Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

Testicular function and Leydig cell ultrastructure in long-term bilaterally cryptorchid rams

This study was conducted to investigate the effects of bilateral cryptorchidism induced in adult rams on testicular function and Leydig cell ultrastructure. The results indicated that long-term bilateral cryptorchidism resulted in decreased testicular size, degeneration of seminiferous tubules, elevated serum LH levels, maintenance of normal testosterone concentrations in peripheral and spermatic vein serum, impairment of the magnitude and duration of androgen response to exogenous luteinizing hormone (LH), a 13-fold reduction in total number of Leydig cells/paired testes, and a 3-fold hypertrophy in the average size of remaining Leydig cells. Based on quantitative morphometry, the hypertrophied Leydig cells exhibited significant increases in the volume of intracellular organelles, including the cell nucleus, mitochondria, smooth and rough endoplasmic reticulum, lysosome-like bodies and lipid vesicles. Quantitatively, the hypertrophy alone was not enough to offset the loss in number of Leydig cells and was insufficient to explain the maintenance of normal levels of testosterone in jugular and spermatic venous blood. The additional mechanisms responsible for production of normal serum testosterone levels in the cryptorchid ram remain to be elucidated.     

Changes in Leydig cell ultrastructure and function during pubertal development in the boar

Changes in the ultrastructure of Leydig cells during pubertal development in the boar (40 to 250 days of age) were assessed using quantitative morphometric procedures, and the results were compared to the in vitro steroid-producing capacity and gonadotropin sensitivity of testicular tissue obtained from the same boars. Volume of individual Leydig cells declined through 100 days of age, increased rapidly to a peak at 130-160 days (i.e., puberty), and then declined to intermediate levels by 220-250 days of age. The pattern of change in the number of intracellular organelles per Leydig cell was very similar to the change that occurred in Leydig cell volume. Changes in the total intracellular volume occupied by each type of organelle were highly correlated with changes in Leydig cell volume (r = 0.40-0.99, p less than 0.01), and this was particularly true for the nucleus (r = 0.63), mitochondria (r = 0.88), smooth endoplasmic reticulum (SER; r = 0.97), and total cytoplasm (r = 0.99) of the boar Leydig cell. In vitro production of testosterone and estradiol, expressed per Leydig cell, also peaked at 130-160 days, and was highly correlated to average Leydig cell volume, volume of SER, and number and total volume of mitochondria (r = 0.63-0.84; p less than 0.01). Observations in the present study indicated that onset of puberty in boars coincides with a dramatic increase in average Leydig cell size and SER volume per Leydig cell, accompanied by an increase in number of other intracellular organelles, including mitochondria, lysosomes, and lipid droplets, and a peak in the steroid-producing capacity per Leydig cell. A decline in Leydig cell size, intracellular organelles, and sensitivity to gonadotropin stimulation occurred postpubertally.     

Release of poly a(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg++, (b) resuspension in a high salt medium lacking Mg++, and (c) chelation of Mg++ by EDTA or pyrophosphate. Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [(3)H] poly U for in situ hybridization in glutaraldehyde-fixed RM. Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg++. Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes.     

Causes for decreased fertility in out-of-season mated ewes

The interval from onset of estrus to preovulatory luteinizing hormone (LH) release, conception and fertilization rates, and number of accessory spermatozoa per ovum at 48 hr postmating in untreated cyclic ewes and in progestogen-pregnant mare serum gonadotropin (PMSG) treated, anestrous ewes were compared in efforts to identify sources of lowered fertility for matings induced in anestrous ewes with exogenous hormones. Blood samples for LH determination were collected at 0, 2, 4, 6, 8, 10, 12, and 24 hr after the onset of estrus. Conception and fertilization failure rates were determined at 48 hr, 12 days, or parturition. The progestogen-PMSG treated ewes had a shorter interval from onset of estrus to preovulatory LH release, lower conception rates, and fewer accessory spermatozoa than cyclic ewes had. Conception failure, rather than embryonic mortality, was the major cause of reduced fertility for the out-of-season mated ewes and apparently resulted from insufficient viable spermatozoa in the oviducts to fertilize the ova.     

Cortisol and prolactin concentrations during three different seasons in relocated Brahman and Hereford bulls

The objective of this study was to evaluate seasonal changes of cortisol and prolactin (PRL) concentrations in Brahman and Hereford bulls moved to locations that differ in geographical and environmental conditions. Postpubertal Hereford bulls from Montana (n = 15) and Nebraska (n = 15) and Brahman bulls from Texas (n = 18) were located in or relocated to Montana, Nebraska or Texas so that each location had 5 Montana Herefords, 5 Nebraska Herefords and 6 Texas Brahman bulls. Blood samples were collected at 20-minute intervals for 8 hours in November (Fall 1), April (Spring) and November (Fall 2) of the next year. These dates corresponded to 6, 12 and 18 months, respectively, after relocation in May of the first year. Cortisol concentrations were higher (P<0.05) in Fall 1 than in Fall 2 and were higher (P<0.05) for bulls in Montana than for bulls in Texas. The decrease in cortisol concentrations from Fall 1 to Fall 2 was negatively related (P<0.05) to age and weight. There was a three-way interaction (P<0.05) of breed-type origin, location and season for PRL concentrations. Seasonal patterns of PRL concentrations differed between relocated Texas Brahman and Hereford bulls, and patterns for relocated bulls differed from those of the nonrelocated bulls. Seasonal patterns of PRL were influenced to a greater extent by relocation in Texas Brahman bulls than in Hereford bulls.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Evidence for retrograde flow of spermatozoa into the urinary bladder of bulls during electroejaculation

Spermatozoa were not found in the urine collected from 12 bulls before electroejaculation, whereas spermatozoa were found in the urine collected after electroejaculation. The concentration of spermatozoa in four consecutive samples of urine collected during the first postelectroejaculation micturition did not differ (P > 0.80) within bulls, suggesting that the spermatozoa found in the urine were those that had flowed into the urinary bladder during electroejaculation. The mean percentage of retrograde flow was 21% and ranged from 1 to 50% for the 12 bulls. These findings demonstrate that there was a significant urinary loss of spermatozoa during electroejaculation.     

Losing the desire: selection can promote obligate asexuality

Whilst parthenogenesis has evolved multiple times from sexual invertebrate and vertebrate lineages, the drivers and consequences of the sex-asex transition remain mostly uncertain. A model by Stouthamer et al. recently published in BMC Evolutionary Biology shows a pathway by which obligate asexuality could be selected for following endosymbiont infection.     

Repetitive testicular biopsy in the ram during pubertal development

A procedure for testicular biopsy was developed and tested in rams at 14, 18 and 22 wk of age. The biopsy procedure produced a tissue sample with minimal cell-to-cell disruption and caused no detectable detriment to testicular development in rams. At least three biopsies from the same testis were obtained at 4-wk intervals without influencing the developmental patterns of either the biopsied or nonbiopsied testicle. Data obtained by biopsy indicated that the testes of Suffolk rams reached a comparable stage of development (22 wk of age) approximately 4 wk later than the testes of Finnsheep rams (18 wk of age). These results demonstrate that repetitive testicular biopsy can be performed successfully in the ram during pubertal development. The biopsy procedure allows repeated sampling of the same animal and offers a reasonable alternative to castration.