Preservation of oocyte and granulosa cell morphology in bovine preantral follicles cultured in vitro

Described in the present paper is a culture system that preserves oocyte and granulosa cell morphology in bovine preantral follicles during 5 d in vitro. The effects of additional hypoxanthine and energy substrata (i.e., pyruvate and glutamine) on the morphology of cultured preantral follicles were investigated. It was shown that addition of a mixture of pyruvate, glutamine and hypoxantine to the culture medium increased the percentage of follicles with an intact oocyte from 29.4 to 78.6%. Morphological criteria are described to discriminate between normal and degenerated preantral follicles during culture by inverted microscopy. In addition, the importance of histological evaluation to judge the quality of oocyte and granulosa cells is demonstrated.     

Effect of adiponectin on bovine granulosa cell steroidogenesis,oocyte maturation and embryo development

Background  

Adiponectin is an adipokine, mainly produced by adipose tissue. It regulates several reproductive processes. The protein expression of the adiponectin system (adiponectin, its receptors, AdipoR1 and AdipoR2 and the APPL1 adaptor) in bovine ovary and its role on ovarian cells and embryo, remain however to be determined.     

Mouse antral oocytes regulate preantral granulosa cell ability to stimulate oocyte growth in vitro

In this study we evaluated whether mouse oocytes derived from early antral or preovulatory follicles could affect the ability of preantral granulosa cells to sustain oocyte growth in vitro. We found that early antral oocytes with a diameter > or =75 microm did not grow any further during 3 days of culture on preantral granulosa cell monolayers in vitro, while most of the oocytes with a smaller diameter increased significantly in size. Similarly, about 65% of growing oocytes isolated from preantral follicles grew when cultured on preantral granulosa cells. By coculturing with growing oocytes fully grown early antral or preovulatory oocytes, a small proportion (about 10%) of growing oocytes increased in diameter, and changes in granulosa cell morphology were observed. Such effects occurred as a function of the fully grown oocyte number seeded and were not associated with a decrease in coupling index values. By avoiding physical contact between antral oocytes and granulosa cells, the proportion of growing oocytes undergoing a significant increase in diameter was about 36%. These results indicate that fully grown mouse oocytes can control preantral granulosa cell growth-promoting activity through the production of a soluble factor(s) and the maintenance of functional communications with surrounding granulosa cells.     

Identification of novel oocyte and granulosa cell markers

Here we present novel gene expression patterns in the ovary as part of an ongoing assessment of published micro-array data from mouse oocytes and embryos. We present the expression patterns of 13 genes that had been determined by micro-array to be expressed in the mature egg, but not during subsequent preimplantation development. In-situ hybridization of sectioned ovaries revealed that these genes were expressed in one of two distinct patterns: (1) oocyte-specific or (2) expressed in both the oocyte and surrounding granulosa cells. Despite the fact that micro-array data demonstrated expression in the egg, several of these genes are expressed at low levels in the oocyte, but strongly expressed in granulosa cells. Eleven of these genes have no reported function or expression during oogenesis, indicating that this approach is a necessary step towards functional annotation of the genome. Also of note is that while some of these gene products have been well characterized in other tissues and cell types, others are relatively unstudied in the literature. Our results provide novel gene expression information that may provide insights into the molecular mechanisms of follicular recruitment, oocyte maturation and ovulation and will direct further experimentation into the role these genes play during oogenesis.     

Effect of IGF-I on pig oocyte maturation,fertilization, and early embryonic development in vitro,and on granulosa and cumulus cell biosynthetic activity

Porcine granulosa cells have been shown previously to both secrete and respond to insulin-like growth factor-I (IGF-I), suggesting an autocrine function of this peptide in the follicle. The present work was undertaken to determine possible effects of IGF-I on in vitro maturation, in vitro fertilization, and early embryonic development in culture. Granulosa and cumulus cell proliferation and differentiation based on ³H-thymidine uptake and progesterone production, respectively, were also assessed. The results showed that the cleavage rate of oocytes was markedly stimulated in a dose-dependent manner by the addition of IGF-I to the oocyte maturation medium (P < 0.05). Embryo development beyond the 8-cell stage was improved by IGF-I, reaching a maximum of 22% at 200 ng/ml IGF-I. Treatment with IGF-I after fertilization increased the percentage of total oocyte cleavage (P < 0.05) to approximately 52%, 43%, and 57% at, respectively, 25, 50, and 100 ng/ml IGF-I. ³H-thymidine incorporation by granulosa cells was significantly increased in cultures treated with FSH (3-fold) or IGF-I (6-fold) compared to the control. For the cumulus cells, FSH caused a similar increase (3-fold) in ³H-thymidine incorporation while IGF-I stimulated a 15-fold increase. Progesterone production by the granulosa cells was increased to the same extent by treatment with FSH or IGF-I (4.7 and 5.1-fold, respectively). However, for the cumulus cells, while FSH caused a marked 16-fold increase in progesterone production, IGF-I caused only a marginal increase of 2.5-fold. These results indicate a beneficial effect of IGF-I on in vitro porcine oocyte maturation and pre-implantation embryo development, suggesting a physiological role for IGF-I in vivo. The in vivo effect of IGF-I may be indirect via autocrine stimulation of cumulus and/or granulosa cells resulting in enhanced oocyte maturation and fertilization. © 1994 Wiley-Liss, Inc.     

Effects of trehalose co-incubation on in vitro matured prepubertal ovine oocyte vitrification

Our aim was to evaluate if loading prepubertal ovine oocyte with trehalose would impact on their further developmental potential in vitro and if it would improve their survival to vitrification procedures. COCs matured in vitro with (TRH) or without (CTR) 100mM trehalose were tested for developmental potential after in vitro fertilization and culture. Trehalose uptake was measured by the antrone spectrophotometric assay. No differences were recorded between the two experimental groups in fertilization rates (91.1 CTR vs 92.5% TRH), cleavage rates calculated on fertilized oocytes (96.1 CTR vs 95.4% TRH), first cleavage kinetic (56.1 CTR vs 51% TRH), and blastocyst rates (14.3 CTR vs 13.0% TRH). Anthrone assay revealed that in TRH group trehalose concentration/oocyte was 2.6microM. MII oocytes were then vitrified using cryoloops in TCM 199 containing 20% FCS, sucrose 0.5M, 16.5% Me(2)SO, 16.5% EG and plunged in LN(2). After warming, oocytes from TRH and CTR groups were tested for membrane integrity using the propidium iodide (PI)/Hoechst differential staining, and for developmental ability after in vitro fertilization. Trehalose in maturation medium affected membrane resistance (P<0.01) to vitrification/warming but not fertilization and cleavage rates. The differential staining showed a lower number of PI positive cells in TRH group compared to CTR one (14.3 vs 24.7%, respectively). Fertilization rates and cleavage rates did not differ between the two groups (55.3 and 41% for TRH and 47.7 and 41.7% for CTR, respectively). In conclusion trehalose in maturation medium stabilizes cell membranes during vitrification/warming of prepubertal ovine oocytes but does not affect fertilization and cleavage rates after warming.     

Mammalian oocyte growth and development in vitro

This paper is a review of the current status of technology for mammalian oocyte growth and development in vitro. It compares and contrasts the characteristics of the various culture systems that have been devised for the culture of either isolated preantral follicles or the oocyte-granulosa cell complexes from preantral follicles. The advantages and disadvantages of these various systems are discussed. Endpoints for the evaluation of oocyte development in vitro, including oocyte maturation and embryogenesis, are described. Considerations for the improvement of the culture systems are also presented. These include discussions of the possible effects of apoptosis and inappropriate differentiation of oocyte-associated granulosa cells on oocyte development. Finally, the potential applications of the technology for oocyte growth and development in vitro are discussed. For example, studies of oocyte development in vitro could help to identify specific molecules produced during oocyte development that are essential for normal early embryogenesis and perhaps recognize defects leading to infertility or abnormalities in embryonic development. Moreover, the culture systems may provide the methods necessary to enlarge the populations of valuable agricultural, pharmaceutical product-producing, and endangered animals, and to rescue the oocytes of women about to undergo clinical procedures that place oocytes at risk. © 1996 Wiley-Liss, Inc.     

Effects of granulosa cell mitochondria transfer on the early development of bovine embryos in vitro

The objective of this study was to determine the effect of exogenous mitochondria obtained from granulosa cells on the development of bovine embryos in vitro. We classified cumulus oocyte complexes (COCs) as good (G)- and poor (P)-quality oocytes based on cytoplasmic appearance and cumulus characteristics, and assessed mtDNA copy numbers in the G and P oocytes with real-time polymerase chain reaction (PCR). The mitochondria were isolated by fractionation and suspended in mitochondria injection buffer (MIB). Part one of the experiment consisted of the following treatments: (1) G-oocytes + sperm, (2) P-oocytes + mitochondria + MIB + sperm, (3) P-oocytes + MIB + sperm, and (4) P-oocytes + sperm. In part 2, oocytes were parthenogenetically activated. The treatments were: (1) G-oocytes, (2) P-oocytes + mitochondria + MIB, (3) P-oocytes + MIB, and (4) P-oocytes alone. The results indicated a significant difference in mtDNA copy number between G (361 113 +/- 147 114) and P (198 293 +/- 174 178) oocytes (p < 0.01). The rates of morula, blastocyst, and hatched blastocysts derived from P-oocytes + mitochondria were similar to those of G-oocytes, but significantly higher than P-oocytes without exogenous mitochondria in both the ICSI and parthenogenetic activation experiments. We found no difference in blastomere numbers between G-oocytes and P-oocytes + mitochondria in either experiment, but blastomere numbers in these two groups were significantly higher than in P-oocyte groups without exogenous mitochondria. These data suggest that mtDNA content is very important for early embryo development. Furthermore, the transfer of mitochondria from the same breed may improve embryo quality during preimplantation development.     

A comparison between oocyte growth in coculture with granulosa cells and oocytes with granulosa cell-oocyte junctional contact maintained in vitro

Evidence is presented that strongly supports the hypothesis that the junctional association between oocytes and granulosa cells must be maintained to promote oocyte growth and development in vitro and that the coculture of oocytes with granulosa cells is not a sufficient condition for oocyte development. Furthermore, it is shown that incorporation of uridine and leucine by oocytes into TCA-insoluble material is significantly greater in granulosa cell-enclosed oocytes than in cocultured oocytes.     

In vitro models for oocyte development

The mammalian ovary has a large store of primordial follicles, which are a potential source of oocytes for in vitro production of embryos. Several culture systems have been developed to support the growth and development of oocytes from rodent primordial and preantral follicles and progress is slowly being made in modifying these techniques to support the in vitro growth of porcine and bovine follicles. Oocytes from porcine preantral follicles can acquire competence to resume meiosis and proceed to Metaphase II after in vitro growth (IVG) but fertilisation has yet to be demonstrated. This paper presents the current status of technology for the in vitro growth and development of immature mammalian oocytes. Culture systems used successfully to grow immature rodent oocytes are compared and adaptations of these methods to support porcine and bovine oocyte growth discussed.     

Expression of insulin-like growth factor binding protein-5 by ovine granulosa cells is regulated by cell density and programmed cell death in vitro

In vivo, in the sheep ovary, the expression of insulin-like growth factor binding protein (IGFBP)-2 and particularly IGFBP-5 has been shown to increase dramatically in apoptotic granulosa cells from atretic follicles. The aim of this work was to study the relationship between apoptosis induced by serum starvation in vitro and expression of IGFBP-2 and -5 by ovine granulosa cells. For this purpose, granulosa cells from follicles 1–3 mm in diameter were cultured in the presence of serum for 2 days, then cultured in the presence or absence of serum for 24, 48, or 72 hr. At the end of the culture, cells were counted, cell viability was assessed by studying DNA fragmentation, and IGFBPs expression was studied by quantitative autoradiography, Western-ligand blotting, immunoblotting, and quantitative in situ hybridization. In vitro, IGFBP-2 and particularly IGFBP-5 were the main IGFBPs secreted by ovine granulosa cells. Serum starvation provoked (i) apoptosis of granulosa cells within 48 hr, (ii) a marked decrease in cell density, and (iii) a marked increase in the amount of IGFBP-5 associated with cell membranes and with the walls of culture wells, but no change in culture medium. The increase in the amount of cell- and wall-associated IGFBP-5 after serum starvation was essentially due to the consecutive decrease in cell density rather than to an increase in cell apoptosis. Indeed, irrespective of the presence or absence of serum, the amount of IGFBP-5 associated to cell membranes was inversely correlated to cell density. In contrast, the amount of IGFBP-5 present in culture medium was positively correlated to cell density. Furthermore, expression of IGFBP-5 mRNA was shown to increase with both cell density and cell death. Indeed, the expression of IGFBP-5 mRNA dramatically increased with cell density, irrespective of the presence or absence of serum, but at a similar cell density, expression was higher in serum-free than in serum conditions. Overall, these results indicate that, in vitro, the localization of IGFBP-5 on ovine granulosa cell membranes and in culture medium, respectively, was mainly dependent on cell density, whereas expression of IGFBP-5 mRNA was related to both cell density and cell death. These data suggest that IGFBP-5 is involved in both growth arrest and apoptosis of granulosa cells in the sheep. J. Cell. Physiol. 177:13–25, 1998. © 1998 Wiley-Liss, Inc.     

The effects of plasminogen on in vitro ovine embryo development

Plasminogen activator production by ovine embryos and the effects of plasminogen on ovine embryo development and zona pellucida integrity were evaluated. Eight-cell to sixteen-cell embryos were cultured in Whitten's medium containing 0, 60, or 120 micrograms/ml plasminogen. Plasmin and plasminogen activator concentrations in the medium were determined by a caseinolytic assay. More blastocysts hatched in medium containing 60 and 120 micrograms/ml plasminogen (33 and 21%, respectively) than 0 microgram/ml plasminogen (0%; p less than 0.05). Zona pellucida dissolution time in acidified phosphate-buffered saline was less after incubation in medium with 60 and 120 micrograms/ml plasminogen (7.2 and 5.9 min, respectively) than 0 microgram/ml plasminogen (9.4 min; p less than 0.05). Plasminogen activator production was low until the morula stage, increased during morula-blastocyst transition, and remained elevated through blastocoelic expansion and hatching. Zona pellucida solubility, plasminogen activator production, and plasminogen conversion to plasmin increased as embryonic stage advanced; however, plasminogen activator production and plasmin conversion to plasmin were poorly correlated with zona pellucida solubility. The results indicate that ovine embryos produce plasminogen activator, and plasmin can increase zona pellucida solubility; however, other factors may also be involved in altering zona pellucida integrity prior to hatching.     

Cumulus cell function during bovine oocyte maturation,fertilization, and embryo development in vitro

Several contemporary micromanipulation techniques, such as sperm microinjection, nuclear transfer, and gene transfer by pronuclear injection, require removal of cumulus cells from oocytes or zygotes at various stages. In humans, the cumulus cells are often removed after 15–18 hr of sperm-oocyte coincubation to assist the identification of the fertilization status. This study was designed to evaluate the function of cumulus cells during oocyte maturation, fertilization, and in vitro development in cattle. Cumulus cells were removed before and after maturation and after fertilization for 0,7,20, and 48 hr. The cumulus-free oocytes or embryos were cultured either alone or on cumulus cell monolayers prepared on the day of maturation culture. Percentages of oocyte maturation, fertilization, and development to cleavage, morula, and blastocyst stages and to expanding or hatched blastocysts were recorded for statistical analysis by categorical data modeling (CATMOD) procedures. Cumulus cells removed before maturation significantly reduced the rate of oocyte maturation (4–26% vs. 93–96%), fertilization (0–9% vs. 91–92%), and in vitro development at all stages evaluated. Cumulus cells removed immediately prior to in vitro fertilization (IVF) or 7 hr after IVF reduced the rates of fertilization (58–60% and 71%, respectively, vs. 91–92% for controls), cleavage development (40–47% and 53–54% vs. 74–78% for controls), and morula plus blastocyst development (15% and 24% vs. 45%, P < 0.05). Cumulus cell co-culture started at various stages had no effect on fertilization and cleavage development but significantly improved rates of embryo development to morula or blastocyst stages (P < 0.05). Cumulus cell removal at 20 hr after IVF resulted in similar development to controls (P > 0.05) at all stages tested in this study. The intact state of surrounding cumulus cells of oocytes or embryos appears to be beneficial before or shortly after insemination (at or before 7 hr of IVF) but not essential at 20 hr after IVF. © 1995 Wiley-Liss, Inc.     

Effects of cytokines on porcine granulosa cell steroidogenesis in vitro

Recent evidence indicates that factors produced by immune cells (cytokines) may play a role in ovarian function. To explore this possibility, we examined the effects of conditioned medium obtained from cultures of either unstimulated splenocytes (splenocyte-conditioned medium; SCM) or concanavalin A-stimulated splenocytes (CAS) on estrogen and progesterone production by porcine granulosa cells. Granulosa cells were obtained from small (less than 3 mm) or large (greater than 7 mm) follicles and treated with increasing doses of SCM or CAS in the presence or absence of pFSH (100 ng/ml) for 24 h at 37 degrees C. In granulosa cells obtained from small follicles it was found that both SCM and CAS evoked a dose-dependent increase in estrogen but not progesterone production. Estrogen production was no further enhanced by the presence of FSH. Additionally, SCM was able to augment FSH-stimulated progesterone production by these cells, whereas CAS had no effect. Identical treatment of granulosa cells obtained from large follicles demonstrated that both SCM and CAS caused dose-dependent increases in estrogen as well as progesterone production. In response to CAS, FSH augmented progesterone production but exerted a biphasic on estrogen production (inhibiting at lower doses while stimulating at higher doses). In contrast, SCM had no effect on FSH-stimulated estrogen production. Additional controls indicated that the above results could not be attributed to either concanavalin A or serum. Taken together, these findings suggest that cytokines can exert significant effects over granulosa cell steroidogenesis and further imply that these factors may play an important role in the differentiation and developmental regulation of granulosa cell function.     

Heterologous cell contacts and metabolic coupling in bovine cumulus oocyte complexes

The integrity of the cumulus cell processes were studied in four categories of bovine cumulus oocyte complexes (COCs) selected on their morphological characteristics. Three different types of cumulus cell process endings (CCPEs) were identified, one penetrating the cortex, another not penetrating the cortex, and a third form was intermediate and more rare in appearance. The process endings that penetrated the cortex frequently made gap junctions with the oolemma. The division of the three types of CCPEs over the four different COC categories was specific for three of the four categories. The first-category COC predominantly possessed the penetrating CCPE, the fourth-category COC possessed predominantly the nonpenetrating CCPE, and the second and third categories had both types of CCPEs. The metabolic coupling of the cumulus-oocyte contacts was assessed by means of incorporation of 3H-choline into the oocyte. The majority of category 4 COCs transferred low levels of choline into the oocyte while the majority of the oocytes of the other three categories transferred high levels of choline into the oocyte. Category 4 includes a smaller proportion of oocytes capable of cleaving after fertilization than the other three categories. This reduced developmental capacity is probably due to the loss of metabolic coupling before the onset of culture.     

Effects of resazurin on bovine oocyte fertilization and embryo development in vitro

Resazurin is a redox dye (7-hydroxy-3H-phenoxazin-3-one-10-oxide) used for assessing potential fertility of spermatozoa and functional status of eukaryotic cells. In this study, the fertilizing capacity of spermatozoa treated with resazurin and effects of resazurin on bovine embryo development in vitro was examined. Abattoir-derived bovine oocytes were collected and subjected to in vitro maturation (IVM), fertilization (IVF) and culture (IVC). In Experiment 1, bovine oocytes (n=2767) were fertilized with spermatozoa exposed to resazurin (17.6 μg/ml) for 0, 15, 30, 60 min, respectively. There was no significant (P>0.05) difference with respect to oocyte cleavage, morula and blastocyst production between treatments. In Experiment 2, oocytes (n=1671) were treated with resazurin (1.8 μg/ml) during IVM, IVF, IVC, respectively, or during the entire IVM, IVF and IVC procedures. There was no significant (P>0.05) difference in cleavage rates. However, the proportion of embryos that developed into blastocysts, expanded and hatched blastocysts in those groups in which oocytes/embryos were treated with resazurin during IVC or IVM/IVF/IVC was significantly (P<0.05) less than those exposed to resazurin during IVM only, or during IVF only. We conclude that resazurin did not have significant adverse effects on fertilizing capability of bovine spermatozoa; however, extended treatment of embryos with resazurin may be detrimental to embryonic development.     

Delay on the in vitro kinetic development of prepubertal ovine embryos

In the present study we characterize the developmental potential of prepubertal and adult ovine oocytes, analyzing the developmental speed to two-cell and blastocyst stages and its relationship with hatching from the zona pellucida, development after vitrification and the number and allocation of inner mass and trophoblastic cells. Prepubertal and adult ovine oocytes were matured and fertilized in vitro and first cleavage rates at 22, 26 and 32 h were recorded. Cleaved oocytes were cultured and blastocyst production was assessed at 6-9 days post-fertilization (dpf). Blastocysts from the two sources obtained on different days were divided into two groups: the first was vitrified, warmed and cultured in vitro to evaluate re-expansion of the blastocoelic cavity; blastocysts of the second were cultured separately to allow for hatching and count of trophoblastic and inner mass cells of hatched blastocysts by differential staining. We observed a significantly lower rate (P < 0.01) of cleaved prepubertal oocytes at 22 and 26 h after fertilization while it was higher (P<0.01) at 32 h than in the adult ones. Adult blastocyst production was significantly lower (P < 0.01) in prepubertal than in adult groups and began on the seventh dpf, later (P < 0.01) than in the adult group, where they appeared on the sixth dpf. Prepubertal blastocysts hatched at a lower rate than the adult ones (P < 0.01) and in both experimental groups faster blastocysts showed a higher (P < 0.01) hatching rate. Similarly, prepubertal derived blastocysts showed lower viability after vitrification (P < 0.01) compared to the adult counterparts, and in particular slower embryos had reduced viability after vitrification compared to the fastest (P < 0.01). Cell number was not different between blastocysts of both groups obtained at 6 and 7 dpf, which were higher (P < 0.01) than those obtained at 8 and 9 dpf. The ICM/trophoblast cell ratio was similar in 6- and 7-day obtained blastocyst and increased (P < 0.01) in those obtained 1 or 2 days later. These findings show that differences in kinetic development between prepubertal and adult derived embryos reflect differences in developmental capacity of the oocytes from which they derive and could be indicative of embryo quality.     

Influence of somatotropin and nutrition on bovine oocyte retrieval and in vitro development

The objective of this study was to determine the effects of supplemental bovine somatotropin (bST) and limit feeding on follicular growth and oocyte competence in yearling beef heifers. Sixteen growing heifers (424+/-4 kg) were randomly assigned to 1 of 4 treatments in a 2 x 2 factorial arrangement, with main effects of bST (0 or 33 microg/kg BW/d) and feeding regimen (ad libitum or 0.75 ad libitum intake). Animals were treated for 100 d prior to follicular aspiration, and treatments continued for the 42-d period that follicles were aspirated. Follicles were observed ultrasonically then aspirated, and recovered oocytes were matured, fertilized and developed in vitro. The number of follicles observed ultrasonically was greater with bST treatment (P<0.01) but was unchanged by plane of nutrition. The number and quality of recovered oocytes were similar among treatments, as was the number of oocytes resulting in blastocyst formation.