A comparison of bone strain measurements at anatomically relevant sites using surface gauges versus strain gauged bone staples

Instrumented bone staples were first introduced as an alternative to surface-mounted strain gauges for use in human in vivo bone strain measurements because their fixation to bone is secure and requires not only minimally invasive surgery. Bench-top bone bending models have shown that the output from strain gauged bone staples compares favorably to that of traditional mounted gauges. However their within- and across-subject performance at sites typically instrumented in vivo has never been examined. This study used seven human cadaver lower extremities with an age range of 23-81 years old and a dynamic gait simulator to examine and compare axial strains in the mid tibial diaphysis and on the dorsal surface of the second metatarsal as measured simultaneously with strain gauged bone staples and with traditional surface-mounted gauges. Rosette configurations were used at the tibial site for deriving principal compression and tension, and shear strains. Axial outputs from the two gauge types demonstrated strong linear relationships for the tibia (r(2)=0.78-0.94) and the second metatarsal (r(2)=0.96-0.99), but coefficients (slopes) for the relationship were variable (range 7-20), across subjects and across sites. The apparent low reliability of strain gauged staples may be explained by the fact that both strain gauged staples and surface strain gauges are inexact to some degree, do not measure strains from exactly the same areas and strain gauged staples reflect surface strains as well as deformations within the cortex. There were no relationships for the principal tibia compression, tension or shear strain measurements derived from the two rosette gauge types, reflecting the very different anatomical areas measured by each of the constructs in this study. Strain gauged bone staples may be most useful in comparing relative axial intra-subject differences between activities, but inter-subject variability may require larger sample sizes to detect differences between populations.     

Tensile properties of the in vivo human gastrocnemius tendon

In the present experiment we obtained the tensile properties of the human gastrocnemius tendon, a high-stressed tendon suitable for spring-like action during locomotion. Measurements were taken in vivo in six men. The gastrocnemius tendon elongation during tendon loading−unloading induced by muscle contraction−relaxation was measured using real-time ultrasonography. Tendon forces were calculated from the moment generated during isometric plantarflexion contraction, using tendon moment arm length data obtained in vivo with the tendon travel method. Tendon stiffness data were calculated from the slope of the tendon force−elongation curve, and were then normalized to the tendon's original dimensions, obtained from morphometric analysis of sonographs, to estimate the tendon Young's modulus. Mechanical hysteresis values were obtained from area calculations by numerical integration. The elongation of the tendon increased curvilinearly with the force acting upon it, from 1.7±1 mm (0.8±0.3% strain) at 87.5±8.5 N to 11.1±3.1 mm (4.9±1% strain) at 875±85 N. The tendon Young's modulus and mechanical hysteresis were 1.16±0.15 GPa and 18±3%, respectively. These values fall within the range of values obtained from in vitro experiments and are very similar to the respective values recently obtained from in vivo measurements in the less highly stressed human tibialis anterior tendon (1.2 GPa and 19%), thus indicating that the material properties of tendon are independent of physiological loading and function. Combining the present tendon force−elongation data with previously reported Achilles tendon force data recorded during walking indicates that the gastrocnemius tendon would provide 6% of the total external work produced by the locomotor system. This estimate illustrates the contribution of passive elastic mechanisms on the economy and efficiency of walking. The contributions would be greater in more active exercise such as running.     

Estimates of genetic parameters and genetic trends for live weight and fleece traits in Menz sheep

Menz sheep are indigenous to the highlands of Ethiopia, and highly valued for their meat and wool production. The area is characterized as a low input mixed barley-sheep production system. In 1998, a selection experiment was set up to evaluate the response of Menz sheep to selection for yearling live weight (WT12) and greasy fleece weight (GFW) combined in an economic index. In this paper, we report the results of this breeding program obtained between 1998 and 2003. Average annual genetic selection responses for WT12 and GFW were 1.506 and 0.043 kg in the selected flock and 0.392 and −0.008 kg in the control flock. Annual genetic trends in the selected flock, estimated by regressing BLUP estimated breeding values on year of birth, were 0.495 ± 0.053 kg for WT12, 0.012 ± 0.002 kg for GFW, and Birr 5.53 ± 0.55 for the aggregate breeding value (1 Ethiopian Birr = 0.115 USD). Corresponding values for the control flock were 0.276 ± 0.065 kg, 0.003 ± 0.002 kg and Birr 2.93 ± 0.69. Correlated responses in birth weight (WT0), weaning weight (WT3), 6-month weight (WT6) and staple length (STPL) in the selected flock were 0.038 ± 0.005 kg, 0.271 ± 0.03 kg, 0.388 ± 0.039 kg and 0.011 ± 0.017 cm, respectively. Heritabilities, estimated by fitting a multitrait individual animal model were 0.464 ± 0.014, 0.477 ± 0.016, 0.514 ± 0.017, 0.559 ± 0.019, 0.393 ± 0.016 and 0.339 ± 0.014 for WT0, WT3, WT6, WT12, GFW and staple length (STPL), respectively. Phenotypic and genetic correlations between all traits were positive, except for STPL and WT12. Estimates of genetic parameters and observed genetic trends confirm that selective breeding can lead to significant genetic improvement in Menz sheep.     

Identification of acridinyl hydrazides as potent aspartic protease inhibitors

We have identified acridinyl derivatives as potent aspartic protease inhibitors by virtual screening of in-house library of synthetic compounds. Enzyme inhibition experiments showed that both compounds inhibit human cathepsin D and Plasmodium falciparum plasmepsin-II in nanomolar ranges. The IC₅₀ values against cathepsin D and plasmepsin-II of compound-Nar103 were found to be 9.0 ± 2.0 and 4.0 ± 1.0 nM and of compound-Nar110 were 0.5 ± 0.05 and 0.13 ± 0.03 nM, respectively. Ligand docking predicted the binding of acridinyl derivatives at the substrate-binding cleft, where hydrazide part of the inhibitors interact with the S1–S1′ subsite residues including catalytic aspartates. The phenyl ring and acridinyl moiety of the inhibitors were predicted to interact with S2/S3 and S2′/S3′ subsite residues.     

Influence of sucrose on the rheology and granule size of cross-linked waxy maize starch dispersions heated at two temperatures

Cross-linked waxy maize (CWM) starch dispersions (STDs) of concentration 50 g kg⁻¹ were heated in sucrose solutions containing 0–600 g kg⁻¹ (g sucrose/kg dispersion) at 85 °C at low shear and in intermittently agitated cans at 110 °C. The STDs heated in 0–300 g kg⁻¹ sucrose exhibited antithixotropic behavior, while those heated in 400–600 g kg⁻¹ sucrose exhibited thixotropic behavior. The mean starch granule diameter of the starch dispersions did not show strong dependence on sucrose concentration. The dispersions, especially those with high sucrose concentrations and heated at 110 °C, exhibited G′ versus frequency (ω) profiles of gels. The STDs exhibited first normal stress differences that increased in magnitude with the concentration of sucrose. Values of the first normal stress coefficient of canned dispersions calculated from dynamic rheological data plotted against ω and experimental values plotted against shear rate of some of the STDs overlapped.     

Effect of joint rotation correction when measuring elongation of the gastrocnemius medialis tendon and aponeurosis

It is well known that during maximal plantar flexion contractions the ankle joint rotation overestimates the actual elongation of the tendon and aponeurosis. The aim of this study was to examine the influence of the curve length changes of the Achilles tendon on the joint rotation corrected elongation and strain of the gastrocnemius medialis (GM) tendon and aponeurosis. Nine subjects (age: 29.4 ± 5.7 years, body mass: 78.8 ± 6.8 kg, body height: 178 ± 4 cm) participated in the study. The subjects performed maximal voluntary isometric plantarflexion contractions in the prone position on a Biodex-dynamometer. Ultrasonography (Aloka SSD 4000) was used to visualize the muscle belly of the GM muscle-tendon unit. To calculate the curve length changes of the Achilles tendon its surface contour was reconstructed using a series of small reflective skin markers having a diameter of 2.5 mm. The elongation of the GM tendon and aponeurosis was calculated (a) as the difference of the measured and the passive (due to joint rotation) displacement of the tendon and aponeurosis and (b) as the difference of the measured displacement and the length changes of the reconstructed Achilles tendon surface contour. The absolute difference between the elongation obtained by both methods were 1.2 ± 0.4 mm. These differences were due to the higher changes in length obtained by the reconstruction of the tendon curved surface contour as compared to the changes observed in the passive displacement of the digitised point at the aponeurosis. Without correcting for angle joint rotation, the measured elongation clearly overestimates the actual elongation of the GM tendon and aponeurosis. After the passive displacement correction the calculated elongation still overestimates the actual elongation of the GM tendon and aponeurosis. However, this overestimation has a negligible effect on the examined in vivo strain (0.3%) of the tendon and aponeurosis.     

Patterns of strain in the macaque ulna during functional activity

In vivo bone strain experiments were performed on the ulnae of three female rhesus macaques to test how the bone deforms during locomotion. The null hypothesis was that, in an animal moving its limbs predominantly in sagittal planes, the ulna experiences anteroposterior bending. Three rosette strain gauges were attached around the circumference of the bone slightly distal to midshaft. They permit a complete characterization of the ulna's loading environment. Strains were recorded during walking and galloping activities. Principal strains and strain directions relative to the long axis of the bone were calculated for each gauge site. In all three animals, the lateral cortex experienced higher tensile than compressive principal strains during the stance phase of walking. Compressive strains predominated at the medial cortex of two animals (the gauge on this cortex of the third animal did not function). The posterior cortex was subject to lower strains; the nature of the strain was highly dependent on precise gauge position. The greater principal strains were aligned closely with the long axis of the bone in two animals, whereas they deviated up to 45° from the long axis in the third animal. A gait change from walk to gallop was recorded for one animal. It was not accompanied by an incremental change in strain magnitudes. Strains are at the low end of the range of strain magnitudes recorded for walking gaits of nonprimate mammals. The measured distribution of strains in the rhesus monkey ulna indicates that mediolateral bending, rather than anteroposterior bending, is the predominant loading regime, with the neutral axis of bending running from anterior and slightly medial to posterior and slightly lateral. A variable degree of torsion was superimposed over this bending regime. Ulnar mediolateral bending is apparently caused by a ground reaction force vector that passes medial to the forearm. The macaque ulna is not reinforced in the plane of bending. The lack of buttressing in the loaded plane and the somewhat counterintuitive bending direction recommend caution with regard to conventional interpretations of long bone cross-sectional geometry. Am J Phys Anthropol 106:87–100, 1998. © 1998 Wiley-Liss, Inc.     

Butanol production by Clostridium beijerinckii. Part I: use of acid and enzyme hydrolyzed corn fiber

Fermentation of sulfuric acid treated corn fiber hydrolysate (SACFH) inhibited cell growth and butanol production (1.7 ± 0.2 g/L acetone butanol ethanol or ABE) by Clostridium beijerinckii BA101. Treatment of SACFH with XAD-4 resin removed some of the inhibitors resulting in the production of 9.3 ± 0.5 g/L ABE and a yield of 0.39 ± 0.015. Fermentation of enzyme treated corn fiber hydrolysate (ETCFH) did not reveal any cell inhibition and resulted in the production of 8.6 ± 1.0 g/L ABE and used 24.6 g/L total sugars. ABE production from fermentation of 25 g/L glucose and 25 g/L xylose was 9.9 ± 0.4 and 9.6 ± 0.4 g/L, respectively, suggesting that the culture was able to utilize xylose as efficiently as glucose. Production of only 9.3 ± 0.5 g/L ABE (compared with 17.7 g/L ABE from fermentation of 55 g/L glucose-control) from the XAD-4 treated SACFH suggested that some fermentation inhibitors may still be present following treatment. It is suggested that inhibitory components be completely removed from the SACFH prior to fermentation with C. beijerinckii BA101. In our fermentations, an ABE yield ranging from 0.35 to 0.39 was obtained, which is higher than reported by the other investigators.     

Maternal serum progesterone concentration during pregnancy and lamb birth weight at parturition in Javanese Thin-Tail ewes with different litter sizes

Pregnant ewes, (N=38), with similar body weight and age (19, 13 and 6 carrying 1, 2 and 3 fetuses, respectively) were used to study the relationship between maternal serum progesterone concentration during pregnancy and lamb birth weight at parturition. Average maternal serum progesterone concentration in the ewes carrying 1, 2, and 3 fetuses was: 5.3±0.3, 6.2±0.7 and 6.6±0.5 ng/ml, during weeks 0 to 7 of gestation; 16.9±1.4, 25.3±1.5, and 26.8±2.5 ng/ml, during weeks 8 to 20; and 13.2±1.0, 18.7±1.0, and 19.8±1.7 ng/ml, during the entire gestation period respectively. Total lamb birth weight in ewes carrying 1, 2, and 3 fetuses was 1.9±0.1, 3.2±0.2, and 4.2±0.4 kg, respectively. In the respective litter sizes, ewes with higher mean serum progesterone concentration during the whole pregnancy gave birth to lambs with higher birth weight (r²=0.76, 0.42 and 0.46, for ewes carrying 1, 2 and 3 fetuses, respectively). The results of the study indicated that prenatal growth could probably be improved by increasing endogenous secretion of maternal progesterone during pregnancy.     

High temperature-induced changes in exopolysaccharides, lipopolysaccharides and protein profile of heat-resistant mutants of Rhizobium sp. (Cajanus)

A thermosensitive wild-type strain (PP201) of Rhizobium sp. (Cajanus) and its 14 heat-resistant mutants were characterized biochemically with regard to their cell surface (exopolysaccharides (EPSs) and lipopolysaccharides (LPSs)) properties and protein profile. Differences were observed between the parent strain and the mutants in all these parameters under high temperature conditions. At normal temperature (30 °C), only half of the mutant strains produced higher amounts of EPSs than the parent strain, but at 43 °C, all the mutants produced higher quantities of EPS. The LPS electrophoretic pattern of the parent strain PP201 and the heat-resistant mutants was almost identical at 30 °C. At 43 °C, the parent strain did not produce LPS but the mutants produced both kinds of LPSs. The protein electrophoretic pattern showed that the parent strain PP201 formed very few proteins at high temperature, whereas the mutants formed additional new proteins. A heat shock protein (Hsp) of 63–74 kDa was overproduced in all mutant strains.     

Observations from the UK Supra-Regional Assay Service laboratory for the measurement of Vitamin D metabolites

As a Supra-Regional Assay Service (SAS) laboratory, we receive samples from all over the UK. Of these some are sent frozen and others by post or courier. We have examined transport and storage conditions to see whether they affect the measurement of Vitamin D metabolites and potentially contribute to the variation in measurement of 25-hydroxyvitamin D (25OHD) seen in the DEQAS scheme. We have also examined the samples received during 2005. We found that different transport and storage conditions did not contribute significantly to the normal variation seen in measuring Vitamin D metabolites (CV% (±S.E.) for stored versus assay controls: 5.1 ± 0.06% versus 4.5 ± 0.04% for 25OHD and 10.8 ± 1.0% versus 12.3 ± 1.0% for 1,25D). A review of the service showed a 240% increase in samples received over the last 5 years. Despite an increased awareness of the need to measure Vitamin D status, in this cross-section of patient samples 92% of Asian and 86% of white patients were found to be Vitamin D-insufficient (<30 ng/ml) and 27% of Asian and 14% of white patients were profoundly deficient (<5 ng/ml) and at risk of bone disease.     

Using digital image correlation to determine bone surface strains during loading and after adaptation of the mouse tibia

Previous models of cortical bone adaptation, in which loading is imposed on the bone, have estimated the strains in the tissue using strain gauges, analytical beam theory, or finite element analysis. We used digital image correlation (DIC), tracing a speckle pattern on the surface of the bone during loading, to determine surface strains in a murine tibia during compressive loading through the knee joint. We examined whether these surface strains in the mouse tibia are modified following two weeks of load-induced adaptation by comparison with contralateral controls. Results indicated non-uniform strain patterns with isolated areas of high strain (0.5%), particularly on the medial side. Strain measurements were reproducible (standard deviation of the error 0.03%), similar between specimens, and in agreement with strain gauge measurements (between 0.1 and 0.2% strain). After structural adaptation, strains were more uniform across the tibial surface, particularly on the medial side where peak strains were reduced from 0.5% to 0.3%. Because DIC determines local strains over the entire surface, it will provide a better understanding of how strain stimulus influences the bone response during adaptation.     

Role of dexamethasone on vasopressin release during endotoxemic shock

The present study was designed to assess the hypothesis that dexamethasone (DEX) through the control of nitric oxide (NO) synthesis could regulate the release of vasopressin (AVP), which plays an important role in the regulation of arterial pressure and plasma osmolality. Endotoxemic shock was induced by intravenous (i.v.) injection of 1.5 mg/kg lipopolisaccharide (LPS) in male Wistar rats weighing 250–300 g. After LPS administration, a group of animals were treated with DEX (1.0 mg/kg of body weight), whereas saline-injected rats served as controls. The LPS administration induced a significant decrease in mean arterial pressure (MAP) with a concomitant increase in heart rate (HR) (ΔVMAP: − 16.1 ± 4.2 mm Hg; ΔVHR: 47.3 ± 8.1 bpm). An increase in plasma AVP concentration occurred and was present for 2 h after LPS administration (11.1 ± 0.9 pg/mL) returning close to basal levels thereafter and remaining unchanged until the end of the experiment. When LPS was combined with i.v. administration of a low dose of DEX, we observed an attenuation in the drop of MAP (ΔVMAP: − 2.2 ± 1.9 mm Hg) and a decrease in NO plasma concentration [NO] after LPS administration (1098.1 ± 68.1 µM) compared to [NO] after DEX administration (523.4 ± 75.2 µM). However, this attenuation in the drop of MAP was accompanied by a decrease in AVP plasma concentration (3.7 ± 0.4 pg/mL). These data suggest that AVP does not participate in the recovery of MAP when DEX is administered in this endotoxemic shock model.     

Patterns of strain in the macaque tibia during functional activity.

The strain environment of the tibial midshaft of two female macaques was evaluated through in vivo bone strain experiments using three rosette gauges around the circumference of the bones. Strains were collected for a total of 123 walking and galloping steps as well as several climbing cycles. Principal strains and the angle of the maximum (tensile) principal strain with the long axis of the bone were calculated for each gauge site. In addition, the normal strain distribution throughout the cross section was determined from the longitudinal normal strains (strains in the direction of the long axis of the bone) at each of the three gauge sites, and at the corresponding cross-sectional geometry of the bone. This strain distribution was compared with the cross-sectional properties (area moments) of the midshaft. For both animals, the predominant loading regime was found to be bending about an oblique axis running from anterolateral to posteromedial. The anterior and part of the medial cortex are in tension; the posterior and part of the lateral cortex are in compression. The axis of bending does not coincide with the maximum principal axis of the cross section, which runs mediolaterally. The bones are not especially buttressed in the plane of bending, but offer the greatest strength anteroposteriorly. The cross-sectional geometry therefore does not minimize strain or bone tissue. Peak tibial strains are slightly higher than the peak ulnar strains reported earlier for the same animals (Demes et al. [1998] Am J Phys Anthropol 106:87-100). Peak strains for both the tibia and the ulna are moderate in comparison to strains recorded during walking and galloping activities in nonprimate mammals.     

Thermal stabilization of collagen molecules in bone tissue

Differential thermal calorimetry (DSC) analysis of partially dehydrated bovine bone, demineralized bone and bovine tendon collagen was performed up to 300 °C to determine factors influencing stability of mineralized collagen in bone tissue. Two endothermal regions were recognized. The first, attributed to denaturation of collagen triple helix, was hydration dependent and had a peak at 155–165 °C in bone, 118–137 °C in tendon and 131–136 °C in demineralized bone. The second region extended from 245 to 290 °C in bone and from 200 to 280 °C in tendon and was connected with melting and decomposition of collagen. Differences in thermodynamic parameters between cortical and trabecular bone tissue were stated. Activation energy of collagen unfolding in native bone tissue increased with dehydration of the bone. From the results of the present study we conclude that dehydrated bone collagen is thermally very stable both in native and in demineralized bone. Presence of mineral additionally stabilizes bone tissue.     

Multi-channel surface EMG classification using support vector machines and signal-based wavelet optimization

The study proposes a method for supervised classification of multi-channel surface electromyographic signals with the aim of controlling myoelectric prostheses. The representation space is based on the discrete wavelet transform (DWT) of each recorded EMG signal using unconstrained parameterization of the mother wavelet. The classification is performed with a support vector machine (SVM) approach in a multi-channel representation space. The mother wavelet is optimized with the criterion of minimum classification error, as estimated from the learning signal set. The method was applied to the classification of six hand movements with recording of the surface EMG from eight locations over the forearm. Misclassification rate in six subjects using the eight channels was (mean ± S.D.) 4.7 ± 3.7% with the proposed approach while it was 11.1 ± 10.0% without wavelet optimization (Daubechies wavelet). The DWT and SVM can be implemented with fast algorithms, thus, the method is suitable for real-time implementation.     

Modulation of ouabain-insensitive Na(+)-ATPase activity in the renal proximal tubule by Mg(2+), MgATP and furosemide

In addition to the (Na⁺+K⁺)ATPase another P-ATPase, the ouabain-insensitive Na⁺-ATPase has been observed in several tissues. In the present paper, the effects of ligands, such as Mg²⁺, MgATP and furosemide on the Na⁺-ATPase and its modulation by pH were studied in the proximal renal tubule of pig. The principal kinetics parameters of the Na⁺-ATPase at pH 7.0 are: (a) K_0.5 for Na⁺=8.9±2.2 mM; (b) K_0.5 for MgATP=1.8±0.4 mM; (c) two sites for free Mg²⁺: one stimulatory (K_0.5=0.20±0.06 mM) and other inhibitory (I_0.5=1.1±0.4 mM); and (d) I_0.5 for furosemide=1.1±0.2 mM. Acidification of the reaction medium to pH 6.2 decreases the apparent affinity for Na⁺ (K_0.5=19.5±0.4) and MgATP (K_0.5=3.4±0.3 mM) but increases the apparent affinity for furosemide (0.18±0.02 mM) and Mg²⁺ (0.05±0.02 mM). Alkalization of the reaction medium to pH 7.8 decreases the apparent affinity for Na⁺ (K_0.5=18.7±1.5 mM) and furosemide (I_0.5=3.04±0.57 mM) but does not change the apparent affinity to MgATP and Mg²⁺. The data presented in this paper indicate that the modulation of the Na⁺-ATPase by pH is the result of different modifications in several steps of its catalytical cycle. Furthermore, they suggest that changes in the concentration of natural ligands such as Mg²⁺ and MgATP complex may play an important role in the Na⁺-ATPase physiological regulatory mechanisms.     

Isolation of a thermostable uricase-producing bacterium and study on its enzyme production conditions

A uricase-producing bacterium was isolated from soil with a medium containing uric acid as the only carbon source. Based on its morphological and physiological characteristics, as well as 16S rDNA sequence and phylogenetic tree analysis, this new isolate belong to the genus Microbacterium. After heat treatment at 70 °C for 30 min, the uricase retained about 100% of the initial activity. The enzyme activity remained largely unchanged when it was stored in borate buffer at pH 8.5 at 37 °C for 40 days. The effects of different factors on the enzyme production were studied. Maize milk was the best C and N resources, and the uric acid showed to be an inducer for uricase production. When the strain was cultured at 30 °C at pH 7.5 for 30–36 h, the uricase activity peaked at 1.0 U/ml.     

Production, purification and properties of endoglucanase from a newly isolated strain of Mucor circinelloides

A newly isolated strain of the fungus, Mucor circinelloides (NRRL 26519), when grown on lactose, cellobiose, or Sigmacell 50 produces complete cellulase (endoglucanase, cellobiohydrolase, and β-glucosidase) system. The extracellular endoglucanase (EG) was purified to homogeneity from the culture supernatant by ethanol precipitation (75%, v/v), CM Bio-Gel A column chromatography, and Bio-Gel A-0.5 m gel filtration. The purified EG (specific activity 43.33 U/mg protein) was a monomeric protein with a molecular weight of 27 000. The optimum temperature and pH for the action of the enzyme were at 55 °C and 4.0–6.0, respectively. The purified enzyme was fully stable at pH 4.0–7.0 and temperature up to 60 °C. It hydrolysed carboxymethyl cellulose and insoluble cellulose substrates (Avicel, Solka-floc, and Sigmacell 50) to soluble cellodextrins. No glucose, cellobiose, and short chain cellooligosaccarides were formed from these substrates. The purified EG could not degrade oat spelt xylan and larch wood xylan. It bound to Avicell, Solka-floc, and Sigmacell 50 at pH 5.0 and the bound enzyme was released by changing the pH to 8.0. The enzyme activity was enhanced by 27±5 and 44±14% by the addition of 5 mM MgCl₂ and 0.5 mM CoCl₂, respectively, to the reaction mixture. Comparative properties of this enzyme with other fungal EGs are presented.     

The effect of follicle-stimulating hormone on follicular development, granulosa cell apoptosis and steroidogenesis and its mediation by insulin-like growth factor-I in the goat ovary

The effect of FSH on goat follicular development, granulosa cell apoptosis and steroidogenesis and its mediation by insulin-like growth factor (IGF)-I were studied through both in vivo and in vitro experiments. The FSH treatment was begun on Day 9 after estrus and consisted of injections twice a day for 3 days in decreasing doses (7.5–7.5–5.0–5.0–2.5–2.5 mg). Does in both treatment and control groups were slaughtered for ovaries on Day 12. Granulosa cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Expression of IGF-I and IGF-II mRNA was determined by RT–PCR, while concentrations of progesterone (P4), estradiol (E2), IGF-I and IGF-II were measured by radioimmunoassay (RIA). Following parameters increased significantly (P<0.05) after the FSH treatment: follicle number (5.0±1.5 versus 9.0±2.0 per ovary), the level of E2 (0.1±0.1 ng/ml versus 0.7±0.2 ng/ml), the E2/P4 ratio (0.7±0.4 versus 4.7±3.0) and the concentrations of IGF-I (0.5±0.2 ng/ml versus 119.4±15.1 ng/ml) and IGF-II (0.12±0.03 ng/ml versus 40.9±18.7 ng/ml) in follicular fluid of the medium sized (3–5 mm) follicles and in the ovarian cortex the relative quantity of IGF-I mRNA (0.37±0.17 versus 0.90±0.12 Max OD). In contrast, the ratio of apoptotic granulosa cells in these follicles was reduced significantly (0.53±0.1 versus 0.10±0.01, P<0.05). In large (>5 mm) follicles, however, only the follicle number (2.3±0.7 versus 7.0±1.5 per ovary) and the level of IGF-I (38.4±11.0 ng/ml versus 87.3±13.9 ng/ml) increased significantly (P<0.05), whereas other values did not change. In vitro culture of granulosa cells showed that FSH significantly (P<0.05) enhanced IGF-I production (12.7±2.1 ng/ml versus 26.±21.9 ng/ml) by these cells, and both FSH and IGF-I reduced the ratios of apoptotic cells (from 0.7±0.07 to 0.3±0.1 and 0.2±0.04, respectively) and the effect was additive when both were used together. H89, the PKA pathway inhibitor, blocked the effect of FSH on granulosa cell apoptosis and IGF-I production in vitro. These results indicated that FSH mainly enhanced the development of medium sized follicles in the goat by suppressing the apoptosis of granulosa cells via increasing production of IGF-I and steroids, possibly through the PKA pathway.