U. Besenfelder

Molecular Mechanisms and Pathways Involved in Bovine Embryonic Genome Activation and Their Regulation by Alternative In Vivo and In Vitro Culture Conditions

ABSTRACT Understanding gene expression patterns in response to altered environmental conditions at different time points of the preimplantation period would improve our knowledge on regulation of embryonic development. Here we aimed to examine the effect of alternative in vivo and in vitro culture conditions at the time of major embryonic genome activation (EGA) on the development and transcriptome profile of bovine blastocysts. Four different blastocyst groups were produced under alternative in vivo and in vitro culture conditions before or after major EGA. Completely in vitro- and in vivo-produced blastocysts were used as controls. We compared gene expression patterns between each blastocyst group and in vivo blastocyst control group using EmbryoGENEs bovine microarray. The data showed that changing culture conditions from in vivo to in vitro or vice versa, either before or after the time of major EGA, had no effect on the developmental rates; however, in vitro conditions during that time critically influenced the transcriptome of the blastocysts produced. The source of oocyte had a critical effect on developmental rates and the ability of the embryo to react to changing culture conditions. Ontological classification highlighted a marked contrast in expression patterns for lipid metabolism and oxidative stress response between blastocysts generated in vivo versus in vitro, with opposite trends. Molecular mechanisms and pathways that are influenced by altered culture conditions during EGA were defined. These results will help in the development of new strategies to modify culture conditions at this critical stage to enhance the development of competent blastocysts.

Contribution of the female reproductive tract to low fertility in postpartum lactating dairy cows.

Infertility in dairy cattle is a multifactorial problem that may be linked to follicle development and the quality of the ovulated oocyte, to sperm transport and fertilization, to the reproductive tract environment, or to a combination of these factors. Using a state-of-the-art endoscopic embryo transfer technique, the aim of this study was to compare the ability of the reproductive tract of postpartum dairy cows and nulliparous heifers to support the development of early embryos to the blastocyst stage. Bovine embryos of 2 to 4 cells (n=1,800) were produced by in vitro maturation and fertilization of oocytes derived from the ovaries of slaughtered cattle. The estrus cycles of nulliparous Holstein heifers (n=10) and postpartum Holstein cows (n=8, approximately 60 d postpartum) were synchronized using an 8-d controlled internal drug release device coupled with prostaglandin injection. On d 2, one hundred 2- to 4-cell embryos were endoscopically transferred to the oviduct ipsilateral to the corpus luteum. Five days later, on d 7, the oviduct and uterus were flushed nonsurgically to recover the embryos. The number of embryos developing to the blastocyst stage was recorded immediately at recovery and following overnight culture in vitro. A representative number of blastocysts from heifers and cows were stained to assess cell number. Progesterone concentrations were lower in cows than in heifers on d 5, 6, and 7 (d 7=2.39+/-0.33 vs. 5.34+/-0.77ng/mL, respectively). More embryos were recovered from heifers than cows (79.0+/-7.0 vs. 57.2+/-11.4%). Of the embryos recovered, 33.9+/-3.6% had developed to the blastocyst stage in the heifer oviduct compared with 18.3+/-7.9% in the postpartum cow oviduct. There was no evidence of a difference in blastocyst quality as evidenced by total cell number in the blastocysts (71.2+/-5.7 vs. 67.0+/-5.3, respectively). In conclusion, the reproductive tract of the postpartum lactating dairy cow may be less capable of supporting early embryo development than that of the nonlactating heifer, and this may contribute to the lower conception rates observed in such animals.

Repeated endoscopic ovum pick-up in sheep

Endoscopy is an effective and minimally invasive technique which offers the possibility of repeated ovum pick-up (OPU). In this study 4 different treatment programs (Groups A, B, C and D) for repeated endoscopic OPU in sheep were investigated. The number of follicles and oocytes, quality of cumulus-oocyte-complexes (COCs), and detectable effects on fertility of the donor ewes were compared. Each group consisted of 5 East Friesian Milksheep. In Group A, follicles were punctured twice a week, in Group B once a week, and in Group C once a week followed by administration of 1500 IU PMSG 48 h prior to OPU. In Group D follicles were punctured and the sheep stimulated with 1500 IU PMSG 48 h prior to OPU once every 2 weeks. The PMSG-stimulated sheep received anti-PMSG immediately after OPU. Over a period of 10 weeks 216 OPU-sessions were performed. A total of 1978 follicles was punctured, and 1098 oocytes were recovered, for a collection rate of 55.5%. In the Groups A, B, C and D an average of 6.8, 8.6, 12.2 and 14.9 follicles per animal and session was aspirated, and an average of 3.8, 4.9, 7.0 and 7.6 COCs per animal per session was recovered, respectively. No significant differences between groups were observed in the collection rates (51.1 to 57.1%) or in the quality of the COCs, and 65 to 70% of the COCs were suitable for in vitro production of ovine embryos. Seven sheep developed small adhesions between the ovary and infundibulum. After the study 15 ewes became pregnant following natural mating with the same fertile ram (5 from Group A, 1 from Group B, 4 from Group C and 5 from Group D). In conclusion, OPU once a week in PMSG/anti-PMSG treated ewes was found to be the most effective treatment program for oocyte collection.

Expression of synthetic cDNA sequences encoding human insulin-like growth factor-1 (IGF-1) in the mammary gland of transgenic rabbits

We have developed an expression system where foreign proteins are synthesized specifically in the mammary gland of transgenic rabbits and secreted into the milk. Regulatory elements were isolated from the bovine alpha S1-casein-encoding gene and combined with a synthetic DNA coding for human IGF-1 and for [Gln58]IGF-1, an IGF-1 analogue. The resulting hybrid DNA constructs were used to generate transgenic rabbits. Females of seven transgenic lines tested were positive for synthesis of IGF-1. Transmission of the transgene to progeny and IGF-1 production in female offspring was observed in all transgenic lines analysed. As expected, expression of transgene mRNA could only be detected in the mammary gland. Production levels of transgenic protein were as high as 1 g IGF-1 per liter rabbit milk. IGF-1, as well as [Gln58]IGF-1, when secreted into rabbit milk, was correctly processed and biologically active. IGF-1 was purified from the milk of transgenic rabbits to a nearly homogenous active form.

Effect of Elevated Circulating Progesterone Concentration on Bovine Blastocyst Development and Global Transcriptome Following Endoscopic Transfer of In Vitro Produced Embryos to the Bovine Oviduct

Elevated concentrations of circulating progesterone in the immediate postconception period have been associated with an increase in embryonic growth rate, interferon-tau production, and pregnancy rate in cattle and sheep. Much of this effect is likely mediated via downstream effects of progesterone-induced changes in gene expression in the uterine tissues. Using state-of-the-art endoscopic techniques, this study examined the effect of elevated progesterone on the development of in vitro produced bovine zygotes transferred to the oviducts of heifers with high or normal circulating progesterone concentrations and on the transcriptome of blastocysts developing under such conditions. Simmental heifers (n = 34) were synchronized using a controlled internal drug release (CIDR) device for 8 days, with a prostaglandin F2alpha analogue administered 3 days before removal of the CIDR device. Only animals exhibiting a clear standing estrus (Day 0) were used. To produce animals with divergent progesterone concentrations, half of the animals received a progesterone-releasing intravaginal device (PRID) on Day 3 of the estrous cycle; the PRID was left in place until embryo recovery. All animals were sampled for blood daily from Day 0 to Day 7. Cleaved embryos were transferred by endoscopy to the ipsilateral oviduct of each recipient on Day 2 and then recovered by nonsurgically flushing the oviduct and the uterus on Day 7. The number of embryos developing to the blastocyst stage was recorded at recovery and following overnight culture in vitro. Potential effects of elevated progesterone on transcript abundance were examined using the Affymetrix GeneChip Bovine Genome Array. Insertion of a PRID on Day 3 resulted in a significant elevation of progesterone concentration (P < 0.05) from Day 3.5 until Day 6. Elevated progesterone did not affect the proportion of embryos developing to the blastocyst stage. Genomewide gene expression analysis identified 194 differentially expressed genes between embryos collected from heifers with normal or elevated progesterone, and quantitative real-time PCR validation with a subset of selected genes and an independent sample confirmed the microarray results. Interaction network analysis indicated a significant interaction between progesterone-regulated genes in the blastocyst and in the maternal endometrium. These results suggest that elevated concentrations of progesterone do not affect the ability of the early embryo to reach the blastocyst stage in vivo but do result in subtle changes to the transcriptome of the embryo that may be associated with advanced elongation posthatching.

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.

Stable Production of Human Insulin-like Growth Factor 1 (IGF-1) in the Milk of Hemi- and Homozygous Transgenic Rabbits Over Several Generations

One transgenic rabbit line was generated carrying a fusion gene consisting of the cDNA for human IGF-1 fused to a mammary gland specific expression cassette derived from bovine alpha-S1-casein sequences. Transgene expression was shown to be strictly tissue and lactation period specific. The transgenic rabbit line was bred for six generations. All transgenic animals showed stable production of biologically active IGF-1 over the generations and no apparent effect on the physiological or reproductive performance was observed. The absence of adverse effects on homozygous transgenic rabbits suggested the absence of insertional mutagenesis. Eight hemizygous transgenic offspring analysed produced on average 363 ± 12μg/ml (ranging from 223 ± 61 to 484 ± 39 μg/ml) mature human IGF-1 in their milk, whereas three homozygous animals produced on average 543 ± 41 μg/ml (ranging from 360 ± 15 to 678 ± 80 μg/ml). Homozygous huIGF-1 females clearly showed a significantly increased production performance of the recombinant protein.

Tyrosinase gene variants in different rabbit strains

Tyrosinase (EC 1.14.18.1) encoded by the c-locus is known to be the key enzyme in the melanin production. It is expressed in melanocytes, occurring mainly in the skin and the pigment cell layers of the eyes. The mammalian tyrosinase coding sequence has a length of 1.6 kb and is composed of 5 exons. Tyrosinase gene mutations in humans and mice were shown to give rise to different pigmentation phenotypes, ranging from complete albinism to intermediate phenotypes (Jackson 1994; Oetting and King 1999). In this study, eight European rabbit ( Oryctolagus cuniculus ) strains covering a wide range in the extent of coat pigmentation were examined for the presence of strain-specific tyrosinase coding sequence variants. The different rabbit strains are described in Table 1 and shown in Fig. 1. Two strains (R1: German Giant, and R2: Belgian Hare) showed complete pigmentation of coat and eyes. Using comparative sequence analysis, specific nucleotide exchanges in the coding region of the tyrosinase gene were suggested to cause the phenotypes ‘chinchilla’ (R4), ‘california’ (R5, adult animals showing coat pigmentation only at the extremities) and ‘albino’ (R7: White New Zealand, and R8: ZIKA t-Hybrid). In addition, rabbits showing only eye pigmentation (R6: Vienna White) were examined. Direct sequencing analysis of PCR/RTPCR amplification products of three individuals of each strain was carried out to avoid false interpretation of the nucleotide sequences owing to the possible occurrence of artificial failures in the enzymatic amplification of PCR/RT-PCR products. In addition, individual clones of the PCR/RT-PCR amplificates were examined. The coding sequence of the rabbit tyrosinase gene has a length of 1593 nt (GenBank Accession No. AF210660). The designation of the signal peptide, the six putative N-glycosylation sites, and of the exon boundaries of exons 2 to 5 of the rabbit tyrosinase coding sequence was carried out by comparing the sequence to the wildtype coding sequences of humans and mice (human: GenBank Access. No. AH003020, homologous to M27160; mouse: GenBank Access. No. D00440, homologous to M24560). The mature rabbit tyrosinase contains 512 aa (human: 511 aa; mouse: 515 aa) led by the 18-aa signal peptide. The polyadenylation signal (AATAAA) was observed 261 nt after the translation stop codon and 24 nt before the poly(A) tail. Compared with other mammalian tyrosinase gene sequences, 82% (mouse) and 88% (human) identities were observed in the rabbit coding nucleotide sequence of the mature protein; the rabbit tyrosinase amino acid sequence showed 85% (mouse) and 90% (human) identity. The differences in the length of the protein as well as a major part of the higher rate of non-homology in the mouse amino acid sequence are caused by differences in the C-terminal end of the protein. Comparing the different rabbit strains, the following observations were made (Table 1): The albino strains R7 and R8 showed a specific nucleotide exchange leading to an amino acid exchange (Thr, conserved in human and mouse → Lys) in the last N-glycosylation site. The nucleotide exchange at nt 881 of the tyrosinase coding sequence (Glu , conserved in human and mouse→ Gly) was observed in both the ‘chinchilla’ (R4) and the ‘california’ (R5) rabbit strains, whereas R4 showed an additional specific amino acid exchange (Thr 358 → Ile). At all three positions described above, no heterozygosity was observed in any of the animals examined. In strain R6, showing only eye but not coat pigmentation, tyrosinase transcripts were found in the pigment cell layers of the eyes, but not in the skin. Absence of tyrosinase gene transcription products in the coat of the animals of strain R6 was proven by repeated nested PCR analysis. In addition, few non-strain-specific coding sequence missense mutations were observed. Nucleotide exchanges were seen at nt 91 G → A (Val → Met, conserved in humans) in all three animals of strain R2 (homozygous), and in one of three animals of strain R1 (homozygous) and R6 (heterozygous) each. Absence of differences in the pigmentation of strain R1 indicated that this mutation does not affect the function of the tyrosinase. The nucleotide exchange at nt 860 G→ C (Ser, conserved in humans → Thr) was revealed in two of three rabbits of strain R2; the third animal of strain R2 was homozygous for the wild-type sequence at this position. Both non-strain-specific missense mutations have not yet been described in mutant humans or mice. In a minority of the examined animals, a silent polymorphism at nt 576 C → T (conserved in human and mouse) was found, thereby altering the BamHI restriction site. Fig. 2 shows the position of both strainspecific and non-strain-specific amino acid exchanges revealed in the eight rabbit strains in relation to the structural domains of the rabbit tyrosinase. The wild-type nucleotide sequence of the rabbit tyrosinase mRNA was determined in the animals of strain R1, which excluded the non-strain-specific nucleotide exchange cited in Table 1 (GenBank Access. No. AF210660). With the rabbit sequence as the third complete mammalian tyrosinase coding sequence in hand, the ‘classical’ tyrosinase mouse mutations resulting in aberrant pigmentation phenotypes may be reviewed. The substituted amino acids in the mouse mutants c, c, c andc (Jackson 1994) are conserved both in the wild-type sequences of rabbits and humans. The strain-specific rabbit tyrosinase coding sequences were compared with the complete mammalian wild-type tyrosinase coding sequences of humans and mice known to date to evaluate the consequences of the polymorphisms for the function of the enzyme. Mutations and polymorphisms of the tyrosinase gene of humans and mice are collected in various databases (human: http:// www.cbc.umn.edu/tad; mouse: http://www.informatics.jax.org). Rabbit strains are usually bred in an outbred breeding management. The different features of the coat pigmentation are the main characteristics for the definition, classification, and breeding regimen of the various strains. Therefore, tyrosinase as the key enzyme for the melanin production was suggested to show less The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession number AF210660.

Effect of reproductive tract environment following controlled ovarian hyperstimulation treatment on embryo development and global transcriptome profile of blastocysts: implications for animal breeding and human assisted reproduction

BACKGROUND In mammals, the reproductive tract plays a crucial role in the success of early reproductive events and provides an optimal microenvironment for early embryonic development. However, changes in the reproductive tract environment associated with controlled ovarian hyperstimulation and the influence on the embryo transcriptome profile have not been investigated. Therefore, we investigated differences in the development rate and the transcriptome profile of bovine blastocysts developing in the reproductive tract of unstimulated or superovulated heifers. METHODS Nineteen Simmental heifers were synchronized, superovulated and artificially inseminated; nine heifers were flushed on Day 2 after insemination and 2-4-cell stage embryos were recovered and endoscopicaly transferred to the ipsilateral oviduct of unstimulated (i.e. single-ovulating) synchronized recipients (n= 4 recipients; 25-50 embryos per recipient). The remaining 10 superovulated heifers and the unstimulated recipients were then non-surgically flushed on Day 7 to collect embryos. The blastocyst transcriptome profile was examined using the Affymetrix GeneChip Bovine Genome Array. RESULTS The proportion of embryos, which developed to the blastocyst stage, was lower in superovulated heifers than unstimulated heifers (P< 0.05). Blastocysts that developed under the abnormal endocrine conditions associated with ovulation induction showed higher cellular and metabolic activities, as genes involved in the oxidative phosphorylation pathway, different metabolic processes and translation and transcription processes, in addition to genes expressed in response to stress, were highly expressed compared with embryos that developed in the oviduct of unstimulated animals. CONCLUSIONS The environment in which the embryo develops in the oviduct/uterus significantly alters gene expression patterns, especially those genes that regulate metabolic activity in the embryo.

Influence of lactation on metabolic characteristics and embryo development in postpartum Holstein dairy cows

The aim of this study was to examine the direct effect of lactation on the ability of the reproductive tract of postpartum dairy cows to support early embryo development. Twenty-one primiparous Holstein heifers were used. Immediately after calving, half of the cows were dried off (i.e., never milked), and the other half entered the milking herd and were milked twice daily. Jugular blood samples were taken twice per week from 15 d before calving to approximately 100 d postpartum to measure nonesterified fatty acids, β-hydroxybutyrate, glucose, insulin, and insulin-like growth factor-I. At the same time, body weight and body condition score were recorded for each cow. At approximately 60 d postpartum (experiment 1), approximately 65 two- to four-cell embryos, produced by in vitro maturation and fertilization, were endoscopically transferred to the oviduct ipsilateral to the corpus luteum of all cows on d 2 of the estrous cycle. Five days later (d 7), the oviduct and uterus were flushed nonsurgically and the number of embryos developing to the blastocyst stage was recorded. At approximately 90 d postpartum (experiment 2), the estrous cycles of the same cows were resynchronized and 15 to 20 in vitro-produced blastocysts were transferred to the uterus of each recipient on d 7. All cows were slaughtered on d 14 to assess embryo survival and dimensions. Body weight and body condition score were significantly different between groups for the entire postpartum period of the study. Concentrations of nonesterified fatty acids and β-hydroxybutyrate were higher and concentrations of glucose, insulin, and insulin-like growth factor-I were lower in lactating compared with nonlactating cows. Embryo recovery rates from lactating and dry cows were similar. In experiment 1, fewer embryos developed to the blastocyst stage in the lactating cows compared with the nonlactating cows. In experiment 2, embryo survival and conceptus dimensions were not different between lactating and nonlactating cows. In conclusion, the data indicate that the reproductive tract of the lactating dairy cow is compromised in its ability to support early embryo development compared with that of matched dry cows and this may contribute to early embryo mortality observed in such animals.