Biomedical subjects

B D Murphy

Publications and source records attributed to B D Murphy.

At least 37 records · Page 2Linked to original sources

PubMed1997

The effects of progesterone, 4,16-androstadien-3-one and MK-434 on the kinetics of pig testis microsomal testosterone-4-ene-5alpha-reductase activity.

The enzyme 3-oxo-steroid: NADP+ 4-oxidoreductase (EC 1.3.1.22; 5alpha-reductase) was assayed in testicular microsomes of pigs of 3, 20 and 24 weeks of age. The activity was very low in 3-week-old animals and approximately 10-fold higher in 5- and 6-month-old pigs. The pH optimum was 6.3 in 6-month-old animals, 5.7 in 5-month-old animals, but could not be reliably determined in 3-week-old animals. The kinetic parameters for 5alpha-reductase in testis microsomes from 6-month-old animals were; K((m)(app)), 8.0 micromol/l, V((max)(app)), 6.7 nmoles/90 min/mg protein. Progesterone was a competitive inhibitor of testosterone 5alpha-reduction with an apparent K((i)(app)) of 0.86 micromol/l. However, 4,16-androstadien-3-one (dienone), which undergoes 5alpha-reduction in the biosynthesis of the pheromonally active 16-androstenes, was a comparatively poor inhibitor with a K((i)(app)) of 4.9 micromol/l. Similarly, MK434, which is a selective inhibitor of the human type 2 5alpha-reductase, but which inhibits both types 1 and 2 in the rat, was also a poor competitive inhibitor of testosterone 5alpha-reductase in the pig testis (K((i)(app)), 3.1 micromol/l). It would appear from these studies that the pig testis microsomal 5alpha-reductase corresponds to a type 1 isozyme that is not capable of reducing dienone other than under conditions where the dienone concentration would be in considerable excess of testosterone. It is, therefore, probable that substrate-specific 5alpha-reductases exist in the pig testis for the 5alpha-reduction of testosterone and dienone.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed1997

Follicle-stimulating hormone and intracellular second messengers regulate steroidogenic acute regulatory protein messenger ribonucleic acid in luteinized porcine granulosa cells.

Ligand- and second messenger-regulated expression of the gene for steroidogenic acute regulatory protein (StAR) was evaluated in luteinized porcine granulosa cells. For comparison, cytochrome P450 side-chain cleavage (P450scc) was examined. Northern hybridization with homologous cDNA probes demonstrated three StAR mRNA species, of 2.7, 1.6, and 0.8 kilobases (kb), with the smallest variably present, and a single P450scc band at 1.9 kb. FSH elevated both STAR and P450scc messages in a dose-dependent manner over 6 h and continually stimulated both over 24 h (p < 0.001). STAR message induction depended on transcription, as did that of P450scc. Over 6 h, actinomycin D eliminated constitutive StAR message and reduced that of P450scc by two thirds, indicating briefer persistence of StAR. Pretreatment with cycloheximide prevented FSH induction of StAR and P450scc mRNA, implicating intermediate protein synthesis in expression of both genes. Dibutyryl cAMP caused time-dependent increases in StAR and P450 mRNAs over 24 h (p < 0.001), indicating the importance of the protein kinase A (PKA) pathway in their gene expression. Activation of the protein kinase C (PKC) pathway by a phorbol ester eliminated FSH induction of STAR mRNA increases (p < 0.01) while only reducing P450scc induction (p < 0.05). Thus, StAR gene expression, as reflected in mRNA abundance, is regulated by FSH via the PKA pathway and is dependent on transcription and translation. Conversely, the PKC pathway inhibits induction of these important steroid synthetic genes in luteinized granulosa cells.

Animals↗

PubMed1997

Luteal and placental characteristics of carnivore gestation: expression of genes for luteotrophic receptors and steroidogenic enzymes.

Experiments were carried out to investigate the abundance of mRNA for luteotrophic receptors and steroidogenic elements in the ovaries and corpora lutea of mink during the embryonic diapause, peri-implantation and postimplantation pregnancy. The second aim was to determine whether the mink placenta synthesized progesterone. Homologous cDNA probes for the mink LH and prolactin receptors were generated by the polymerase chain reaction. Heterologous cDNA probes for steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase-delta 4-delta 5 isomerase (3 beta HSD) were also used. The abundance of mRNA encoding the prolactin receptor was low during the period of embryonic diapause and increased concurrent with circulating progesterone. The abundance of LH receptor message reached peak values during the peri-implantation period followed by maintenance of a steady-state after implantation. The abundance of StAR and P450scc messages appeared not to vary during gestation, while that for 3 beta HSD was correlated with changes in circulating progesterone. There was no evidence of 3 beta HSD activity or transcripts in the placenta. These results indicate that prolactin and LH are necessary for activation of the corpus luteum during the period of embryonic diapause, and for its maintenance during postimplantation gestation. The mink placenta does not synthesize progesterone.

3-Hydroxysteroid Dehydrogenases↗

PubMed1996

Steroidogenic properties of a spontaneously established porcine granulosa cell line (PGC-2).

A spontaneously established porcine granulosa cell line (PGC-2) was cloned through the continuous culturing of primary granulosa cells collected from equine chorionic gonadotropin (eCG)-treated prepubertal gilts. This established cell line has undergone approximately 100 passages and shows contact-inhibition of growth. PGC-2 stained with a monoclonal antibody (mAb) directed against cytokeratin, indicating its epithelial nature, but not with a mAb directed against vimentin, suggesting that it is not fibroblast-derived. Immunoblotting revealed that PGC-2 expresses cadherin, an epithelial Ca+2-dependent cell adhesion molecule. The cells were dependent on serum for growth and had a doubling time of approximately 20 hr when cultured with 10% fetal bovine serum. The cell line was examined for the presence of FSH receptors, cAMP responses, and steroidogenic capabilities. The cell line lacks FSH receptors as assessed by radiolabelled-ligand binding, and no transcripts for FSH receptor were detected by Northern blotting of total cellular RNA. Neither FSH nor cholera toxin (0.5 ng/mL) stimulated increases in cAMP levels in these cells, whereas forskolin (10 microM) induced a fivefold increase in cAMP production. When a higher concentration of cholera toxin (300 ng/mL) was used, however, cAMP levels doubled by 2 hr. Despite a lack of responsiveness to purified of SH or oLH, the cells were capable of progesterone and estradiol production when provided with the appropriate substrates. We conclude that PGC-2 display properties that are similar to immature granulosa cells and may provide a suitable in vitro model for the study of granulosa cell function.

Animals↗

PubMed1996

Steroidogenic acute regulatory protein in bovine corpora lutea.

Steroidogenic acute regulatory protein (StAR) transfers cholesterol from the outer to the inner mitochondrial membrane to initiate steroidogenesis. Our purpose was to determine the tissue distribution of StAR mRNA and the occurrence of StAR gene products in the bovine corpus luteum (CL). Tissues were taken from the slaughterhouse or by ovariectomy of cattle at specific times after estrus or ovulation. StAR mRNA was identified by Northern analysis employing a 1.6-kb cDNA mouse StAR probe, and polyclonal antiserum against mouse StAR was used in Western analysis of StAR protein in bovine luteal tissue. The mRNA for cytochrome P450 side-chain cleavage enzyme (P450scc) was also evaluated by means of a homologous cDNA probe. Two isoforms of StAR mRNA, approximately 2.9 and 1.8 kb, were present in bovine CL, adrenal, theca, and granulosa cells and caruncles and cotyledons. One, or sometimes two, protein bands were recognized by the mouse StAR antiserum. P450scc mRNA colocalized in all sites where StAR mRNA was found, and in bovine liver. StAR mRNA was low in developing CL, increased 9- to 15-fold during the mid- to late-luteal phase, and disappeared in CL that had regressed. StAR protein concentrations were highly correlated with StAR mRNA throughout the estrous cycle (r = 0.93, p < 0.05). P450scc mRNA abundance did not vary through the luteal phase except for its disappearance in regressed CL. Corpora lutea from intact animals treated with prostaglandin F2 alpha displayed a 50% decline in StAR mRNA over 12 h while P450scc mRNA remained unchanged. At 24 h StAr mRNA was undetectable, while P450scc mRNA had declined to 50% of pretreatment values. We conclude that StAR mRNA and protein are tightly coupled in the bovine CL, being present at low levels during CL development and in elevated concentrations during the midluteal phase, and disappearing in regressed CL within 24 h of prostaglandin-induced luteolysis. We have further shown, for the first time, that StAR mRNA is present in the mammalian placenta.

Animals↗

PubMed1995

Ultrasonographic analysis of gestation in mink (Mustela vison).

Mink are seasonal breeders that display an obligate delay preceding implantation and a post implantation gestation of 31 d. The purpose of this study was to evaluate gestational parameters in mink by ultrasonography. A total of 92 female mink were mated twice during the period from March 2 to 20. The mink were scanned once and allowed to whelp (n=55); or scanned at 3 to 5-d intervals until parturition (n=13); or immediately subjected to autopsy (n=24) after scanning. Embryonic age was calculated from the date of parturition or from crown rump length. Uterine swelling diameter and fetal head size were correlated with embryonic age. The gestational sac grew rapidly once implantation had occurred. Uterine swellings of 4 to 5 mm in diameter were found on Days 2 to 4 post implantation and increased through Days 18 to 20, at which time they began to elongate due to the longitudinal growth of the fetus. Fetal cardiac activity could be detected on Days 10 to 12 post implantation in live embryos. The heart frequency was 198 +/- 3.0 beats per minute and did not vary from Days 12 to 30 post implantation. Fetal head diameter of 5 mm was first detected on Day 19 post implantation and grew gradually to 9 to 10 mm at parturition. It was not possible to accurately assess the number of conceptuses in utero. We conclude that ultrasonography can be employed in mink to diagnose pregnancy, to predict the parturition date and to determine the presence of live fetuses.

Journal Article↗

PubMed1995

Development of immortalized endometrial epithelial and stromal cell lines from the mink (Mustela vison) uterus and their effects on the survival in vitro of mink blastocysts in obligate diapause.

Mink endometrial cell lines were established by stable transfection of a plasmid vector encoding the SV40 large T antigen driven by the human beta-actin promoter. A second plasmid vector, pSV2neo, was employed for selection of transfected cells. Specificity and homogeneity of consequent cell lines were evaluated by immunocytochemistry employing antibodies against cytokeratin, desmin, and vimentin. Cytokeratin was found exclusively in epithelial cells, whereas vimentin appeared primarily in stromal cells. Neither cell line showed detectable desmin activity. These cell lines along with Buffalo rat liver (BRL) cells were employed in coculture with mink embryos in obligate diapause. Mink stromal and BRL cell lines were most effective in enhancing embryo survival in vitro. The percentages of cocultured embryos that survived for 72 h or more were 65% with epithelial cells, 75% with stromal cells, 68% with the combination of stromal and epithelial cells, and 93% with BRL cells. Only 23% of the embryos cultured without cells survived beyond 48 h. Embryo growth was also observed; some embryos in coculture showed trophoblastic outgrowth and adhesion to the cell surfaces. These results demonstrate that mink embryos in obligate delay can survive and develop in culture and that coculture with uterine or BRL cells increases the length and frequency of survival.

Alkaline Phosphatase↗

PubMed1995

Angiotensin II interferes with steroidogenesis in porcine granulosa cells.

The elements for the synthesis and activity of the vasopressor angiotensin II (AII) are present in the mammalian ovary. In the present investigation, the effects of AII were determined on three parameters of steroidogenic function in porcine granulosa cells in vitro: the accumulation of progesterone, the cellular content of the enzyme 3 beta-hydroxysteroid dehydrogenase delta 5-4 isomerase (3 beta-HSD), and the accumulation of mRNA for 3 beta-HSD. Cells were incubated with LH (200 ng/ml) in the presence or absence of AII (10(-7) M) or phorbol 12-myristate 13-acetate (PMA, 10(-7) M); doses of AII from 10(-10) to 10(-6) M in the presence or absence of LH; the AII reactor antagonist saralasin (10(-6) M) in the presence of AII or in combination of AII and LH; and AII in the presence or absence of (Bu)2 cAMP. The results demonstrate that LH increased progesterone, 3 beta-HSD message, and 3 beta-HSD content. Both PMA and AII interfered with the LH-induced progesterone accumulation, reducing the response by 50% or more. All also abrogated the LH-induced increases in 3 beta-HSD mRNA and 3 beta-HSD enzyme content in porcine granulosa cells. The AII inhibition was dose-dependent. The AII receptor antagonist saralasin blocked the inhibitory effects of AII on LH-induced steroidogenic events. AII interfered with the (Bu)2 cAMP induction of steroidogenesis and 3 beta-HSD mRNA and enzyme accumulation when (Bu)2 cAMP was present at a concentration of 30 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗

PubMed1994

Ovarian follicular development in mink (Mustela vison).

Ovarian follicular dynamics were studied during the breeding season, before and after ovulation in mink. Nulliparous female mink were stimulated to ovulate with an injection of 4 micrograms GnRH. Ovaries from three animals were collected on days 0, 2, 3, 4, 5, 6 and 7 after hormone treatment. A second dose of GnRH was administered on day 8 and ovaries were collected from three animals on day 9. Corpora lutea and follicles were identified in histological sections and follicles were classified by stage of development, healthy versus atretic, and by diameter. Preovulatory follicles (diameter 0.7-1.0 mm) were present in the ovaries of all animals on day 0 and these responded to GnRH treatment by ovulating. A synchronized wave of follicular development occurred following ovulation. Changes in follicle populations indicated that follicles are recruited from the small antral follicle class (0.2-0.4 mm) into the 0.4-0.6 mm class, with the first defined changes occurring between days 2 and 4. From the recruited group, a smaller cohort of follicles is selected to become the dominant follicles between days 4 and 6, and these acquire the ability to respond to a stimulus which induces ovulation at diameters of > 0.7 mm. The ovaries of unmated mink also contained substantial numbers of large, degenerating, luteinized, unruptured follicles. These degenerating, luteinized follicles are considered to represent the demise of large follicles that failed to receive an ovulatory stimulus.

Animals↗

PubMed1994

Regulation of follistatin gene expression in the ovary and in primary cultures of porcine granulosa cells.

Experiments were designed to test the hypotheses that (1) follistatin gene expression in granulosa cells is regulated during follicular growth, and (2) that alteration of follistatin mRNA concentration can be hormonally induced in primary cultures of porcine granulosa cells. RNA isolated from granulosa cells from small (1-3 mm diameter), medium (3-5 mm) and large (> 5 mm) follicles of prepubertal and postpubertal sows was analysed by hybridization to a porcine follistatin cDNA probe. Amounts of follistatin mRNA increased with follicular diameter, but no differences in follicular follistatin mRNA were detected between prepubertal and postpubertal sows. Treatment of cultured porcine granulosa cells with FSH or LH for 20 h stimulated follistatin mRNA concentration by a factor of two (100 ng FSH ml-1) and a factor of 1.5 (10 ng LH ml-1), respectively, over untreated controls. Treatment of cultured granulosa cells with 200 ng FSH ml-1, 200 ng LH ml-1, 10 mumol dibutyryl cAMP l-1, 30 mumol forskolin l-1 and 100 ng cholera toxin ml-1 stimulated follistatin mRNA accumulation in granulosa cells by factors of 4.9, 3.7, 1.6, 13.7 and 3.5, respectively, compared with control cultures. Stimulation of follistatin mRNA accumulation in cultured granulosa cells by dibutyryl cAMP (30, 100 and 300 mumol l-1) and forskolin (3, 10 and 100 mumol l-1) was dose dependent. FSH and forskolin induced time-dependent increases in follistatin mRNA concentration in cultured granulosa cells, with maximal induction occurring 72 h after treatment (a factor of 4.5 for FSH and 15.5 for forskolin).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Roles of melatonin and prolactin in testicular crudescence in mink (Mustela vison).

Peripubertal male mink (Mustela vison) were treated with prolactin, melatonin or antibodies against melatonin to determine the effects of altered circulating concentrations of prolactin and melatonin throughout one season of testicular development. Treatment began on 1 October and continued until 4 March. Administration of 0.5 mg ovine prolactin day-1 by minipump increased the circulating concentration of prolactin for the duration of the study and increased serum concentrations of LH. This treatment had no effect on the testosterone concentration or on testis size. Neither chronic treatment with melatonin throughout the period of crudescence nor passive immunization against melatonin for 79 days affected the circulating concentrations of prolactin, LH, testosterone or testis size. These results show clearly that, unlike in other seasonally breeding species, prolactin does not play a significant role in testis growth in the mink. Administration of melatonin to male mink in October did not affect testis growth, presumably because the melatonin signal that cues photoperiodic events had already been received. Administration of antibodies against melatonin did not affect any of the features measured, suggesting that melatonin may have neural but not peripheral effects. Further support for this view can be found in the absence of an influence of melatonin on testis growth or on the plasma concentration of testosterone.

Animals↗

PubMed1994

Superovulation in the cow with pregnant mare serum gonadotrophin: effects of dose and antipregnant mare serum gonadotrophin serum.

The effects of pregnant mare serum gonadotrophin (PMSG) dose and PMSG antiserum on superovulation in crossbred beef cows were studied. In experiment I, three groups were treated with 1200, 2400 or 3600 IU of PMSG and 48 h later with prostaglandin (PGF). The mean numbers of corpora lutea (CL), unovulated follicles, and total ova/embryos collected increased as the PMSG dose increased. The percent of fertilized ova and transferable embryos was lowest in the highest dose group (p < 0.05). In experiment II, all cows received 2500 IU of PMSG; groups 1 and 2 were treated with sheep anti-PMSG serum at 48 h or 60 h after PGF; group 3 cows were PMSG-only controls. The number of CL was lowest and the number of unovulated follicles highest in the PMSG-only group (p < 0.05). The number of CL was higher in group 2 (anti-PMSG at 60 h) than in the control group, with the anti-PMSG at 48 h not different from the other groups. Numbers of total ova/embryos, fertilized ova, and transferable embryos were higher (p < 0.05) in both antiserum-treated groups relative to the PMSG-only group. We conclude that superovulation of beef cows with PMSG and treatment with PMSG antiserum will induce a higher superovulatory response and will result in higher CL numbers and fewer unovulated follicles. Further, the variability in the superovulatory response to PMSG treatment was still evident when PMSG antiserum was administered.

Animals↗

PubMed1993

Alterations in 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) mRNA accumulation induced by beta-adrenergic stimulation in cultured porcine granulosa cells.

1. In primary cultures of porcine granulosa cells incubation with isoproterenol (10(-7)-10(-5) M) produced a dose-dependent increase in 3 beta-HSD mRNA, 3 beta-HSD content and progesterone production which ranged from 1.5- to 5-fold. 2. These effects were completely blocked by alprenolol. Terbutaline (10(-6) M) increased 3 beta-HSD mRNA, 3 beta-HSD content and progesterone production (1.5- to 3-fold), an effect which could be prevented by alprenolol. 3. Treatment with dobutamine (10(-6) M) produced no significant change in 3 beta-HSD mRNA accumulation. 4. The results suggest that beta-adrenergic agonists have the capacity to regulate transcription and translation of 3 beta-HSD mRNA in granulosa cells and this effect is mediated by the beta 2 adrenergic receptor.

3-Hydroxysteroid Dehydrogenases↗

PubMed1993

Effects of bovine follicular fluid and partially purified bovine inhibin on FSH and LH release by bovine pituitary cells in culture.

We have established a dispersed bovine pituitary cell culture system to study the effects of charcoal-extracted bovine follicular fluid (BFF) or bovine inhibin, partially purified by immunoaffinity chromatography (IPI), on the spontaneous release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Pituitary cells were plated at 0.25, 0.5 or 1 x 10(6) viable cells/well (c/w) and incubated for 48 h. The medium was replaced and BFF (0, 0.54, 2.7, 13.7, 68.7 or 343.5 micrograms protein) or IPI (0, 0.01, 0.06, 0.29, 1.45 or 7.25 micrograms protein) added to the cultures and the incubation was continued for 48 h. Concentrations of FSH and LH in spent medium were determined by RIA and data analyzed by ANOVA with means compared by Student-Neuman-Keuls (SNK) test. We have shown an increase in spontaneous FSH and LH release attributable to both number of bovine pituitary cells plated and to the length of incubation. The addition of BFF reduced spontaneous FSH release over 48 h incubation. The dose-dependent inhibition curves observed in culture in which different numbers of cells were plated, indicates that inhibition was greater when 1 x 10(6) c/w were plated compared to 0.25 or 0.5 x 10(6) c/w. Bovine follicular fluid at 0.45 micrograms of protein (equivalent to 0.01 microliters of BFF) incubated with 1 x 10(6) c/w, suppressed FSH release by 10.6% compare to control. Maximal suppression of 34.1% was obtained with 50 micrograms (equivalent to 1.56 microliters of BFF). Immunopurified bovine inhibin at 1.45 micrograms tended to suppress FSH release and at 7.25 micrograms significantly reduced FSH release. Neither BFF nor IPI had a measurable effect on LH release. We conclude that BFF and IPI suppress the spontaneous release of FSH from bovine pituitary cells in culture in a dose-dependent manner, without concomitant suppression of LH release.

Animals↗

PubMed1993

Prostaglandins alter the abundance of messenger ribonucleic acid for steroidogenic enzymes in cultured porcine granulosa cells.

The effects of prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E2 (PGE2) on the accumulation of progesterone and mRNA for the steroidogenic enzymes 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and cytochrome P450 side-chain cleavage (P450scc) were determined in luteinized granulosa cells. These cells were aspirated from 3-5-mm follicles of prepubertal pigs and cultured 48 h with 10% serum and then 48 h under serum-free conditions. Cells were then incubated with medium (control), ovine LH (20 ng/ml), (bu)2cAMP (0.5 mM) and doses of PGF2 alpha (0.08-2.0 micrograms/ml) or PGE2 (0.1-1.0 micrograms/ml), or combinations of PGF2 alpha (0.1-1.0 micrograms/ml) or PGE2 (0.1-1.0 micrograms/ml) with and without 20 ng/ml LH. Cultures were terminated after 12 h of incubation, and the abundance of mRNA for 3 beta-HSD and P450scc was determined by hybridization with specific cDNA probes. Parallel cultures were terminated at 24 h for analysis of progesterone in the medium by RIA. The results demonstrated that LH and (bu)2cAMP elevated progesterone by 3- to 6-fold and the accumulation of mRNA for 3 beta-HSD by 1.5 to 3.5 or 4-fold and for P450scc by 2-4-fold. PGF2 alpha reduced the basal level of progesterone accumulation, as well as the steady-state concentrations of mRNA for both 3 beta-HSD and P450scc. PGF2 alpha interfered with LH and (bu)2cAMP induction of 3 beta-HSD and P450scc message, and this interference was dependent on the PGF2 alpha dose employed.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxysteroid Dehydrogenases↗

PubMed1993

Regulation of follistatin messenger ribonucleic acid in porcine granulosa cells by epidermal growth factor and the protein kinase-C pathway.

Follistatin is a 35-kilodalton monomer isolated from follicular fluid by virtue of its ability to suppress FSH secretion from cultured pituitary cells. Experiments were designed to test the hypothesis that the accumulation of follistatin RNA in the ovary is regulated by epidermal growth factor (EGF) and activation of the protein kinase-C (PKC) pathway. Follistatin mRNA was quantitated by slot blot hybridization of total RNA from primary cultures of porcine granulosa cells treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA), an activator of PKC. PMA (0.1, 1.0, 10, and 100 nM) induced a dose-dependent increase in follistatin mRNA accumulation after 2 h, with a maximal increase of 40-fold over that in untreated control cultures at a dose of 10 nM. PMA (10 nM) induced a time-dependent increase in follistatin mRNA levels, with a maximal response at 2 h. Follistatin gene expression was induced by a 2-h incubation with EGF (3 nM), but not by LH (100 ng/ml), GnRH (10 nM) or prostaglandin F2 alpha (80 micrograms/ml). EGF (0.01, 0.1, 1, and 10 nM) induced a dose-dependent induction of follistatin gene expression in granulosa cells after 2-h incubation, with maximal stimulation of 33-fold at a dose of 1 nM. The time course of induction of follistatin mRNA by EGF was very similar to that induced by PMA, with maximal stimulation occurring at 2 h and declining thereafter. Pretreatment of granulosa cells for 24 h with PMA abrogated the EGF-induced stimulation of follistatin mRNA accumulation. However, cotreatment of granulosa cells with EGF and PMA for 2 h resulted in additive stimulation of follistatin mRNA. These results demonstrate that 1) follistatin gene expression in cultured porcine granulosa cells is acutely stimulated by PMA and EGF in a time- and dose-dependent manner; 2) follistatin gene expression may be regulated by the PKC pathway; and 3) the stimulatory effect of EGF on follistatin gene expression may require PKC.

Animals↗

PubMed1993

Control of luteal function in the mink (Mustela vison).

The ranch mink was studied to determine the role of pituitary luteotrophins on corpus luteum (CL) function before and after implantation. Twelve mink were treated with monoclonal antiserum against gonadotrophin-releasing hormone (GnRH), and 12 with an irrelevant monoclonal antibody during embryonic diapause. Activation of the CL, plasma progesterone concentration and embryo implantation were unaffected by this treatment. In a second trial, groups of ten mink were treated with GnRH antibodies, bromocriptine, bromocriptine plus 0.5 mg prolactin per day per animal, or ethanol vehicle. Comparison of the consequent profiles of progesterone indicated that both bromocriptine and anti-GnRH compromised postimplantation CL function by inducing regression of the corpus luteum. Incubation of dissociated luteal cells from ovaries of mink at 21-24 days after implantation with either LH or prolactin increased the accumulation of progesterone over 2 h. Addition of 25-hydroxy-cholesterol (25OHC) as substrate increased basal levels and the progesterone accumulation stimulated by LH and prolactin; the increases induced by luteotrophins were additive. There was an apparent synergistic interaction between prolactin and canine low-density lipoproteins (LDL) in the stimulation of progesterone secretion in vitro. The results are interpreted to indicate that LH/FSH are not required for luteal support during embryonic diapause, or for luteal activation. Prolactin is necessary for luteal activation, and LH and/or FSH and prolactin are obligate luteotrophins during the postimplantation period in the mink.

Animals↗

PubMed1993

Induction of follicular development in silver foxes (Vulpes vulpes) with equine chorionic gonadotrophin (eCG) and antibodies against eCG.

Experiments were conducted that demonstrated that 1000 iu equine chorionic gonadotrophin (eCG) was effective in induction of follicular development and ovulation in silver foxes during anoestrus. This treatment resulted in large, unovulated follicles; thus, trials in which the effects of eCG were reduced or abrogated by antibodies against eCG were carried out. Passive immunization against eCG on days 3 and 4 after eCG treatment interfered with subsequent follicular development and prevented ovulation. Treatment with eCG antibodies on days 5 or 7 after eCG treatment did not prevent ovulation and neutralization beginning on day 5 appeared to provide for the best ovulatory yield. The results suggest that combinations of eCG and anti-eCG antibody may provide a useful means of inducing ovarian activity in anoestrous foxes.

Anestrus↗