Biomedical subjects

D T Armstrong

Publications and source records attributed to D T Armstrong.

At least 55 records · Page 3Linked to original sources

PubMed1995

Localization of epidermal growth factor (EGF) receptor in the rat corpus luteum, and EGF and transforming growth factor-alpha stimulation of luteal cell steroidogenesis in vitro.

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) have potent mitogenic effects on granulosa and theca cells. However, their effects on steroidogenesis by these cells is controversial, and there is limited information regarding their effects on luteal cell steroidogenesis. The present study investigated the cellular distribution of the EGF receptor (EGF-R) in the rat corpus luteum (CL) by immunocytochemical staining, and the effects of EGF and TGF-alpha on progesterone and 20 alpha-dihydroprogesterone (20 alpha-OH-P) production in cultures of luteal cells. Using a primary antibody directed against the human EGF-R peptide, specific EGF-R staining was obtained in the CL. Both small and large luteal cells had EGF-R staining. In initial cell culture experiments, treatment of freshly isolated luteal cells with EGF or TGF-alpha (0.5-50 ng/ml) for 24 h had no effect on progesterone and 20 alpha-OH-P accumulation. Addition of LH (250 ng/ml) alone caused a 3.5-fold increase in both progestins, but co-treatment with EGF or TGF-alpha produced no further enhancement of progestin accumulation. However, when cells were seeded overnight and the attached cells were washed prior to growth factor treatment for 3 days with media change every 24 h, both EGF and TGF-alpha caused dose-dependent increases in progesterone accumulation/24 h period (up to 2-fold at 50 ng/ml growth factor) on days 1 and 2 but not day 3 of treatment. 20 alpha-OH-P accumulation was similarly stimulated (up to 2.5-fold) by EGF and TGF-alpha under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

20-alpha-Dihydroprogesterone↗

PubMed1995

Epidermal growth factor and its receptor gene expression and peptide localization in porcine ovarian follicles.

The present study was undertaken to determine the expression of genes for epidermal growth factor (EGF) and its receptor (EGF-R) in various components of medium-sized porcine ovarian follicles by reverse transcription-polymerase chain reaction (RT-PCR), and to localize their peptides during folliculogenesis by immunocytochemistry. A strong band for EGF mRNA transcript was detected in the oocyte, whereas the signal in cumulus, granulosa, and theca cells was very weak but detectable. In contrast, a very strong EGF-R mRNA signal was observed in cumulus, granulosa, and theca cells, whereas the signal in the oocyte was very weak. EGF peptide was localized in the oocyte, cumulus, and granulosa cells of all stages of follicle. In the oocyte, the intensity of immunostaining was more pronounced in primordial and primary follicles, compared to atrial follicles. In large antral follicles, immunostaining was pronounced in granulosa cells, whereas theca cells showed little or no detectable staining for EGF. EGF staining was also observed in the cumulus and granulosa cells of follicles undergoing atresia. EGF-R immunostaining was observed in the oocytes of primordial and primary follicles, and in cumulus, granulosa, and theca cells of all stages of follicle, including atretic follicles. In large antral follicles, the intensity of immunostaining was more pronounced in theca cells than in granulosa cells, and the oocyte showed little or no detectable staining. No immunostaining was observed when the primary antibody was replaced with preimmune serum (EGF), or preabsorbed with the control peptide (EGF-R), confirming the specificity of the staining procedures.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Transforming growth factor alpha gene expression and peptide localization in porcine ovarian follicles.

The present study was undertaken to determine transforming growth factor alpha (TGF alpha) gene expression in various components of medium-sized porcine ovarian follicles by reverse transcription-polymerase chain reaction, and to localize the peptide during folliculogenesis by immunocytochemistry. A strong signal for TGF alpha transcript was detected in cumulus and granulosa cells, whereas the signal in theca cells was very weak but detectable. No TGF alpha mRNA was detected in the oocyte. Immunolocalization studies revealed intense TGF alpha staining in cumulus and granulosa cells of all stages of follicular development. TGF alpha immunostaining was also observed in the oocytes of primordial and primary follicles. In antral follicles, immunostaining was observed only in cumulus and mural granulosa cells, whereas theca cells and oocytes exhibited very little or no detectable staining. TGF alpha immunostaining was absent in follicles undergoing atresia. No TGF alpha immunostaining was observed when the antibody was omitted from the procedure, and the staining was substantially reduced when the primary antibody was preabsorbed with the same mass of TGF alpha peptide, suggesting the specificity of the staining procedure. These results suggest a local follicular production of TGF alpha in the porcine ovary and thus a role for TGF alpha of follicular origin in the regulation of follicular development in an autocrine/paracrine fashion.

Animals↗

PubMed1995

Seminal fluid and the expression of MHC class I antigens in the placenta of the rat.

PROBLEM: To determine whether seminal fluid influences the expression of MHC class I antigens on the surface of basal trophoblast cells in the placenta of the rat. METHODS: Transfer of DA x DA embryos into a WF (allogeneic) or DA (syngeneic) recipient made pseudopregnant by hormonal treatment followed by mating with a vasectomized male (seminal fluid) or by mechanical stimulation (no seminal fluid). Antigen expression was determined by electron microscopic immunocytochemistry using the appropriate gold-labeled monoclonal antibodies. RESULTS: Seminal fluid did not affect the expression of MHC class I antigens on the surface of the basal trophoblast in either allogeneic or syngeneic matings. CONCLUSIONS: The suppression of the expression of paternal class I antigens on the surface of the basal trophoblast cells in allogeneic pregnancies most likely occurs at the genome level shortly after fertilization.

Animals↗

PubMed1994

Purification of a cell-cell adhesion regulator from porcine seminal vesicle fluid.

Seminal plasma derived factors are implicated in mediating inflammation in the female reproductive tract following insemination at mating. During inflammation, leukocytes are activated to express adhesion receptors resulting in adherence to each other and for the ECM as well as for various cell types. The present study describes the purification of a leukocyte cell-cell adhesion regulator derived from seminal vesicle fluid. Seminal vesicle fluid proteins were chromatographed by cation exchange, hydrophobic interaction and reversed phase. Chromatography on Phenyl Superose resolved two distinct forms of cell-cell adhesion regulation, type I and II. Reversed phase chromatography of fractions inducing type I adhesion resulted in the isolation of a 15kDa adhesion inducing protein (pAIF-1). The N-terminal sequence contained a hydrophobic consensus sequence which exists in: two bovine seminal vesicle proteins (BSPA3, PDC 109); IGF-II receptor; fibronectin; and the cation independent mannose-6-phosphate receptor.

Amino Acid Sequence↗

PubMed1994

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Gonadotropin stimulation regimens for follicular aspiration and in vitro embryo production from calf oocytes.

Crossbred beef x dairy calves were randomly allocated at 3 wk of age to different gonadotropin treatment regimens for stimulation of follicle development and induction of oocyte maturation in vivo. Follicular responses were assessed laparoscopically, and oocytes were aspirated for assessment of maturational state or for in vitro fertilization (IVF) and culture to determine developmental capacity. Follicle-stimulating Hormone (FSH), administered in a single subcutaneous injection together with a low dosage of PMSG, was as effective as the same total dosage of FSH administered in 6 injections over a 3-d period. Without accompanying PMSG, this dose of FSH was ineffective in stimulating follicle development. The mean number of preovulatory follicles (> 5mm, with hyperemic appearance) doubled with each successive stimulation at 3-wk intervals, reaching 35 follicles per calf at 9 wk of age. Oocyte yields ranged from 55 to 81% of follicles aspirated, and did not differ significantly among age, FSH regimen and oocyte maturation stimulus. A combination of LH + FSH was more effective in stimulating cumulus cell expansion than LH by itself (73 vs 22% of recovered oocyte-cumulus cell complex (OCC) respectively; P<0.05). Of 33 unselected immature oocytes (cumulus unexpanded) subjected to in vitro maturation (IVM) and IVF, 30% developed to blastocysts during co-culture with bovine oviduct epithelial cells, which was not significantly different from 25% of 36 oocytes from adult ovaries which reached the blastocyst stage under similar conditions. The results indicate that follicle responses of calf ovaries to FSH stimulation increase progressively from 3 to 9 wk of age, and that oocytes recovered laparoscopically from these follicles produce blastocysts in culture at rates similar to oocytes from adult cattle ovaries collected at slaughter. The approach offers promise for embryo production from donor calves of superior genetic merit for embryo transfer, thereby enhancing the rate of genetic gain above that attainable by conventional breeding or by embryo transfer in adult cattle.

Journal Article↗

PubMed1994

Catecholestrogen modulation of steroid production by rat luteal cells: mechanism of action.

This study investigated the mechanisms underlying 2-hydroxyestradiol (2-OHE2) effect on luteal steroidogenesis using serum-free cultures of mixed luteal cells from day 8 pseudopregnant rats. Initially, interactions between 2-OHE2 and LH or dibutyryl (db)cAMP on progesterone production were investigated. LH (250 ng/ml) and 2-OHE2 (2.5 microg/ml) had comparable effects on progesterone accumulation, while dbcAMP (5 mM) was more stimulatory. When applied together, 2-OHE2 did not synergize with LH or dbcAMP to further enhance progesterone accumulation. Furthermore, in time course experiments, the dose-dependent effect of 2-OHE2 was to reduce and eventually abolish the time-dependent increase in cAMP accumulation. In contrast LH stimulated cAMP accumulation at all times. Experiments in which cells were co-treated with 2-OHE2, 22-OH-cholesterol and cyanoketone, or with 2-OHE2 and 22-OH-cholesterol or pregnenolone indicated that 2-OHE2 not only had a stimulatory effect on the cholesterol side-chain cleavage and 3beta-hydroxysteroid dehydrogenase enzymes, but it also appeared to inhibit the 20alpha-hydroxysteroid dehydrogenase leading to a relative increase in progesterone accumulation. Experiments with hormone antagonists suggested that the actions of 2-OHE2 were not mediated by the estrogen, alpha- or beta-adrenergic receptors. The results of this study support the concept of a physiological role for catecholestrogens in rat luteal steroidogenesis.

Animals↗

PubMed1994

Response of peripheral blood leucocytes to mitogenic factor(s) in porcine seminal plasma.

Porcine seminal plasma (PSP) contains a potent mitogenic substance capable of causing proliferation and extensive agglutination in cultured porcine peripheral blood leucocytes (PBL). In order to determine the specific leucocyte cell population affected by this mitogen, lymphocyte, monocyte and polymorphonuclear (PMN)-enriched cell fractions were separated from PBL and treated with increasing concentrations of PSP (1-8%, v/v). For monocyte cell populations enriched through elutriation and adherence to plastic, cell proliferation in response to PSP treatment was consistently higher than PSP-treated unfractionated PBL. Monocyte-depleted PBL and lymphocyte populations enriched through elutriation demonstrated a decrease in proliferation compared to PSP-treated unfractionated PBL. PMN populations separated from PBL by discontinuous Percoll gradient centrifugation were unresponsive to PSP. Agglutination was observed for the unfractionated PBL and each enriched cell population. These results demonstrate that PSP contains a potent mitogen which induces proliferation in monocyte-enriched cell populations and may reflect the potential of PSP to act as an immune regulator in the uterine environment during early embryo development and implantation.

Agglutination↗

PubMed1994

Preimplantation development of in vitro-matured and in vitro-fertilized ovine zygotes: comparison between coculture on oviduct epithelial cell monolayers and culture under low oxygen atmosphere.

The roles of medium composition, serum source, embryo coculture, and culture under low O2 conditions on the development of in vitro-matured and in vitro-fertilized (IVMF) ovine zygotes were investigated in three separate experiments. In the first experiment, the proportion of cocultured IVMF zygotes developing to the blastocyst stage was significantly higher (38.0% vs. 3.5%; p < 0.05) than that of non-cocultured zygotes treated within three embryo culture media (TCM-199 + 10% fetal bovine serum [FBS]; bicarbonate-buffered, glucose-free synthetic oviduct fluid medium [mod-SOFM] + 10% FBS; and bicarbonate-buffered BSA-free Tyrode's salt solution [mod-TALP] + 10% FBS) under a 5% CO2 atmosphere in air. In a second experiment, a significantly higher (p < 0.05) proportion of cocultured zygotes placed in TCM-199 medium survived to the blastocyst stage (37.4% blastocysts vs. 23.4% in mod-SOFM). No significant effect of serum (FBS vs. human serum [HS]) was observed on embryonic development, but coculture was confirmed to exert a significant influence on development to the blastocyst stage. In the final experiment, survival of the embryo under a reduced oxygen (5% CO2:5% O2:90% N2) atmosphere was investigated. In contrast to results in the initial experiments, embryonic survival was significantly higher (p < 0.05) in the non-cocultured treatment groups (21.9% blastocysts vs. 0.4% for cocultured zygotes). Serum source also had a significant (p < 0.05) influence upon the development of non-cocultured zygotes: 32.3% of zygotes cultured with HS progressed to the blastocyst stage vs. 11.5% of zygotes cultured in FBS-supplemented medium. These results have characterized two distinct culture environments, each capable of supporting the development of high frequencies of unselected IVMF zygotes to the blastocyst stage in vitro.

Animals↗

PubMed1994

A growth factor phenotype map for ovine preimplantation development.

The reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the patterns of expression for several growth factor ligand and receptor genes during ovine preimplantation development. Transcripts for insulin-like growth factor (IGF)-I, IGF-II, and the receptors for insulin and IGF-I were detected throughout ovine preimplantation development from the 1-cell to the blastocyst stage. Transforming growth factor alpha (TGF alpha) transcripts were also detected throughout ovine preimplantation development. The mRNAs encoding basic fibroblast growth factor (bFGF) were detected in all stages of the ovine preimplantation embryo, although the relative abundance of this transcript consistently decreased from the 1-cell to the blastocyst stage, suggesting that it may represent a maternal transcript in early sheep embryos. Transcripts encoding ovine trophoblast protein (oTP) were detected only within blastocyst-stage embryos. Primary ovine oviduct cell cultures express the transcripts for IGF-II, IGF-I, TGF alpha, bFGF, TGF beta 1, and the receptors for insulin and IGF-I, suggesting that paracrine growth factor circuits may exist between the oviduct epithelium and the early ovine embryo. Transcripts for insulin, epidermal growth factor (EGF), and nerve growth factor (NGF) were not detected in any stage of the ovine preimplantation embryo or within the oviduct cell preparations. The expression of growth factor transcripts very early in mammalian development would predict that these molecules fulfil a necessary role(s) in supporting the progression of early embryos through the preimplantation interval. Our future efforts will be directed to understanding the nature of these putative regulatory pathways.

Animals↗

PubMed1994

Possible roles of insulin and insulin-like growth factors in rat preimplantation development: investigation of gene expression by reverse transcription-polymerase chain reaction.

The sensitive mRNA phenotyping technique of reverse transcription-polymerase chain reaction was used to demonstrate that insulin receptor mRNA is present in rat embryos during the preimplantation period. In addition, mRNA encoding insulin-like growth factor (IGF) type I and type II receptors have also been detected in rat preimplantation embryos. IGF-I mRNA was not detected in preimplantation embryos but was found in oviducts and uteri of prepubertal and early pregnant rats. IGF-II mRNA was present in both embryos and in oviducts and uteri during the preimplantation period. These findings suggest that insulin and IGF-I could influence early embryo development in endocrine or in paracrine fashions, whereas IGF-II may have an additional autocrine mode of action in affecting preimplantation embryos in rats.

Animals↗

PubMed1994

Expression of plasminogen activator genes and enzymatic activities in rat preimplantation embryos.

Plasminogen activator has been implicated in tissue invasion and remodelling because of its role in the degradation of the extracellular matrix. Its activity can be detected in mouse embryos as early as day 6 of pregnancy, suggesting that plasminogen activator is involved in the process of implantation. The present study determined the time course of expression of the genes encoding tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) during the preimplantation period in rats by the sensitive mRNA phenotyping procedure of reverse transcription-PCR. The tPA mRNA was present in rat oocytes and two-cell embryos, but was not detected between the four-cell and blastocyst stages. The uPA mRNA was first detected in two-cell rat embryos, and was present through to the blastocyst stage. In chromogenic assays, plasminogen activator activity was detected in oocytes and embryos between two-cell and blastocyst stages. Most plasminogen activator activity present in preimplantation embryos appeared to be uPA, as it could be inhibited by anti-uPA antibody and a specific uPA inhibitor, amiloride, but not by anti-tPA antibody. The present data demonstrate the expression of uPA gene and uPA activity in preimplantation rat embryos, suggesting that embryonic uPA may be involved in early embryo development and implantation.

Amino Acid Sequence↗

PubMed1993

Factors influencing resumption of meiotic maturation and cumulus expansion of porcine oocyte-cumulus cell complexes in vitro.

The present study was undertaken to examine effects of various combinations of epidermal growth factor (EGF), transforming growth factor-beta 1 (TGF-beta 1), follicle-stimulating hormone (FSH), luteinizing hormone (LH), androstenedione (A4), and estradiol-17 beta (E2) on meiotic maturation and cumulus expansion in the pig using an in vitro model system. Oocyte-cumulus cell complexes (OCC) were cultured in the media containing the above-mentioned agents for 24 hr and were observed for germinal vesicle breakdown (GVBD), indicative of initiation of meiotic maturation, and for expansion of their cumulus cells. Treatment with EGF significantly increased (P < 0.05) incidence of GVBD, with maximal stimulation occurring at 1 ng/ml (55% vs. 12% in the control). Concentrations of EGF as low as 100 pg/ml significantly stimulated GVBD over control (37% vs. 12%). Addition of EGF (1 ng/ml) and FSH (1.5 micrograms/ml) together and LH (2 micrograms/ml) and FSH (1.5 micrograms/ml) together resulted in significantly higher (P < 0.01) GVBD levels than were observed in response to EGF, FSH, or LH alone. Addition of E2 (1 microgram/ml) had no effect by itself but significantly decreased the incidence of GVBD in the presence of FSH and of LH + FSH. Addition of A4 (1 microgram/ml) significantly reduced the percentage of oocytes undergoing GVBD when added alone or with FSH. Although both EGF and LH stimulated cumulus expansion, FSH was more effective in stimulating cumulus expansion than EGF or LH. TGF-beta 1 had no effect on GVBD or cumulus expansion.(ABSTRACT TRUNCATED AT 250 WORDS)

Androstenedione↗

PubMed1993

Human menopausal gonadotropin during in vitro maturation of human oocytes retrieved from small follicles enhances in vitro fertilization and cleavage rates.

OBJECTIVES: To compare the IVF rates of oocytes retrieved from small follicles (< 2 mL in volume) with those of oocytes retrieved from large follicles and to test the effect of adding gonadotropins to the IVF medium on the fertilization rates of oocytes from small follicles. DESIGN: Oocytes were retrieved with endovaginal ultrasound (US) guidance from patients undergoing infertility treatment in our IVF program. Oocytes were grouped according to the volume of the originating follicle and subjected to our routine procedure for IVF. HMG was added to the IVF medium for some of the oocytes from small follicles. SETTING: Toronto Fertility and Sterility Institute is affiliated with the University of Western Ontario and University of Toronto and is equipped for RIA, endovaginal US monitoring and oocyte retrieval, and for processing and culturing gametes and embryos. PATIENTS: Infertile patients admitted to our IVF program. INTERVENTIONS: Patients underwent ovarian stimulation with hMG before oocyte retrieval. No other interventions were introduced to the processing and culturing the gametes and embryos except the addition of hMG to the medium of some of the small follicle-originated oocytes with the informed consent from the patients. MAIN OUTCOME MEASURES: Rates of fertilization, cleavage of the fertilized embryos before replacement, and meiotic status of some of the oocytes from small follicles. RESULTS: Most of the oocytes from small follicles did not complete the first meiotic division; they had low rates of fertilization and cleavage compared with oocytes from large follicles, and these rates were improved by the addition of hMG to the IVF medium. CONCLUSIONS: Oocytes from small follicles are probably less mature and require a more physiological environment to achieve normal rates of fertilization and cleavage.

Adult↗

PubMed1993

Differential modulation of porcine theca, granulosa, and luteal cell steroidogenesis in vitro by tumor necrosis factor.

The role of tumor necrosis factor alpha (TNF alpha) in ovarian function was investigated using in vitro culture of theca and granulosa cells isolated from gilt follicles (4-6 mm) and small (SLC) and large (LLC) luteal cells from mid-cycle corpora lutea. TNF alpha did not affect basal accumulation of progesterone (P) by theca cells after 72 h of culture. However, TNF alpha (0.1-100 ng/ml) caused a marked dose-dependent noncytotoxic inhibition (p < 0.05) of LH or LH+insulin (I)-stimulated P accumulation by theca cells after 72 h. Maximal inhibitions averaged 87 +/- 6% at 5 ng/ml TNF alpha for LH-stimulated P and 69 +/- 4% at 50 ng/ml TNF alpha for LH+I-stimulated P. The inhibitory effect of TNF alpha, evident by 24 h after culture, progressively increased on Days 2 and 3 of culture. The effect of TNF alpha on theca cells was mediated by cAMP generation as evidenced by TNF alpha inhibition of LH-induced cAMP accumulation and P accumulation in response to LH and forskolin but not dibutyryl cAMP. Consistent was this, TNF alpha had no effect on increased P accumulation by theca cells in the presence of 22-hydroxycholesterol or pregnenolone alone, but inhibited further increases in P accumulation stimulated by LH plus sterol substrates. Unlike that in theca cells, FSH-induced P accumulation in granulosa cell cultures was slightly enhanced (p < 0.05) by low doses of TNF alpha (0.1, 0.5, and 1.0 ng/ml) after 72 h, while higher doses (5-50 ng/ml) did not alter P accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Androstenedione↗

PubMed1993

Porcine oocytes release cumulus expansion-enabling activity even though porcine cumulus expansion in vitro is independent of the oocyte.

In the mouse, the oocyte secretes a factor that enables cumulus cells to undergo expansion in response to epidermal growth factor (EGF) or FSH while expansion of the porcine cumulus oophorus has been shown to be independent of the oocyte. This study was undertaken to confirm independence of the porcine cumulus oophorus of the oocyte for its expansion and to determine if the porcine oocyte secretes the putative cumulus expansion-enabling factor that is required for FSH-stimulated mouse cumulus expansion. Porcine oocyte-cumulus cell complexes (P-OCC), oocytectomized oocyte-cumulus cell complexes (P-OOX) and intact clumps of cumulus cells (P-CCC) were cultured at 39 C in TCM-199 medium containing EGF (1 ng/ml) or FSH (1.5 micrograms/ml). After 24h culture periods, cumulus expansion was scored on an arbitrary scale of 0 to +4. EGF stimulated similar cumulus expansion (expansion score +3 compared to 0 for controls) in all the three groups. FSH stimulated cumulus expansion in significantly higher number of complexes in all groups compared to EGF. However, there was no difference in cumulus expansion among the three groups with EGF or FSH. To determine the production of the factor by the porcine oocyte, isolated clumps of cumulus cells of the mouse (M-CCC) were cocultured with porcine denuded oocytes (P-DO) in TCM-199 with or without 1.5 micrograms/ml FSH at 37 C for 24h. No expansion was observed when M-CCC were cultured alone in the presence of FSH or cocultured with P-DO in the absence of FSH. However, coculture of M-CCC with P-DO in the presence of FSH resulted in expansion of M-CCC similar to that observed in intact mouse oocyte-cumulus cell complexes (M-OCC) in response to FSH. These studies indicate that even though porcine cumulus expansion in vitro is not dependent on the oocyte, the porcine oocyte is capable of secreting cumulus expansion-enabling factor(s).

Animals↗

PubMed1993

Decontamination of rat embryos and transfer to specific pathogen-free recipients for the production of a breeding colony.

When animals are introduced to a specific pathogen-free (SPF) facility, care must be taken to avoid the possibility of disease transmission to the local colony. This study investigated the application of a combination of reproductive biotechnologies to establish a new disease-free colony of two rat strains, DarkAgouti(DA/Pit) and Wistar Furth(WF/Pit), from a stock known to be chronically infected with the following pathogens: Mycoplasma pulmonis, Kilham's rat virus, sialodacryoadenitis/coronavirus, and reovirus type 3. To eliminate the pathogens and optimize the use of animals, superovulation, embryo washing and trypsinization, and embryo transfer were used. Donors (DA/Pit and WF/Pit) were treated as follows: the mature females were synchronized by subcutaneous (s.c.) injection with 40 micrograms luteinizing hormone-releasing hormone agonist/animal on day -4. All immature and mature females were induced to superovulate by continuous s.c. infusion with a commercial porcine follicle-stimulating hormone (FSH) preparation (3.4 or 6.8 mg NIH-FSH-P1 units per day, respectively), beginning on the morning of day -2. On the afternoon of day 0, the animals received 30 IU human chorionic gonadotropin injected intraperitoneally and mated. From a total of 213 ova flushed from the oviducts of 16 programmed donors, 195 transferrable two-cell embryos were recovered. Two outbred strains of SPF rats, Long-Evans (LE) and Wistar (W), were used as recipients. These mature females (LE and W) were synchronized by using luteinizing hormone-releasing hormone agonist as described and made pseudopregnant by cervical stimulation. Two-cell embryos (DA/Pit and WF/Pit) were washed and trypsinized, then transferred to the oviducts of the pseudopregnant recipients (LE and W).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗