Growth of normal human ovarian surface epithelial cells in reduced-serum and serum-free media

Summary The human ovarian surface epithelium (OSE) is believed responsible for over 85% of ovarian cancers, yet little is known about the normal biology of these cells. To date, culture of OSE has only been reported in media with high serum supplements. We have developed two media, one with less than 1% of serum (OSEM-1) and the other comprised of highly purified and defined materials (OSEM-2), which allow us to study OSE under relatively defined conditions. By substituting 0.05% of Pedersen’s fetuin for 15% fetal bovine serum (FBS) with Medium 199/MCDB105 basal medium, the cell numbers reached 50 to 60% of those in the presence of 15% FBS over 7 days. However, over several weeks, the total number of population doublings achieved were comparable to those in 15% FBS. Addition of insulin, transferrin, ethanolamine, lipoic acid, and phosphatidylcholine to the medium with Pedersen’s fetuin (OSEM-1) enhanced growth up to 20% more than in their absence. Supplementation of M199/105 with highly purified (>99%) fetuin, alpha₂-macroglobulin, and hydrocortisone resulted in a defined medium (OSEM-2) that permitted 1 to 2 doublings/7 days. In addition, cells maintained a more normal, epithelial-like morphology in culture for a longer period in the presence of Pedersen’s or purified fetuin than in M199/105/15% FBS, thus increasing the number of morphologically normal cells available for experimentation. Addition of 0.05% Pedersen’s fetuin to M199/105 in the presence of 6 to 8% FBS resulted in levels of growth equivalent to those in M199/105/15% FBS alone. We are now able to study the effects of various compounds on the growth and differentiation of OSE under defined conditions, and have reduced the requirement for FBS to produce large numbers of OSE cells.     

Human ovarian surface epithelium in primary culture

The ovarian surface epithelium (OSE) represents a minute fraction of the cell mass of the ovary but gives rise to over 80% of human ovarian carcinomas. No experimental models for the study of human OSE exist. To characterize OSE cells in culture, explants of ovarian surface from normal ovary of premenopausal women were grown on plastic, glass, and collagen gel in 25% fetal bovine serum/Waymouth's medium 752/1. About 25% of explants produced epithelial outgrowths. Morphologically, these outgrowths resembled OSE in vivo and endothelial and mesothelial cells in culture, but they differed from cultured ovarian stromal, granulosa, and luteal cells. Only OSE among ovarian cell types were intensely keratin positive by immunofluorescence. Keratin also distinguished OSE cells from the keratin-negative endothelial cells. Most but not all OSE colonies tested showed 17 beta-hydroxysteroid dehydrogenase (HSD) activity, which was absent in peritoneal mesothelial cells. Colonies from most patients were limited to a few millimetres and became stationary within a few weeks. Changes that accompanied cessation of growth included senescence, increased keratin content, or the formation of multicellular papillary aggregates. With time, OSE cells tended to assume a fibroblast-like morphology but remained keratin positive and continued to resemble OSE by scanning electron microscopy (SEM). Subcultured OSE cells persisted in a stationary keratin-positive form for many weeks. Throughout this study, all pavementlike epithelial outgrowths that were contiguous with an explant stained for keratin; thus, such colonies can be assumed to be OSE. Conversely, fibroblast-shaped cells may represent OSE as indicated by keratin content and SEM appearance. The methods presented here permit culture of normal human OSE under conditions in which the cells exhibit morphologic plasticity, variable 17 beta-HSD activity, and presence of keratin.     

Human ovarian surface epithelium in primary culture

Summary The ovarian surface epithelium (OSE) represents a minute fraction of the cell mass of the ovary but gives rise to over 80% of human ovarian carcinomas. No experimental models for the study of human OSE exist. To characterize OSE cells in culture, explants of ovarian surface from normal ovary of premenopausal women were grown on plastic, glass, and collagen gel in 25% fetal bovine serum/Waymouth's medium 752/1. About 25% of explants produced epithelial outgrowths. Morphologically, these outgrowths resembled OSE in vivo and endothelial and mesothelial cells in culture, but they differed from cultured ovarian stromal, granulosa, and luteal cells. Only OSE among ovarian cell types were intensely keratin positive by immunofluorescence. Keratin also distinguished OSE cells from the keratin-negative endothelial cells. Most but not all OSE colonies tested showed 17β-hydroxysteroid dehydrogenase (HSD) activity, which was absent in peritoneal mesothelial cells. Colonies from most patients were limited to a few millimetres and became stationary within a few weeks. Changes that accompanied cessation of growth included senescence, increased keratin content, or the formation of multicellular papillary aggregates. With time, OSE cells tended to assume a fibroblast-like morphology but remained keratin positive and continued to resemble OSE by scanning electron microscopy (SEM). Subcultured OSE cells persisted in a stationary keratin-positive form for many weeks. Throughout this study, all pavementlike epithelial outgrowths that were contiguous with an explant stained for keratin; thus, such colonies can be assumed to be OSE. Conversely, fibroblast-shaped cells may represent OSE as indicated by keratin content and SEM appearance. The methods presented here permit culture of normal human OSE under conditions in which the cells exhibit morphologic plasticity, variable 17β-HSD activity, and presence of keratin. Supported by a grant and a research associateship to N. A. by the National Cancer Institute of Canada.     

Optimization of culture conditions for endothelial progenitor cells from porcine bone marrow in vitro

Objectives: The aim of this study was to determine an optimal culture method for porcine bone marrow‐derived endothelial progenitor cells (EPCs). Materials and methods: Mononuclear cells (MNCs) were isolated by density centrifugation and differentiated into EPCs in in vitro. At first‐passage, EPCs were cultured at different cell densities (5 × 10³, 1 × 10⁴, 2 × 10⁴ or 5 × 10⁴/cm²) and in basic medium (EGM, medium 199, DMEM or 1640) supplemented with FBS (2%, 5%, 10% or 20%) and different combinations of cytokines (VEGF, VEGF + bFGF, VEGF + bFGF + EGF, or VEGF + bFGF + EGF + IGF), the experiment being based on L₆₄ (4²¹) orthogonal design. Results and conclusions: This demonstrated that the optimal culture method for our EPCs displayed higher expansion and migration rates as compared to other groups, by analysis of variance; that is, cultured at 1 × 10⁴/cm² in M199 supplemented with 10% FBS and VEGF + bFGF + IGF + EGF. Furthermore, percentage of positive cells stained by Dil‐ac‐LDL and FITC‐UEA‐1 was more than 65%, and as shown by immunohistochemistry, these cells also stained positively for CD133, CD34 and KDR. The present study indicates that the number and function of porcine EPCs significantly increased when using our optimized culture parameters.     

Effects of epidermal growth factor and transforming growth factor-beta 1 on rat heart endothelial cell anchorage-dependent and -independent growth

This report describes the effects of epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGF-beta 1) on the anchorage-dependent and -independent growth of rat heart endothelial cells (RHE-1A). When RHE-1A cells were grown in monolayer culture with medium containing 10% fetal bovine serum (FBS) supplemented with epidermal growth factor (0.1-100 ng/ml), growth was stimulated fivefold when compared to that of cells grown in medium containing 10% FBS alone. The stimulatory effect of EGF on RHE-1A cell monolayer growth was dose-dependent and half-maximal at 5 ng/ml. The addition of TGF-beta 1 in the range 0.1-10 ng/ml had no effect on RHE-1A cell monolayer growth when added to medium containing 10% FBS alone or 10% FBS supplemented with EGF (50 ng/ml). RHE-1A cells failed to grow under anchorage-independent conditions in 0.3% agar medium containing 10% FBS. In the presence of EGF, however, colony formation increased dramatically. The stimulatory effect of EGF was dose-dependent in the range 0.1-100 ng/ml and was half-maximal at 5 ng/ml. In contrast to its effects under anchorage-dependent conditions, TGF-beta 1 (0.1-10 ng/ml) antagonized the stimulatory effects of EGF on RHE-1A cell anchorage-independent growth. The inhibitory effect of TGF-beta 1 was dose-dependent and half-maximal at 0.1 ng/ml. EGF-induced RHE-1A soft agar colonies were isolated and reinitiated in monolayer culture. They retained the cobblestone morphology and contact-inhibition characteristic of normal vascular endothelial cells. Each of the clones continued to express Factor VIII antigen. These findings suggest that TGF-beta may influence not only endothelial cell proliferation but also anchorage dependence. These effects may in turn be of relevance to endothelial cell growth and angiogenesis in vivo.     

Ovarian surface epithelium: autonomous production of connective tissue-type extracellular matrix.

The ovarian surface epithelium (OSE) is known to contribute to postovulatory repair of the ovarian cortex by proliferation and migration over the site of follicular rupture, and by deposition of a basement membrane. We examined the production of other extracellular matrix components in culture by OSE cells of the rat (ROSE), using immunofluorescence microscopy, electron microscopy, and proline incorporation. We compared recently explanted cells in low passage, the immortal line ROSE 239, whose growth pattern resembles low passage cultures, and the immortal line ROSE 199, which forms ridges and papillae. The epithelial nature of all three cell types was confirmed by the presence of keratin and laminin. All three cell types secreted collagen types I and III and at least one (ROSE 199) produced highly polymerized banded fibrils, which are characteristic for stromal or interstitial extracellular matrix. Simultaneous production of collagen types I and III, keratin, and laminin by cloned subpopulations ruled out an origin of the lines in mixed epithelial/fibroblast populations. The results demonstrate that OSE has the capacity to synthesize major components of connective tissue stroma. They suggest that this epithelium, in addition to its postulated proteolytic role, may also express synthetic activity in the remodelling of the ovarian cortical stroma. A capacity of OSE cells to produce stromal components autonomously might be an important factor in the formation of ovarian surface papillae and in neoplastic progression of OSE-derived ovarian carcinomas.     

Effect of a serum extender containing growth factors on development of IVM and IVF bovine embryos

The present experiments were conducted to determine if supplementation of the culture medium with a serum extender containing growth factors would increase development of bovine embryos into morulae or blastocysts, following in vitro maturation (IVM) and in vitro fertilization (IVF). In Experiment 1, bovine zygotes were cultured in CR1 medium supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 2, bovine zygotes were cultured in the presence of cumulus cells in CR1 medium supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 3, bovine oocytes were matured in Medium 199 supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 4, oocytes were matured in Medium 199 with 10% fetal bovine serum (FBS) or 5% FBS with serum extender. Following maturation, zygotes were cultured in CR1 medium with 10% FBS or 5 % FBS and serum extender. In all 4 experiments, the embryos were cultured in vitro until Day 7 after IVF, and development to the morula or blastocyst stage was assessed. The findings of the first 2 experiments showed that the serum extender did not directly influence embryo development but did stimulate development when cumulus cells were included in the culture system. The remaining 2 experiments showed that the serum extender did influence development through its interactions with cumulus cells during maturation and/or culture. These findings suggest that although growth factors or other products do not directly stimulate bovine embryo development their effects may be mediated through secondary cell systems.     

Human macrovascular endothelial cells: Optimization of culture conditions

Summary The purpose of this study is to identify optimal culture conditions to support the proliferation of human macrovascular endothelial cells. Two cell lines were employed: human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells (HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) were investigated in relation to cell proliferation. Additionally, a variety of extracellular matrix (ECM) substrate-coated culture dishes were also tested. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly used M199 medium, MCDB 131 resulted in a 2.3-fold increase in cell proliferation. Media containing 20% FBS increased cell proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 μg/ml) and endothelial cell growth supplement (ECGS) (50μg/ml) significantly improved cell proliferation. Epithelial growth factor (EGF) provided no improvement in cell proliferation. There were no statistical differences in cell proliferation or morphology when endothelial cells were grown on uncoated culture plates compared to plates coated with ECM proteins: fibronectin, laminin, gelatin, or collagen types I and IV. The culture environment yielding maximal HSVEC and HUVEC proliferation is MCDB 131 nutrient medium supplemented with 2 mM glutamine, 20% FBS, 50 μg/ml heparin, and 50 μg/ml ECGS. The ECM substrate-coated culture dishes offer no advantage.     

Effects of carp serum on the growth of goldfish fin cells in early passage

The culture medium supplemented with carp serum and fetal bovine serum (FBS) promoted cell growth significantly and induced morphological change of goldfish fin cells in early passage as compared to the medium containing FBS alone. However, these effects were not observed in RBCF-1, a cell line established from the goldfish fin. The sensitivity of the cells in early passage to carp serum suggests the following possibilities: (1) cells in early passage retain the ability to respond to growth-promoting factors specifically included in carp serum; and (2) this ability is lost during the process of long-term culture and/or long-term culture in FBS eliminates cell groups showing high dependency of cell growth on carp serum.     

Isolation and characterization of rabbit ovarian surface epithelium,granulosa cells,and peritoneal mesothelium in primary culture

Summary Mammalian ovarian surface epithelial (OSE) cells and peritoneal mesothelial (PM) cells have a common embryologic origin, yet certain morphologic and histochemical characteristics are different in the adult. In this study, a two-step culture method was developed to examine the characteristics of these two cell types in vitro. OSE, PM, and ovarian granulosa (GC) cells were isolated from estrous rabbits and cultured for 6 d in 5% serum-supplementedd-valine medium (to inhibit fibroblast growth), then incubated for a further 2 d in serum-free McCoy's 5A medium. This study showed that rabbit OSE and PM cells in vitro maintained certain in vivo morphologic characteristics; OSE cells exhibited distinct cell borders and abundant microvilli of homogeneous size and shape, whereas PM cells were characterized by obscure cell borders and abundant microvilli of heterogeneous form. GC in vitro exhibited overlapping cell borders and sparse microvilli of homogeneous structure. This study showed for the first time that cultured rabbit OSE and PM cells, but not GC, contain distinct filaments of cytokeratin 18. In addition, rabbit OSE cells and GC, but not PM cells, contained 17β-hydroxysteroid dehydrogenase. However, only GC contained delta 5-3β hydroxysteroid dehydrogenase. OSE, PM, and GC maintained their ultrastructural and histochemical characteristics in serum-free medium. These results suggest that rabbit OSE cells in vitro could be distinguished from PM cells by histochemical and ultrastructural differences. Furthermore, because these characteristics were not altered in serum-free medium, the two-step culture method will be valuable in further hormonal studies of these cells in vitro. This work was supported in part by Grant No. 202-3192 from the University of South Dakota Parsons Fund     

Expression and function of HOXA genes in normal and neoplastic ovarian epithelial cells

We studied the roles of three HOXA genes in cultured normal ovarian surface epithelial (OSE) cells and ovarian cancer cells. They included HOXA4 and HOXA7 because, by cDNA microarray analysis, these were more highly expressed in invasive ovarian carcinomas than in benign or borderline (noninvasive) ovarian tumors, and HOXA9 because it characterizes normal oviductal epithelium, which resembles ovarian serous adenocarcinomas. The three HOXA genes were more highly expressed when OSE cells were dividing and motile than when they were confluent and stationary, and also when they dispersed in response to EGF treatment or to reduced calcium concentrations in culture media. The expression of the HOXA genes varied among ovarian cancer cell lines, but was highest in lines with compact epithelial morphologies. We focused on HOXA4 as the most highly expressed in the ovarian carcinoma array. HOXA4 expression did not parallel proliferative activities of either OSE or ovarian cancer lines. Moreover, modifying HOXA4 expression in ovarian cancer cell lines did not alter either E-cadherin expression or CA125 secretion. However, HOXA4 downregulation enhanced EGFR phosphorylation and migration in serum-starved OSE and ovarian cancer cells in response to EGF, and enhanced migration of all ovarian cancer lines in 5% serum even without EGF treatment. Thus, HOXA4 expression does not correlate with proliferation or with epithelial differentiation, but it increases in response to OSE cell dispersion and negatively regulates EGFR activation and the motility of OSE and of ovarian cancer cells. HOXA4 expression was highest in cancer lines with compact epithelial growth patterns, suggesting, again, an anti-dispersion function. In summary, increased HOXA4 expression in ovarian cancer appears to constitute a tumor-suppressive, homeostatic response to aberrant cell behavior, and, in particular, to cell dispersion and migration.     

A serum-free medium for use in a cumulus cell co-culture system for bovine embryos derived from in vitro maturation and in vitro fertilization

The objective of this study was to develop a serum-free medium for the co-culture of bovine embryos that would yield a percentage of blastocysts equal to that obtained with fetal bovine serum (FBS)-supplemented medium. Cumulus cell-enclosed oocytes (CEO) matured and inseminated in vitro were cultured in a tissue culture medium (TCM)-199 or in a serum-free medium (bovine embryo culture medium; BECM) until 240 h post insemination. Replacement of 10% (v/v) FBS with either 3 mg crystallized bovine serum albumin (BSA)/ml or 3 mg fatty acid-free BSA/ml in TCM-199 had no effect (P > 0.14) on embryo development to the >or= 2-cell (51 to 60%), >or= 8-cell (24 to 33%), blastocyst (16 to 19%) and hatched-blastocyst (7 to 10%) stages at 48, 96, 192 and 240 h post insemination, respectively. Oocyte-enclosing cumulus cells in BSA-supplemented medium grew in clusters rather than in layers, as was noted in FBS-supplemented medium. When CEO were cultured in fatty acid-free BSA-supplemented media (TCM-199 and BECM), a significantly (P < 0.001) higher percentage of oocytes developed to blastocysts after culture with (22%) or without (18%) a cumulus cell monolayer than after denuding the oocytes (7%). Glucose in concentrations of 0 to 5.56 mM added for periods of 18 and 120 h post-insemination had neither a stimulatory nor a deleterious effect on preimplantation development. In conclusion, a serum-free medium supplemented with BSA can be successfully used in a cumulus cell co-culture system for bovine embryos.     

In vitro maturation, in vitro fertilization and embryonic development of canine oocytes

In this study we have investigated the efficiency of in vitro maturation (IVM) as a basic way to study the development of canine oocytes after in vitro fertilization (IVF). We decided, therefore, to perform two-part experiments. Firstly, experiment I compared the effects of TCM199 without fetal bovine serum (FBS) with TCM199 supplemented with 5% FBS on the in vitro nuclear maturation rate of canine oocytes. For the efficiency of meiotic development to the metaphase II (MII) stage, we found that 4.7% (4/64) of all oocytes grown in TCM199 without FBS developed to the MII stage compared with only 1.7% (1/59) of those grown in TCM199 with 5% FBS for 48 h. Therefore, FBS did not increase in vitro nuclear maturation. In experiment II, the cleavage rate of canine oocytes used for IVF was investigated following heparin treatment. Canine oocytes were fertilized in four groups: Fert-TALP medium without heparin (Fert I) or Fert-TALP medium supplemented with 10, 20 or 30 microg/ml heparin (Fert II, Fert III, Fert IV, respectively). Oocytes that were grown for 24 h in Fert I following fertilization showed the highest rate of all of the groups, 6.5% (5/77) and developed to the early morula stage. Markedly, the oocytes cultured in Fert I for 24 h following insemination had a higher rate of embryonic development than other groups. We can assert that, unlike findings in other mammals, heparin treatment in canine IVF does not increase the efficiency of the fertilization rate and is therefore not an important factor.     

Growth requirements and neoplastic transformation of two types of normal human breast epithelial cells derived from reduction mammoplasty

Summary A chemically defined culture medium was developed to support the growth of two distinctly different types of normal human breast epithelial cells (HBEC) derived from reduction mammoplasty. Type I cells expressed luminal epithelial cell markers and were deficient in gap junctional intercellular communication (GJIC), whereas Type II cells expressed basal epithelial cell markers and were efficient in GJIC. In this study, we examined and compared the growth factor and hormone requirements of these two types of cells and a series of cell lines that were obtained by sequential transfection with SV40 DNA (extended lifespan, nontumorigenic), treatment with 5-bromodeoxyuridine (BrdU)/black light (immortal and weakly tumorigenic), and infection of a virus carrying the neu oncogene (highly tumorigenic). Growth of Type I cells was inhibited by withdrawing epidermal growth factor (EGF), hydrocortisone (HC), or insulin (INS) from the culture media, but was enhanced by fetal bovine serum (FBS) supplementation. Growth of Type II cells was inhibited by withdrawal of EGF, HC, or INS from the media, and was inhibited by FBS supplementation. Withdrawal of human transferrin (HT) or 17β-estradiol (E₂) from the media did not alter the growth of Type I or Type II cells. SV40 transfected Type I cell lines still required EGF, HC, or INS for optimal growth. However, the highly tumorigenic cell line did not show a growth dependence on EGF, HC, or INS but did appear to require HT and 3,3′,5-triiodo-D.L. thyronine (T₃) for optimal growth. In addition, FBS stimulated the growth of these cell lines. Thus, this study shows that Type I HBEC are distinctly different from Type II HBEC in growth response to FBS and that neoplastically transformed Type I cells could become growth factor and hormone independent.     

Derivation and characterization of a zebrafish liver cell line

ZF-L cells were derived from normal adult zebrafish liver, and have been growing in culture for more than 100 generations. The cells were derived in basal nutrient medium supplemented with fetal bovine serum (FBS), trout serum, trout embryo extract, bovine insulin and mouse epidermal growth factor. After 50 generations in culture, optimal growth of the cells was achieved in medium supplemented with FBS (5%) and trout serum (0.5%). ZF-L cells were hypodiploid (modal chromosome number= 46) and exhibited an epithelial morphology. ZF-L cell homogenates exhibited alanine and aspartate aminotransferase, glucose-6-phosphatase and alkaline phosphatase enzyme activities. The cells synthesized and released several proteins into the culture medium, including a 70 kDa protein recognized by anti-bovine serum albumin IgG.Abbreviations NF -naphthoflavone - EGF epidermal growth factor - EROD 7-ethoxyresorufinO-deethylase - FBS fetal bovine serum - PBS phosphate-buffered saline - PMSF phenylmethylsulfonyl fluoride - TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin     

Survival of oocytes recovered from vitrified sheep ovarian tissues

The objective of this work was to develop an effective vitrification technique for cryopreserving oocytes in sheep ovarian tissues. Ovaries were surgically recovered from 15 pubertal ewes and the ovarian cortex was cut into sections. Ovarian tissues were placed in equilibration medium consisting of 4% (v/v) ethylene glycol (EG) and 20% (v/v) FBS in TCM-199 on ice for 30 min and transferred to vitrification solution (35% EG, 5% polyvinylpyrrolidone, 0.4M trehalose and 20% FBS in TCM-199) for 5 min. Ovarian tissues were vitrified by dropping the tissue on the surface of a steel cube cooled by liquid nitrogen. Cumulus-enclosed oocyte complexes (COC) were also collected and vitrified following the procedure used for ovarian tissues. After 2-3 weeks of storage in liquid nitrogen, ovarian tissues and COC were thawed at 37 degrees C in 0.3M trehalose and COC in ovarian tissues were mechanically and enzymatically isolated. Vitrified COC and freshly collected COC were washed twice in maturation medium (TCM-199 supplemented with 0.255 mM pyruvate and 10% heat-treated estrus cow serum) and cultured in 50 microl drops of maturation medium under paraffin oil for 23-25h at 39 degrees C in a humidified atmosphere of 5% CO(2) in air. After culture, cumulus cells were removed by hyaluronidase treatment and vortexing and oocytes were fixed and stained. No significant differences were observed between vitrified oocytes, oocytes recovered from vitrified ovarian tissues and non-vitrified control oocytes in the percentage of oocytes with acceptable staining per total number of oocytes fixed or with visible chromatin per total number of oocytes with acceptable staining. However, fewer (P<0.05) oocytes obtained from vitrified ovarian tissues (70%) reached metaphase II compared to vitrified oocytes (88%) and non-vitrified control oocytes (90%). In contrast, when oocytes with at least 3-5 layers of cumulus cells were considered from each of the three groups, no differences (P>0.05) were observed due to treatment in the percentages of oocytes developing to metaphase II. These results demonstrate that sheep oocytes can be successfully cryopreserved by vitrification of ovarian tissues and exhibit in vitro maturation rates similar to that of vitrified and non-vitrified oocytes.     

Effect of sperm exposure time on in vitro fertilization and embryo development of bovine oocytes matured in vitro

This study was designed to investigate the effect of sperm exposure time on the fertilization rate and subsequent developmental capacity of bovine oocytes matured in vitro. Cumulus oocyte complexes (COCs) obtained from 2 to 6 mm follicles were matured for 24 h in TCM-199 supplemented with fetal bovine serum (FBS) and hormones (FSH, LH and estradiol 17-beta). In vitro fertilization (IVF) was performed by incubating 15 to 20 matured oocytes with 1 x 10(6) percoll separated frozen-thawed spermatozoa in 1 ml of IVF-TL medium for either 4, 8, 12, 16, 20, 24 or 28 h. Following sperm exposure for different periods of times, the presumptive zygotes were co-cultured with Buffalo Rat Liver cells (BRLC) monolayers in CZB medium without glucose, a simple semi-defined medium developed for mouse embryo culture, for 3 d post-insemination and then in M199/FBS (TCM-199-HEPES supplemented with 20% heat-treated FBS and 1 mM sodium pyruvate) for 5 d. The fertilization rates differed significantly among the 7 treatment groups, with higher frequencies obtained by co-incubation of gametes for 20, 24 or 28 h (67 to 76%) than for 4, 8 and 12 h (26 to 54.5%), with 16 h (57%) being intermediate. However, the length of sperm exposure time did not significantly affect subsequent embryo development, although an increasing trend was noted from 4 h to 20 h. The number of fertilized oocytes at 3 d post-insemination cleaving to 2- to 4-cell vs 8-cell stage was not different among treatment groups. Development of 8-cell embryos to morulae and blastocysts did not differ among the treatment groups. These data suggest that the optimum duration of sperm-oocyte incubation is 24 h, and periods shorter than 16 h may result in a reduced fertilization rate.     

Development of bovine embryos in a cell-free culture medium: Effects of type of serum, timing of its inclusion and heat inactivation

Bovine embryos, derived from in vitro matured (IVM)/in vitro fertilized (IVF) ova, were used to investigate the effects of timing of serum inclusion in the culture medium and different types of blood sera and heat inactivation of the serum on embryo development. In Experiment 1, oocytes at 18 h post insemination were allocated to 1 of the following 4 treatments: 1) TCM-199 + 0.1 mg/ml polyvinylalcohol (PVA), 2) TCM-199 supplemented with 10% bovine calf serum (BCS), 3) PVA medium followed by BCS medium at 47 h, or 4) PVA medium followed by BCS medium at 82 h. Supplementation with BCS at 18 h post insemination suppressed (P<0.05) development of morulae/blastocysts (17.6%) when compared with PVA (30.5%) or with serum supplementation at 47 or 82 h post insemination (32.4 and 27.6%, respectively). However, inclusion of BCS at 18, 47 or 82 h post insemination produced more blastocysts (16.8, 29.3 and 22.1%, respectively; P<0.05) than medium +PVA (8.8%). In Experiment 2, ova were cultured from 18 h to 42 h post insemination in PVA-medium, then >/=2-cell embryos were transferred into serum-supplemented medium for another 168 h. Fetal bovine serum (FBS) +/- heat-inactivation (56 degrees C for 30 min, = heated FBS) suppressed morula/blastocyst development compared with medium + PVA, medium + BCS or medium + heated BCS (P<0.05). Bovine calf serum was superior to FBS in supporting blastocyst development (35.1 and 15.2%, respectively), but there was no difference between BCS and heated BCS. However, heated FBS increased the proportion of blastocysts/>/=8-cell embryos compared with that of FBS (51.0 and 31.4%, respectively; P<0.05). These results indicate that the type of serum supplementation and the timing of its inclusion in the culture medium markedly affect bovine embryo development in vitro, and that heat inactivation of serum with high embryotrophic properties is not necessary.     

Effect of including growth factors and antioxidants in maturation medium used for in vitro culture of buffalo oocytes recovered in vivo

This study examined the effect of including one of two growth factors (100 ng/ml IGF-1 or 20 ng/ml EGF) in combination with one of two antioxidants (50 microM cysteamine or 50 microM beta-mercaptoethanol) in maturation, fertilization and subsequent development of buffalo oocytes. The oocytes were recovered by in vivo ovum pick-up technique from six Murrah buffalo heifers twice a week over a period of 16 weeks. Immediately after ovum pick-up oocytes recovered from six donors were allocated randomly to five different maturation treatments. The control treatment was the basic maturation medium (MM; TCM-199 supplemented with 10% FBS, 10 IU/ml LH, 0.5 microg/ml FSH, 1 microg/ml estradiol-17beta and 50 microg/ml gentamicin). The other four treatments consisted of the control maturation medium (MM) plus one combination of a growth factor and an antioxidant viz. IGF-1+cysteamine; IGF-1+beta-ME; EGF+cysteamine or EGF+beta-ME. The total number of oocytes assigned to each maturation treatment ranged from 31 to 66. After maturation in different maturation medium, media used for in vitro fertilization and subsequent development of embryo was same for all groups. Data were analysed using Chi-square test. The maturation rate observed for the growth factor plus antioxidant treatments was similar to that for the control (90.4%). The highest cleavage rate recorded in the IGF-1+cysteamine treatment (71.9%) which was significantly higher (P<0.05) than the IGF-1+beta-ME (45.2%) and EGF+beta-ME (46.4%) treatments, but not significantly differ from the control (63.8%) and EGF+cysteamine treatment (60.7%). The proportion of cleaved oocytes those developed to blastocyst stage was significantly higher in the IGF-1+cysteamine treatment (52.2%; P<0.05) than in the control (23.3%), the EGF+cysteamine (13.5%) or the EGF+beta-ME (7.7%) treatments, but did not differ significantly from the IGF-1+beta-ME (28.6%) treatment. Following non-surgical transfer of 15 embryos to 14 synchronized recipients, four became pregnant and only one recipient sustained the pregnancy as long as 4.5 months when spontaneous abortion occurred. It was concluded that supplementing the maturation medium with IGF-1+cysteamine improved the production of buffalo embryos significantly in vitro culture.