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Biomedical subjects

S Lun

Publications and source records attributed to S Lun.

At least 37 records · Page 2Linked to original sources
PubMed1997

Isolation, characterization and radioimmunoassay of luteinizing hormone in the brushtail possum.

Luteinizing hormone (LH) was purified from brushtail possum (Trichosurus vulpecula) pituitary glands. The purification procedure consisted of ammonium sulfate precipitation followed by triazinyl-dye chromatography, hydrophobic interaction chromatography and gel filtration. A yield of 10 microg LH g-1 pituitary with a recovery of 20% was obtained from 1400 pituitary glands (20.3 g). Contamination with possum follicle-stimulating hormone (FSH) was < or =0.05%. The amino acid analysis and the N-terminal sequencing for 10 cycles revealed close homology with LH from other mammals. Minor amounts of LH that had been truncated near the N-terminal were also detected. No contaminating proteins were found by amino acid sequencing. The potency of possum LH was 20% that of ovine LH in a receptor assay using possum testicular receptors and 4% that of ovine LH when bovine corpora lutea receptors were used. Possum LH was able to stimulate production of cyclic adenosine 3',5'-monophosphate by bovine granulosa cells. A radioimmunoassay (RIA) for possum LH using 125I-possum LH and an antiserum raised against ovine LH was developed. The RIA has a sensitivity of 0.15 ng mL-1, a 50% displacement of 1.9 ng mL-1 and a cross-reactivity of <0.02% against possum FSH. Plasma concentrations were 0.24+/-0.04 ng mL-1 (n = 8) and 0.39+/-0.12 ng mL-1 (n = 8) in female and male possums respectively. Administration of mammalian gonadotrophin-releasing hormone (GnRH) and chicken GnRH II stimulated increases in plasma LH concentrations in male and female possums. When comparing LH responses with administration of mammalian GnRH or chicken GnRH II, plasma LH concentrations remained elevated for a longer period of time in males than in females (P < 0.01); plasma LH concentrations also remained elevated for longer after mammalian GnRH than after chicken GnRH II (P < 0.01). Gonadectomy stimulated an increase in plasma concentrations of LH in both male (P < 0.01) and female (P < 0.05) possums. The rate of increase in plasma LH concentrations in males was faster than that in females. In summary, we have purified, partially characterized, and developed a RIA for possum LH.

Amino Acids↗
PubMed1997

Granulosa cell apoptosis, aromatase activity, cyclic adenosine 3',5'-monophosphate response to gonadotropins, and follicular fluid steroid levels during spontaneous and induced follicular atresia in ewes.

The aims of the present study in ewes were 1) to test the hypothesis that apoptosis in granulosa cells is one of the processes involved in the structural demise of follicles and 2) to define the temporal relationships among the occurrence and degree of apoptosis in granulosa cells, aromatase activity, production of cyclic AMP (cAMP) by granulosa cells in response to FSH or LH, concentrations of estradiol 17 beta (E2) and progesterone in follicular fluid, and the characteristic morphometric changes associated with the process of follicular atresia. To address these aims, ewes were treated with either saline or steroid-free bovine follicular fluid (bFF) at 60 h after estrus, and ovarian follicles > or = 3 mm diameter were recovered at 0, 12, 18, or 24 h later. Apoptotic granulosa cells were identified by the presence of oligonucleosomes after 3'-end labeling of extracted DNA with [32P]alpha dideoxy ATP (ddATP). The degree of oligonucleosome formation, based on the intensity of radiolabeling, was given an apoptosis score (AP) of 0 (nondetectable), 1 (slight), 2 (moderate), or 3 (marked). Moreover, a labeling index (LI) was calculated from the amount of radiolabeled ddATP incorporated into low-molecular weight (< 4.2 kb) DNA fragments. On the basis of morphometric criteria, 73% (141 of 194) of the follicles classified as healthy had apoptotic granulosa cells compared to 86% (18 of 21) of the follicles classified as atretic. In the bFF-but not saline-treated ewes, the concentrations of plasma FSH had declined to basal values at 12 h after treatment. At the beginning of the treatment period, the degree of granulosa cell apoptosis was either undetectable (AP = 0, 47% of follicles) or slight (AP = 1, 44% of follicles) in the majority of follicles. After 12 h from the bFF but not the saline injection, there was a significant increase in the proportion of follicles (> or = 3 mm diameter) per ewe containing apoptotic granulosa cells (p < 0.001) and a significant decrease in the number of follicles per ewe with aromatase activity (p < 0.05) and with follicular fluid E2 > 20 ng/ml (p < 0.05). By 24 h after bFF treatment, apoptosis was evident in all follicles (> or = 3 mm diameter), fewer follicles contained FSH-responsive granulosa cells in terms of cAMP production (p < 0.05), and none were LH-responsive. A significant negative relationship was found between the degree of granulosa cell death as measured by L1 and follicular fluid E2 concentrations. In summary, the presence of apoptotic granulosa cells in an appreciable number of follicles considered to be healthy by morphometric criteria and before their commitment to preovulatory enlargement and ovulation suggests that apoptosis may be a physiological process in developing follicles and/or a very early event in atresia. Collectively, these data provide strong evidence that granulosa cells may die by apoptosis before there is an appreciable decrease in the capacity of the granulosa cell layer as a whole to respond to gonadotropins or to produce E2.

Animals↗
PubMed1997

Morphological evidence of apoptosis and the prevalence of apoptotic versus mitotic cells in the membrana granulosa of ovarian follicles during spontaneous and induced atresia in ewes.

Apoptosis is a process by which granulosa cells are thought to be deleted during ovarian follicular atresia. The aims of the present studies, using sheep as the experimental model, were to determine 1) whether morphological changes in cells composing the membrana granulosa during the process of atresia conformed with the general criteria of apoptotic cell death as assessed using tissue sections stained with hematoxylin and eosin; 2) whether cells classified as apoptotic on the basis of their morphology contained fragmented DNA using an in situ 3' end-labeling technique; and 3) the degree of apoptosis and mitosis within the granulosa cell populations of large antral follicles (> or = 3 mm in diameter) during both spontaneous and experimentally induced atresia using stereological methods. The results showed that most degenerate granulosa cells in follicles undergoing atresia display the morphological characteristics of apoptosis, suggesting that this is the most common pathway of cell deletion. Typical features were cells containing nuclei with marginated chromatin; cells with a single small densely staining nucleus (pyknotic appearance); cells with multiple smaller, densely staining nuclear fragments; and densely staining membrane-bound bodies (apoptotic bodies) either singly or in clusters. Cells with morphological features more typical of oncosis or necrosis were sometimes observed, but mainly during the later stages of atresia. All cells classified as apoptotic on the basis of morphological criteria contained fragmented DNA as measured by 3' end-labeling. Apoptotic bodies and/or cells were found in all follicles examined, including those classified as healthy. The overall prevalence of apoptotic cells plus apoptotic bodies expressed as a percentage of the total granulosa cell number per follicle varied from 0.02% to 0.20% in healthy follicles, varied from 0.21% to 2.00% in follicles in early (primary) atresia, and was > 2.0% in follicles in later (secondary) atresia. Percentages of mitotic cells in healthy follicles were > 0.5% in all but one of those examined and were < 1.0% in all follicles classified as atretic. Both morphological and 3' end-labeling results indicated that apoptotic cells were widely disseminated throughout the membrana granulosa, including the cell layer adjacent to the basement membrane. Collectively, these observations indicate that during early atresia, apoptosis occurs randomly and is not limited to specific areas within follicles. Our finding that apoptotic cell death and mitosis occur simultaneously within the same follicle is consistent with the notion that atresia is determined by a dynamic equilibrium between cell division, differentiation, and death.

Animals↗
PubMed1997

Fed-batch culture strategy for high yield of baker's yeast with high fermentative activity.

Based on the determination of the operational conditions for batch culture and fed-batch culture of baker's yeast by process analysis and continuous culture, a correlation, which describes the relationship between fermentative activity (FA) and specific growth rate (mu), was obtained. Combining this correlation and the exponential fed-batch culture equation, a fed-batch culture strategy was developed to obtain high yield and high fermentative activity by controlling mu at different stages. The results showed that when the initial and residual sugar concentrations were controlled to be 15-30 g/L and 3-6 g/L, respectively, different stirring speeds were applied at different stages to provide optimal oxygen transfer conditions. When this proposed fed-batch culture strategy was used in a fed-batch culture, the yield of baker's yeast reached 0.432 g/g with high fermentative activity (1180 ml). Thus, the combined bed-batch culture of baker's yeast with high yield and high fermentative activity was realized.

Biotechnology↗
PubMed1995

Expression of gonadotrophin subunit genes in sheep that were homozygous carriers and non-carriers of the Booroola fecundity gene FecB.

Homozygous carriers (BB) of the Booroola fecundity gene FecB are characterized by high plasma concentrations of immunoreactive or biologically active FSH and, to a lesser extent, of immunoreactive LH, relative to non-carriers (++). Bovine cDNA probes for the alpha gonadotrophin, FSH beta and LH beta genes were used to investigate FecB-specific differences in the mRNA species for the gonadotrophin subunits in pituitaries obtained from ++ and BB mid-luteal phase ovary-intact ewes, ovariectomized ewes and ovary-intact or ovariectomized ewes with hypothalamic-pituitary disconnection (HPD) given the same regimen of pulsatile GnRH. No FecB-specific differences in the number or size of mRNA transcripts detected by northern blotting were noted for any of these genes. Densitometry of the northern blots revealed no significant FecB-specific differences in the relative amounts of mRNA encoding alpha gonadotrophin, FSH beta or LH beta in the pituitaries from any of the experimental groups of ++ and BB sheep. Furthermore, there were no significant FecB-specific differences in the pituitary content of FSH or LH in these animals, despite significantly higher plasma concentrations of FSH in the ovary-intact and ovariectomized HPD groups. These data show that whereas the FecB gene causes increased plasma concentrations of FSH, no consistent effects can be demonstrated on pituitary gonadotrophin content or on gonadotrophin subunit gene transcription, using northern analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Effects of ovariectomy and FecBB genotype on the median charge and circulating half-life of pituitary FSH isoforms of ewes.

This study investigated the effects of short-term (20 days) ovariectomy, the effects of FSH assay (radioimmunoassay, receptor assay or in vitro bioassay) and of FecBB genotype on the characteristics of pituitary FSH from Booroola ewes. Pituitary extracts were obtained from ovariectomized homozygous carriers (BB) and non-carriers (++, n = 8 per genotype) and ovary-intact controls (n = 4 per genotype). The extracts (n = 4 per genotype per treatment) were subjected to agarose suspension electrophoresis and the eluates were assayed by the three FSH methods. There were significant effects of ovariectomy (P < 0.01) and assay system (P < 0.05) but not of genotype on the median charge of FSH isoforms. The mean +/- SEM migration rates for FSH in intact and ovariectomized ewes were 0.469 +/- 0.006 and 0.439 +/- 0.006 albumin mobility units, respectively (P < 0.01), indicating a shift to more basic isoforms after short-term ovariectomy. When the pituitary extracts were subjected to anion-exchange HPLC, there was a significant (P < 0.01) shift to more basic isoforms in the ovariectomized ewes as shown using agarose electrophoresis, and no gene effects were noted. When the pituitary extracts (n = 4-8 per group) were injected into mature female mice, there were no significant effects of ovariectomy or genotype on the circulating half-lives of the pituitary FSH isoforms. These results indicate that after short-term ovariectomy, the increase in plasma FSH concentrations is accompanied by a shift in the median charge of pituitary FSH isoforms without any observable change in their metabolic clearance rates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Pituitary and plasma levels of growth hormone in Booroola sheep that are either homozygous carriers or non-carriers of the FecBB fecundity gene.

The aim of this study was to examine the effect of the FecBB fecundity gene on plasma concentrations and pituitary content of growth hormone (GH) in sheep. No differences were found between homozygous carriers (BB) and non carriers (++) of the FecBB gene with regard to pituitary GH contents in both ovariectomized and intact ewes. However, ovariectomized ewes had higher levels of pituitary GH than intact ewes (P < 0.01). There were no differences between FecBB genotypes with respect to plasma concentrations of GH in 6-year-old ovariectomized ewes bled every 10 min for 12 h or in ram lambs bled weekly during their first year of life. GH levels in the rams decreased until week 27, increased to a peak at week 31 then decreased before increasing again at week 43. Mean plasma GH concentrations in the ewe lambs bled weekly for a year decreased until week 19 then remained at approximately this level for the remainder of the year. Mean GH plasma concentrations in the ram lambs were higher than in the ewe lambs (P < 0.001). Ewe lambs that were homozygous for the FecBB gene had lower body weights (P < 0.05) and had higher levels of GH (P < 0.01) than non carrier ewe lambs during their first year. Before the average age of first behavioural oestrus (36 weeks) GH levels in the ewe lambs were negatively correlated with body weights (r = -0.69, P < 0.001, n = 22). When body weight was included as a covariate in analysis of variance the genotype difference in ewe lamb plasma GH concentrations was no longer significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1994

Apoptosis in bovine granulosa cells in relation to steroid synthesis, cyclic adenosine 3',5'-monophosphate response to follicle-stimulating hormone and luteinizing hormone, and follicular atresia.

Apoptosis is a process of selective cell deletion implicated as a mechanism underlying the process of ovarian follicular atresia. The aims of this study were 1) to test the hypothesis that granulosa cell death during follicular (> or = 4 mm diameter) atresia in cows occurs by apoptosis and 2) to define relationships between the occurrence and degree of granulosa cell apoptosis, cAMP response to FSH or LH, extant aromatase activity, and other previously established biochemical and morphometric indices of granulosa cell function and follicular atresia in this species. Granulosa cells and follicular fluid from individual follicles 4-18 mm in diameter were collected from luteal-phase cow ovaries. Follicles were classified by morphometric criteria as "healthy" (n = 45) or atretic (n = 34). Apoptosis in granulosa cells from each follicle was inferred from detection of internucleosomal DNA cleavage by 3'-end radiolabeling; it was quantitated both subjectively from intensity of oligonucleosome labeling (apoptosis [AP] score = 0, 1, 2, or 3) and objectively by beta-counting of low-molecular weight gel fragments (labeling index; LI). Extant aromatase activity (ng estradiol produced/10(6) cells/3 h) and cAMP response (pmol/10(6) cells) to different doses of FSH or LH (1-10,000 ng/ml) was determined for granulosa cells from most healthy follicles (n = 39). Apoptosis was detected in granulosa cells from all atretic follicles as well as from 76% of healthy follicles, from 80% (16 of 20) of follicles with follicular fluid estradiol to progesterone ratios > 1, and from 71% (10 of 14) of follicles with extant aromatase activity (> 2 ng/10(6) cells/3 h). For healthy and atretic follicles, degree of DNA fragmentation was inversely related to the number of granulosa cells recovered (as percentage maximum by follicle size). In healthy follicles, FSH stimulated cAMP synthesis is a dose-dependent manner in granulosa cells from all follicles examined (> or = 4 mm), but only 36% of these had appreciable aromatase activity. The cAMP response to FSH (per cell) increased with follicle size from 4-7 mm in diameter and remained high in granulosa cells from follicles > or = 8 mm with aromatase activity; in cells without aromatase activity, cAMP response to FSH decreased with increasing follicle size > or 8 mm. The cAMP response to LH was generally low or undetectable in granulosa cells from 4-8-mm follicles; it then increased linearly with increasing follicle diameter > or = 8 mm, but to a greater degree in cells with aromatase activity than in cells without.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Biopotency in vitro and metabolic clearance rates of five pituitary preparations of follicle stimulating hormone.

Five pituitary preparations of follicle stimulating hormone (FSH), namely NIDDK-oFSH-17, Bioscan oFSH, Ovagen, Folltropin-V and F.S.H.-P., were examined for biological activity in terms of their potency in an in vitro bioassay, receptor assay and heterologous radioimmunoassay and in terms of their metabolic clearance rates. In the three assays, Bioscan oFSH was the most potent (P < 0.05) (3- to 5-fold the potency of NIDDK-oFSH-17), with Ovagen being 25-50% the potency of the NIDDK standard (P < 0.05). Folltropin-V and F.S.H.-P. had the lowest potencies in all three assays. For each preparation, the ratio of activities between the assays was not consistent, suggesting that the preparations behaved differently in each assay. In 9 of 10 cases, potency estimates in the heterologous radioimmunoassay were greater than those in the in vitro bioassay or receptor assay. Polyacrylamide gel electrophoresis of the preparations showed banding consistent with the molecular weight of FSH, but also indicated that the preparations were contaminated with other proteins to varying extents. The half-lives of these preparations when injected into the bloodstream of mature female mice were 28.0, 8.6, 13.4, 11.6 and 17.4 min for NIDDK-oFSH-17, Bioscan oFSH, Ovagen, Folltropin-V and F.S.H.-P. respectively. The slopes of the decay rates were significantly different from each other (P < 0.05) except between Ovagen and Folltropin-V. The results of these studies show that a number of widely available FSH preparations have differing biopotencies. Moreover, the biopotency of a preparation in vitro is not related to its metabolic clearance rate, and not all FSH preparations behave identically in different assays. Measures of biopotency in vitro combined with those of metabolic clearance rate may provide useful information on the properties of FSH preparations used for research purposes and for superovulation of farmed livestock.

Animals↗
PubMed1993

Plasma gonadotropin concentrations and ovarian characteristics in Inverdale ewes that are heterozygous for a major gene (FecX1) on the X chromosome that influences ovulation rate.

Ewes heterozygous (I+) for the Inverdale prolificacy gene (FecX1) located on the X chromosome have ovulation rates approximately 1.0 times higher than noncarriers (++). The aims of this study were to examine, in I+ and ++ ewes, the peripheral plasma concentrations and/or ovarian secretion rates of FSH, LH, inhibin, estradiol, and progesterone during anestrus and the estrous cycle and after ovariectomy. Also examined were aspects of ovarian morphology and functions of granulosa cells in vitro. No FecX1-specific differences were noted for the ovarian hormones or for FSH or LH. However, I+ animals contained significantly more ovarian antral follicles (p < 0.05) and their granulosa cells had a higher mean LH responsiveness in vitro with respect to cAMP synthesis at smaller follicular diameters relative to ++ ewes (I+ = > 2.5 mm; ++ = > 4.5 mm). Moreover, nonatretic follicles in I+ animals compared to the ++ genotype contained fewer granulosa cells and smaller CL. Although the I+ animals had a higher ovulation rate than the controls (p < 0.05), the total weight of CL was not different between the genotypes. These findings suggest that the FecX1 gene affects ovarian function without altering ovarian hormone secretion. The findings also suggest that there are no FecX1-specific differences in the mean concentrations of gonadotropins, although further studies on temporal changes in gonadotropin secretion are warranted.

Animals↗
PubMed1993

Gonadotrophin-releasing hormone and the control of ovulation rate by the FecB gene in Booroola ewes.

Booroola sheep carry a FecB gene that confers high fecundity. The aims of the present studies were to determine in homozygous carriers (BB) and non-carriers (++) of the Booroola FecB gene whether there are FecB differences in the secretory characteristics of GnRH in hypophyseal-portal blood of ovariectomized ewes (Expt 1) and whether differences in ovulation rate would occur following the administration of PMSG and pulsatile GnRH to ovary-intact Booroola ewes with hypothalamic-pituitary disconnection (HPD) (Expt 2). In Expt 1, no FecB gene-specific differences were noted for GnRH with respect to pulse frequency, pulse amplitude or overall secretion rate. Irrespective of genotype there were 1.9 +/- 0.2 GnRH pulses h-1 (n = 20 ewes; 10 BB and 10 ++ animals) and 1.9 +/- 0.1 pulses of immunoreactive (i) LH h-1. In Expt 2, ovulation was induced in the HPD ovary-intact animals on three occasions (e.g. 56, 393 and 423 days after HPD surgery) using a PMSG (200 or 400 iu)-GnRH pulse (250 ng i.v. for 96 h)-GnRH bolus (10 micrograms i.v.) regimen after pretreating the animals with GnRH pulses (250 ng i.v. for 14 days). On all occasions the ovulation rates were significantly higher (P < 0.05) in BB ewes (n = 6 or 7) than in ++ animals (n = 7). No differences between the genotypes were noted with respect to the mean concentrations of progesterone in plasma notwithstanding the differences in ovulation rates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Effect of chronic FSH administration on ovarian follicular development, ovulation rate and corpora lutea formation in sheep.

This study in ewes examined the effects on ovarian function of a pulsatile regimen of ovine FSH (NIADDK-oFSH-17) administered over a 24- to 28-day period beginning on day 1 of the oestrous cycle (day 0 = oestrus). The FSH (1.66 micrograms or 5.00 micrograms) was administered i.v. over a 1-min interval once every hour throughout the treatment period. In other ewes ovine LH (NIDDK-oLH-23) was administered (10 micrograms once every 2 h) for 24-28 days together with oFSH (1.66 micrograms/h). Compared with untreated controls (n = 19 ewes), FSH alone at both doses (n = 10 ewes/dose) as well as the FSH + LH treatment (n = 10) led to significant increases in the plasma concentrations of FSH (P < 0.01), ovarian weight (P < 0.05) and ovulation rate (P < 0.01) but there was no change in the mean weight of individual corpora lutea (CL). Exogenous FSH at the high but not the low dose alone or with LH stimulated a significant overall increase in plasma inhibin concentrations (P < 0.05). The geometric mean (and 95% confidence limits) ovulation rates in the high FSH (i.e. 5.00 micrograms/h), low FSH (i.e. 1.66 micrograms/h), low FSH + LH, and control treatment groups were 15.3 (9.3, 24.8), 3.7 (2.1, 6.0), 3.7 (2.5, 5.8) and 1.4 (1.2, 1.7) respectively. The FSH or FSH + LH treatments did not alter the total numbers of antral follicles (> or = 1 mm diameter). However, the high but not the low FSH or low FSH + LH treatment led to significant increases in the mean numbers of large follicles (i.e. > 4.5 mm diameter; P < 0.01) and a higher proportion of non-atretic antral follicles. Highly significant linear relationships were found between the mean plasma concentrations of FSH or inhibin and the ovulation rate (FSH: r = 0.74, P < 0.0001; inhibin: r = 0.93, P < 0.0001). Highly significant linear relationships were also found between the plasma concentrations of FSH or inhibin and the number of large follicles (i.e. > 4.5 mm diameter; FSH, r = 0.78, P < 0.0001; inhibin, r = 0.80, P < 0.0001) and between the plasma concentrations of inhibin and the number of granulosa cells in large follicles (r = 0.78, P < 0.0001). After the high FSH but not the low FSH treatment there were significant increases in both FSH- and LH-induced responsiveness in granulosa cells with respect to cyclic AMP synthesis in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Inhibin production in vitro by granulosa cells from Booroola ewes which were either homozygous or non-carriers of a fecundity gene influencing their ovulation rate.

The production of inhibin by granulosa cells was studied in vitro using cells from follicles of various sizes and health. Follicles were recovered on Days 10-13 of the oestrous cycle, from Booroola x Romney ewes which were homozygous (FF) carriers or non-carriers (++) of the fecundity (F) gene. Inhibin was measured using a bioassay based on the suppression of follicle-stimulating hormone (FSH) output by cultured pituitary cells from ovariectomized Romney ewes and, in some instances, for comparative purposes, by radioimmunoassay also. Geometric mean inhibin production by granulosa cells from nonatretic follicles increased with increasing follicle diameter, during the first 24 h of culture, for both genotypes. The geometric mean production of inhibin by cells from nonatretic 3-4.5 mm diameter FF follicles (the largest follicles found in FF ewes), was significantly higher (P less than 0.05) than that by cells from non-atretic 3-4.5 mm diameter ++ follicles, but similar to that of cells from non-atretic greater than or equal to 5 mm diameter ++ follicles. The production of oestradiol-17 beta by cells cultured in the presence of testosterone (1 microgram/ml) followed a pattern similar to cellular inhibin production. There was a positive linear correlation between inhibin and oestradiol-17 beta production during the first 24 h of culture, for both genotypes. In addition to acting as a substrate for oestradiol-17 beta synthesis, testosterone generally had a slight, stimulatory effect on inhibin production.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Oocyte production and ovarian steroid concentrations of immature rats in response to some commercial gonadotrophin preparations.

Four commercial gonadotrophin preparations, namely Folligon, F.S.H.-P., Folltropin and Ovagen, were examined for their effects on oocyte production and ovarian steroid concentrations in immature rats. The ratios of the FSH to LH concentrations of the preparations, determined by radioreceptor assays, were Folligon 5, F.S.H.-P. 18, Folltropin 49 and Ovagen 1090. Forty-eight hours after administering each gonadotrophin preparation to immature rats, ovulation was induced by injection of chorionic gonadotrophin. Twenty-four hours later, oocytes were recovered from the oviducts and counted. Oocytes were produced after injection of chorionic gonadotrophin following a single injection of Folligon (10-50 i.u.). However, no oocytes were produced in response to the other gonadotrophin preparations unless they were administered by continuous infusion (30-1000 micrograms day-1). When given by injection (Folligon) or infusion (others), the gonadotrophin preparations all promoted a dose-dependent increase in mean oocyte production, except at the highest doses when mean oocyte numbers either remained unchanged or declined significantly in the cases of Folligon and F.S.H.-P. The highest mean numbers of oocytes produced in response to Folltropin (48 +/- 9 oocytes, mean +/- s.e.m.) and Ovagen (47 +/- 7) were greater than those attained with Folligon (21 +/- 6) or F.S.H.-P. (31 +/- 5). Mean ovarian weights also increased in a dose-dependent fashion in response to each of the gonadotrophin preparations. Measurements of ovarian steroid concentrations 48 h after the onset of gonadotrophin treatment (i.e. immediately prior to ovulation induction with chorionic gonadotrophin) showed that the gonadotrophin preparations markedly influenced the ratios of ovarian oestradiol-17 beta and androgen (androstenedione plus testosterone) concentrations. At low doses the gonadotrophin preparations increased the ratio of oestradiol-17 beta to androgens, but at the highest doses, with the exception of Ovagen, the ratio was reduced relative to peak values. Co-infusion of ovine LH (NIADDK-oLH-25; 10-20 micrograms day-1) with Ovagen (250 micrograms day-1) or ovine FSH (10 micrograms day-1, NIADDK-oFSH-17), both low in LH content, increased the mean number of oocytes produced and also the ovarian oestradiol-17 beta:androgen concentration ratio. However, with 40 micrograms LH day-1, the oestradiol-17 beta:androgen ratio fell due to a continued increase in mean ovarian androgen concentrations and a decrease in mean ovarian oestradiol-17 beta concentration. The mean number of oocytes produced also fell significantly.(ABSTRACT TRUNCATED AT 400 WORDS)

Androstenedione↗
PubMed1990

Effects of follicle stimulating hormone, cholera toxin, pertussis toxin and forskolin on adenosine cyclic 3',5'-monophosphate output by granulosa cells from Booroola ewes with or without the F gene.

The cAMP outputs by granulosa cells from 3-4.5 mm diameter (medium) follicles of Booroola FF ewes were similar to those by cells from greater than or equal to 5 mm diameter (large) follicles of ++ ewes with respect to time or dose of FSH, cholera toxin or forskolin. Likewise, the cAMP outputs by cells from 1-2.5 mm diameter (small) FF follicles were similar to those by cells from small and medium ++ follicles with respect to time or dose of FSH, cholera toxin or forskolin. At FSH, cholera toxin or forskolin doses of 1 microgram/ml, 0.5 microgram/ml and 10(-4) M respectively, the granulosa cell cAMP outputs of medium FF or large ++ follicles were approximately 2-fold (P less than 0.05) higher than in the respective small FF and medium ++ follicles. The effects of cholera toxin plus forskolin or FSH plus forskolin were additive irrespective of genotype or follicle size, with significant differences (P less than 0.05) observed between follicle sizes but not genotype. No differences were noted between cholera toxin plus forskolin or FSH plus forskolin on granulosa cell cAMP output. For the FSH and forskolin treatments, increased mean cAMP outputs were evident after 10 min, whereas after cholera toxin treatment they were not evident until after 20 min incubation. For all treatments the rate of cAMP production tended to slow down after 40-60 min. Pre-incubation of granulosa cells with pertussis toxin subsequently resulted in a significantly greater (P less than 0.05) FSH-induced output of cAMP relative to the untreated controls irrespective of follicle size. However, no gene-specific differences were noted when the cAMP outputs of cells from medium or small FF follicles were compared with cells from large or small-medium ++ follicles respectively. These results indicate that the activity (or composition) of the regulatory and catalytic components of adenylate cyclase in the FF granulosa cells change in a manner similar to those observed in ++ cells with the only difference being that the increases in cyclase in FF ewes occurs as follicles enlarge from 1-2.5 to 3-4.5 mm in diameter, whereas in ++ ewes they occur as follicles enlarge from 3-4.5 to greater than or equal to 5 mm in diameter. No evidence was found to link the F gene to the granulosa cell cAMP response independently of follicle size. It is suggested that the association between the F gene and the size-specific difference in follicle maturation may be unrelated to the FSH receptor/cAMP generating system.

Adenylate Cyclase Toxin↗
PubMed1989

Binding characteristics of 125I-labelled human FSH to granulosa cells from Booroola ewes which were homozygous, heterozygous or non-carriers of a major gene(s) influencing their ovulation rate.

At 37 degrees C 125I-labelled human (h) FSH (NIAMDD-hFSH-I-3) bound rapidly to granulosa cells from Booroola and Romney ewes with 50% maximum binding achieved after 3 min and equilibrium being reached within 45 min, irrespective of whether the cells were obtained from the FF, F+ or ++ Booroola genotypes or from Romney ewes. Binding of 125I-labelled FSH followed second order kinetics and there was no effect of follicle diameter (1-2.5 mm vs greater than or equal to 3 mm). Irrespective of breed, genotype or follicle size, the mean (+/- s.e.m.) calculated association rate constant, (ka) was 7.3 (+/- 0.8) x 10(5) litres mol-1 sec-1 (n = 12). Dissociation of receptor bound 125I-labelled hFSH was less than 5% after 30 min and low but variable (i.e. between 0 and 30%) after 2-6 h irrespective of breed, genotype or follicle size. No gene-specific differences were noted in binding specificity between F+ and ++ genotypes: studies were not performed with cells from FF ewes because of insufficient cells. The binding of 125I-labelled hFSH could be displaced with sheep FSH (NIH-FSH-S16; 10% cross-reaction) and FSH-P (2.5% cross-reaction) but other sheep pituitary hormones and hCG showed little or no cross-reaction (less than or equal to 0.1%). The calculated binding capacities (Bmax) and equilibrium dissociation constants (Kd) for 125I-labelled hFSH binding to granulosa cells did not differ between the Booroola genotypes or between Booroola or Romney follicles of different diameter (i.e. 1-2.5 mm; or greater than or equal to 3 mm). The overall mean +/- s.e.m. (n = 24) Bmax and Kd values were 16.7 +/- 0.8 fm/mg protein (i.e. approximately 800 available receptor binding sites/cell) and 1.1 +/- 0.1 nM respectively. Collectively, these findings suggest that the earlier maturation of follicles in FF or F+ ewes compared to ++ ewes is unlikely to be due to gene-specific differences in the FSH binding characteristics of the granulosa cells.

Animals↗
PubMed1989

Adenosine 3',5-cyclic monophosphate phosphodiesterase activity in granulosa cells from Booroola x Romney ewes with and without the F gene.

Granulosa cells from ovarian follicles (greater than or equal to 1 mm diameter) in Booroola ewes which are homozygous (FF) or heterozygous (F+) for the F gene have previously been shown to produce significantly more cAMP in response to FSH or LH than those from similar sized follicles in ewes without the F gene (++). The aim of these studies was to test whether these F gene-specific differences arose because of differences in cAMP-phosphodiesterase (cAMP-PDE) activity. In the first study using 1 mumol cAMP/l as substrate, no F gene-specific effects were noted in cAMP-PDE activity in granulosa cells from small (1-2.5 mm diameter, n = 4 per genotype) or large (greater than or equal to 3 mm diameter, n = 4 per genotype) follicles from FF, F+ or ++ ewes, despite F gene-specific effects in FSH (1 microgram/ml)- and LH (0.1 microgram/ml)-induced cAMP accumulation in these same cell preparations. The overall mean levels of cAMP-PDE across all genotypes in cells from small and large follicles were 0.47 +/- 0.04 (S.E.M., n = 12) and 0.28 +/- 0.03 pmol cAMP/10(6) cells per min respectively; the mean PDE activity in cells from small follicles was significantly (P less than 0.05) higher compared with that in cells from large follicles. In a second study, granulosa cells from each genotype were pooled over all follicle sizes (greater than or equal to 1 mm diameter, one pool per genotype) and the rates of cAMP hydrolysis tested over a range of substrate concentrations (0-16 mumol/l) but no gene-specific differences with respect to the Michaelis constant and maximum velocity were noted.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1987

Ovarian follicular activity in Booroola lambs with and without a fecundity gene.

The ovaries of 3-month-old Booroola lambs which were heterozygous carriers of a major gene (F) influencing the ovulation rate in mature ewes (i.e. F + lambs) were compared to those ofsimilarly-aged Booroola lambs which were non-carriers of the F-gene (i.e. ++ lambs). The ovaries of the F + Booroola lambs were significantly lighter (P less than 0.01) than those of ++ lambs even though the mean +/- s.e.m. number of follicles (greater than or equal to 1 mm diam.) in the F + lambs was greater than that in the ++ lambs (i.e. F + lambs, 30.2 +/- 2.5 follicles; ++ lambs, 18.4 +/- 1.2 follicles; P less than 0.01). In granulosa cells from non-atretic follicles (greater than or equal to 1 mm diam.) from F + and ++ Booroola lambs, FSH (NIAMDD-FSH-S16) doses of 100 and 1000 ng/ml caused significant stepwise increases (P less than 0.05) in cyclic adenosine 3',5'-monophosphate (cAMP) production compared to that achieved at FSH doses of 0 and 1 ng/ml or at any FSH dose in cells from atretic follicles. However, no significant differences in FSH-induced cAMP production were noted with regard to Booroola genotype or follicular diameter. None of the granulosa cell preparations from non-atretic follicles of 1-2.5 mm diameter from F + lambs (N = 13) or from non-atretic follicles of 1-4.5 mm diameter from ++ lambs (N = 16) responded to LH (NIAMDD-LH-S24; 10 or 1000 ng/ml) to produce significantly more cAMP than did the controls. In contrast, the granulosa cell preparations from non-atretic follicles of 3-4.5 mm diameter from F + lambs (N = 4) and from non-atretic follicles of greater than or equal to 5 mm diameter of ++ lambs (N = 4) produced significantly more cAMP (P less than 0.05) in response to LH (1000 and/or 10 ng/ml) relative to that in the controls. The theca interna from follicles of lambs of both genotypes had functional LH receptors as judged by the androstenedione responses to exogenous LH although no genotypic differences were noted. In F + lambs, the follicular fluid concentrations of testosterone but not oestradiol (i.e. in 1-4.5 mm diam. follicles) and granulosa cell aromatase activity (i.e. in 3-3.5 mm diam. follicles) were significantly higher (both P less than 0.05) than in corresponding follicles or cells from ++ lambs. Collectively the results suggest that the Booroola F-gene influences the composition and function of sheep ovaries before puberty.

Animals↗