K. Schellander

Artificial insemination in cattle with DNA‐treated sperm

Abstract We have investigated the use of sperm cells as vectors for transferring exogenous DNA into the genome of cattle by artificial insemination with DNA‐treated sperm. First we demonstrated the DNA‐binding ability of cattle sperm with radioactively labeled DNA. For artificial insemination ejaculated semen was washed and incubated with 1 μg DNA/106 sperm for one hour at 37°C. Three hundred synchronized heifers were inseminated once with a dose of 40×106 sperm. Forty‐five calves and 41 fetuses were obtained. Southern analysis revealed in one calf a signal after probing with the 1 kb Pst I fragment of pSV2‐cat.

In vitro fertilization and cleavage of bovine oocytes matured in medium supplemented with estrous cow serum

Bovine follicular oocytes were matured in TCM 199 supplemented with: (1) fetal calf serum (FCS, 20% v/v), Luteinizing Hormone (LH, 10 ug/ml), and Estradiol-17-beta (E(2), 1 ug/ml) in Experiment 1; (2) 20% cow serum recovered at standing estrus (Experiment 2); or (3) 20% FCS (Experiment 3). Maturation, fertilization, and initial cleavage development were evaluated at 16 and 48 h after in vitro insemination. The proportions of oocytes fertilized after maturation in the presence of added hormones (78.5%, Experiment 1) or estrous serum (71.3%, Experiment 2) were significantly higher (p < 0.01) than after use of FCS alone (39.3%, Experiment 3). Cleavage of zygotes within 48 h post-insemination differed significantly (p < 0.01) between maturation treatments, 27.3%, 75.5% and 6.6% for Experiments 1, 2, and 3, respectively. Results demonstrate a beneficial influence of estrous cow serum, characterized by an elevated concentration of LH, on bovine oocyte maturation in vitro.

Secretion of cumulus expansion-enabling factor (CEEF) in porcine follicles

The objective of this study was to find out whether porcine cumulus and mural granulosa cells can secrete cumulus expansion‐enabling factor (CEEF). Culture drops of M‐199 medium were conditioned with denuded porcine oocytes (1 oocyte/μl), cumulus cells from oocytectomized complexes (1 OOX/μl), pieces of mural granulosa isolated from preantral to preovulatory follicles (1000 cells/μl), or oviductal cells (1000 cells/μl) for 24 hr. The production of CEEF was assessed by the addition of mouse OOX and follicle‐stimulating hormone (FSH) (1 μg/ml) to microdrops of the conditioned medium. After 16–18 hr, expansion of the mouse OOX was scored on a scale of 0 to 4 by morphologic criteria. Mouse OOX did not expand in nonconditioned FSH‐supplemented medium. Immature porcine oocytes produced +3 to +4 expansion of the mouse OOX. Granulosa cells isolated from preantral and early antral follicles and cumulus cells isolated from all stages of follicle development constitutively secreted CEEF under in vitro conditions. Mural granulosa cells of small, medium, and preovulatory (PMSG) follicles also secreted CEEF in vitro; however, FSH or leutenizing hormone (LH) stimulation was essential for this secretion. Hormonally induced secretion of CEEF was accompanied by expansion of the mural granulosa itself. Granulosa cells isolated from follicles of gilts 20 hr after PMSG and human chorionic gonadotropin (hCG) administration did not produce CEEF and did not expand in response to FSH and LH in vitro. CEEF activity also was found in the follicular fluid of small antral follicles, was reduced in medium follicles, and was not detectable in PMSG‐stimulated follicles. However, CEEF activity was reestablished in the follicular fluid of preovulatory follicles by hCG injection, conceivably due to increased production of CEEF by cumulus cells. We conclude that (1) porcine cumulus and mural granulosa cells are capable of CEEF production in vitro and (2) autocrine secretion of CEEF by cumulus cells is involved in regulation of porcine cumulus expansion both in vitro and in vivo. Mol. Reprod. Dev. 49:141–149, 1998.

Effects of different cryoprotectants and carbohydrates on freezing of matured and unmatured bovine oocytes

Cumulus cell-enclosed bovine oocytes in germinal vesicle (GV) and in metaphase II (MII) stages were cryopreserved. Different concentrations (1 M; 1.5 M) of various cryoprotectants (glycerol, PROH, DMSO) were tested. After thawing, the oocytes were exposed to various carbohydrates (sucrose, lactose, trehalose) at a concentration of 0.1 M and 0.25 M for cryoprotectant removal. Developmental capacity of the frozen-thawed oocytes was studied by in vitro maturation, fertilization and culture. We found no difference in subsequent development using glycerol or PROH for GV and MII oocytes. The DMSO treatment led to significantly better cleavage and development up to 4-cell stage in MII oocytes. Development beyond the 8-cell stage was obtained only when unmatured oocytes were frozen. No difference in the efficiency of the 3 cryoprotectants was detected in MII oocytes. However, in GV oocytes, glycerol and PROH yielded significantly better cleavage and 4-cell rate compared to DMSO (P<0.001). Influence of the concentration of a cryoprotectant on development was not observed in GV or MII oocytes. Among the 3 cryoprotectants, DMSO was less suitable, at both concentrations, than PROH and glycerol for the development of 6- to 8-cell stage embryos in the GV group. In the MII group, 1.5 M DMSO was as efficient as PROH and as glycerol at a 1.5-M concentration, and it was more efficient than 1 M glycerol. The use of carbohydrates during rehydration did not render a beneficial effect at either of the 2 concentrations, and when no carbohydrates were used in the MII group the oocytes cleaved better than GV oocytes.

A detailed analysis of pronucleus development in bovine zygotes in vitro: Cell‐cycle chronology and ultrastructure

The aim of the present experiment was to analyze the chronology of pronucleus development and DNA synthesis, as well as the ultrastructure of intranuclear bodies, in bovine zygotes produced in vitro. Bovine oocytes were matured and fertilized in vitro, and sperm penetration and pronucleus development were examined. DNA synthesis was investigated by sequential incubation with [3H]‐ and [14C]thymidine followed by autoradiography on semithin sections. Ultrathin sections for transmission electron microscopy were prepared from the same zygotes. Sperm penetration was noted for the first time at 4 hr after in vitro insemination and reached a maximum at 6 hr. Pronucleus formation was initiated at 4 hr, and up to at least 11 hr the maternal pronucleus was more developed than its paternal counterpart. DNA synthesis was initiated at 14–15 hr, and the S‐phase lasted for 8–10 hr. The most prominent ultrastructural entities of the pronuclei were the nucleolus precursor bodies (NPBs). During the S‐ and G2‐phases, the NPBs spatially associated with clusters of interchromatin‐like granules. The two components were firmly attached to each other by an electron‐dense reticulum. During the late G2‐phase, the NPBs were apparently detached from the interchromatin‐like granules and the electron‐dense reticulum again. The interaction between the intranuclear bodies and granules appears to be comparable with the situation previously described for in vivo‐produced bovine zygotes (J Laurinčík et al., Mol Reprod Dev 43:62–69, 1996), except for the lack of vacuolization of the NPBs during the S‐phase in vitro. Mol. Reprod. Dev. 50:192–199, 1998.

OMEC II: A new ovine mammary epithelial cell line

We have established three independent ovine mammary epithelial cell lines which arose from primary cultures of ovine mammary epithelial cells by spontaneous immortalization. One of them, OMEC II, was characterised in greater detail. The cells grow rapidly on plastic dishes in medium containing 10% FCS without any requirement for additional growth factors or hormones. Immunofluorescence staining of this cell line showed expression of cytokeratin (46 kDa) and ZO‐1, a tight‐junction associated protein, but negative immunostaining for an anti‐vimentin antibody. In confluent cell monolayers ‘domes’ became visible indicating the development of a polarised phenotype and the ability of directed secretion. When grown in collagen gels typical ducts with end‐buds were observed. Treatment with lactogenic hormones increased the frequency of dome formation, but no expression of β‐lactoglobulin was found. To our knowledge this is the first report on an ovine mammary epithelial cell line.

Variation of the growth hormone gene in ryr 1 genotyped Austrian pig breeds.

SUMMARY Polymorphism in the second intron of the porcine growth hormone gene of 273 Austrian Landrace and 81 Austrian Edelschwein pigs was investigated with a PCR-RFLP-technique. Results showed significantly different genotype patterns between the two breeds. The frequency of the Hae II(-) allele was significantly (P < 0,001) higher in the landrace than in the Edelschwein population (0,83 and 0,47 resp.). The Msp I(+) allele was predominant in both breeds but signifanctly higher in the Landrace (0,98 versus 0,69; P < 0,01). Analyses the Hae II/Msp I locus combination revealed also in breed specific difference. In the Landrace a very low interaction was found between the Hae II and ryr 1 locus, and between Msp I and ryr 1 locus (c. c. = 0,181 and 0,186 resp.). The correlation was slightly stronger (c. c. = 0,266) between the ryr 1 and Hae II/Msp I genotypes. No correlation was detected among the three loci in the Edelschwein population. ZUSAMMENFASSUNG: Variabilität des Somatotropin Gens in österreichischen Schweinerassen genotypisiert hinsicbtlich ryr 1 An 273 Österreichischen Landrasse Schweinen und 81 Österreichischen Edelschweinen wurde der Polymorphismus am zweiten Intron des Schweinewachstumshormons mittels PCR-RFLP-Technik untersucht. Genotypen-und Genfrequenzen waren zwischen den beiden Rassen signifikant verschie- den. Die Hae II(-) Allelfrequenz war bei den Landrassetieren signifikant höher (P < 0,001) als bei den Edelschweinen (0,83 bzw. 0,47). In beiden Rassen überwiegte das Msp I(+) Allel, das aber signifikant öfters bei der Landrasse auftrat (0,98 bzw. 0,69; P < 0,01). Die Verteilung der Locuskombination von Hae II/Msp I zwischen den beiden Rassen war ebenfalls unterschiedlich. Bei der Landrasse konnte nur ein sehr geringer Zusammenhang zwischen dem Hae II und ryr 1 Locus (c. c. = 0,181 bzw. 0,186) und auch zwischen dem Msp I und ryr 1 Locus festgestellt werden. Die Korrelation zwischen dem ryr 1 und Hae II/Msp I Genotyp war geringfügig größ;er (c. c. = 0,266). Bei den Edelschweinen konnte kein Zusammenhang zwischen den drei untersuchten Loci festgestellt werden.

Comparison of aggregation and injection techniques in producing chimeras with embryonic stem cells in mice

We analyzed embryonic stem cell lines for their capacity to produce aggregation chimeras with diploid or developmentally compromised tetraploid embryos. Descendants of embryonic stem cells which contributed to midgestation fetuses at high levels were capable of supporting fetal development also with tetraploid partners. Different numbers of embryonic stem cells were introduced into diploid and tetraploid morulae as well as into blastocysts by microinjection. There were no differences in the frequency of embryonic stem cell-containing fetuses when comparing aggregation or injection into morulae versus blastocysts. However, the distribution pattern of embryonic stem cell derivatives in chimeric fetuses suggested that pre-compaction embryos are more suitable for generating fetuses with high embryonic stem cell contribution. Injection of embryonic stem cells into tetraploid embryos showed that completely embryonic stem cell-derived fetuses can also be produced by this technique. Totally embryonic stem cell derived fetuses were observed in each group, when embryonic stem cells were injected into diploid embryos. However, the rate of chimeras and chimerism was lower when 1 or 3 embryonic stem cells were used versus 8 or 15 cells. This suggests that the number of embryonic stem cells introduced might play a role in the colonization ability.

A DNA-Specific Technique for Pig and Cattle Oocytes using Counterstain-Enhanced Fluorescence

SUMMARYPig and cattle follicular oocytes were stained by a counterstain- enhanced-fluorescence technique with Chromomycin A3-Distamycin A-DAPI (4,6- Diamidino-2-phenylindole) (CCD) as DNA-specific staining dyes. This method provided a clear visualization of the nuclear structures in pig and cattle oocytes. The cytogenetic stages found consisted of two major types; type I, with fine chromosomal filaments (bivalents), predominant in oocytes originating from small follicles ( 2 mm). The majority of the porcine and bovine Giemsa stained chromomeres displayed DAPI bright and CMA3 pale staining. Thus, they resemble mitotic G- and Q-bands in their staining behaviour.