Hanne Skovsgaard Pedersen

Combinatorial synthesis of benztropine libraries and their evaluation as monoamine transporter inhibitors

A combinatorial synthesis of benztropine analogues is presented. Radical azidonation of 3-benzyloxy-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester 3 to 3-(1-azidobenzyloxy)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester 4 was used as a key step in the synthesis. This step was optimized by adding 10% DMF to the reaction. Reaction of 4 with phenyl magnesium bromide followed by Boc removal and N-methylation gave benztropine 1. Reaction of five-component Grignard reagents with 4 was used to create a two-dimensional library of 25 N-normethylbenztropine analogues. Further reaction of this library with five alkyl bromides was carried out to create a three-dimensional library containing 125 compounds. Screening of the libraries towards binding and inhibition of uptake of the human dopamine (hDAT), serotonin (hSERT) and norepinephrine transporters (hNET) was carried out. None of the synthesized compounds were found to be stronger than benztropine, and none were selective for inhibition of binding over monoamine uptake.

Developmental potential of pig embryos reconstructed by use of sow versus pre-pubertal gilt oocytes after somatic cell nuclear transfer

In this study, the developmental ability of cloned embryos using gilt versus sow oocytes was evaluated under the hypothesis that the efficiency of nuclear transfer using gilt oocytes was lower than that of sow oocytes, but that it could be optimized. Five experiments were performed with routine production of cloned embryos with sow oocytes serving as the control. Results showed that: Experiment 1: Blastocyst rates of cloned embryos with gilt oocytes was about half compared with control. Experiment 2: An extended maturation time of 48 h used for gilt oocytes resulted in lower blastocyst rates after cloning. Experiment 3: Development of cloned embryos with gilt oocytes was improved by co-culture with sow oocytes. Experiment 4: After maturation of gilt oocytes using follicular fluid from gilt instead of sow, the oocytes were sorted into large and small oocytes, and after cloning, blastocyst rates were higher using large gilt oocytes compared with small oocytes; however, the rate remained lower compared with control. Experiment 5: Six sow recipients received a total of 503 morulae and blastocysts cloned from gilt oocytes (four recipients) and 190 cloned from sow oocytes (two recipients). All recipients became pregnant and went to term, resulting in 26 (gilt oocytes) and six (sow oocytes) piglets. In conclusion, results confirmed that nuclear transfer efficiency was higher using sow versus gilt oocytes, but the use of gilt oocytes can be optimized by sorting after ooplasm size following maturation and by maturing gilt and sow oocytes together.

Effect of ambient light exposure of media and embryos on development and quality of porcine parthenogenetically activated embryos.

Light exposure is a common stress factor during in vitro handling of oocytes and embryos that originates from both microscope and ambient light. In the current study, the effect of two types of ambient light (daylight and laboratory light) on porcine parthenogenetically activated (PA) embryos was tested in two experiments: (1) ambient light on medium subsequently used for embryo in vitro development; and (2) ambient light exposure on activated oocytes before in vitro development. The results from Experiment 1 showed that exposure of culture medium to both types of ambient light decreased the percentage of blastocysts that showed good morphology, only after 24 h exposure. The results from Experiment 2 revealed a reduction in both blastocyst formation and quality when activated oocytes were exposed to both types of ambient light. This effect was seen after only 1 h exposure and increased with time. In conclusion, exposure to ambient light can be harmful to embryo development, both when medium is exposed for a long period of time and, to a greater extent, when the embryo itself is exposed for >1 h. In practice, it is therefore recommended to protect both culture medium and porcine embryos against ambient light during in vitro handling in the laboratory.

Identification of potential biomarkers in donor cows for in vitro embryo production by granulosa cell transcriptomics

The Ovum Pick Up-In vitro Production (OPU-IVP) of embryos is an advanced reproductive technology used in cattle production but the complex biological mechanisms behind IVP outcomes are not fully understood. In this study we sequenced RNA of granulosa cells collected from Holstein cows at oocyte aspiration prior to IVP, to identify candidate genes and biological mechanisms for favourable IVP-related traits in donor cows where IVP was performed separately for each animal. We identified 56 genes significantly associated with IVP scores (BL rate, kinetic and morphology). Among these, BEX2, HEY2, RGN, TNFAIP6 and TXNDC11 were negatively associated while Mx1 and STC1 were positively associated with all IVP scores. Functional analysis highlighted a wide range of biological mechanisms including apoptosis, cell development and proliferation and four key upstream regulators (COX2, IL1, PRL, TRIM24) involved in these mechanisms. We found a range of evidence that good IVP outcome is positively correlated with early follicular atresia. Furthermore we showed that high genetic index bulls can be used in breeding without reducing the IVP performances. These findings can contribute to the development of biomarkers from follicular fluid content and to improving Genomic Selection (GS) methods that utilize functional information in cattle breeding, allowing a widespread large scale application of GS-IVP.

Ultrastructure and mitochondrial numbers in pre- and postpubertal pig oocytes

Prepubertal pig oocytes are associated with lower developmental competence. The aim of this experiment was to conduct an exhaustive survey of oocyte ultrastructure and to use a design-unbiased stereological approach to quantify the numerical density and total number of mitochondria in oocytes with different diameters from pre- and postpubertal pigs. The ultrastructure of smaller prepubertal immature oocytes indicated active cells in close contact with cumulus cells. The postpubertal oocytes were more quiescent cell types. The small prepubertal oocytes had a lower total mitochondrial number, but no differences were observed in mitochondrial densities between groups. Mature postpubertal oocytes adhered to the following characteristics: presence of metaphase II, lack of contact between cumulus cells and oocyte, absence of rough endoplasmic reticulum and Golgi complexes, peripheral location of cortical granules and central localisation of mitochondria, vesicles and lipid droplets. Prepubertal oocytes displayed more variation. The ultrastructure of large pre- and postpubertal oocytes was compatible with higher developmental competence, whereas that of smaller prepubertal oocytes could explain their reduced capacity. The higher number of mitochondria in large pre- and postpubertal oocytes could have an influence on oocyte competence, by increasing the pool of mitochondria available for early embryonic development.

Effects of post-mortem storage conditions of bovine epididymides on sperm characteristics: investigating a tool for preservation of sperm from endangered species

To preserve genetic diversity in threatened populations of domestic and wild animals, cryopreservation combined with reproductive technologies are useful. Here, we present a reliable and efficient protocol enabling preservation of epididymal sperm using domestic cattle as a model species. We propose that this technique has great potential in conservation of threatened species because it can be used to rescue genetic variation in the event of unexpected death, castration or in situations where obtaining an ejaculate is not feasible.

Analysis of nucleolar morphology and protein localization as an indicator of nuclear reprogramming.

When a cell is reprogrammed to a new phenotype, the nucleolus undergoes more or less dramatic modulations, which can be used as a marker for the occurrence of the reprogramming. This phenomenon is most pronounced when differentiated cells are reprogrammed to totipotency when they are submitted to cloning by somatic cell nuclear transfer. However, when cells are reprogrammed by less fundamental means, as for example treatment by Xenopus extract or expression of pluripotency genes, more subtle nucleolar modulations can also be noted. The monitoring and understanding of the reprogramming-related nucleolar modulations are based upon detailed knowledge about the nucleolar changes that occur during normal development from the developing oocyte over oocyte maturation and fertilization to the activation of the embryonic genome in the early embryo. Below, the ultrastructural and molecular modulations of the nucleolus are summarized in this developmental context, but also as they occur in assisted reproductive technologies such as in vitro fertilization and somatic cell nuclear transfer. Moreover, detailed protocols for monitoring the nucleolar changes by transmission electron microscopy and immunocytochemistry are presented.

96 IN VITRO FERTILIZATION IN MOUSE AS A REPROTOXICITY MODEL FOR XENOBIOTICS

To assess reprotoxicity of pesticides and other xenobiotics, rodents are often used with natural mating and litter size as end-points. However, because a male mouse can produce normal-size litters even with >50% reduced sperm count (Ecotox. Env. Saf. 73, 1092), this model could approve some chemical substances despite their reprotoxic effect. Thus, we aimed to establish a mouse IVF system to evaluate reprotoxicity of xenobiotics, illustrated by 2 pesticides [vinclozolin (vin), chlormequat (ccc)]. Experimental mice were given 2 pesticide doses by mixing into the feed: low (L) or high (H), equivalent to no or lowest observed adverse effect level, respectively (Env. Health Perspect. 117, 1272). To produce experimental males, mated NMRI females were fed low dose (vinL, cccL), high dose (vinH, cccH), or no pesticide from 1-cell stage through birth and until weaning, from where experimental males continued pesticide feeding. Each experimental male at 8 to 10 weeks old was naturally mated with 2 females having normal feed for 5 days, then continued with pesticide or control feed for at least 35 days before use for IVF. Naturally mated females had normal feed until birth to evaluate litter size. For IVF, oocytes were collected after superovulation of 3- to 4-week-old C57BL/6J females (2-5/male). Based on our previous experiment (Anim. Reprod. 13, in press), 25,000 sperm/mL was within the responsive range and selected for IVF (Theriog. 65, 1716). The IVF embryos were in vitro cultured for 4 days. Pronuclei were evaluated 9h after IVF start (Day 0), and 2-cell/blastocyst were evaluated on Day 1/4. Mean litter size was estimated by normal linear mixed-effects model, and mean pronuclei, 2-cell, and blastocyst rates were estimated by binomial generalized linear mixed-effects models with identity link function, included a male-subject random intercept accounting for correlation between multiple fertilizations by same male, and a factor defining groups as fixed effect. Preliminary results (Table 1) are based on current data from half of our full experiment. No signs of different litter sizes after natural mating were observed between pesticide groups and control. Compared with control, rates of pronuclei, 2-cell, and blastocyst tended to decrease in vinH and increase in vinL group. No clear differences between cccL, cccH, and control groups were found. These results could indicate a pesticide effect (vinH) on mouse male reproductive system that can be detected in an IVF system but not by natural mating. Our data show a large variation in IVF results between individual males and females, so our full dataset is required before conclusions are made.

In vitro production of bovine embryos: cumulus/granulosa cell gene expression patterns point to early atresia as beneficial for oocyte competence: Cumulus/granulosa cell gene expression patterns point to early atresia as beneficial for oocyte competence

In vitro production (IVP) of bovine embryos has become widespread technology implemented in cattle breeding and production. Here, we review novel data on cumulus/granulosa cell gene expression, as determined by RNAseq on cellular material from pooled follicular fluids at the single animal level, and relate these finding to previous data on oocyte developmental competence and ultrastructure. The cumulus/granulosa cell gene expression patterns indicate that early follicular atresia is associated with increased blastocyst yield and this hypothesis is supported by previous data on oocyte competence and ultrastructure.

Basic and practical aspects of pregnancy establishment in cattle

Bovine embryos are increasingly produced using reproductive technologies, e.g. ovum pick-up (OPU), in vitro embryo production (IVP) and embryo transfer (ET). Such in vitro manipulated embryos are known to deviate in several aspects compared to in vivo derived embryos. Pregnancy establishment in cattle involves timed biological events including fine-tuned communication, initiated and carried out by both the embryo and the endometrium. This stimulates research to increase the understanding of events and interactions taking place in the uterus after embryo transfer, both from a biological and systems biology point of view. This review will focus on the biological events taking place during early embryonic development, implantation and beginning of placentation, with focus on transfer of in vitro produced embryos, including a systems biology approach for selection of superior embryo recipients.