Biomedical subjects

D T Armstrong

Publications and source records attributed to D T Armstrong.

At least 19 recordsLinked to original sources

PubMed2003

Comparison of oocyte factors and transforming growth factor-beta in the regulation of DNA synthesis in bovine granulosa cells.

Oocytes are powerful local modulators of follicular cell functions and many of the activities of oocytes are attributed to members of the transforming growth factor-beta (TGF-beta) superfamily. Whilst in the mouse it is known that members of this family are able to mimic many of the effects of oocytes on follicular cells, the relative importance of any of these factors is unknown in bovine follicles. The objectives of this study were to determine if bovine oocytes express and secrete TGF-beta and to compare oocyte-secreted factor(s) to TGF-beta in terms of their capacities to stimulate mural granulosa cell (MGC) DNA synthesis. Bovine ovaries were collected from an abattoir and RNA was extracted from isolated MGC, cumulus cells, cumulus-oocyte complexes and denuded oocytes (DO). Using RT-PCR, all cell types were found to express TGF-beta1 and TGF-beta2 mRNA transcripts. However, no TGF-beta bioactivity could be detected from DO using a sensitive (40 pg/ml) and specific mink lung fibroblast cell bioassay. MGC were cultured with various combinations and doses of TGF-beta2 and DO for 18 h, followed by a 6-h pulse of [3H]-thymidine as an indicator of cellular DNA synthesis. MGC DNA synthesis was stimulated by both TGF-beta2 and DO. However in response to increasing doses of TGF-beta2, [3H]-thymidine levels plateaued at <2-fold above control levels, whereas levels continued to increase over the dose range of DO tested (up to 3.4-fold). Addition of a TGF-beta pan-specific neutralising antibody to MGC cultures eliminated the TGF-beta2-stimulatory effects on DNA synthesis and the TGF-beta2-suppressive effects on progesterone production, but the antibody was unable to neutralise the same responses when induced by DO. These results support a role for TGF-beta1, TGF-beta2 and DO in paracrine/autocrine regulation of bovine granulosa cell function, but indicate that neither TGF-beta1 nor TGF-beta2 can account for the actions of bovine oocytes on granulosa cells.

Animals↗

PubMed2002

Effects of leptin administration and feed restriction on thecal leucocytes in the preovulatory rat ovary and the effects of leptin on meiotic maturation, granulosa cell proliferation, steroid hormone and PGE2 release in cultured rat ovarian follicles.

Leptin is expressed by adipocytes and is thought to play a role in regulating food intake and in reproduction. It has been demonstrated that acute leptin administration to immature gonadotrophin-primed rats in vivo inhibits ovulation and causes a decline in food intake. However, feed restriction alone does not inhibit ovulation. Two experiments were designed to investigate the mechanism of leptin-induced inhibition of ovulation. In the first experiment, which was prompted by the importance of ovarian leucocytes in ovulation, the role of leucocytes in leptin-induced inhibition of ovulation was investigated. The second experiment investigated whether high leptin concentrations could inhibit other factors important to ovulation, such as meiotic competence of oocytes, granulosa cell proliferation, steroid or PGE(2) release, and interleukin 1beta production, in vitro. In the first experiment, the populations of neutrophils and monocytes-macrophages in the preovulatory follicles of gonadotrophin-primed, leptin-treated and -untreated rats were examined. A decrease in food intake, as a result of either leptin treatment or feed restriction, specifically reduced the numbers of neutrophils and monocytes-macrophages infiltrating the theca interna of preovulatory follicles without affecting the numbers found in the stroma. The findings show that reduced infiltration of thecal neutrophils and macrophages into preovulatory follicles is a response to reduced food intake. Furthermore, this reduction is not the direct cause of the leptin-induced inhibition of ovulation. In the second experiment, ovarian follicles were cultured for 4 or 12 h in the presence or absence of the following hormones: FSH (500 miu), insulin-like growth factor I (IGF-I) (50 ng ml(-1)), LH (100 ng ml(-1)) and leptin (300 ng ml(-1)). The results demonstrated that high concentrations of leptin in follicle culture do not affect meiotic maturation or steroid release, but tend to inhibit release of PGE 2 (although this result was not significant). DNA synthesis in granulosa cells was not inhibited by leptin in FSH- and IGF-I-supplemented culture media. These results are in agreement with previous studies that have shown that leptin inhibits the stimulatory effects of IGF-I on FSH-stimulated oestradiol production in rat granulosa cells without affecting progesterone production. In summary, leptin does not appear to have an adverse effect on the components of ovulation tested in this study, and therefore must impact on the ovulatory cascade in a way that remains to be defined.

Animals↗

PubMed2001

Mouse oocyte mitogenic activity is developmentally coordinated throughout folliculogenesis and meiotic maturation.

Oocytes secrete soluble factors that regulate the growth and differentiation of follicular cells, including maintenance of the distinctive cumulus cell phenotype. This study determines whether the mitogenic activity of oocytes is developmentally regulated and examines the responsiveness of follicular cells to oocytes at different stages of follicular development. Prepubertal SV129 mice of varying ages were primed with 5 IU equine chorionic gonadotropin (eCG) and oocytes/zygotes collected either 46 h post-eCG (immature oocytes), 12 h after administration of 5 IU human CG (hCG; ovulated ova), or 12 h post-hCG and mating (zygotes). Mural granulosa cells (MGC) from antral follicles and GC from preantral follicles were cultured +/- denuded oocytes (DO) for 18 h, followed by a 6-h pulse of [(3)H]thymidine as an indicator of cellular DNA synthesis. Coculturing MGC with meiotically maturing oocytes led to a dose-dependent increase in [(3)H]thymidine incorporation (20-fold above control levels at 0.5 DO/microl). However, [(3)H] counts remained unchanged from control levels when cultured with meiotically incompetent DO from 11- to 15-day-old mice (3% germinal vesicle breakdown; GVB), irrespective of dose of DO or developmental status of GC (MGC or preantral GC). In some treatments, spontaneous meiotic resumption of competent oocytes was prevented by culturing with 5 microM milrinone, a selective inhibitor of oocyte-specific cyclic nucleotide phosphodiesterase. The mitogenic capacity of oocytes was found to decline during and after oocyte maturation. [(3)H]Thymidine incorporation in MGC was highest (11-fold above controls) when cultured with meiotically inhibited (milrinone-treated) GV DO, stimulated 5.5-fold by culture with maturing oocytes, 3-fold with ovulated ova, and unstimulated by zygotes. [(3)H]Thymidine incorporation in MGC was not altered by the dose of milrinone, either in the presence or absence of DO. Metaphase I marked the beginning of the decline in the capacity of oocytes to promote MGC DNA synthesis. These results demonstrate that the capacity of oocytes to promote proliferation of granulosa cells follows a developmental program, closely linked to oocyte meiotic status, increasing with the acquisition of meiotic competence and declining during and after oocyte maturation.

Animals↗

PubMed2001

Effects of maternal age on oocyte developmental competence.

The widespread use of a variety of assisted reproductive technologies has removed many of the constraints that previously restricted mammalian reproduction to the period between onset of puberty and reproductive senescence. In vitro embryo production systems now allow oocytes from very young animals to undergo fertilization and form embryos capable of development to normal offspring, albeit at somewhat reduced efficiencies compared to oocytes from adult females. They also can overcome infertility associated with advanced age of animals and women. This review examines oocyte developmental competence as the limiting factor in applications of assisted reproductive technologies for both juvenile and aged females. Age of oocyte donor is a significant factor influencing developmental competence of the oocyte. Age-related abnormalities of oocytes include a) meiotic incompetence or inability to complete meiotic maturation resulting in oocytes incapable of fertilization; b) errors in meiosis that can be compatible with fertilization but lead to genetic abnormalities that compromise embryo viability; and c) cytoplasmic deficiencies that are expressed at several stages of development before or after fertilization. In general, oocytes from juvenile donors and the embryos derived therefrom appear less robust and may be less tolerant to suboptimal handling and in vitro culture conditions than are adult oocytes. Research to identify specific cytoplasmic deficiencies of juvenile oocytes may enable modifications of culture conditions to correct such deficiencies and thus enhance developmental competence. Use of oocytes from aged donors for assisted reproduction can have a variety of applications such as extending the reproductive life of individual old females whose offspring still have high commercial value, and conservation of genetic resources such as rare breeds of livestock and endangered species. In general, female fertility decreases with advancing age. Studies of women in oocyte donation programs have established reduced oocyte competence as the major cause of declining fertility with age, although inadequate endometrial function can also be a contributing factor. Most research has emphasized the importance of chromosomal abnormalities because of the well established increase in aneuploidy with increasing maternal age but little is known about the underlying cellular and molecular mechanisms. Research aimed at identifying the specific developmental deficiencies of oocytes from juvenile donors and abnormalities of oocytes from aged females will assist in overcoming present bottlenecks that limit the efficiency of assisted reproduction technologies. Such research will also be crucial to the development of new oocyte-based technologies for overcoming infertility and possibly subverting chromosomal abnormalities in women approaching menopause.

Animals↗

PubMed2001

Growth-inhibitory effects of CD40 ligand (CD154) and its endogenous expression in human breast cancer.

CD40 binding produces multifaceted growth signals in normal and malignant B cells, whereas its physiological role is less well characterized in epithelial cancers. We examined the growth outcome of CD40 ligation in human breast cancer cells, using CD40+ (T47D and BT-20) and CD40-negative (MCF-7, ZR-75-1) cell lines as defined by flow cytometric analysis, immunohistochemistry, and reverse transcription-PCR. Treatment with the soluble recombinant CD40 ligand (CD40L) molecules gp39 or CD40L-trimer significantly reduced [3H]thymidine uptake in BT-20 and T47D cells by up to 40%, but did not affect the growth of CD40-negative MCF-7 or ZR-75-1 cells. Similarly, significant growth inhibition was observed after co-incubation with CD40L-transfected murine L cells (55.0 +/- 8.9%, P < 0.001) that express membrane CD40L constitutively, or with paraformaldehyde-fixed, CD3+ CD40L+ PBLs from three different HLA-mismatched donors (39.7 +/- 3.7%, P < 0.01). Untransfected L cells and non-CD40L-expressing lymphocytes did not produce significant growth inhibition. The in vivo antitumorigenic effects of CD40L were examined using a s.c. severe combined immunodeficient-hu xenograft model. Pretreatment with two different soluble recombinant CD40L constructs (CD40L and gp39) produced similar xenograft growth-inhibitory effects [67 +/- 24% (n = 4), and 65 +/- 14% (n = 8) inhibition, respectively], which were reversed by co-treatment with the CD40L-neutralizing antibody LL48. In vitro analysis indicated that CD40L-induced growth inhibition was accompanied by apoptotic events including cell shrinkage, rounding, and detachment from the adherent T47D culture monolayer. Thirty-one and 27% of gp39-treated T47D and BT-20 cells underwent apoptosis, respectively, as compared with 56 and 65% from the same cell lines after treatment with the Fas agonistic antibody CH-11. An up-regulation of the proapoptotic protein Bax in T47D and BT-20 cells was observed, which indicated that this Bcl-2 family member may contribute to this growth-inhibitory effect. To explore the clinical relevance of CD40L-CD40 interaction, retrospective immunohistochemical analysis was carried to characterize in situ CD40- and CD40L-expression in breast cancer patient biopsies. All of the infiltrating ductal (5 of 5 cases tested) and lobular (4 of 4 cases) breast carcinomas, carcinomas in situ (6 of 6 cases), and mucinous carcinoma tested (1 case) expressed CD40. Varying proportions of tumor cells also expressed CD40L in the majority of infiltrating ductal (3 of 5 cases) and lobular (3 of 4 cases) carcinomas, and carcinomas in situ (4 of 6 cases), as determined by immunohistochemistry and validated by RT-PCR detection of the CD40L message in only CD40L positive-staining cases. Tumor infiltrating mononuclear cells from infiltrating carcinomas and carcinomas in situ expressed CD40 (10 of 10 cases), but less commonly CD40L (1 case of infiltrating lobular carcinoma, 2 cases of carcinoma in situ). Our findings indicate that the CD40 signaling pathway is active in human breast carcinoma cells. However, tumor-infiltrating lymphocytes from primary tumor tissues may be limited in their capacity to directly modulate tumor growth through the CD40L-CD40 loop.

Animals↗

PubMed2000

Differential regulation by FSH and IGF-I of extracellular matrix IGFBP-5 in bovine granulosa cells: effect of association with the oocyte.

Inhibition of insulin-like growth factor (IGF)-I induced DNA synthesis in bovine oocyte-cumulus complexes (OCCs) caused by follicle-stimulating hormone (FSH) has been linked to changes in the extracellular matrix which do not occur in mural granulosa cells (MGCs). We investigated regulation by IGF-I and FSH of secreted and extracellular matrix entrapped IGF-binding proteins. OCCs and MGCs from bovine ovaries were cultured in media supplemented with IGF-I and FSH for 24 h. Culture media and extracellular matrix were analysed for IGF-binding proteins by Western ligand blot and immunoblot and found to contain principally IGFBP-3 and -5. The combined treatment of IGF-I and FSH increased the concentration of IGFBP-3 in OCC and MGC conditioned media by 4- and 6-fold, respectively. Treatment of OCCs and not MGCs with IGF-I and FSH together increased extracellular matrix IGFBP-5 by 2.5-fold. The differential regulation of extracellular matrix IGFBP-5 in OCCs compared to MGCs suggest involvement of changes in the extracellular matrix brought about by IGF-I and FSH in overall regulation of IGF-I in the ovarian follicle.

Animals↗

PubMed2000

Oocyte-secreted factor(s) determine functional differences between bovine mural granulosa cells and cumulus cells.

Cumulus cells and mural granulosa cells (MGC) are phenotypically different and there is now evidence suggesting that the oocyte plays an active role in determining the fate of follicular somatic cells. This study investigates the role of oocyte-secreted factor(s) in the regulation of the growth and differentiation of cumulus and MGC. Bovine cumulus-oocyte complexes (COC) and MGC were cultured with various hormones for 18 h followed by a further 6-h pulse of [(3)H]thymidine as an indicator of follicular cell DNA synthesis. The COC incorporated 11 to 14 times more [(3)H]thymidine than MGC in either the absence or presence of 50 ng/ml insulin-like growth factor (IGF)-I. Purified porcine FSH (450 ng/ml) added together with IGF-I marginally increased (3)H incorporation in MGC relative to IGF-I alone but dramatically decreased incorporation in COC sixfold. Conversely, mean progesterone production in the presence of IGF-I + FSH was 13-fold higher from MGC than from COC, confirming a distinctive phenotype of cumulus cells. However, this phenotype was found to be dependent on the presence of the oocyte, as microsurgical removal of the oocyte (oocytectomy) resulted in an 11-fold decrease in [(3)H]thymidine incorporation in cumulus cells treated with IGF-I, elimination of the inhibitory effect of FSH on IGF-I-stimulated DNA synthesis, and led to a 2-fold increase in progesterone production in medium with IGF-I and FSH. All of these markers were completely restored to COC levels when oocytectomized complexes were cocultured with denuded oocytes (DO) at a concentration of 0.5 oocytes/microl, demonstrating that oocytes secrete a soluble factor(s) that promotes growth and attenuates cumulus cell progesterone secretion. In the presence of IGF-I, [(3)H]thymidine incorporation in MGC increased ninefold above control levels with the addition of DO. The addition of FSH to IGF-I-increased (3)H counts in MGC, however, led to a decrease in counts in MGC + DO as is also observed in COC. Furthermore, progesterone production was halved when DO were added to MGC cultures, most notably in the presence of IGF-I and/or FSH. These results provide further evidence that MGC and cumulus cells have distinctive phenotypes and that the oocyte is responsible for some of the characteristic features of cumulus cells. Bovine oocytes secrete a soluble factor(s) that simultaneously promotes growth and attenuates steroidogenesis in follicular somatic cells.

Animals↗

PubMed2000

Effect of cell-to-cell contact on in vitro deoxyribonucleic acid synthesis and apoptosis responses of bovine granulosa cells to insulin-like growth factor-I and epidermal growth factor.

Follicle development is the result of a balanced ratio between cell proliferation and cell death. Previous studies demonstrated differential mitotic responses to insulin-like growth factor (IGF)-I and epidermal growth factor (EGF) of cumulus cells (CC) and mural granulosa cells (MGC). Because cell-to-cell contact seems to modulate the occurrence of programmed cell death, the present experiments investigated the role of cell association in mediating apoptosis and the mitogenic responses to these growth factors of CC and MGC. Cumulus cells were cultured either as intact cumulus-oocyte complexes (COC) or after dissociation with EGTA + sucrose, in the presence of 50 ng/ml IGF-I, 5 ng/ml EGF, or both. Mural granulosa cells from the same follicles were similarly cultured either as cell aggregates or as dissociated cells. Synthesis of DNA was assessed by measurement of [(3)H]thymidine incorporation during the last 6 h of a 24-h culture in TCM199. Percentages of cells undergoing apoptosis were determined immunohistochemically in intact COC and GC aggregates, before and after dissociation as well as after the culture period. Epidermal growth factor and IGF-I stimulated DNA synthesis in both cell types; however, EGF inhibited the action of IGF-I in intact COC but not in MGC. Compared to nondissociated cells, dissociation resulted in a reduction of the mitogenic response of CC to both growth factors and of MGC to EGF. Unlike the response of intact COC to combined treatment with the two growth factors, dissociated CC displayed additive responses to the two growth factors in combination. Addition of denuded oocytes to cultures of dissociated CC enhanced both basal and growth factor-stimulated DNA synthesis but did not restore the inhibitory effect of EGF on the IGF-I response characteristic of intact COC. A significant proportion of intact MGC aggregates underwent apoptosis after 24 h of culture, while no increase of apoptotic cells was observed in intact COC. A dramatic increase in the percentage of apoptotic cells was observed in both CC and MGC when cell-cell contact was interrupted, and EGF and IGF-I were able to partially prevent its occurrence. Taken together these data showed that CC and MGC exhibit qualitatively and quantitatively different responses to IGF-I when cultured in the presence of EGF both in terms of DNA synthesis and onset of apoptosis. Moreover, the disruption of cell-cell contact was a major factor reducing cell proliferation and inducing apoptosis among both subsets of GC.

Animals↗

PubMed2000

The in vivo and in vitro effects of exogenous leptin on ovulation in the rat.

Leptin, a hormonal product of the Lep gene, is expressed by adipocytes and is thought to play a role in regulating food intake and reproduction. The leptin protein has been localized in many reproductive tissues, including the ovary. Several publications indicate that the ovary is directly affected by leptin and that leptin may be a factor linking obesity and reproductive dysfunction. In this study, the effect of systemic leptin administration on ovulation in the rat ovary, both in vivo and in vitro, was investigated. Ip administration of leptin (30 microg at 3 hourly intervals for 15 h) to immature gonadotropin-primed rats caused a decline in ovulation in vivo, from 15.9+/-2.0 oocytes in the control animals to 5.3+/-1.6 oocytes in the leptin-treated animals (P < 0.001). Plasma progesterone and estradiol levels were analyzed immediately before ovulation, and neither was altered significantly in animals receiving the leptin treatment. Food consumption and body weight decreased following leptin treatment; however, a loss in body weight alone (pair-fed controls) was insufficient to explain the decrease in ovulation observed in the leptin-treated animals. In vitro perfusion of FSH-primed whole ovaries showed that treatment with leptin in combination with LH significantly decreased ovulations from 5.7+/-1.6 per ovary perfused with LH alone to 1.3+/-0.6 in those with LH and 1 microg/ml leptin (P < 0.05). Progesterone and estradiol levels in the samples taken during the perfusion period were unaffected by leptin treatment. In summary, leptin administration resulted in fewer ovulations, both in vivo and in vitro, but did not influence steroid levels. Systemic leptin administration at these doses can therefore inhibit ovulation, a process that occurs through a direct effect on the ovary.

Animals↗

PubMed2000

Effect of granulocyte-macrophage colony-stimulating factor deficiency on ovarian follicular cell function.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine secreted by lymphohaemopoietic and other cell lineages, is known to influence ovarian cyclicity and embryo development. The aim of this study was to examine the effect of GM-CSF on ovarian follicular cell function using GM-CSF-deficient (GM -/-) mice. Immature GM -/- and GM +/+ mice were stimulated with eCG, and cumulus-oocyte complexes and mural granulosa cells were collected 48 h later. Expression of GM-CSF receptor (GM-CSFR) alpha and beta mRNA subunits by cumulus-oocyte complexes and mural granulosa cells was examined using RT-PCR. Cumulus-oocyte complexes from both genotypes were found to express mRNA for the GM-CSFRalpha-subunit only, while the mural granulosa cells expressed both the alpha and beta receptor subunits. Cumulus-oocyte complexes recovered from GM -/- mice had approximately twice the number of cumulus cells per cumulus-oocyte complex than did those of GM +/+ mice (P < 0.05), even though the growth-promoting activity of denuded GM -/- oocytes was found to be equivalent to that of wild-type oocytes. GM-CSF deficiency was associated with marginally increased DNA synthesis in cumulus cells and significantly (P < 0.05) lower progesterone production by mural granulosa cells recovered from GM -/- compared with those recovered from GM +/+ mice. The addition of rec-mGM-CSF in vitro did not affect DNA synthesis in either cell type or progesterone production by mural granulosa cells, irrespective of GM-CSF status. There was no effect of GM-CSF deficiency on the capacity of FSH and insulin-like growth factor I to stimulate DNA synthesis in cumulus-oocyte complexes (approximately 15- and threefold, respectively) and in mural granulosa cells (approximately two- and threefold, respectively). Taken together, these data show that GM-CSF influences events associated with follicular maturation in mice. The effects of GM-CSF are not exerted directly in granulosa or cumulus cells, but appear to be mediated indirectly, perhaps through the agency of steroidogenesis-regulating secretions of local macrophage populations residing in the theca.

Analysis of Variance↗

PubMed1998

Comparative analysis of calf and cow oocytes during in vitro maturation.

To determine possible causes of reported differences between developmental competence of oocytes isolated from prepubertal (10- to 14-week-old calves) and adult cows, three parameters were analysed, comparatively, during in vitro maturation (IVM): (1) oocyte diameter, (2) oocyte energy metabolism, and (3) protein synthesis of oocytes and cumulus cells. Cumulus-oocyte complexes were isolated from follicles of 3-5 mm in diameter in both age groups. Mean oocyte diameter was smaller (P < 0.02) in calves than in cows (118.04 +/- 1.15 versus 122.83 +/- 0.74 microns). During the first 3 hr of IVM, calf oocytes metabolised glutamine and pyruvate at lower rates than adult oocytes, but after 24 hr of culture, both molecules were metabolised at the same rate as for adult oocytes. A significant decrease in protein synthesis, as measured by [35S]methionine and [35S]cysteine incorporation was recorded after 9 hr of IVM in calf oocytes, while in adult oocytes a significant decrease in protein synthesis was detected only after 24 hr. After the first 3 hr of maturation, proteins of 130, 26, and 24 kDa were more abundant in adult than in calf oocytes, while a protein of 55 kDa was more visible in calf than in adult oocytes. At the same time, among proteins newly synthesised by cumulus cells, molecules of 405, 146, 101, and 77 kDa were more abundant in adults than in calves. In conclusion, calf oocytes and cumulus cells showed several differences when compared with their adult counterparts, which are consistent with their reported lower developmental competence.

Aging↗

PubMed1997

Effects of epidermal growth factor and follicle-stimulating hormone during in vitro maturation on cytoplasmic maturation of porcine oocytes.

The present study was undertaken to investigate the influence of epidermal growth factor (EGF) and follicle-stimulating hormone (FSH) during in vitro maturation on cytoplasmic maturation of porcine oocytes as revealed by the success of fertilization and by the changes in the pattern of protein synthesis in oocytes and cumulus cells. For fertilization studies, oocyte-cumulus cell complexes (OCC) were cultured in media containing human recombinant EGF (1 ng/ml) or FSH (1.5 microg/ml) or both for 44 hr prior to fertilization with fresh sperm for 6-8 hr. The oocytes were then fixed, stained, and examined as whole mounts following an additional 14 hr of culture. Addition of EGF, FSH, and EGF + FSH significantly increased the proportion of oocytes reaching MII stage. The addition of EGF alone significantly decreased the percentage of polyspermic oocytes and increased the proportion of monospermic oocytes forming 2 normal pronuclei. FSH abolished these effects of EGF and significantly increased the percentage of polyspermic oocytes forming more than 2 pronuclei when added alone or with EGF. For protein analysis, OCC were cultured in media containing the above hormones for 6, 24, and 44 hr and exposed to 0.5 mCi/ml L-[35S]methionine during the last 3 hr of cultures. The oocytes and cumulus cells were separated prior to lysis in SDS sample buffer, and denatured polypeptides were separated by 1-dimensional SDS-PAGE. In the oocyte, addition of EGF and FSH alone stimulated the synthesis of 34, 45, and 97 kDa proteins after 6 hr of culture; however, the addition of EGF and FSH together was without any effect. After 24 hr, EGF alone inhibited the synthesis of these peptides, whereas FSH alone and with EGF maintained the stimulation of synthesis of 34 and 45 kDa proteins. Two additional peptides corresponding to 66 and 200 kDa appeared at this time as a result of exposure to FSH alone or with EGF. After 44 hr of culture, these 2 new peptides were observed in all groups and the stimulatory effect of FSH and FSH + EGF was still evident. An additional peptide of 26 kDa appeared at this time as a result of FSH and EGF + FSH treatments. In the cumulus cells, EGF and FSH each alone induced the synthesis of a new peptide of 26 kDa after 6 hr of culture. FSH when added alone or with EGF induced the synthesis of an additional peptide of 29 kDa, the synthesis of which remained unchanged at 24 and 44 hr. After 24 hr, FSH alone and in combination with EGF induced the synthesis of an additional 38 kDa peptide and its synthesis was still maintained at 44 hr. EGF alone had no effect on protein synthesis in cumulus cells at 24 and 44 hr. These studies indicate that EGF may have a physiological role in the regulation of cytoplasmic maturation of porcine oocytes.

Animals↗

PubMed1997

Bovine parthenogenesis is characterized by abnormal chromosomal complements: implications for maternal and paternal co-dependence during early bovine development.

The present study was conducted to examine the karyotypes of parthenogenetic bovine embryos arising from the application of standard oocyte activation and diploidization methods. Bovine cumulusoocyte complexes were collected and matured in vitro for 24 hr prior to oocyte activation with either 5 microM ionomycin or 7% ethanol for 5 min. Groups of activated oocytes were further treated with 5 micrograms/ml cytochalasin D or 1.9 mM 6-dimethylaminopurine (DMAP) for 6 hr. Cleavage varied significantly (P < .05) among the treatment groups with 68.0% of the ethanol- and DMAP-treated oocytes dividing. Blastocyst development did not vary with 18.4 +/- 2.5% of all treated oocytes progressing to this stage. Blastocyst development did not occur in groups subjected to oocyte activation alone. Blastocysts displayed haploid (2.3%), diploid (11.4%), tetraploid (40.9%), octaploid (4.5%), and mixoploid chromosomal complements (40.9%). Two-cell stage parthenogenotes resulting from ethanol or ionomycin treatment alone displayed haploid (66.7%), diploid (16.7%), tetraploid (4.2%), and mixoploid (12.5%) complements. Our results demonstrate that diploid bovine parthenogenotes arising from these procedures are a minority, with the majority of parthenogenotes displaying polyploid and mixoploid chromosomal complements. The events contributing to these abnormal chromosomal complements occur as early as completion of the first cell cycle, possibly linking these events with the absence of a paternally supplied centrosome.

Adenine↗

PubMed1997

Effects of using vasectomized bulls in artificial insemination practice on the reproductive efficiency of Italian buffalo cows.

The effects of the presence or absence of vasectomized male buffaloes on the reproductive efficiency of buffalo cows (n = 396) undergoing artificial insemination (AI) was studied on six farms owned and operated by a single consortium. Lactating animals were separated into two groups of various sizes on each farm and kept under semi-range conditions. Vasectomized bulls were present in one group at a bull/empty-cow ratio of 1:30. No bulls were present in the other group. Reproductive efficiency between the two groups over a period of 3.5 months was compared and evaluated on the basis of: 1) the number of spontaneous overt estruses associated with either feeble or intense signs of estrous behaviour; 2) the number of functional estrous cycles, i.e. estrous cycles with luteal phases defined as normal, based on specified progesterone concentrations in milk or blood plasma 8-10 days after estrus; 3) the number of consecutive functional estrous cycles in cases of induced estrus; and 4) pregnancy rate. Groups with bulls present demonstrated a significantly higher reproductive efficiency than groups without them. There was a higher incidence of spontaneous estrus (92 versus 69%; P < 0.01); spontaneous estrus of high intensity (62.2 versus 31.1%; P < 0.01); and higher incidence of functional estrous cycles following both spontaneous (65.8 versus 57.1%) and induced (77.0 versus 59.5%; P < 0.05) estrus. Exposure to vasectomised bulls also increased the incidence of consecutive functional estrous cycles (90.5 versus 68.1%; P < 0.01), and the pregnancy rate in cows inseminated at spontaneous (42.5 versus 18.9%; P < 0.01) or induced (51.1 versus 33.3%; P < 0.05) estrus. Overall pregnancy rate did not differ significantly between cows inseminated at induced or spontaneous estrus, although in the absence of bulls, pregnancy rate per AI was higher in cows inseminated at induced than at spontaneous estrus (33.3 versus 18.9%).

Animals↗

PubMed1997

Advances in production of embryos in vitro from juvenile and prepubertal oocytes from the calf and lamb.

The use of juvenile donors in embryo-transfer (ET) programmes offers considerable potential for accelerated genetic gain in domestic livestock through reduced generation interval. The present paper reviews recent research aimed at optimizing embryo production from oocytes collected from young calves and lambs using in vitro methods of embryo production. Emphasis is placed on criteria for donor selection, oocyte-collection methods, and hormone-stimulation methods designed to produce maximum yields of viable oocytes. In vitro fertilization (IVF) rates of calf and lamb oocytes did not differ significantly whether matured in vivo or in vitro, and rates of development of blastocyst stages in culture were similar to those observed for embryos derived from adult donors. Blastocysts produced by IVF of lamb and calf oocytes established ET pregnancies at rates of 30-45%. Pregnant recipients have reached full term and delivered normal offspring at rates similar to those expected following ET of embryos produced in vivo from superovulated donors. On the basis of current follicle-stimulation protocols, on rates of blastocyst production in vitro under optimal conditions, and on observed pregnancy rates from fresh transfer of IVF embryos, 8-10 pregnancies may be expected per oocyte collection from 10-12-week-old calves and from 6-8-week-old lambs.

Animal Husbandry↗

PubMed1997

Insulin-like growth factor-1, a component of serum that enables porcine cumulus cells to expand in response to follicle-stimulating hormone in vitro.

The present study was aimed at determining whether insulin-like growth factor-1 (IGF-1) is a component of fetal bovine serum (FBS) that enables porcine cumulus cells to expand in response to FSH in vitro. Cumulus-oocyte complexes (COC) obtained from 4- to 6-mm follicles of prepubertal gilts were cultured at 39 degrees C for 24 h in media that contained human recombinant IGF-1 (50 ng/ml), FBS (15% v:v), or their combination, with or without FSH (1.5 microg/ml), and cumulus expansion was scored microscopically. Expansion was FSH dependent and was observed only when IGF-1, FBS, or both were present. The proportion of FSH-stimulated COC exhibiting full expansion in response to IGF-1 alone did not differ significantly (p > 0.05) from the proportion in those cultured with FBS or IGF-1+FBS (79 +/- 2.8% vs. 84 +/- 2.2% or 76 +/- 6.2%, respectively). In a concentration-response study, FSH-stimulated expansion was observed in a significant proportion of COC (32 +/- 2.8% vs. 0% control) at 1 ng/ml IGF-1, with the proportions increasing dose-dependently to maximal values between 10 and 75 ng/ml IGF-1, and decreasing at higher IGF-1 concentrations. Exposure of COC to an IGF-1 receptor (IGF-1R)-neutralizing antibody (Ab) for 90 min before addition of FSH and FBS dose-dependently inhibited cumulus expansion, with maximal inhibition at 10 microg/ml (1 +/- 1.0% vs. 68 +/- 1.1% control). In the absence of FBS or IGF-1, some COC had a tendency toward slight expansion when cultured with FSH, and the Ab completely inhibited that effect, suggesting that this may be due to endogenous IGF-1 production by the COC. The Ab effect was reversible and was eliminated by washing twice with fresh medium followed by culture for an additional 24 h in the presence of FSH and FBS. Expression of IGF-1R mRNA in the isolated oocyte and cumulus cells was determined by reverse tramscriptase polymerase chain reaction using sequence-specific primers. The IGF-1R message was detected in both the oocyte and cumulus cells. Collectively, these observations suggest that IGF-1 is a component of serum that enables cumulus cells to expand in response to FSH in vitro, and that the effect is receptor mediated. Since IGF-1 is present in the follicle in vivo, it may have a physiological role during gonadotropin-induced cumulus expansion.

Animals↗

PubMed1997

Bovine oviductal and embryonic insulin-like growth factor binding proteins: possible regulators of "embryotrophic" insulin-like growth factor circuits.

Bovine oviductal monolayer and vesicle primary cultures express insulin-like growth factor (IGF)-I and -II mRNAs and polypeptides. Early bovine embryos also express IGF-I, IGF-II, IGF-I receptor, IGF-II receptor, and insulin receptor mRNAs. This study reports the expression of IGF binding protein (IGFBP) mRNAs and polypeptides in bovine oviduct primary cultures and IGFBP mRNAs in preattachment embryos. Release of immunoreactive IGF-I and IGF-II by oviduct cultures and bovine blastocysts was also determined. IGFBP-2, -3, -4, and -5 transcripts were observed in oviduct primary cultures throughout an 8-day interval. IGFBP-1 and -6 mRNAs were consistently not detected in the oviduct. Messenger RNAs encoding IGFBPs -2, -3, and -4 were detected throughout bovine preattachment development, while transcripts encoding IGFBP-5 were detected only in blastocysts. IGFBP-1 and -6 transcripts were not detected in early embryos. Ligand blot analysis with 125I-labeled IGF-II revealed the presence of four prominent polypeptide bands of approximate molecular masses 24, 31, and 36 kDa, and a broad band extending from 46 to 53 kDa, in conditioned media samples prepared from oviduct primary cultures. Western immunoblot analysis confirmed the identity of the 24-kDa, 31-kDa, and 36-kDa species as IGFBP-4, -5, and -2, respectively. Levels of the release of IGF-II from oviductal vesicle cultures were significantly greater than levels observed for monolayer cultures (p < 0.005). No significant difference in the levels of IGF-I release between monolayer and vesicle cultures was observed. Pools of 10 blastocysts released on average 36.2 +/- 3.9 pg of IGF-II per embryo, while the release of embryonic IGF-I was below the levels of detection for our assay. The results suggest that maternally derived IGF may be regulated by IGFBPs to support bovine preattachment development.

Animals↗

PubMed1997

Interactions between follicle-stimulating hormone and growth factors in regulation of deoxyribonucleic acid synthesis in bovine granulosa cells.

Follicle growth is regulated by the combined actions and interactions of pituitary gonadotropic hormones and local intraovarian paracrine and/or autocrine agents, including the peptide growth factors, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF). The present study was undertaken to determine a) whether the previously demonstrated inhibitory effect of FSH on DNA synthesis was related to its ability to cause cumulus mucification as a differentiated response incompatible with continued proliferation, and b) whether increased IGF-binding proteins (IGFBP) secreted in response to FSH competed with IGF receptors, thereby inhibiting response to exogenous IGF-I. To determine the effects of cumulus mucification in modulating the mitogenic response to IGF-I, two other agents that induce cumulus mucification by different mechanisms, EGF and dibutyryl cAMP (dbcAMP), were compared with FSH. To determine the possible role of IGFBP in modulating the mitogenic response to IGF-I, an IGF-I analogue that does not bind to IGFBP, long arg3-IGF-I (LR3-IGF-I), was compared with native IGF-I for efficacy in stimulating DNA synthesis in the absence and presence of each of the above agonists. Both IGF-I and LR3-IGF-I stimulated [3H]thymidine incorporation in cumulus cells to a much greater extent than in mural granulosa cells. Incorporation in mural cells was increased by each of FSH, EGF, and dbcAMP acting by itself, and in most instances was considerably enhanced by the combined action of these agents with each of the IGF-I forms. In contrast, the considerably greater stimulatory effect of both IGF-I and LR3-IGF-I on cumulus cells was markedly decreased by each of FSH, EGF, and dbcAMP. These findings suggest that the inhibition of IGF-I-stimulated DNA synthesis in cumulus cells is a consequence of induction of cumulus cell differentiation (mucification) by FSH and EGF rather than through competition between IGF-I receptor and IGFBP secretion induced by these agents.

Animals↗