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Biomedical subjects

F L Barnes

Publications and source records attributed to F L Barnes.

At least 19 recordsLinked to original sources
PubMed2003

Presence of LH receptor mRNA in granulosa cells as a potential marker of oocyte developmental competence and characterization of the bovine splicing isoforms.

As the expression of the LH receptor (LH-R) in granulosa cells is thought to be associated with later stages of folliculogenesis, this study was undertaken to evaluate the presence of LH-R mRNA as a suitable marker for developmental competence of oocytes. Granulosa cells and cumulus-oocyte complexes (COCs) were recovered from cows that had received ovarian stimulation. The COCs were subjected to embryo production procedures in vitro to assess the embryonic potential of the oocyte, and the corresponding granulosa cells were used to evaluate the presence of LH-R mRNA by RT-PCR. The presence of LH-R transcripts in granulosa cells is not a key characteristic of a follicle bearing a competent oocyte, although a higher proportion of oocytes reach the blastocyst stage when LH-R mRNA is detected in the granulosa cells. Different LH-R isoforms were cloned and sequence discrepancies among six of the isoforms enabled the design of specific oligonucleotides to study the presence of the isoforms in different follicular cells. All LH-R transcripts studied and the 80 kDa protein product corresponding to the full length receptor were found in granulosa cells of small (< 4 mm) and large (> 5 mm) follicles. When the granulosa cells were cultured, the transcripts were downregulated by the culture conditions; downregulation was more acute in granulosa cells from small follicles. The addition of LH to the culture media enhanced LH-R mRNA downregulation. The presence of several LH-R transcript isoforms was tissue specific and in the theca cells LH-R mRNA was restricted mainly to cells from larger follicles. This finding indicates that the expression and the splicing of LH-R mRNA are regulated in a cell-specific and follicular size-specific manner.

Alternative Splicing↗
PubMed2001

Differential display and suppressive subtractive hybridization used to identify granulosa cell messenger rna associated with bovine oocyte developmental competence.

The main objective of this study was to identify mRNA expressed in the granulosa cells characterizing differentiated follicles bearing developmentally competent bovine oocytes. Analytical comparisons were made on mRNA pools of granulosa cells using differential display reverse transcription polymerase chain reaction (DDRT) analysis and suppressive subtractive hybridization (SSH). With DDRT, mRNA patterns of granulosa cells from small (< 4 mm) and large (> 8 mm) follicles cultured in the presence or absence of LH were compared to identify mRNA associated with follicular size or with the LH response. Nine clones were sequenced, and two were identified. One of the clones, DRAK 1, was associated with the presence of LH in the medium. Other comparisons directed toward the identification of mRNA associated with the presence of a competent oocyte were done on granulosa cells collected in vivo from superstimulated heifers. With the DDRT analysis, four clones associated with the oocyte developmental competence status were identified. With the SSH analysis, four clones specific to the presence of an incompetent oocyte were sequenced and none were identified, whereas 49 clones specific to the presence of a competent oocyte were sequenced and 18 were identified. Among these clones, early growth response 1, sprouty 2, cytochrome C oxidase, matrix metalloproteinase inducer, matrix metalloproteinase, epiregulin, prostaglandin receptor, and progesterone receptor were the most relevant to the ovarian physiology being examined.

Adenosine Triphosphatases↗
PubMed2000

The effects of the early uterine environment on the subsequent development of embryo and fetus.

Synchrony between the embryo and the uterine endometrium is essential for the establishment of pregnancy and birth in people and livestock. When asynchronous conditions occur a variety of complication result that include failure of the embryo to implant, early embryonic mortality, retarded development and growth, and accelerated development and growth. These complications all appear to be induced within the first week of embryo development and not withstanding the immediate endpoint of large or small size at birth, may alter the course of development throughout the life of the animal. Progesterone appears to play a causative role in establishing the abnormal growth of the fetus by decelerating or accelerating embryonic development. This may act through increasing the transport of blood born growth factors into the uterine lumen or by stimulating the release of growth factors from the endometrium directly. It can not be ruled out that progesterone mediated abundance of, or absence of, appropriate nutrition may bring about the same lifelong outcome. In vitro culture situations that include serum and/or co-culture can also bring about these abnormalities of growth. It is hypothesized that exposure to growth factors "out of phase" may result in an irreversible induction of abnormal development. The described abnormalities that occur in sheep and cattle have not yet been described for children resulting from IVF.

Animals↗
PubMed2000

Subtractive hybridization used to identify mRNA associated with the maturation of bovine oocytes.

The main objective of this project is to identify mRNA associated with oocyte maturation and embryonic developmental competency. The knowledge of genes and their accumulated mRNA is essential to better understand the mechanisms involved in the oocyte maturation and the survival of the in vitro produced embryo. We used bovine slaughterhouse-recovered ovaries and collected the oocytes from two follicle size categories: <2 mm and 3-5 mm. The mRNA content of oocytes from follicles 3-5 mm where considered to be more competent when compared to the content of oocytes from follicles <2 mm. In this report we compare two different technical approaches both involving PCR to compare the mRNA pools of the oocytes. In the first approach we performed the differential display (DDRT) technique to amplify and display side by side the cDNAs of groups of 10 denuded oocytes. From this approach, we isolated 28 different bands. After analysis, three of those bands had strong homology with known genes. In the second approach pools of 50 denuded oocytes were submitted to suppressive subtraction hybridization (SSH). We identified several known genes like cyclin B1, splicing factor ccl.4, cytochrome c oxidase, and mineralocorticoid receptor while numerous other clones remain unidentified. The cyclin B1 clone was used as a probe to evaluate its follicular size specificity on virtual Northern blot. The PCR basis of these techniques allows comparison of mRNA from tissues of low abundance such as oocytes. In this study the SSH resulted in longer clones than DDRT and showed high specificity.

Animals↗
PubMed2000

Oocyte maturation.

The oocyte is dependent on granulosa cells to provide nutrients and regulatory signals. Granulosa cells must be at the appropriate stage of differentiation to initiate these signals and transmit them to the oocyte. Studies have shown that in vitro-matured oocytes from follicles in early stages of atresia are more competent to support embryonic development than those from actively growing follicles. The acquisition of developmental competence appears to occur prior to in vitro maturation and can be induced by gonadotropin-free coasting in vivo or postmortem ovary incubation in vitro. The acquisition of developmental competence is probably a common signaling or differentiation pathway that occurs in the oocyte and/or associated granulosa regardless of whether the oocyte is destined to ovulate or degenerate. Early follicle atresia is the visually discernible characteristic in vitro that is associated with increased developmental potential.

Animals↗
PubMed1996

Nucleolar and mitochondrial morphology in bovine embryos reconstructed by nuclear transfer.

The nucleolar and mitochondrial morphology of developing reconstructed bovine nuclear transfer (NT) embryos and stage-matched in vivo-produced control embryos were examined under the electron microscope. Each reconstructed embryo at the one-cell (n = 12), two-cell (n = 5), three-cell (n = 3), four-cell (n = 5), 5-8-cell (n = 5) and blastocyst (n = 3) stages was produced by fusion of a 16-32-cell-stage blatomere with an aged enucleated bovine oocyte. The normal and reconstructed embryos showed similar mitochondrial morphology. However, NT embryos produced several pleiomorphic forms not seen in controls, and were more heterogeneous at early stages of development. Control embryos exhibited nucleolar features considered indicative of rRNA synthesis from the eight-cell stage onwards. In contrast, the NT embryos presented nucleoli with morphology consistent with rRNA synthesis in all embryos examined, except in the three-cell and in two of the five four-cell embryos. From this nucleolar morphology, it was concluded that nuclear reprogramming does not occur immediately following nuclear transfer, but occurs gradually over the first two or three cell cycles.

Animals↗
PubMed1996

Production of embryos from in vitro-matured primary human oocytes.

OBJECTIVE: To determine the factors that influence the number and quality of embryos produced from primary oocytes collected from untreated regularly ovulating and irregular or anovulatory polycystic women. DESIGN: A direct comparison between two patient groups whose oocytes were matured in vitro and a comparison of the embryo development of in vitro-matured oocytes from untreated patients with in vivo-matured oocytes of superovulated IVF-ET patients obtained during the same period. SETTING: The Monash IVF Clinic, involving patients who expressed the desire to avoid super-ovulation with fertility drugs. MAIN OUTCOME MEASURES: The completion of nuclear maturation of oocytes after 36 or 48 hours culture, fertilization in vitro, and embryo development ratio. RESULTS: Oocytes from regular cycling patients matured and fertilized at significantly higher rates than irregular cycling and anovulatory women and their embryos had significantly higher mean embryo development ratio. The mean embryo development ratio of embryos of regular cycling patients was similar to superovulated IVF patients but irregular cycling and anovulatory patients had a significantly lower embryo development ratio. Culture of oocytes for 48 hours increased maturation of oocytes from 57% to 82% but did not affect fertilization or cleavage rates. Embryo development was not affected significantly by the grade of follicular cell cover of oocytes. CONCLUSIONS: The developmental capability of primary oocytes is higher in regular cycling women than in irregular cycling and anovulatory women with polycystic ovary disease.

Anovulation↗
PubMed1995

Blastocyst development and birth after in-vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching.

Immature oocyte recovery followed by in-vitro oocyte maturation and in-vitro fertilization is a promising new technology for the treatment of human infertility. The technology is attractive to potential oocyte donors and infertile couples because of its reduced treatment intervention. Immature oocytes were recovered by ultrasound-guided transvaginal follicular aspiration. Oocytes were matured in vitro for 36-48 h followed by intracytoplasmic sperm injection (ICSI). Embryos were cultured in vitro for 3 or 5 days before replacement. Assisted hatching was performed on a day 5 blastocyst stage embryo. Embryo and uterine synchrony were potentially enhanced by luteinization of the dominant follicle at the time of immature oocyte recovery. Mature oocyte and embryo production from immature oocyte recovery were similar to the previous IVF results of the patients. A blastocyst stage embryo, produced as a result of in-vitro maturation, ICSI, in-vitro culture and assisted hatching, resulted in the birth of a healthy baby girl at 39 weeks of gestation.

Blastocyst↗
PubMed1994

Nuclear transplantation by microinjection of inner cell mass and granulosa cell nuclei.

The developmental potential of bovine inner cell mass (ICM) and somatic differentiated (granulosa cell) nuclei was investigated using nuclear transplantation. ICM blastomeres were isolated after immunosurgery of day 7 in vitro produced blastocysts and cumulus granulosa cells recovered from in vitro matured oocytes. Nuclear transplantation was carried out by microinjection of the lysed donor cells into enucleated mature oocytes. Oocytes were activated by three 0.2 kVcm-1/20 microseconds pulses in mannitol containing 100 microM Ca2+, with each pulse 22 min apart. Embryos were cultured in vitro for 7 days and blastocysts were transferred into recipients. ICM and granulosa cell donor nuclei directed 7% (20/304) and 9% (19/213) development to blastocysts, respectively. Fifteen blastocysts from ICM donors resulted in four pregnancies (27%) and two births. No pregnancy was detected with granulosa cell donors. The results illustrate the totipotency of ICM nuclei and indicate that granulosa cell nuclei promote preimplantation development of nuclear transplant embryos.

Animals↗
PubMed1993

Electrically induced calcium elevation, activation, and parthenogenetic development of bovine oocytes.

The influence of electrical stimulation on the level of intracellular Ca2+ in bovine oocytes, as well as activation and extent of parthenogenetic development, was investigated. Mature oocytes were electrically stimulated at 29 hr of maturation, and intracellular Ca2+ concentration was determined with the Ca2+ indicator fura-2 dextran (fura-2 D). The Ca2+ response of oocytes to a given electrical pulse was variable. Oocytes responded with either no Ca2+ rise from baseline (approximately 12 nM), a short-duration Ca2+ rise (from 12 nM to 300 nM) that returned to baseline within 2 min of the pulse, or a long-duration Ca2+ rise (from 12 nM to 1,000-2,000 nM) that never returned to baseline during the 8 min period over which the oocytes were monitored. In these oocytes, Ca2+ level returned to baseline when oocytes were removed from 0.30 M mannitol and placed in an ionic medium. Increasing field strength or pulse duration tended to increase the proportion of oocytes displaying a Ca2+ rise, and at 1.0 kVcm-1 for 40 microseconds, all oocytes displayed a long-duration Ca2+ elevation. Direct transfer of oocytes from culture medium to mannitol also triggered a Ca2+ rise. Multiple stimulations, either electrical or by transferring to mannitol, produced multiple Ca2+ rises. This mannitol-induced Ca2+ rise could be inhibited by first washing the oocytes in medium containing equal parts of 0.30 M mannitol and phosphate buffered saline (PBS). The level of Ca2+ stimulation affected activation and development of oocytes. Insufficient, or, conversely, excessive Ca2+ stimulation impaired development. Optimum development was obtained with 1) three pulses of 0.2 kVcm-1 for 20 microseconds, each pulse 22 min apart, after direct transfer of oocytes from culture medium to mannitol (22% blastocysts) or 2) three pulses of 1.0 kVcm-1 for 20 microseconds after transfer of oocytes from culture medium to medium containing equal parts mannitol and PBS, then to mannitol (24% blastocysts). This procedure avoided induction of a Ca2+ rise prior to the pulse. The results indicate that the level of Ca2+ stimulation can be regulated by incubation conditions prior to the pulse and, to some extent, by field strength and pulse duration. The level of electrical stimulation influenced oocyte Ca2+ response, activation, and parthenogenetic development.

Animals↗
PubMed1993

Histone H1 kinase activity in bovine oocytes following calcium stimulation.

The influence of number of Ca2+ stimulations on the profile of histone H1 kinase activity in bovine oocytes was investigated. A CA2+ stimulation consisted of transferring oocytes directly from culture medium to mannitol containing 100 microM Ca2+ and pulsing oocytes with a 0.2 kVcm-1, 20 microseconds discharge. One, three, or six Ca2+ stimulations were given, each 22 min apart. Oocytes were frozen from 0 to 8 hr after the first stimulation at indicated time points and assayed for histone H1 kinase activity. H1 kinase activity was quantified using a densitometer and expressed as a percent of activity in nonpulsed metaphase II oocytes. Stimulating oocytes in the absence of Ca2+ in the pulsing medium did not inactivate H1 kinase. In the presence of Ca2+, however, H1 kinase was rapidly inactivated after stimulation. A single stimulation decreased H1 kinase activity to 44% +/- 11% of its initial level in 1 hr. However, H1 kinase was dramatically reactivated at 2 hr after the stimulation and reached 122% +/- 22% of the initial activity at 6 hr. With three stimulations, basal H1 kinase activity was 21% +/- 3% and was obtained in 30 min. H1 kinase reactivation started at 4 hr after the first stimulation and level of activity reached 38% +/- 15% at 8 hr. Six stimulations also led to rapid H1 kinase inactivation and to a basal activity of 14% +/- 0.4%. With six stimulations, however, basal H1 kinase activity was maintained over at least 8 hr, and no reactivation occurred during this period. Basal H1 kinase activity obtained after six stimulations was similar to that of fertilized oocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Influence of recipient oocyte cell cycle stage on DNA synthesis, nuclear envelope breakdown, chromosome constitution, and development in nuclear transplant bovine embryos.

Nuclear transplantations into metaphase II (MII) and S phase oocyte cytoplasm were performed to investigate the influence of recipient cell cycle stage on nuclear function and development of bovine nuclear transplant (NT) embryos. Rate of inactivation of histone H1 kinase and duration of DNA synthesis in activated oocytes were determined. The proportion of S phase blastomeres in in vivo produced day 5.5 bovine embryos was measured. DNA synthesis was also assessed in NT embryos after transfer into MII and S phase cytoplasm. MII NT embryos were produced by fusing a blastomere into a MII oocyte; the fusion pulse served to activate the oocyte. S NT embryos were produced by fusing a blastomere into an early S phase oocyte electrically activated 4 h prior to fusion. Nuclear envelope structure, chromosome constitution, and extent of development were examined in MII and S NT embryos. Histone H1 kinase activity dropped to baseline within 2 h of electrical activation. A second electrical pulse did not alter H1 kinase activity when delivered 4 h after the first pulse. The frequency of S phase blastomeres in day 5.5 bovine embryos ranged from 79% to 100%, depending on the duration of culture in 3H-thymidine. Nuclear transplantation into MII cytoplasm resulted in a transient drop in DNA synthesis over 3.5 h. DNA synthesis resumed at 4.5 h post activation (hpa), concomittantly with initiation of DNA replication in activated oocytes. In contrast, DNA synthesis was not interrupted after transfer into S phase cytoplasm. DNA synthesis persisted until 13.5 hpa, as in activated oocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

The kinetics of oocyte activation and polar body formation in bovine embryo clones.

The kinetics of polar body formation were examined in parthenogenetically activated, in vitro matured and aged bovine oocytes. Subsequently, the presence or absence of polar body formation was determined in bovine embryo clones. Polar body formation, defined as telophase II, occurred by 1 (13/40, 43%) and 2 h (15/21, 71%) postparthenogenetic activation of metaphase II stage oocytes. Parthenogenetically activated oocytes readily formed pronuclei by 4 h. Some oocytes had chromatin in a highly condensed state at 1, 2, and 4 h postactivation (13/72, 18%). These oocytes often (10/13, 77%) appeared to be "self-enucleated," as the condensed chromatin was found in a membrane-bound extrusion. The phenomenon was most prevalent when oocytes were handled at room temperature (25-27 degrees C). Nuclear transfer procedures were established to bring about synchronous blastomere fusion and oocyte activation conditions. Synchronous conditions were achieved only when oocytes were handled and manipulated at 37-39 degrees C. Embryo clones examined 2 h postfusion did not form a polar body. Conversely, nucleate demi-oocyte controls were at the late telophase II stage of meiosis. The results are discussed in relation to cell cycle effects on bovine nuclear transfer.

Animals↗
PubMed1991

Embryonic transcription in in vitro cultured bovine embryos.

The rate of cleavage and the onset of embryonic transcription of bovine embryos cultured in vitro (IVC) has been investigated. Embryos were derived from in vitro matured, in vitro fertilized oocytes (IVM/IVF) to improve developmental synchrony. The rate of cleavage was assessed by morphological evaluation between the one- and eight- to 16-cell stage. The rate of cleavage was found to be equivalent to that reported for in vivo recovered embryos. To assess the onset of embryonic transcription, embryos were cultured to the eight- to 16-cell stage in the presence of alpha-amanitin for various periods of time followed by two-dimensional polyacrylamide gel electrophoresis. Embryos readily cleaved to the eight- to 16-cell stage in the presence of inhibitor. alpha-Amanitin-sensitive protein synthesis was first detected at 36-48 h post-insemination (hpi) and continued up to 84 hpi. We conclude that bovine embryos produced by IVM/IVF/IVC are competent to initiate embryonic transcription at 36-48 h post-insemination and suggest that in vitro-induced cleavage arrest is not due to failure of the embryonic genome to initiate transcription.

Amanitins↗
PubMed1989

Age dependence of bovine oocyte activation.

The ability of bovine oocytes to undergo parthenogenetic activation using either a Ca++-Mg++-H+ ionophore (A23187) or electric shock was investigated, as a prelude to understanding activation potential following nuclear transfer into ooplasm. Oocytes were collected from slaughterhouse ovaries by aspiration of 1-5-mm follicles. The time of placement into maturation medium was noted, and maturational age (time in culture) measured from that point. After exposure to activating conditions eggs were cultured for a further 12-16 hours, fixed, and stained with aceto-orcein. Oocytes that progressed to telophase or pronuclear formation were considered activated. Concentrations of A23187 ranging from 100 pM to 100 microM showed that 1-100 microM levels resulted in 94-100% activation at 30 hours maturation. Frequency of activation differed from controls (no ionophore) at 100 nM (49%; P less than 0.05). With A23187 maximum response occurred between 26 and 30 hours of maturation (77% and 92%, respectively). A short pulse electric shock, capable of causing oocyte membrane fusion, gave similar results relative to maturational age (82% and 90% activation for 26 and 30 hours, respectively). Therefore, maximum response to the two activating stimuli occurred in oocytes at similar maturational ages. Exposure to activating conditions prior to onset of activating ability (18 hours) followed by another exposure at 26 hours showed that the oocytes were still fully able to activate upon reaching maturational activation competence. Because cytochalasin B is present in the medium used for nuclear transfer, oocytes were incubated with cytochalasin B prior to exposure to an activating stimulus. Frequency of activation was similar to the control treatment (61% and 73%). The effect of mechanical stress of cytoplasm removal and replacement by electrofusion on activation was also not significant. Overall, maturational age of the oocyte was the main determinant of activation ability.

Aging↗
PubMed1989

Timing of nuclear progression and protein synthesis necessary for meiotic maturation of bovine oocytes.

In cows, protein synthesis is required for germinal vesicle breakdown (GVBD). This study examines more closely the need for protein synthesis and the nuclear changes in the bovine oocyte during 24 h of culture. Bovine oocytes with compact and complete cumulus were washed and incubated in groups of 10 for up to 24 h in 50-microliters drops of TCM-199 supplemented with follicle-stimulating hormone (NIAMADD, 0.5 micrograms/ml), luteinizing hormone (LH) NIAMADD, 5 micrograms/ml), estradiol-17 beta (1 microgram/ml), pyruvate (20 microM), and 10% heat-treated fetal calf serum. Medium was overlaid with paraffin oil. Oocytes (n = 891) were fixed at the end of each 3-h interval from 0 to 24 h of culture, or at 24 h after addition of cycloheximide (10 micrograms/ml at 10 different times during maturation (0, 1, 2, 3, 6, 9, 12, 15, 18, 21 h; n = 175). At each time point, the chromosomal status of oocytes was evaluated, frequencies were computed, and the time spent on each step was determined. The germinal vesicle (GV) was present from 0 to 6.6 h, GVBD at 6.6 to 8.0 h, chromatin condensation at 8.0 to 10.3 h, metaphase I at 10.3 to 15.4 h, anaphase I at 15.4 to 16.6, telophase I at 16.6 to 18.0 h, and metaphase II at 18.0 to 24 h. Cycloheximide blocked oocyte maturation at GVBD, if added from 0 to 3 h; at chromatin condensation, if present from 6 to 24 h; and at metaphase I, when present from 9 to 12 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Nuclear transplantation in mouse embryos: assessment of nuclear function.

Enucleated mouse zygotes receiving eight-cell nuclei rarely develop beyond the two-cell stage, whereas enucleated two-cell embryos with eight-cell nuclei develop to blastocysts, and a few develop to midgestation. In this study the function of eight-cell nuclei in either the zygote or two-cell recipient was assessed by monitoring several nuclear-dependent events. These included methionine uptake, qualitative changes in protein synthesis, the time of blastocoele formation, and changes in nuclear volume. Although enucleated zygotes with nuclei from eight-cell embryos did not develop past the two-cell stage and had abnormally low levels of methionine uptake, they did show a normal zygote-to-two-cell transition in the types of polypeptides synthesized. Enucleated two-cell embryos with eight-cell nuclei formed blastocysts on schedule with the recipient cell stage and reached levels of methionine uptake equivalent to control embryos. The types of proteins synthesized by two-cell embryos with eight-cell nuclei indicated that the nucleus was developing partly in accord with its own developmental program. Transplanted nuclei did not enlarge to acquire the volume of the recipient cell's nucleus in either the zygote or the two-cell recipient. These results indicate that the difference in development between zygotes and two-cell embryos (each with eight-cell nuclei) may be the result of a greater overlap of function between the two-cell and eight-cell stages, rather than the extent of nuclear reprogramming. Nuclear function was not completely normal in either type of embryo, which may explain their failure to develop to term.

Animals↗