Biomedical subjects

S Lun

Publications and source records attributed to S Lun.

At least 19 recordsLinked to original sources

PubMed2005

Oocyte-expressed genes affecting ovulation rate.

From examination of inherited patterns of ovulation rate in sheep, several breeds have been identified with point mutations in two growth factor genes (BMP15 and GDF9) and a related receptor (ALK6) that are expressed in oocytes. Five different point mutations have been identified in the BMP15 gene, one in GDF9 and one in ALK6. Animals heterozygous for these mutations or heterozygous for two of these mutations or homozygous for the ALK6 mutation have higher ovulation rates (i.e. +0.6-10) than their wild-type contemporaries. Animals homozygous for the BMP15 or GDF9 mutations are sterile due to arrested follicular development from the primary stage of growth. The BMP15 and GDF9 mutations are thought to result in reduced levels of mature protein or altered binding to cell-surface receptors. In sheep, GDF9 mRNA is present in germ cells before and after ovarian follicular formation as well as throughout follicular growth, whereas BMP15 mRNA is found in oocytes only from the primary stage of growth. Also ALK6 together with related cell-surface receptors such as ALK5 and BMPRII mRNA are present in oocytes at most, if not all, stages of follicular growth. Both GDF9 and BMP15 proteins are present in follicular fluid indicating that they are secreted products. Immunisation of sheep with GDF9 or BMP15 peptides shows that both growth factors are essential for follicular development, ovulation and/or corpus luteum formation. In animals with the ALK6 mutation, ovarian follicles undergo precocious maturation leading to three to seven follicles ovulating at smaller diameters without any increase above wild-types in the ovarian secretions of steroid or inhibin. One important consequence of the ALK6 mutation appears to be a decreased ability of some BMPs to inhibit differentiation of follicular cells. Current findings in sheep suggest that BMP15, GDF9 and ALK6 are targets for new methods of fertility regulation in some mammals.

Animals↗

PubMed2005

Prolactin in the brushtail possum (Trichosurus vulpecula): development of homologous radioimmunoassay using recombinant possum prolactin.

We report the production of recombinant possum prolactin (posPrl), and its use in the development and validation of a highly specific homologous radioimmunoassay for the measurement of prolactin (Prl) in brushtail possums. This enabled the subsequent investigation of some basic mechanisms involved in the regulation of Prl secretion in this species. Recombinant posPrl spanning the entire coding region was expressed in Escherichia coli, resulting in a 199 amino acid protein with a molecular weight approximately 23 kDa. The potency of posPrl was 45.3 +/- 4.8% that of ovine Prl in a radioreceptor assay using possum mammary gland receptors and induced a 3.4 +/- 0.8-fold increase in progesterone secretion in primary possum granulosa cells. Antiserum (G27) was raised against recombinant posPrl and was highly specific for possum Prl (approximately 30% binding at 1:60,000 final dilution), and exhibited negligible cross-reactivity (<0.0001%) with possum growth hormone. Serial dilutions of pituitary gland extracts, and plasma samples from male and female possums gave parallel inhibition curves to recombinant posPrl standards in the assay. Biological validation of the RIA included treating possums with drugs known to alter Prl secretion in other mammals. In seasonally anoestrous female possums, administration of 20 microg thyrotropin-releasing hormone (TRH) resulted in a 15-fold increase (P < 0.01) in plasma Prl concentrations. In mid-late lactating female possums, a bolus of cabergoline (dopamine agonist; 75 microg) reduced (P < 0.05) plasma Prl levels to baseline for 24 h, while repeated administration (6 x 75 microg at 12 h intervals) suppressed (P < 0.01) plasma Prl concentrations until 24h after the last injection. Prolonged inhibition of Prl levels subsequently caused marked (P < 0.01) attenuation in rate of bodyweight increase of pouch young. The amplitude of the Prl surge in response to a bolus of TRH (15 microg) was 5-fold lower in cabergoline-treated, compared to control mid-late lactating possums. In conclusion, we report the development and validation of a robust and sensitive RIA for measuring Prl concentrations in the plasma of brushtail possums.

Animals↗

PubMed2003

Cytokines in mammary lymph and milk during endotoxin-induced bovine mastitis.

Cytokine kinetics were examined in milk and in afferent and efferent lymph of the supramammary lymph node after intramammary infusion of endotoxin from Escherichia coli. Cows were sampled 0, 2 and 4h after infusion (p.i.). Neutrophils appeared in afferent lymph 2h p.i., and in efferent lymph and milk 4h p.i. The milk contained high concentrations of interleukin-8 (IL-8) at 2 and 4h p.i. IL-8 was also found in lymph, but at lower concentrations. The tumor necrosis factor-alpha (TNF-alpha) concentration tended to increase in afferent lymph at 2h p.i., and increased in milk at 4h p.i. The level of IL-1beta increased at 4h p.i. in milk, but was not detected in lymph. Interferon-gamma was not detected in any sample, at any time. The results indicate a primary role for IL-8 in the recruitment of neutrophils into the gland, and suggest that IL-1beta and TNF-alpha are not necessary for IL-8 production and release in response to endotoxin.

Animals↗

PubMed2003

Production of biologically active tethered ovine FSHbetaalpha by the methylotrophic yeast Pichia pastoris.

The pituitary-derived glycoprotein hormone FSH plays a central role in controlling vertebrate gonadal function. In female mammals the maturation of ovarian follicles is critically dependent upon stimulation by FSH. Moreover, injection of exogenous FSH is used extensively to stimulate increased numbers of follicles to ovulate. Structurally FSH is a heterodimeric glycoprotein composed of two non-covalently associated polypeptide subunits. The tertiary structures of both the alpha- and beta-subunits are constrained by intramolecular disulphide bonds and are post-translationally modified with two N-linked carbohydrate moieties, the structure of which appears to modulate in vivo biological activity. Here we report the expression of ovine FSH (oFSH) as a biologically active single-chain polypeptide using the methylotrophic yeast Pichia pastoris. Sequences encoding the mature oFSH alpha- and beta-proteins were fused to form a gene encoding a fusion protein with the C-terminus of the beta-chain joined to the N-terminus of the alpha-chain, with the chains separated by a two amino acid linker sequence. This fusion gene was itself fused to two alternative Pichia leader sequences (mating factor alpha and acid phosphatase) and transformed into the Pichia strains GS115 and SMD1168. The recombinant fusion protein (oFSHbetaalpha) was expressed at approximately 0.1 microg/ml in 'shake-flask' cultures. The Pichia-expressed tethered protein was biologically active in an in vitro bioassay, had a molecular mass of 28 kDa, as determined by SDS-PAGE, and bound the bovine FSH receptor with a binding profile similar to that of native oFSH.

Animals↗

PubMed2002

A novel beta-N-acetylglucosaminidase of Clostridium paraputrificum M-21 with high activity on chitobiose.

A beta- N-acetylglucosaminidase gene ( nag3A) from Clostridium paraputrificum M-21 was cloned in Escherichia coli. The nag3A gene consists of an open reading frame of 1,239-bp, encoding 413 amino acids with a deduced molecular weight of 45,531 Da. Nag3A is a single domain enzyme containing a family 3 glycoside hydrolase catalytic domain. Nag3A was purified from recombinant E. coli and characterized. The enzyme hydrolyzed chitooligomers such as di- N-acetylchitobiose, tri- N-acetylchitotriose, tetra- N-acetylchitotetraose, penta- N-acetylchitopentaose, hexa- N-acetylchitohexaose, ball-milled chitin, and synthetic substrates such as 4-methylumbelliferyl N-acetyl beta- D-glucosaminide [4-MU-(GlcNAc)], but had no activity at all against p-nitrophenyl-beta- D-glucoside, p-nitrophenyl-beta- D-xyloside, or p-nitrophenyl-beta- D-galactosamine. The enzyme was optimally active at 50 degrees C and pH 7.0, and the apparent K(m) and V(max) values for 4-MU-(GlcNAc) were 7.9 micro M and 21.8 micro mol min(-1) mg protein(-1), respectively. SDS-PAGE, zymogram, and immunological analyses suggested that this enzyme is induced by ball-milled chitin.

Acetylglucosaminidase↗

PubMed2001

Continuous production of poly-3-hydroxybutyrate by Ralstonia eutropha in a two-stage culture system.

Poly-3-hydroxybutyrate (PHB) is mainly accumulated by Ralstonia eutropha under unbalanced growth conditions, which limits its production in batch or fed-batch modes. The continuous production of PHB was investigated in a two-stage continuous culture system. The first-stage produced cell mass giving the maximal cell dry weight of 27.1 g l(-1) at 0.21 h(-1) of dilution rate. High specific cell growth rate results in the decrease of PHB synthesis under glucose-limited and nitrogen-rich conditions in the first-stage. The second-stage produced PHB giving the maximal PHB concentration of 47.6 g l(-1) at 0.14 h(-1) of dilution rate. Specific PHB synthetic rate reached highest value at low dilution rate under nitrogen-limited condition in the second-stage, and decreased with the increase of ammonium concentration in the culture. In the continuous culture system, the maximal PHB productivity could reach 1.43 g l(-1) h(-1) at a dilution rate of 0.12 h(-1), but with relatively low PHB content of 47.6%. Maximal yield of PHB on glucose could reach 0.36 g g(-1) glucose at 0.075 h(-1) of dilution rate with relatively high PHB productivity of 1.23 g l(-1) h(-1) and PHB content of 72.1%, respectively.

Cupriavidus necator↗

PubMed2001

Language mapping in less than 15 minutes: real-time functional MRI during routine clinical investigation.

Neurosurgical interventions often require the presurgical determination of language dominance or mapping of language areas. Results obtained by fMRI are closely correlated with invasive procedures such as electrical stimulation mapping or the intracarotid amobarbital test. However, language fMRI is not used routinely, because postprocessing is time-consuming. We utilized a real-time analysis software installed directly on the MR console computer and SPM99 as reference postprocessing software. We assessed the reliability of the immediate determination of language dominance based on individual activation maps by comparing the results of the visual analysis of images derived from conventional postprocessing with those produced by the real-time tool. All images were rated independently by six senior neurologists blinded to other data. We validated the robustness of the real-time method statistically by comparing global and regional lateralization indices derived from real-time and postprocessing analysis. Functional MRI was performed with a standard 1.5-T whole-body scanner. Brain activity was contrasted between an alternating semantic judgment and letter matching task. Twelve right-handed, healthy control subjects and 12 consecutive patients with drug-resistant, localization-related epilepsy were investigated. The semantic condition induced almost invariably left hemispheric activations in Broca's area, the premotor cortex, the dorsolateral prefrontal cortex, and the temporoparietal region. Although real-time analysis reduced noise less effectively than SPM99, visual ratings and lateralization indices produced highly concordant results with both methods. In conclusion, real-time fMRI, as used here, allowed reliable language lateralization and mapping in less than 15 min during routine clinical MRI investigation with no need for postprocessing.

Adult↗

PubMed2001

Ontogeny of steroidogenesis in the fetal sheep gonad.

The aim of this study was to determine 1) the time of onset and cellular localization of gene expression for steroidogenic factor-1 (SF-1), steroidogenic acute regulatory protein, 3beta-hydroxysteroid dehydrogenase/Delta(5),Delta(4) isomerase (3beta-HSD), and the cytochrome P450 enzymes for cholesterol side-chain cleavage (P450(scc)), 17alpha-hydroxylase (P450(17alphaOH)), and aromatase (P450(arom)) during gonadal development; and 2) the amount of progesterone, androstenedione, testosterone, and 17beta-estradiol present in the fetal sheep gonad. Fetuses were collected on Days 24, 26, 28, 30, 32, 35, 40, 55, and 75 of gestation, and gene expression was determined by in situ hybridization. The steroid content of gonads collected on Days 30, 35, 55, and 75 of gestation was determined by RIA. Developing gonads collected from both male and female fetuses were steroidogenically active around the time of morphological sexual differentiation. In the female, the steroidogenic cells were initially located at the boundary of the cortex and medulla but become increasingly restricted to the mesonephric-derived cell streams. In the male, once tubules were identifiable, steroidogenesis was restricted to the interstitial regions. Interestingly, expression of both SF-1 and 3beta-HSD was observed prior to morphological sexual differentiation. In addition, expression of both of these genes was more widespread than the other genes in both males and females.

3-Hydroxysteroid Dehydrogenases↗

PubMed2000

Role of ovarian failure in reproductive senescence in aged red deer (Cervus elaphus) hinds.

Physiological and endocrine factors associated with reproductive senescence were assessed in a group of 19 ageing red deer hinds. Reproductive success, defined as the percentage of hinds weaning a calf successfully, decreased gradually from 89% at 6-7 years of age to 50% at 17 years, and subsequently decreased markedly; only one hind reared a calf at 19-20 years of age. When the 12 surviving hinds were approaching 21 years of age, they were compared with ten mature 7-year-old females over the onset of the breeding season. All hinds were subsequently killed, the reproductive tracts were recovered and antral (>/= 2 mm in diameter) and preantral follicle populations were determined by dissection (n = 7 hinds per age group) or stereological analysis (n = 2 ovaries per age group), respectively. Cyclical ovarian activity (plasma progesterone) was evident in fewer aged hinds compared with mature hinds (3/12 versus 10/10, P < 0.001) and mean plasma LH concentrations were higher in aged animals than in mature animals (0.57 +/- 0.05 and 0.20 +/- 0.05 ng ml(-1), P < 0.001). Mean uterine (44.2 +/- 4.5 and 75.4 +/- 4.2 g; P < 0.001) and ovarian masses (0.88 +/- 0.11 and 1.52 +/- 0.12 g; P < 0.001) were lower in the aged hinds, which also had fewer antral follicles than did mature hinds (0.89 +/- 0.35 and 23.5 +/- 4.5 follicles per hind, respectively; P < 0.001). Only one primordial follicle was observed in one of the ovaries of the aged hinds, compared with 7000-21 000 in the ovaries of mature hinds. The high gonadotrophin concentrations, paucity of primordial and antral follicles and failure of ovulation indicate collectively that waning reproductive performance after 17 years of age is primarily due to ovarian failure.

Aging↗

PubMed2000

[The important role of vitamins in the over-production of pyruvic acid].

The effect of nicotinic acid, thiamine, pyridoxine, biotin and riboflavin on the production of pyruvic acid by Torulopsis glabrata WSH-IP303 with glucose as carbon source and NH4Cl as sole nitrogen source was investigated. By using orthogonal experiment method, thiamine was confirmed to be the most important factor affecting the production of pyruvic acid. Based on a certain concentration range of thiamine (0.01-0.015 mg/L), glucose consumption rate can be enhanced by increasing the concentration of nicotinic acid. When the concentration of nicotinic acid, thiamine, pyridoxine, biotin and riboflavin were 8, 0.015, 0.4, 0.04 and 0.1 mg/L, respectively, the concentration and yield to glucose of pyruvic acid reached 52.4 g/L and 0.525 g/g at 48 h in flask culture, respectively. Batch culture was conducted in a 2.5 L fermentor with initial glucose concentration of 120 g/L. By adopting the optimal concentration combination of vitamins, the concentration and yield to glucose of pyruvic acid reached 69.4 g/L and 0.593 g/g at 57.5 h, which were increased by 32.4% and 13% than the best results in flask culture, respectively.

Ammonium Chloride↗

PubMed2000

[Effects of nitrogen feeding on the accumulation of poly-beta-hydroxybutyrate with Alcaligenes eutrophus].

On the basis of analysis of PHB fermentation processes, the effects of ammonium sulfate feeding rate at PHB formation period on the PHB accumulation by Alcaligenes eutrophus were investigated. It was shown that the complete absence of nitrogen source at PHB formation phase would lead to the decline of PHB synthetic activity, and the obvious influences of different nitrogen feeding rate on PHB synthesis were observed. Higher PHB content, but relative lower cell dry weight, PHB concentration and PHB productivity could be obtained at slower nitrogen feeding rate. The excessive nitrogen feeding rate resulted in the drop of PHB content, which led to the decrease of PHB concentration and PHB productivity. The better results could be achieved when the ammonium sulfate feeding rate was set at around 0.5 g/h.

Ammonium Sulfate↗

PubMed1999

Plasma gonadotropin concentrations in the cyclic female brushtail possum (Trichosurus vulpecula).

Changes in plasma concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), and their relationship to antral follicle development and ovulation, were determined in female brushtail possums (Trichosurus vulpecula) in experiments in which pouch young were removed (RPY) from lactating females to promote ovarian activity. In Experiment 1 (n = 8), the development of preovulatory follicles and ovulation was monitored by laparoscopy. In Experiment 2 (n = 15) estrus and mating were monitored by cytology of urine. Ovulation occurred in 4/8 (Experiment 1) and 9/16 (Experiment 2) possums, and in these animals, plasma FSH concentrations fell progressively over the period of preovulatory follicle development and returned to pretreatment levels after ovulation. With the exception of samples taken at the time of the preovulatory gonadotropin surge, mean plasma LH levels remained basal. In those possums that failed to ovulate, plasma FSH concentrations were elevated while plasma LH concentrations were low; these patterns remained unchanged throughout the sampling period. It was not possible to distinguish between animals that would ovulate and those that would not ovulate after RPY on the basis of gonadotropin profiles at the time of RPY. A further group of possums (Experiment 3, n = 10) were blood-sampled at hourly intervals for 48 h to characterize preovulatory gonadotropin surges, using laparoscopy to monitor preovulatory follicular development and predict ovulation. A preovulatory LH surge (max. conc. 10.2-43.5 ng/ml, duration 7-9 h) was recorded in 4 animals, with a coincident preovulatory FSH surge (max. conc. 1.4-21.4 ng/ml, duration 3-11 h) observed in 3 of these possums. The patterns of gonadotropin secretion in the cycling brushtail possum conform to those reported for eutherians that ovulate spontaneously and appear to be regulated by similar mechanisms.

Animals↗

PubMed1999

[Optimization of fermentation conditions for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with Alcaligenes eutrophus].

A close relationship, between the initial addition time and concentration of propionate and HV fraction, was observed in shaking culture of Alcaligenes eutrophus for the production of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate). The optimal initial addition time of propionate was determined at the onset of PHBV formation period. Although relatively high HV unit could be obtained under high propionate concentration, the growth and product synthetic activities were inhibited obviously. Different ratios of glucose to propionate were fed to stimulate the formation of HV unit and the results were compared. The optimizing feeding strategy of propionate was proposed based on the consideration of several fermentation index. The final cell dry weight, PHB concentration, PHB content and HV fraction in PHBV reached 52.1 g/L, 40.8 g/L, 78.3% and 16.2 mol%, respectively, Yield coefficient of HV unit to propionate and PHBV productivity were obtained to be 0.5 g/g and 0.74 g/(L/h), respectively.

Cupriavidus necator↗

PubMed1999

[Fermentation conditions for production of glutathione by recombinant Escherichia coli].

The fermentation conditions for production of glutathione by recombinant E. coli were investigated, the addition of certain materials, such as yeast extract, precusor amino acids and ATP were mainly focused on. The results showed that the addition of precusor amino acids and ATP could promote intracellular GSH accumulation. DCW and intracellular GSH content would be 24% and 1.4 times higher than that of fermentation without additions when 2.0 g/L ATP and 9 mmol/L precusor amino acids were added at the begining and 12 h of fermentation, respectively. By using the optimized combination of additives obtained from orthogonal experiments, the maximal DCW and total GSH in broth could be improved 10% and 26% higher than the best results in orthogonal experiments, respectively. Based on the analysis of glucose utilizing ability of this strain, an exponential fed-batch culture process was conducted. DCW and total GSH in broth culd be 8.3 and 4.6 times higher than that of shaking flask culture and finally reached 80 g/L and 880 mg/L, respectively.

Adenosine Triphosphate↗

PubMed1998

Steroid contents of and steroidogenesis in vitro by the developing gonad and mesonephros around sexual differentiation in fetal sheep.

The aim of the present study was to establish whether the steroids, progesterone, androstenedione, testosterone and oestradiol, were present in the mesonephric-gonadal complex of female and male sheep fetuses around sexual differentiation (that is, from day 28 to day 45 of gestation, with sexual differentiation occurring at approximately day 32). A second aim was to test whether the mesonephric-gonadal complex, mesonephros (days 35-45 only) and gonad (days 35-45 only) were capable of steroid synthesis in vitro. The steroid contents in the mesonephric-gonadal complex were not detectable before sexual differentiation. However, from day 35 of gestation onwards, the mesonephric-ovarian complex contained mainly oestradiol and the mesonephric-testicular complex contained mainly testosterone: from day 35 until day 45 the increase in content of these two steroids exceeded the increase in the mass of tissue by more than fivefold. From day 40 to day 45 of gestation, the contents of the other steroids in the pathways to oestradiol increased progressively in both sexes but more in parallel with the increase in tissue mass. In contrast to the steroid contents in the tissue at recovery, the mesonephric-gonadal tissue from both sexes in tissue culture was able to synthesize most steroids before and after sexual differentiation and also to metabolise supplementary androstenedione to oestradiol. These findings suggest that many, if not all, of the steroidogenic enzymes in the pathway from cholesterol to oestradiol are present before sexual differentiation. Most of the aforementioned steroids were present in detectable amounts in isolated mesonephros and gonad of both sexes after sexual differentiation. Moreover, for both the isolated mesonephros and gonad, there were increases in the mean contents of most steroids after culture relative to the contents in the tissues at recovery. These data suggest that the mesonephros, as well as the gonad, in both sexes is capable of synthesizing steroid. It is concluded that, in the sheep fetus, the female and male gonads are steroidogenically active after sexual differentiation, that the steroidogenic enzymes develop before sexual differentiation, and that the mesonephros is a site of steroid synthesis.

Androstenedione↗

PubMed1998

Expression and secretion of a biologically active glycoprotein hormone, ovine follicle stimulating hormone, by Pichia pastoris.

The methylotrophic yeast, Pichia pastoris, has been used to co-express recombinant genes formed by fusion of the mating factor-alpha (MFalpha) leader and ovine follicle stimulating hormone (oFSH) alpha and beta subunit coding sequences. Pichia strains carrying single copies of the two fusion genes secreted recombinant oFSH (roFSH) to concentrations of approximately 51.0 ng/ml and 17.5 ng/ml, measured by RIA or in vitro bioassay respectively, whereas a strain with two copies of the alpha and one copy of the beta subunit fusion genes secreted roFSH to concentrations of 61 ng/ml (RIA) and 22 ng/ml (bioassay). It appears that the Pichia-derived roFSH had about one-third the in vitro bioactivity of native oFSH or, alternatively, only one-third of the roFSH is bioactive. Measurements of secreted roFSH alpha and beta subunit concentrations indicated less than 10% of alpha and 25-33% of beta subunits were stably dimerized. The receptor binding properties of the roFSH resemble those of native oFSH. In summary this paper reports the production, by P. pastoris, of a heterodimeric glycoprotein hormone (roFSH) that has in vitro biological activity.

Animals↗

PubMed1998

The effect of environmental conditions and glucose feeding in shaking flask on glutathione (GSH) production.

The effects of pH, broth volume, initial sugar concentration, ratio of carbon to nitrogen and phosphorus, and the glucose feeding method on GSH production in a shaking flask were investigated. The results showed that the proper pH and broth content for GSH production were 6.0 and 60 ml broth per 500 ml flask, respectively. The initial glucose concentration distinctly affected the GSH production; the intracellular GSH content of yeast would decrease when the initial glucose concentration was beyond 12 g/L. A glucose feeding strategy with the purpose of controlling the specific growth rate at an expected value was developed and applied to a 12 hour fermentation with the total glucose concentration 26.2 g/L; the final cell concentration (DCW) and the intracellular GSH content could reach 8.78 g/L and 13.6 mg/g, respectively, while the total GSH in the broth was 119.4 mg/L and the yield of cell to glucose was 0.335 g/g.

Carbon↗

PubMed1997

Follicle-stimulating hormone in the brushtail possum (Trichosurus vulpecula): purification, characterization, and radioimmunoassay.

Follicle-stimulating hormone (FSH) was purified from brushtail possum (Trichosurus vulpecula) pituitary glands by using the following purification techniques: fractional ammonium sulfate precipitation, triazinyl-dye affinity chromatography, hydrophobic interaction chromatography, and gel filtration. A yield of 18 micrograms of FSH per gram of pituitary, with a recovery of 12%, was obtained from 1400 glands (20.3 g wet weight). The purified FSH activity per gram of protein was 1320 times more potent than the initial pituitary homogenate. Contamination with possum luteinizing hormone (LH) was < 0.02%. The amino acid composition of possum FSH was similar to that of ovine FSH. Amino-terminal sequencing for 11 cycles indicated that the alpha subunit has the same sequence as ovine FSH except for residue 7, where the possum FSH alpha subunit contains isoleucine compared to the ovine subunit which contains threonine. The beta subunit has two substitutions in the first 11 residues and does not contain the N terminal serine that is found in ovine FSH. Amino acid sequencing did not detect any contaminating proteins. Possum FSH bound possum and bovine testicular receptors with similar affinities. It was also able to stimulate in vitro cAMP production by Chinese hamster ovary cells which express recombinant FSH receptors. In the receptor assays and the bioassay possum FSH has about 21% of the potency of ovine FSH (USDA-oFSH-19-SIAFP-RP2). An RIA was developed for possum FSH using 125I-possum FSH and an antiserum raised against human FSH. The RIA has a sensitivity of 0.3 ng/ml, a 50% displacement of 2.7 ng/ml, and a cross reactivity of 0.05% against possum LH. Plasma FSH levels in male possums (10.4 +/- 2.4 ng/ml, n = 4) were higher (P < 0.05) than levels in females (1.0 +/- 0.1 ng/ml, n = 4). Five days after gonadectomizing these possums the plasma FSH levels increased (P < 0.05) to 27.1 +/- 0.2 ng/ml in the males and to 6.6 +/- 2.0 ng/ml in the females. In summary, we have purified and partially characterized possum FSH. We have also set up an RIA for the hormone and shown that males have higher levels than females and that plasma FSH increases after gonadectomy.

Amino Acid Sequence↗