Impact of in vitro fertilization of bovine oocytes with sex-sorted frozen-thawed spermatozoa on developmental kinetics, quality and sex ratio of developing embryos.

We studied whether bovine embryos developing after in vitro fertilization (IVF) with sex-sorted spermatozoa differed in developmental kinetics, quality and sex ratio from embryos produced with unsorted spermatozoa. Abattoir-derived oocytes were fertilized with X-sorted, Y-sorted or unsorted spermatozoa from a single bull. To evaluate economical use of the sex-sorted spermatozoa, washed spermatozoa from a single straw (2 million spermatozoa) were used to fertilize each batch of collected oocytes without any further isolation steps. Concentration of the unsorted spermatozoa was adjusted accordingly. Fertilizations were assessed by staining sperm asters at 10 hpi and pronuclei at 20 hpi. Embryo development and morphological quality were monitored on days 2, 7, 8 and 9 of the development (IVF = day 0). All embryos were sexed using PCR. Following fertilization, penetration and subsequent cleavage rates were compromised in the X-sorted group compared with the Y-sorted and unsorted groups (penetration: 58.0% vs. 89.8% and 90.0%, cleavage: 65.3% vs. 81.5% and 75.0%). The use of the sex-sorted spermatozoa did not, however, reduce the proportion of transferable embryos (sex-sorted 29.6% vs. unsorted 27.7%) or their quality (quality 1: sex-sorted 36.0% vs. unsorted 19.9%). The Y-sorted spermatozoa produced more transferable embryos of better quality than the X-sorted spermatozoa (days 7-8: 31.9% vs. 26.4%, quality 1: 38.9% vs. 30.6%). On average, out of 10 transferable embryos, nine were of the predicted sex in the X- and Y-sorted spermatozoa groups. These results indicate that low numbers of X- and Y-sorted spermatozoa can be used successfully for female and male embryo production in vitro.

Effects of serum-free in vitro maturation of bovine oocytes on subsequent embryo development and cell allocation in two developmental stages of day 7 blastocysts.

Maturation of oocytes and the subsequent outcome of the in vitro production (IVP) are affected by the composition of in vitro maturation (IVM) medium. To determine the use of serum interfering with effects of single molecules, we aimed at developing simplified IVM medium. The experimental IVM media were: (1) M199-medium supplemented with hormones and serum (control), (2) as 1 but serum was substituted with fatty acid-free serum albumin (FAFBSA) and (3) M199-medium without hormonal and serum supplementation (M199). The quality of embryos was assessed on day 7 by morphology and cryotolerance, as well as by Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) and differential staining. Results showed that the nuclear maturation was suppressed in M199 group alone. Embryo cleavage and development rates, and the proportion of quality 1 blastocysts were lower in the FAFBSA and M199 groups compared to the control. Differences in the cell allocation of fresh embryos were observed at the blastocyst stage, but not at the expanded blastocyst stage. The control group blastocysts had larger number of cells allocated to the inner cell mass (ICM), and the FAFBSA group blastocysts larger apoptotic cell proportion compared to the blastocysts derived from other groups. After cryopreservation, the reduction of ICM proportion and increase of apoptotic cell proportion of embryos were equal between the experimental groups. In conclusion, exclusion of serum from the IVM media reduces embryo development and may cause perturbations in blastocyst development. Differences in the cell allocation of blastocysts between IVM media may appear only when the developmental stages are taken into account.

Polyamines and regulation of spermatogenesis: selective stimulation of late spermatogonia in transgenic mice overexpressing the human ornithine decarboxylase gene.

Polyamines are believed to participate in the induction of cell growth, differentiation, and proliferation, but their role in spermatogenesis has remained obscure. Two transgenic mouse lines (K2 and K15) that overexpress the human ornithine decarboxylase (ODC) gene coding for a rate-controlling enzyme in polyamine biosynthesis and, hence, contain high levels of tissue putrescine have been used to study the stage-specific role of ODC in spermatogenesis. In K2 mice with 30-fold testicular ODC overexpression, [3H]thymidine incorporation at stages I-VI of the cycle of the seminiferous epithelium was significantly above the control level. This may reflect a specific stimulation of DNA synthesis in type A4, intermediate, and type B spermatogonia. The K15 mice that have about 70-fold ODC overexpression showed an elevation of DNA synthesis only at stage V of the cycle, suggesting a specific dependence of type B spermatogonia on putrescine. In K15 mice, [3H]thymidine incorporation of stage VIII tubule segments was decreased, suggesting that excess amounts of putrescine selectively inhibit meiotic DNA synthesis. We propose that putrescine has strictly selective local stimulatory and inhibitory actions during spermatogenic DNA synthesis, and that its excess amounts ultimately may lead to decreased fertility.

Human ornithine decarboxylase-encoding loci: nucleotide sequence of the expressed gene and characterization of a pseudogene.

Previous studies have shown that human ornithine decarboxylase (ODC)-encoding sequences map to two chromosome regions: 2pter-p23 and 7cen-qter. In the present work we have cloned the expressed human ODC gene from a genomic library of myeloma cells that overproduce ODC protein due to selective gene amplification and determined its entire nucleotide sequence. The gene comprises 12 exons and 11 introns and spans about 8 kb of chromosome 2 DNA. The organization of the human gene is very similar to that of the mouse and rat, with the major difference being the presence of longer intronic sequences in the human gene. Some of these differences can be accounted for by the insertion of four Alu sequences in the human gene. Several potential regulatory elements are present in the promoter region and in 5'-proximal introns, including a TATA box; GC boses; AP-1-, AP-2- and NF-1-binding sites; and a cAMP-responsive element. The 5'-untranslated sequence of ODC mRNA is extremely GC-rich, and computer predictions suggest a very stable secondary structure for this region, with an overall free energy of formation of -225.4 kcal/mol. In addition to the active ODC gene on chromosome 2, ODC gene-related sequences were isolated from human chromosome 7-specific libraries and shown to represent a processed ODC pseudogene.

Molecular cloning of a cDNA encoding human spermine synthase.

We have isolated and sequenced cDNA clones that encode human spermine synthase (EC 2.5.1.22). The total length of the sequenced cDNA was 1,612 nucleotides, containing an open reading frame encoding a polypeptide chain of 368 amino acids. All of the previously sequenced peptide fragments of human and bovine spermine synthase proteins could be located within the coding region derived from the cDNA. An unusual sequence of AATTAA apparently signaled the initiation of polyadenylation. Sequence comparisons between human spermine synthase and spermidine synthases from bacterial and mammalian sources revealed a nearly complete lack of similarity between the primary structures of these two enzymes catalyzing almost identical reactions. A modest similarity found was restricted to a relatively short peptide domain apparently involved in the binding of decarboxylated S-adenosylmethionine, the common substrate for both enzymes. The apparent lack of an overall similarity may indicate that spermine synthase, the enzyme found only in eukaryotes, and spermidine synthase with more universal distribution, although functionally closely related, have evolved separately.

Generation of transgenic dairy cattle from transgene-analyzed and sexed embryos produced in vitro.

We have generated a transgenic calf from in vitro produced bovine embryos which had undergone transgene analysis and sexing prior to the embryo transfer. Bovine oocytes were isolated from slaughter-house-derived ovaries, matured and fertilized in vitro and subsequently microinjected with a dam-methylated gene construct consisting of genomic sequences encoding human erythropoietin and governed by bovine alpha S1-casein regulatory sequences. After 6 to 7 days in culture, the embryos were biopsied and while the embryo remained in culture, the biopsy was subjected to transgene analysis and sexing. The transgene analysis was accomplished with a combined treatment of the embryo lysates with DpnI restriction endonuclease and Bal31 exonuclease followed by polymerase chain reaction (PCR). The transgene analysis was based on the fact that DpnI only cleaves its recognition sequence if the adenine in the sequence is methylated. Pregnancy was induced by the transfer of three viable female embryos with a distinct transgene signal to a hormonally synchronized heifer recipient. Amniotic fluid analysis performed two months after the embryo transfer confirmed the presence of the transgene. The calf born was found to be transgenic by PCR analysis from blood, ear and fetal membranes. The presence of the transgene was also confirmed by Southern blotting.

The effect of ascorbic acid during biopsy and cryopreservation on viability of bovine embryos produced in vivo.

Multiple ovulation embryo transfer (MOET) is used to make more rapid progress in animal breeding schemes. On dairy farms, where female calves are more desired, embryo sex diagnosis is often performed before embryo transfer. Fresh transfers have been favored after biopsy due to cumulative drop in pregnancy rates following cryopreservation. The aim of this study was to explore whether exposure to ascorbic acid (AC) during biopsy and freezing increases the viability of biopsied embryos after cryopreservation. Data on presumptive pregnancy and calving rates of biopsied and cryopreserved/overnight-cultured embryos were gathered. Results showed differences in presumptive pregnancy rates between the groups: 45% for both biopsied-cryopreserved groups (control and AC), 51% for biopsied-overnight-cultured embryos and 80% for intact-fresh embryos. Differences between the groups were also apparent in calving rates: 22% for biopsied-cryopreserved control embryos, 31% for biopsied-cryopreserved AC-embryos, 23% for biopsied-overnight-cultured embryos and 63% for intact-fresh embryos. It is concluded that manipulated embryos are associated with lower presumptive pregnancy and calving rates compared with intact-fresh embryos. The highest calving rates for groups of manipulated embryos were achieved in the AC-group. Therefore, addition of AC can be recommended if biopsy is combined with freezing before transfer.

Nuclear magnetic resonance spectroscopy study on energy metabolism, intracellular pH, and free Mg2+ concentration in the brain of transgenic mice overexpressing human ornithine decarboxylase gene.

We have generated a transgenic mouse line strikingly overexpressing the human ornithine decarboxylase (ODC) gene in their brain. Brain ODC activity was increased in the transgenic animals by a factor of 70 in comparison with their nontransgenic littermates. The content of brain putrescine, the product of ODC, was greater than 60 mumol/g of tissue in the transgenic mice, whereas in the normal animals it was below the level that could be detected by an HPLC method. The concentrations of the higher polyamines (spermidine and spermine) were not significantly different from control values. 31P nuclear magnetic resonance (31P NMR) spectroscopy analyses revealed a significantly reduced (40%) free Mg2+ concentration as calculated from the chemical shift differences of the nucleoside triphosphate alpha and beta peaks in the brains of the transgenic animals. The lower free Mg2+ concentration in the brains of ODC transgenic mice was not a consequence of altered intracellular pH or changes in cellular high-energy metabolites. 1H NMR showed no differences in brain choline/N-acetylaspartate and total creatine/N-acetylaspartate ratios between the two animal groups. These ODC transgenic animals may serve as models in vivo for studies on cerebral postischemic events and on epilepsy, as polyamines are supposed to be involved in these processes.

Enhanced papilloma formation in response to skin tumor promotion in transgenic mice overexpressing the human ornithine decarboxylase gene.

We have studied the induction of papilloma formation in response to skin tumor promotion in transgenic mice overexpressing the human ornithine decarboxylase gene and in their nontransgenic littermates. The transgenic animals displayed a basal epidermal ornithine decarboxylase activity that was nearly 20 times higher than in their nontransgenic littermates. A single topical application of 12-O-tetradecanoylphorbol-13-acetate induced a much more profound and longer-lasting increase in transgene-derived ornithine decarboxylase activity in comparison with the endogenous enzyme activity. Initiation of skin tumorigenesis with a single topical application of dimethylbenz[a]antracene followed by twice-weekly application of 12-O-tetradecanoylphorbol-13-acetate resulted in the appearance of first papillomas both in nontransgenic and transgenic animals by week 7. However, after 11 weeks of 12-O-tetradecanoylphorbol-13-acetate application, the number of papillomas per animal was almost 100% higher in the transgenic animals than in their nontransgenic littermates. These results indicate that an overexpression of epidermal ornithine decarboxylase confers a growth advantage on skin tumors in vivo.

Levels of ornithine decarboxylase genomic sequences, heterogeneous nuclear RNA and mRNA in human myeloma cells resistant to alpha-difluoromethylornithine.

We previously isolated and characterized a human myeloma cell line overproducing ornithine decarboxylase (ODC) due to gene amplification [Leinonen, Alhonen-Hongisto, Laine, Jänne & Jänne (1987) Biochem. J. 242, 199-203]. We have now employed the PCR combined with reverse transcription to determine semiquantitatively ODC gene dosage and the amounts of heterogeneous nuclear (hn) RNA and of mature mRNA of the enzyme in parental and alpha-difluoromethylornithine-resistant human myeloma cells. Experiments with dilution series revealed that the ODC gene copy number and the amount of both hnRNA and mRNA were increased to the same extent (about 100-fold) in the resistant cells. Similar dot-blot analyses of ODC-specific genomic DNA and total RNA indicated that the ODC gene copy number was increased by a factor of 380 and the amount of ODC mRNA by a factor of 700. Our results indicate that the PCR combined with reverse transcription is at least as useful as blot analyses to give semiquantitative assessment of the amounts of specific DNA or RNA sequences. In addition, the use of the PCR enables the analysis of minute sample amounts in extremely short time.

Transgenic mice over-producing putrescine in their tissues do not convert the diamine into higher polyamines.

We recently described a transgenic mouse line over-expressing the human ornithine decarboxylase gene virtually in all tissues. Despite strikingly elevated tissue putrescine concentrations, no or minimal changes were found in the levels of the higher polyamines spermidine and spermine. We have now extended these studies by further increasing tissue putrescine with the aid of 5-fluoromethylornithine, a specific inhibitor of ornithine transaminase and hence the catabolism of L-ornithine. As a result of the treatment with the latter drug, the concentration of putrescine was further increased by a factor of 2-3 without any changes in the concentrations of spermidine and spermine. In the testis of transgenic mice treated with 5-fluoromethylornithine, the concentration of putrescine was nearly 60 times that in non-transgenic untreated animals, yet the concentration of spermidine was only 1.5-fold higher. A similar small increase in brain spermidine was accompanied by a 40-fold elevation in the concentration of putrescine. The apparent blockade between putrescine and spermidine was in all likelihood not attributable to an inhibition of S-adenosylmethionine decarboxylase, the rate-controlling enzyme in the biosynthesis of spermidine and spermine. Our results are more compatible with the view that in non-dividing adult tissues putrescine is sequestered through some unknown mechanisms in a way that makes it unavailable for the synthesis of the higher polyamines.

Transgenic mice over-expressing the human spermidine synthase gene.

We have generated a transgenic mouse line harbouring the functional (chromosome-1-derived) human spermidine synthase (EC 2.5.1.16) gene in their genome. The transgenic animals expressed the human gene-derived mRNA, as revealed by reverse-transcriptase/PCR analysis, in all tissues studied and displayed tissue spermidine synthase activity that was 2-6 times that in their syngenic littermates. The elevated spermidine synthase activity, however, had virtually no effect on tissue putrescine, spermidine or spermine levels. The view that the accumulation of spermidine and spermine is possibly controlled by S-adenosylmethionine decarboxylase was further supported by the finding that tissue spermidine and spermine contents also remained practically normal in hybrid transgenic mice over-expressing both human ornithine decarboxylase and spermidine synthase genes.

Regulation of the expression of human ornithine decarboxylase gene and ornithine decarboxylase promoter-driven reporter gene in transgenic mice.

We have studied the regulation of the expression of ornithine decarboxylase with the aid of transgenic mice harbouring either functional human ornithine decarboxylase genes or the mouse ornithine decarboxylase promoter-driven chloramphenicol acetyltransferase fusion gene in their genome. We used three different stimuli which are well known to enhance ornithine decarboxylase activity in their appropriate target tissues: (i) testosterone in female kidney, (ii) a phorbol ester in epidermis and (iii) partial hepatectomy in liver. Endogenous mouse ornithine decarboxylase activity was strikingly stimulated in response to these treatments. Even though containing the 5' flanking region of the mouse ornithine decarboxylase gene, known to possess full promoter activity, the chloramphenicol acetyltransferase reporter gene was entirely insensitive to any of these stimuli. The human transgene-derived ornithine decarboxylase activity in kidney was unaffected by testosterone treatment, but responded in skin to application of the phorbol ester and likewise was clearly enhanced in regenerating liver. Although mouse endogenous ornithine decarboxylase mRNA levels were distinctly elevated after testosterone, this treatment did not influence the accumulation of the human transgene-derived mRNA. The phorbol ester enhanced the accumulation of mouse endogenous ornithine decarboxylase mRNA and also that derived from the human transgene; however, the enzyme activity was stimulated in regenerating liver without appreciable changes in the levels of endogenous or transgene-derived message. Our present results strongly emphasize the central role of the coding sequence or ornithine decarboxylase gene in the induction of the enzyme activity.

Transgenic animals as bioproducers of therapeutic proteins.

Many human therapeutic proteins are currently produced with the aid of recombinant DNA technology in microbial bioreactors and a few also in large-scale animal cell cultures. Although extremely cost-efficient, the microbial production system has many inherent limitations. Micro-organisms, such as bacteria, can read the universal genetic code and hence produce human proteins with correct amino acid sequence, but cannot carry out post-translational modifications, such as glycosylation, or fold the newly synthesized protein properly to ultimately generate a biologically active entity. Moreover, even though the production of the proteins as such is inexpensive, the downstream processing of the final product may be extremely difficult and costly. Many of these disadvantages, especially the lack of post-translational modifications, can be overcome by employing large-scale animal cell cultures for the production of proteins of pharmaceutical interest. However, due to the long generation time and the requirement for rich culture media, the use of animal cell bioreactors is unacceptably expensive. With the advent of transgenic technology, the production of human pharmaceuticals in large transgenic animals has become more and more attractive. The use of targeted gene transfer, the expression of the transgene of interest can be directed to occur in the mammary gland of large farm animals, such as pigs, sheep, goats or dairy cattle, and hence the transgene product is ultimately being secreted into the milk. Although not yet in commercial use, the last few years have witnessed a remarkable progress in this area and proved the feasibility of the use of 'molecular farming' in high-quantity, low-cost production of valuable therapeutic or industrial proteins. While reviewing the progress of the field over the past few years, we discuss in somewhat greater detail aspects connected with the use of dairy cattle as bioproducers of human therapeutic proteins.

Methylation of human ornithine decarboxylase gene before transfection abolishes its transient expression in Chinese hamster ovary cells.

Different methylations of cloned human ornithine decarboxylase gene with restriction methylases in vitro before transfection greatly reduced the transient expression of ODC in Chinese hamster ovary cells. Single methylation of the gene with Hpa II (CCGG) methylase decreased the transiently expressed peak activity by about 50%, single methylation with Hha I (CCGG) methylase by about 80% whilst a double methylation at both Hpa II and Hha I restriction sites virtually abolished any transiently expressed ornithine decarboxylase activity. These results together with our earlier circumventing evidence indicate that the expression of mammalian ornithine decarboxylase is critically influenced by the methylation state of the gene.

Characterization of a transgenic mouse line over-expressing the human ornithine decarboxylase gene.

We have produced several transgenic mouse lines over-expressing the human ornithine decarboxylase (ODC) gene. We have now characterized one of the transgenic lines as regards the tissue accumulation of the polyamines and the activities of their metabolizing enzymes. Among the tissues analysed, the polyamine pattern was most strikingly changed in testis and brain of the transgenic animals. ODC activity was greatly enhanced in all tissues, except kidney, of the transgenic animals. The most dramatic increase, 80-fold, was found in brain of the transgenic mice. The activities of S-adenosylmethionine decarboxylase and spermidine and spermine syntheses were likewise significantly increased in testis of the transgenic animals. The activities of the enzymes involved in the back-conversion of the polyamines, namely spermidine/spermine acetyltransferase and polyamine oxidase, were similar in the transgenic and non-transgenic animals. As analysed by reverse transcriptase/polymerase chain reaction, all the six tissues of the transgenic animals expressed human-specific ODC mRNA. Determination of the half-life of testicular ODC revealed a stabilization of the enzyme in the transgenic males.

Position-independent, aberrant expression of the human ornithine decarboxylase gene in transgenic mice.

We have generated transgenic mouse lines carrying the human ornithine decarboxylase (ODC) gene in their genome. Six of 7 transgenic lines overexpressed ODC in most of their tissues, which was most strikingly manifested as a highly ectopic enzyme activity in the testis and brain of transgenic mice. A close correlation existed between enzyme activity (or ODC mRNA level) and gene copy number in testis and brain, indicating that the expression occurred independently of the transgene's chromosomal integration site. Transgenic mice carrying the mouse ODC promoter fused to the bacterial chloramphenicol acetyltransferase gene expressed the reporter gene in a similarly aberrant fashion. Even though the human ODC gene construct contained 5'-flanking sequences (800 nt), sufficient to confer maximal promoter activity in transfected cells, and about 1000 nt of 3'-flanking DNA, it is improbable that the observed gene copy number-dependent expression was due to the presence of so-called DNA attachment elements. In contrast, our data suggest that expression of the mammalian ODC gene is governed by distal silencer elements that were missing in the transgene constructs, which permitted an apparently position-independent expression of the transgene.

Activation of polyamine catabolism profoundly alters tissue polyamine pools and affects hair growth and female fertility in transgenic mice overexpressing spermidine/spermine N1-acetyltransferase.

We have generated a transgenic mouse line that overexpresses the rate-controlling enzyme of polyamine catabolism, spermidine/spermine N1-acetyltransferase. Tissues of these mice showed markedly distorted polyamine pools, which in most cases were characterized by the appearance of N1-acetylspermidine, not normally found in mouse tissues, a massive accumulation of putrescine, and decreases in spermidine and/or spermine pools. The most striking phenotypic change was permanent hair loss at the age of 3 to 4 weeks which was typified histologically by the appearance of extensive follicular cysts in the dermis. The effect seemed attributable to putrescine interference with hair development, possibly with differentiation/proliferation of epidermal cells located in hair follicles. Female members of the transgenic line were found to be infertile apparently due to ovarian hypofunction and hypoplastic uteri. The findings demonstrate the utility of spermidine/spermine N1-acetyltransferase overexpression as an effective means for genetically modulating total tissue polyamine pools in transgenic animals and examining the developmental and oncogenic consequences.

Transgenic mice overexpressing ornithine and S-adenosylmethionine decarboxylases maintain a physiological polyamine homoeostasis in their tissues.

Recent work has shown that transgenic mice overexpressing human ornithine decarboxylase display no marked changes in the tissue concentrations of spermidine or spermine in spite of a dramatic increase in putrescine levels. In the tissues of transgenic mice carrying the human spermidine synthase gene and in those of hybrid mice overexpressing both ornithine decarboxylase and spermidine synthase, spermidine and spermine levels remain within normal limits. To test whether the amount of the propylamine group donor, decarboxylated S-adenosylmethionine, limits the conversion of putrescine into the higher polyamines, we have produced transgenic mouse lines harbouring the rat S-adenosylmethionine decarboxylase gene in their genome. However, neither these mice nor the hybrid mice overexpressing both ornithine decarboxylase and S-adenosylmethionine decarboxylase displayed significant changes in their spermidine and spermine tissue levels. To study the mechanism by which cells maintain the constancy of the polyamine concentrations, we have determined the metabolic flux of polyamines in transgenic primary fibroblasts using pulse labelling. The results indicate that the polyamine flow is faster in transgenic primary fibroblasts than in non-transgenic fibroblasts and that the intracellular homoeostasis of higher polyamines is maintained at least partly by the acetylation of spermidine and spermine and their secretion into the medium.

Life-long over-expression of ornithine decarboxylase (ODC) gene in transgenic mice does not lead to generally enhanced tumorigenesis or neuronal degeneration.

Ornithine decarboxylase, the rate-controlling enzyme of the biosynthetic pathway of the polyamines, is one of the most inducible mammalian enzymes showing many of the features common to the oncoproteins. Ornithine decarboxylase activity is likewise strongly induced in response to various neurotoxic stimuli, and the enhanced enzyme activity is believed to be causally related to neuronal damage. We have generated several transgenic mouse lines over-expressing human ornithine decarboxylase gene. We have now subjected the animals to a long-term survival study in order to assess whether constitutively over-expressed ornithine decarboxylase would predispose them to enhanced tumorigenesis and neuronal degeneration. When the transgenic animals were 2 years old, their tissue ornithine decarboxylase activity was 20- to 50-fold that in their syngenic littermates. Macroscopic and microscopic examination of organs revealed no differences between syngenic and transgenic animals as regards spontaneous tumor incidence or age-related changes in brain.