Influence of protein supplements on the secretion of leukaemia inhibitory factor by mitomycin-pretreated Vero cells: possible application to the in vitro production of bovine blastocysts with high cryotolerance.

A co-culture system for bovine embryos using mitomycin-treated Vero cells and serum-supplemented modified synthetic oviduct fluid (mSOF) supports the development of in vitro maturation and fertilization-derived oocytes to hatched blastocysts. In this system, it has been suggested that one contribution made by the co-culture cells to embryo development is production of the cytokine leukaemia inhibitory factor (LIF). However, there are concerns about exposure of early embryos to serum due to its incompatibility with embryo cryosurvival. In this study, the influence of two protein supplements (synthetic serum substitute (SSS), a lipid-free human serum-derived product) and oestrous cow serum (ECS)) on Vero cell LIF secretion was compared, with the aim of designing a co-culture system that is supportive of bovine embryo cryopreservation. Vero cells cultured for 72 h in medium 199 + 5% fetal bovine serum (FBS) (recommended maintenance medium for this cell line) secreted detectable amounts of LIF (13.1 +/- 0.9 pg LIF per 10(5) cells). Culture in mSOF, the medium routinely used in this laboratory for embryo culture, also supported LIF secretion in Vero cells. However, the amount of LIF was tenfold higher (24.7 +/- 6.2 pg LIF per 10(5) cells; P < 0.05) when mSOF was supplemented with 10% (v/v) ECS compared with supplementation with 2% (v/v) SSS. Results of a second series of experiments in which supplementation with each protein was normalized to 10% revealed similar differences in LIF secretion, indicating that LIF secretion was affected by the type, not the amount, of protein. Time course analysis revealed stepwise increases (P < 0.05) in cumulative LIF secretion with every 24 h of culture in mSOF + either SSS or ECS. In terms of embryo development and post-cryopreservation viability, medium supplementation with 2% (v/v) SSS alone versus the two-step system of 2% (v/v) SSS (days 1-4) + 10% (v/v) ECS (days 4-10) had no influence (P > 0.05) on the ability of bovine blastocysts to hatch, with or without intervening cryostorage. However, the rate of blastocyst formation (expressed as the percentage of cleaved embryos) was only 27% in the presence of 2% (v/v) SSS, and increased almost twofold (P < 0.05) when ECS was added beginning on day 4 of co-culture. In summary, Vero cell LIF secretion was increased markedly by ECS. A two-step system of medium supplementation, in which embryos are exposed to ECS beginning on day 4 of in vitro development combined high rates of blastocyst formation with cryotolerance. This effect may be a result of limiting embryo exposure to serum-derived lipid until after the eight-cell stage and providing an increase in LIF during the critical developmental stages of compaction and cavitation.

Evaluation of mitomycin-treated vero cells as a co-culture system for IVM/IVF-derived bovine embryos.

Support of the in vitro development of IVM/IVF-derived bovine embryos by Vero cells was evaluated by comparing the following treatment groups: 1) proliferating (Unt-Vero) vs nonproliferating (Mit-Vero) cells; 2) supplementation of medium with estrous cow serum (ECS) vs bovine serum albumin (BSA); 3) Mit-Vero cells vs bovine oviduct epithelial cells (BOECs); and 4) addition of leukemia inhibitory factor (LIF) to Mit-Vero cell co-cultures at Day 1 vs Day 4. Mit-Vero cells stimulated higher rates of blastocysts (Day 7, 40 vs 27%) and hatched blastocyst (Day 10, 38 vs 12%) formation than Unt-Vero cells. These rates were comparable to those obtained with BOECs; blastocyst hatching was slightly higher following co-culture with Mit-Vero cells (36%) than BOECs (29%). Blastocyst formation was similar in ECS- vs BSA-supplemented medium; however, hatching was greatest (37%) during co-culture in medium +10% ECS. While the addition of LIF throughout the co-culture period was ineffective, addition of the cytokine beginning at Day 4 slightly increased blastocyst formation rates. Evaluation of LIF secretion using ELISA revealed detectable levels of the cytokine in Mit-Vero-conditioned medium (50 pg/10(5) cells); this may explain the minimal influence of exogenous LIF during embryo co-culture. Mit-Vero cells provided comparable support of bovine embryo development when used even up to 2 wk after establishment as monolayers. In conclusion, Mit-Vero cells provide a readily-available, safe and easy-to-use co-culture method which is at least as supporting of bovine embryo development as BOECs. One contribution of these cells may be secretion of the cytokine LIF.

Evaluation of culture systems containing bovine oviduct epithelial cells or granulosa cells to mature and maintain the developmental competence of bovine oocytes in vitro.

The effects of estrous cow serum (ECS), bovine oviduct epithelial cells (BOEC), and bovine granulosa cells (GC) on in vitro maturation (IVM) of immature oocyte-cumulus complexes (OCCs) were evaluated. Selected OCCs were cultured for 24 to 26 h in microdroplets of culture medium (CM; TCM 199 + 25 mM HEPES + 100 mug gentamicin sulfate/ml) or in CM medium supplemented or conditioned with 20% ECS, BOEC +/- 20% ECS or GC + 20% ECS. Supplemented media were incubated for 2 h before addition of OCCs, whereas media were conditioned by incubation with 20% ECS or BOEC +/- 20% ECS for 6 d, or with 20% ECS +/- GC for 24 or 48 h before addition of OCCs. The developmental competence of oocytes after TVM was assessed by insemination with glass wool separated, frozen-thawed bovine spermatozoa in microdroplets of modified medium (TALP) containing heparin (5 mug/ml) and BOEC for 18 h. The presumptive zygotes were cultured in microdroplets of CM medium + 20% ECS + BOEC for 7 to 9 d to assess embryo development to morula and blastocyst stages. The percentages of OCCs undergoing IVM (85 to 94%) and in vitro fertilization (IVF) (66 to 80%) were high, irrespective of the IVM conditions. Only after the IVM of OCCs in CM medium alone was the percentage of oocytes undergoing IVF significantly lower (66%; P<0.05). The proportion of IVF oocytes developing to blastocysts with a normal complement of cells (126 to 138) increased significantly (P<0.05) when the OCCs were matured in supplemented or conditioned CM medium containing ECS and/or somatic cells (18 to 28%) compared with those in CM medium alone (9%). When the CM medium was supplemented or conditioned with GC + 20% ECS, the proportion of fertilized oocytes developing to blastocysts increased significantly (28%; P<0.05). These results indicate that the potential of immature OCCs to be fertilized and to complete embryonic development to the blastocyst stage in vitro is enhanced by maturation in CM medium containing 20% ECS and/or BOEC or GC.

Parthenogenetic development of activated in vitro matured bovine oocytes.

Bovine oocytes matured in vitro for 26 hours were electrically stimulated 1) by a single pulse (Treatment A); 2) by 3 pulses 30 minutes apart (Treatment B); 3) by a single pulse followed by 5 minutes of incubation in the stimulation medium (Treatment C); or 4) by a single pulse at 27 hours of maturation (Treatment D). The oocytes were then cultured for up to 8 days to assess parthenogenetic activation and development. Each electrical stimulation consisted of a 60-mus square wave pulse of 2.5 or 3.6 kV/cm. Treatment A was less effective than the other treatments (P<0.05), activating 47 or 59% of oocytes at 2.5 or 3.6 kV/cm, respectively. However, there were no differences due to voltage nor among the other treatments, which activated 64 to 78% of the oocytes. The cleavage rate, 28 to 38%, was not affected by the activation treatment, but development to the 8-cell stage or beyond was greater after activation with the higher voltage. While the numbers of morulae or blastocysts resulting from any given treatment were too small to support meaningful statistical comparison, the results indicate that bovine parthenogenotes produced in vitro are capable of development to the blastocyst stage.

Estradiol assay by microtitre plate enzyme immunoassay.

Development of a simple enzyme linked immunosorbent assay (ELISA) for estradiol in serum extracts is described. The assay involves use of a 96-well microtitre plate, designed for immunoassay, as the support for a purified, high-titre antiserum, raised against estradiol-6(O)-carboxymethyloxime linked to bovine serum albumin, and using horseradish peroxidase-labelled estradiol-6-(O)-carboxymethyloxime as the labelled species, with 2,2'-azino-bis-(3-ethylbenzthiazoline sulfonic acid) diammonium salt (ABTS) as the chromogenic substrate. The assay characteristics rival those of radio- or chemiluminescence immunoassays for estradiol.

A simple enzyme-linked immunosorbent assay for testosterone.

A new, enzyme-linked, immunosorbent assay for testosterone is described. The assay uses horseradish peroxidase coupled to testosterone as the tracer and offers the same sensitivity and reliability but greater convenience than radioimmunoassay. The assay is also simpler and more rapidly completed than previously described assays for testosterone.