of this study was to determine if angiogenesis in maternal caruncular tissues differs in bovine NT- and IVF-derived pregnancies at the time of placentation. Cloned embryos were produced using cultured ear skin fibroblasts from a 15-year-old Hereford cow; in other experiments, these donor cells resulted in a term live calf and 67% of viable Day-30 embryos dying by Day 51 of gestation. For IVF-derived embryos, IVM oocytes were fertilized with semen from Hereford or Angus bulls. All embryos were cultured in vivo in ligated sheep oviducts, and at Day 8 blastocysts were transferred to synchronized recipient heifers. Whenever possible, one embryo was transferred to each uterine horn. A total of 41 NT and 41 IVF grade 1, 2 and 3 embryos were transferred to 48 recipients. Viable NT (n=9; 22.0% of transferred) and IVF (n=9; 22.0%) embryos were recovered at Day 30, and caruncular tissue adjacent to each embryo was sampled. Expression of genes implicated in angiogenesis was measured by real-time quantitative RT-PCR and normalized to histone 2A expression. In addition, paraformaldehyde-fixed sections were stained by the Periodic Acid-Schiffs method to identify blood vessels. Microvascular density was determined as percentage of total tissue area composed of blood vessels in the luminal caruncular stroma as measured by two observers. Data were analyzed by ANOVA using the GLM procedure of SAS (SAS Institute, Cary, NC, USA). Quantification of gene expression revealed no differences between NT and IVF pregnancies for angiogenesis-promoting growth factors and their receptors: vascular endothelial growth factor-A (VEGF-A), VEGF-C, placental growth factor, VEGF receptor-1 (VEGFR-1/flt-1), VEGFR-2 (flk-1/KDR), VEGFR-3 (flt-4), angiopoietin-1 (Ang-1), and Ang receptor-1 (tie-1). Likewise, NT and IVF pregnancies demonstrated similar mean microvascular densities in the caruncles; however, density differed between individual pregnancies across both groups (P&amp;lt;0.05). For these experiments, the timing of NT and IVF, embryo transfer, and tissue collection on Day 30 was as consistent as possible. Despite these efforts, embryo morphology and developmental stage were highly variable even within the IVF group. It follows that the progress of placentation, and hence the signaling between each embryo and the maternal tissues, may have differed as well, accounting for the variation observed. Our results suggest that failure of maternal tissue to increase angiogenesis during placentation is not a primary cause of aberrant placental development in cloned cattle.

First pregnancies carried to term after transfer of vitrified buffalo embryos entirely produced in vitro.

The application of Ovum Pick-up (OPU) technology, together with multistep embryo production in vitro (IVEP), represents a valid procedure for the recovery of oocytes from live donors and the attainment of a large number of embryos (Looney et al., 1994; Galli et al., 2001). In fact, collection of oocytes by means of ultrasound guided follicular aspiration is routinely performed with success in large prepubescent and adult ruminant species (Galli et al., 2001). In the buffalo species, because of their low response to superovulation treatments (Karaivanov, 1986), OPU technology, together with an improvement in the multistep process of in vitro embryo production, seems to be the best way to enhance genetic progression through the maternal lineage (Galli et al., 2001; Neglia et al., 2003). However, in vitro embryo production in buffalo has always been associated with a lower efficiency as compared to the bovine (Totey et al., 1992), although recently some encouraging results have been reported both from slaughterhouse recovered oocytes (Gasparrini et al., 2000), and from OPUderived oocytes (Neglia et al., 2003). Obviously, in order to optimize the procedure, an efficient method for embryo freezing should be developed. In fact, until now the only pregnancies recorded for the buffalo species were obtained from the transfer of fresh embryos produced in vitro. In 1991 Madan et al. (Madan et al., 1991) reported the birth of the first buffalo calf after the transfer of a fresh blastocyst obtained through in vitro maturation (IVM), in vitro fertilization (IVF) and in co-culture with oviductal cells (IVC). Four other calves were produced in India in 1994, by transfer of fresh blastocysts obtained in vitro (Madan et al., 1994), using the same method. In 1997, Boni et al. (Boni et al., 1997), reported two pregnancies obtained from the transfer of two fresh morulae, produced after OPU/IVEP procedures. Only three calves have been produced from frozen blastocysts (Galli et al., 1998); in this case zygotes (2 days after IVF) were transferred into ligated sheep oviduct before freezing. The possibility of cryopreserving buffalo embryos by vitrification with good efficiency in terms of in vitro survival rate, has recently been reported (Neglia et al., 2001). On

Effects of in vitro production on horse embryo morphology, cytoskeletal characteristics, and blastocyst capsule formation.

Blastocyst formation rates during horse embryo in vitro production (IVP) are disappointing, and embryos that blastulate in culture fail to produce the characteristic and vital glycoprotein capsule. The aim of this study was to evaluate the impact of IVP on horse embryo development and capsule formation. IVP embryos were produced by intracytoplasmic sperm injection of in vitro matured oocytes and either culture in synthetic oviduct fluid (SOF) or temporary transfer to the oviduct of a ewe. Control embryos were flushed from the uterus of mares 6-9 days after ovulation. Embryo morphology was evaluated with light microscopy, and multiphoton scanning confocal microscopy was used to examine the distribution of microfilaments (AlexaFluor-Phalloidin stained) and the rate of apoptosis (cells with fragmented or terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive nuclei). To examine the influence of culture on capsule formation, conceptuses were stained with a monoclonal antibody specific for capsular glycoproteins (OC-1). The blastocyst rate was higher for zygotes transferred to a sheep's oviduct (16%) than for those cultured in SOF (6.3%). Day 7 IVP embryos were small and compact with relatively few cells, little or no blastocoele, and an indistinct inner cell mass. IVP embryos had high percentages of apoptotic cells (10% versus 0.3% for in vivo embryos) and irregularly distributed microfilaments. Although they secreted capsular glycoproteins, the latter did not form a normal capsule but instead permeated into the zona pellucida or remained in patches on the trophectodermal surface. These results demonstrate that the initial layer of capsule is composed of OC-1-reactive glycoproteins and that embryo development ex vivo is retarded and aberrant, with capsule formation failing as a result of failed glycoprotein aggregation.

Cellular and molecular deviations in bovine in vitro-produced embryos are related to the large offspring syndrome.

The large offspring syndrome (LOS) is observed in bovine and ovine offspring following transfer of in vitro-produced (IVP) or cloned embryos and is characterized by a multitude of pathologic changes, of which extended gestation length and increased birthweight are predominant features. In the present study, we used bovine blastocysts to analyze cellular parameters, i.e., the number of cells in Day 7 blastocysts and the size of Day 12 elongating blastocysts, and molecular parameters, i.e., the relative abundance of developmentally important genes: glucose transporter (Glut) 1, Glut-2, Glut-3, Glut-4, heat shock protein (Hsp) 70.1, Cu/Zn-superoxide dismutase (SOD), histone H4.1, basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF) I receptor (R), and IGFII-R. Some blastocysts were produced by in vitro maturation and fertilization followed by in vitro culture in synthetic oviduct fluid medium supplemented with BSA or human serum or by in vivo culture in the sheep oviduct. Other blastocysts were derived in vivo from the uterine horns of superovulated donors. The findings made in the early embryos were related to a representative number of calves obtained from each production system and from artificial insemination (AI). In vitro culture of bovine embryos in the presence of high concentrations of serum or BSA significantly increased the number of cells in Day 7 blastocysts, the size of blastocysts on Day 12, and the relative abundance of the transcripts for Hsp70.1, Cu/Zn-SOD, Glut-3, Glut-4, bFGF, and IGFI-R when compared with embryos from the in vivo production groups. Birthweights of calves derived from IVP embryos were significantly higher than those of calves derived from sheep oviduct culture, superovulation, or AI. The results support the hypothesis that persistence of early deviations in development is causally involved in the incidence of LOS, in particular in increased birthweights. The cellular and molecular parameters analyzed in this study can be considered early markers of LOS in cattle.