The influence of temperature upon polysomes of spermatids of rat testes

Incorporation of [³H]phenylalanine into protein by a reconstituted lysate subcellular system (ribosomes plus high-speed supernatant) from rat spermatids was measured at 34°C after 5 minutes preincubation of one component at 0°C while the other component was incubated at temperatures from 30°C to 40°C. Preincubation at temperatures above 34°C inhibits the ribosomal activity but not the high-speed supernatant activity. The incubation of lysate above 34°C results from a dissociation of polysomes to monosomes. These results indicate that ribosomes are the most sensitive component to the increased temperature on protein synthesis in lysate cell free system by spermatids and that the inhibition of protein synthesis in spermatids above 34°C is at least partly explained by the breakdown of polysomes in these cells.     

Effect of glucose on ATP dephosphorylation in rat spermatids

Round spermatids were isolated from rat testes and the effects of different energy-yielding substrates on the cellular ATP content were estimated. The ATP content was constant and high (6-8 nmol/10(6) cells) during metabolism of exogenous lactate. During incubation for 30 min in the absence of exogenous lactate, there was a remarkably slow decline of the ATP content, indicating ATP production from other substrates. It was shown that this could reflect beta-oxidation of fatty acids, but not the mobilization of an endogenous pool of acetylcarnitine. Glucose metabolism in the absence of exogenous lactate resulted in a rapid decline of the ATP content. This effect of glucose was correlated with a high fructose 1,6-biphosphate content (6-7 nmol/10(6) cells) and could be prevented by the addition of lactate. It is suggested that metabolism of glucose (and also mannose and fructose, but not galactose) in the absence of exogenous lactate can result in ATP dephosphorylation.     

Rescue of infertile transgenic rat lines by intracytoplasmic injection of cryopreserved round spermatids

Transgenic male rats carrying human alpha-lactalbumin with thymidine kinase gene (line name; LAC3) were found to be infertile due to expression of the transgene in the testes. Furthermore, it was not possible to maintain the line even by the use of intracytoplasmic sperm injection (ICSI). Therefore, round spermatids prepared from the LAC3 rats were microinjected into strontium-activated oocytes using a Piezo-driven micromanipulator. Of 263 oocytes microinjected with LAC3 spermatids, 244 (92.8%) survived the injection and 96 (39.3%) developed to the 2-cell stage. Three viable offspring were born after transfer (1.4%, 3/219), and two offspring carried the LAC3 transgene. In the control experiment using spermatids of Wistar rats, similar proportions of post-injection survival (91.3%, 241/264), cleavage (40.2%, 97/241), and development into offspring (0.5%, 1/206) were obtained. Thus, this paper reports not only the first rat offspring derived from round spermatid injection but also the practical application of the microinsemination technique to the rescue of transgenes of infertile transgenic male rats.     

Isolation and viability of presumptive spermatids collected from bull testes by Percoll density gradient

The objective of the present study was to develop a procedure for isolating pure populations of round spermatid(s) (RS) by Percoll density gradient from bull testes. Bull testes were de-capsulated and testicular tissues were dissociated enzymatically to recover RS. After being filtered through a 20 microm nylon mesh, the cells were centrifuged at 650 x g for 25 min through the discontinuous Percoll density gradients (20, 35, 40, 45 and 90% Percoll solution). Isolated cells were analyzed by microscopic observation for survivability and apoptosis. In Experiment 1, both microscopic observation and DNA analysis by flow cytometry showed that approximately 40% of cells collected from 35% Percoll gradient were presumptive RS, whereas in 40% Percoll gradient, mostly primary spermatocytes were observed. Experiment 2 compared the effect of 35% Percoll density isolation on the incidence of apoptosis and necrosis in fresh and frozen-thawed cells to those of untreated cells. The percentage (mean+/-S.E.M.) of necrosis in cells collected from 35% Percoll gradient was less (P<0.05) than in untreated and frozen-thawed cells from 35% Percoll gradient (11.7+/-3.1% compared with 26.3+/-2.0% and 53.5+/-1.3%, respectively), but the rate of apoptosis did not differ (1.2+/-0.49% compared with 2.5+/-0.8% and 0.9+/-0.04%, respectively). The proportional data (mean+/-S.E.M.) of live cells in Percoll treated group were greater (P<0.05) than in untreated and frozen-thawed cells from the 35% Percoll gradient (86.7+/-3.26% compared with 70.8+/-2.73% and 41.9+/-1.69%, respectively). Experiment 3 compared the development rates of embryos injected with RS isolated from fresh and frozen-thawed cells collected with the 35% Percoll gradient to those of untreated cells, and parthenotes as control. There were no significant (P>0.05) differences in the rates of cleavage and blastocyst development between untreated fresh cells and fresh cells collected from the 35% Percoll gradient (75.4 and 10.5% compared with 82.4 and 12.8%). However, there were lesser (P<0.05) cleavage and blastocyst rates in frozen-thawed cells from the 35% Percoll gradient (51.6 and 6.3%) and parthenotes (60.7 and 4.1%) were observed. These results suggest that isolation of presumptive RS by 35% Percoll density gradient is effective in eliminating apoptotic and early necrotic cells. However, the use of RS in improving the developmental potential of embryos merits further studies.     

The role of glucose,pyruvate and lactate in ATP production by rat spermatocytes and spermatids

The ATP content of pachytene spermatocytes and round spermatids, isolated from rat testes, was not maintained during incubation of the germ cells in the presence of glucose. Glucose was metabolized via glycolysis at a considerable rate, but the rate of oxidation of the resulting endogenous pyruvate in the mitochondria was too low to support fully ATP production. Exogenous pyruvate (0.25 mM) or exogenous l-lactate (3–6 mM), however, were effective energy substrates. The lactate dehydrogenase reaction in isolated germ cells favoured the rapid conversion of pyruvate to lactate, at the expense of reducing equivalents from mitochondrial NADH. Hence, to support ATP production by the germ cells via mitochondrial metabolism of endogenous pyruvate, a relatively high concentration of exogenous lactate may be essential. In the spermatogenic microenvironment in vivo, such high concentrations of lactate could result from the net production of lactate by Sertoli cells. The mitochondria of the isolated germ cells produced ATP probably at a close to maximal rate, and spermatogenesis therefore may be extremely sensitive to compounds which interfere with mitochondrial energy metabolism and respiratory control.     

Effect of cytosol fractions from lutropin-stimulated rat testes on pregnenolone production by mitochondria from normal rat testes

The rate limiting step in the production of steroids in the testis is the mitochondrial conversion of cholesterol to pregnenolone. This conversion can be stimulated by lutropin, but the precise interaction between lutropin-induced cytoplasmic factors and the mitochondrial activity in steroid production is as yet unknown. The results described in the present paper concern the steroid production of isolated mitochondrial fractions in recombination experiments with isolated supernatant fractions from total testes homogenates. Cyanoketone as well as SU-10603, an inhibitor of steroid 17α-hydroxylase activity are required to block pregnenolone metabolism. The results show that the cytoplasm contains lutropin-induced factor(s) which can exert its effect in vitro on the cholestorel side-chain cleavage activity in intact mitochondria isolated from control testes.     

The effects of starvation, environmental temperature and injury on the rate of disposal of glucose by the rat

1. The disposal rate of glucose, R, given by R=k(v)Q, where Q is the quantity of plasma glucose and k(v) is a rate coefficient, was determined from the disappearance of [U-(14)C]-glucose from blood after single intravenous injection. Values of R should be close to the carbohydrate oxidation rate in the states investigated. 2. Normal rats (i) experimental methodology was studied. The best (single sampling) method gave the following results. (ii) The plasma glucose concentration (C(p)) and R were temporarily increased by the stress of handling and injection. (iii) R was increased by decreasing the environmental temperature from 29 degrees C to 20 degrees C in line with previously published (Stoner & Marshall, 1971) changes in total body O(2) consumption. (iv) Starvation decreased R such that R=constantxC(p) (2). (v) The results suggested some central control of cell permeability to glucose. 3. Injured post-absorptive rats were studied in the ebb phase after three severe injuries: scalding at 20 degrees and 29 degrees C (non-lethal) and bilateral hind-limb ischaemia at 20 degrees C (85% mortality). (i) Handling and injection did not affect C(p). (ii) The rise in C(p) after injury was not closely correlated with its severity. (iii) The value of R was nearly independent of severity. (iv) Unlike in normal rats R varied little with ambient temperature (in line with O(2) consumption) or with C(p). Values of k(v) varied inversely as C(p). (v) The results were explained in terms of a centrally integrated response to injury involving the hypothalamus which over-rode the controls operating in normal rats. Hormonal factors are discussed.     

Early effects of retinol and retinoic acid on protein synthesis in retinol deficient rat testes

When an [35S] labeled mixture of methionine and cysteine was injected intratesticularly into retinol-deficient rats, two hours later more than 980 cytosolic proteins were detected by computer aided two dimensional gel electrophoresis. Furthermore, two hours after oral refeeding retinyl acetate as the source of retinol to retinol deficient rats, synthesis of 286 proteins was inhibited and that of 101 proteins was activated. Refeeding with retinoic acid leads in two hours to even higher inhibition of protein synthesis and the labeling patterns of proteins are not identical when compared to retinol refed rats. The results indicate that retinol or retinoic acid quickly influence expression of many proteins and suggest that retinol action in the testes is not identical to that of retinoic acid.