Mode of orthophosphate uptake and ATP labeling by mammalian cells.

Incubation of HeLa cells with [32P]orthophosphate results in more rapid labeling of the gamma-phosphorus of ATP than of the intracellular pool of orthophosphate. The specific radioactivity of ATP equals that of extracellular orthophosphate after 2 h of incubation. A similar pattern of labeling is seen with human erythrocytes when incubated at physiological concentrations of orthophosphate (2 mM) and pH 7.4-7.8. At lower pH, 6.8-7.2, the rate of orthophosphate uptake increases and exceeds the rate of labeling of ATP. These data are explained by the existence of a primary system for ATP uptake which involves the mediation of membrane-bound glyceraldehyde-3-phosphate dehydrogenase. Phosphate first enters the cell as 1,3-diphosphoglyceric acid, is then transferred to ATP, and then enters the intracellular orthophosphate pool. At lower pH monovalent orthophosphate also enters the erythrocyte by a process not involving glyceraldehyde-3-phosphate dehydrogenase.

Phosphatidylcholine of blood lipoprotein is the precursor of glycerophosphorylcholine found in seminal plasma.

Glycerophosphorylcholine (GPC) concentration of the male reproductive tract increases from under 0.1 mM in the efferent ducts of the testis to 10-50 mM in the cauda epididymidis. It is reasonable to assume that choline-containing phospholipids serve as precursor(s) of GPC but the identity and source of these precursors had not been established. We performed in vivo labeling experiments in rabbits to distinguish between two types of phospholipid metabolism that might account for epididymal GPC formation: (a) addition of phospholipids to sperm during epididymal transit with degradation of these phospholipids by sperm to yield GPC or (b) degradation of phospholipids by epididymal epithelial cells with excretion of GPC into the duct lumen. We concluded that the latter route accounted for most of the GPC accumulation and that the primary source of the choline moiety of GPC was phosphatidylcholine of blood lipoprotein. We believe that these observations established a specific, vectorial metabolism of phospholipids whereby circulating lipoproteins on one side of the epididymal epithelial cell are transferred to the cell, the lipoproteins degraded by the epithelial cell to GPC, and the GPC moved into the lumen.

Isolation and characterization of the plasma membrane of rat cauda epididymal spermatozoa.

Cauda epididymal rat spermatozoa were isolated by flushing the excised epididymis and the plasma membrane was detached by a nitrogen cavitation treatment (500 psi, 10 minutes equilibration at 4 C). Membrane vesicles were recovered after sucrose gradient centrifugation. Portions of the sperm surface releasing the plasma membrane were assessed by light microscopy of fluoroscein isothiocyanate-succinylated concanavalin A-treated spermatozoa and by transmission electron microscopy. Plasma membrane was detached from the region overlying the acrosome from most spermatozoa and from the middle-piece overlying the mitochondria from some cells. Thus, the fraction analyzed was derived from at least two portions of the sperm surface. The fractions from the sucrose density gradient were analyzed for gross chemical composition (phospholipid, protein and sterol) and the protein components were detected after electrophoresis under denaturing conditions; the peak fractions (at density approximately 1.13 g/ml) were judged homogeneous. Replicate analyses of such preparations established mass ratios of protein to phospholipid of 0.63, total sterol to phospholipid of 0.18, and demosterol to cholesterol of 0.32. The molecular composition of the phospholipid fraction was determined to be 10% phosphatidylserine (mole percent), 3% phosphatidylinosital, 3% sphingomyelin, 31% phosphatidylethanolamine, 27% phosphatidylcholine, 10% diphosphatidylglycerol and 5% of an unknown component. Fatty acyl analyses of the phospholipid fraction revealed that approximately 70% of the residues consisted of palmitoyl (16:0) and stearoyl (18:0) acyl groups, with the balance distributed among various unsaturated acyl groups (18:1, 22:3, 22:4 and 22:5); about 40% of the recovered phospholipids represented ether acyl phosphatides. Differences in the lipid composition of rat vesicles described here and similar vesicles isolated from ram and boar spermatozoa (described previously) are discussed. The partitioning of the nitroxyl spin label 3-doxylheptane into vesicles isolated from rat and ram spermatozoa was assessed by electron paramagnetic resonance spectroscopy at temperatures between 4 C and 26 C; no difference in the response of the spin label in the two vesicle preparations was detected.

A comparison of critical osmolality and hydraulic conductivity and its activation energy in fowl and bull spermatozoa.

Measurements were made of critical osmolality, the osmolality at which 50% of the cells are lysed, and of the permeation time, the time taken to lyse 50% of the cells in an osmotic solution lower than the critical osmolality, for fowl and bull spermatozoa. Cell lysis was determined by means of fluorescent viability stains (carboxyfluorescein diacetate and propidium iodide) using a flow cytometer. The advantages and pitfalls of this approach are addressed. The values obtained have been used to compute the water permeability, or hydraulic conductivity, of the plasma membrane and its activation energy for each species. Fowl spermatozoa were found to have a lower critical osmolality (17 mOsm) than bull spermatozoa (36 mOsm), and this is discussed in relation to the differences in cell shape and size. The hydraulic conductivities of fowl and bull spermatozoa were 2.1 and 10.8 microns x atmosphere x minute, respectively, and the respective activation energies were 4.4 and 3.0 kcal/mol. The relevance of these findings to cryopreservation of spermatozoa is considered.

Exposure of human, boar, or bull sperm to a synthetic peptide increases binding to an egg-membrane substrate.

We evaluated the effects of in vitro exposure of sperm to synthetic FertPlus peptide, which represents a 60-amino acid sequence within rat prosaposin, using a microwell sperm-binding assay (SBA), in which an extract of hen's egg served as the binding substrate. Sperm suspensions were incubated with FertPlus peptide (six to eight concentrations; 0 and 20-1,280 pM) at 37 degrees C for 10 minutes, diluted > or = 20 times, and placed onto SBA plates. After 60 minutes at 37 degrees C, unbound sperm were washed away and the DNA of bound sperm was quantified. Percentage of sperm bound was independent of the percentage of motile sperm, but immotile sperm did not bind. For fresh human sperm (25 ejaculates), the percentage of sperm bound was increased by exposure to 640 pM peptide (P < 0.01). For 11 of 25 samples, the percentage of sperm bound for the aliquot exposed to 640 pM peptide was > or = 1.4 times the value for a 0 pM control aliquot. With frozen-thawed human sperm, for six of seven samples, binding was > or = 1.4 times greater after exposure to 640 pM peptide. For boar sperm held for approximately 24 hours at approximately 18 degrees C before use (28 ejaculates), there was a higher percentage of sperm bound for aliquots previously exposed to 1,280 pM peptide than there was for control aliquots (P < 0.01). For 16 of 28 samples, exposure to peptide increased the percentage of sperm bound by > or = 1.4 times. For frozen-thawed bull sperm, percentage of sperm bound was > or = 1.4 times greater for 4 of 10 samples that were briefly exposed to 160 pM peptide. Clearly, human, boar, and bull sperm were beneficially modified by brief in vitro exposure to FertPlus peptide, so that for many samples a greater percentage of sperm was bound in vitro. As presented in an accompanying paper, fertility of bull sperm was increased by brief exposure to FertPlus peptide.

Cryopreservation of mammalian sperm: what we ask them to survive.

Techniques for freezing bull sperm developed over the past 40 years have not yielded protocols for preserving sperm from other species. Recent advances in our understanding of cell membrane structure function and metabolism now permit alternative modes of investigation. These data will allow development of unique studies which should have a higher probability of yielding successful protocols for sperm from other species. In this review the authors will: (1) provide a general overview of cryopreservation; (2) review emerging concepts of membrane structure and the relationship of membrane composition to water and cryoprotectant movement; (3) emphasize how these parameters affect cell volume and surface areas; (4) focus attention on the concept that cryoprotectants will alter membrane structure and function in addition to their well-recognized effects on bulk solvent; and (5) emphasize the effect of the processing protocol on metabolic balance. These concepts are reintroduced in the context of the established and successful protocol for freezing bull sperm to illustrate the molecular responses that may be necessary to survive a freeze-thaw cycle.

Effects of caudal elevation on testicular function in rats. Separation of effects on spermatogenesis and steroidogenesis.

A variety of biologic processes are perturbed when exposed to microgravity (space flight) for more than 7 days, including testicular function. Suspension of rats in a special harness (caudal elevation) to induce thoracic pooling of blood fluids and remove the support function of the hind limbs is used to mimic, on earth, the effects of microgravity encountered during space flight. Typically, this induces cryptorchidism in male rats. Three experiments were conducted to differentiate the effects of caudal elevation (30 degrees angle) and anatomic location of testes on spermatogenesis and steroidogenesis. Rats were subjected to caudal elevation for 7 days using either a tail harness (experiments 1 and 2) or a whole-body harness (experiment 3). Testes of rats fell into the abdominal cavity when a tail harness was used, but ligation of the inguinal canal prevented this repositioning. For rats with abdominal testes, testicular weight was reduced (P less than 0.05) and histology of testes was abnormal; the number of spermatids per gram parenchyma was lower (P less than 0.05) in tail-suspended rats compared with control rats. In contrast, spermatogenesis was not affected by caudal elevation in most rats in which the inguinal canal was ligated or in rats elevated by whole-body harness. Concentrations of testosterone in serum and testicular interstitial fluid were lower (P less than 0.05) in suspended rats, regardless of the method used for caudal elevation or anatomic location of testes. Concentrations of luteinizing hormone in serum were elevated (P less than 0.05) in rats with intra-abdominal testes.(ABSTRACT TRUNCATED AT 250 WORDS)

A fragment of prosaposin (SGP-1) from rooster sperm promotes sperm-egg binding and improves fertility in chickens.

A protein isolated from the supernatant of cryopreserved rooster sperm was found to increase the capability of cryopreserved rooster sperm to bind in vitro to the perivitelline membrane of a chicken egg and substantially raise fertility after artificial insemination (AI). That activity was partially purified and termed universal primary sperm-egg binding protein (UPSEBP). Insufficient protein remained from 6 x 10(11) sperm, despite retention of bioactivity, to allow sequencing. We deduced that the protein may be related to prosaposin (also termed SGP-1, for sulfated glycoprotein-1), and we used published amino acid sequences of prosaposin as a guide for synthesis of peptides. Certain peptides were found to increase in vitro sperm-egg binding and increase fertility of frozen-thawed or fresh rooster sperm, in a manner similar to semipurified UPSEBP. Active epitopes were in a 60 amino acid sequence, reflecting the intervening sequence between saposins A and B, plus short extensions into saposins A and B. Highest activity was found when this synthetic peptide was oxidized to form a disulfide bond between terminal cysteines. Antibody against a synthetic peptide consisting of 58 of these 60 amino acids bound to a 7-9 kilodalton protein in UPSEBP. Collectively, the data support the conclusion that UPSEBP is a fragment of prosaposin. Because prosaposin is in semen in humans and animals, these observations have broad implications for possible cause and therapy of one type of subfertility.

Maintenance of bioenergetic balance in sperm and prevention of lipid peroxidation: a review of the effect on design of storage preservation systems.

Effective use of encapsulated sperm requires careful review of: (a) the conditions under which the procedure can be effectively used; (b) assessment of the effect of storage conditions on sperm survival; (c) description of the environment of the female tract before, during and after capsule deposition; and (d) economic evaluation of impact and costs of the putative technology. Sperm survival depends on successful sustenance over two periods of storage (at subambient temperatures after collection and extension, then at body temperature when placed in the female tract) and one period of action (after release and until fertilization). The bioenergetic requirements of cauda epididymal and ejaculated bull and ram sperm are reviewed in terms of absolute ATP needs and are discussed in terms of storage needs. In addition, sperm inactivation by lipid peroxidation is discussed and suggestions are provided to minimize the process. Two general types of containers are possible. An open porous form allows free passage of nutrients and metabolic products; the entrapped sperm are thus subjected to the changing environment in the female tract. The other form is a sealed capsule that opens to release sperm before ovulation; it provides a sperm storage environment independent of female tract chemistry but introduces problems of nutrient supply and metabolite release. Potential experimental approaches to evaluate each type of system are discussed.

The epididymis and sperm maturation: a perspective.

In common mammals, sperm leaving the testis are incapable of fertilizing a female gamete. Sperm have limited biosynthetic capability and need to minimize demand for ATP. Hence, modification of sperm to achieve their maturation requires pre-programmed cleavage of integral molecules (planned self-modification) and remodelling by action of molecules found in the suspending fluids. Most of these biocatalysts are secreted by a series of specialized regions in the epididymal epithelium, but some are provided in seminal plasma. The role of the epididymis in sperm maturation is postulated to be 'setting a series of triggers' each capable of initiating cellular changes either at emission or near or in the oocyte, and 'setting a safety' for each trigger to prevent premature occurrence of the event. The attributes required in a spermatozoon for in vitro fertilization and natural mating are different, and their expression is dependent on the site of sperm sampling. Some attributes needed for fertility are probably like an on-off switch, whereas others probably allow a gradually reduced probability of success before going to the off position (analogous to a conventional light switch and a dimmer-type light switch). All essential attributes of a spermatozoon must be expressed in a 'combined effective amount' for that cell to be fertile. Because of mixing, in any segment of the epididymal duct the population of sperm is heterogeneous in age and biological status. Thus, when assessing sperm maturation it is necessary to establish the proportion of sperm that has completed and retained all steps of maturation necessary to achieve fertilization of oocytes under the conditions imposed. In a normal animal, most sperm leaving the epididymis have a 'combined effective amount' of attributes, and the population has a high fertilizing potential.

Changes in epididymal protein anti-agglutinin on ejaculated boar spermatozoa during capacitation in vitro.

This study is a detailed investigation of changes in epididymal protein anti-agglutinin on ejaculated boar spermatozoa during an incubation designed to promote capacitation in vitro. Ejaculated spermatozoa were collected from six mature boars, washed, and incubated to promote capacitation. Sperm samples were subjected to Western blotting-densitometric analyses, flow cytometry after immunostaining and immunocytochemical observation by indirect immunofluorescence. An antiserum to anti-agglutinin was raised in a rabbit by subcutaneous injection of a purified antigen, as described previously (Harayama et al. 1999). Western blotting-densitometric analyses revealed an approximate halving of the amount of sperm-bound anti-agglutinin during the first 45-min incubation, followed by a gradual decrease thereafter. Comparison between immunostained sperm samples by flow cytometry before and after incubation confirmed this decrease in sperm-bound anti-agglutinin during the incubation. Microscopic characterization established that this decrease occurred mainly on the acrosome. Supplementation with seminal plasma (5% or 10%, v/v) attenuated the decrease. These findings are consistent with the conclusion that a large portion of the anti-agglutinin bound to sperm acrosomes is released at an early stage of the capacitation process in vitro.

Evaluation of plasma membrane stability by detergent-induced rupture of osmotically swollen sperm.

A general assay for plasma membrane stability was developed and tested. Osmotically swollen spermatozoa were ruptured with detergents and their volume distribution was monitored with resistance pulse spectroscopy. The extent of cell breakage was determined and expressed as [D]50, the concentration of detergent necessary to lyse 50% of the initially intact cells. Preliminary experiments established the degree to which spermatozoa could be swollen without lysis (no detergent) and the ability of the method to detect known mixtures of intact and membrane disrupted spermatozoa. [D]50 values were determined for caput (immature) and cauda (mature) ram epididymal spermatozoa with four detergents (cetyltrimethylammonium bromide, sodium dodecylsulfate, Zwittergent 3-14, and sodium deoxycholate). [D]50 values for caput spermatozoa were higher than those for cauda spermatozoa (P less than 0.05) for all detergents but cetyltrimethylammonium bromide. These changes are consistent with a qualitative model of membrane structure and stability based on lipid shape and composition and with the compositional changes known to occur during epididymal maturation. Additional studies using rooster spermatozoa established that a typical cryopreservation protocol leaves the surviving spermatozoa with membranes with greater sensitivity to detergent-induced stress. Since osmotic swelling has been microscopically localized to the tail plasma membrane, the changes in membrane stability can be assigned specifically to that region.

A calorimetric method to assess endogenous metabolism and its application to the study of bovine sperm.

Calorimetry was used to assess the importance of endogenous metabolism towards total ATP synthesis in bovine sperm in the presence of extracellular glucose. Sperm were incubated in the calorimeter with D-[U-14C]glucose without or with electron transport inhibitors, rotenone and antimycin A. Steady-state heat production during the incubations was measured for 30 min, the incubations were terminated, and the cell suspensions removed for analysis of radioactive glucose and its metabolic end-products. Heat production (mean +/- S.E.) associated with the metabolism of glucose was calculated, from enthalpies of formation of glucose and its end-products, as -412 +/- 34 mJ/h/10(8) cells in control incubations and -263 +/- 18 mJ/h/10(8) cells in the incubations with electron transport inhibitors. Measured heat production was -455 +/- 36 and =263 +/- 17 mJ/h/10(8) cells, respectively. Thus, heat production by endogenous pathways, the difference between measured total heat production and calculated exogenous heat production, was -43 +/- 14 mJ/h/10(8) cells for control cells and about -6 mJ/h/10(8) cells for inhibited cells. The ratio of heat produced per mol of ATP synthesized is similar for all ATP-producing pathways. Therefore, about 10% of total ATP synthesis in control cells and less than 2% in inhibited cells is provided by endogenous pathways when extracellular glucose is present.

Porcine focal symmetrical poliomyelomalacia: experimental reproduction with oral doses of encapsulated sodium selenite.

Sodium selenite (encapsulated as doses of 1.4 mg, 2.6 mg and 4.2 mg per kilogram of body weight) was given orally on a daily basis to male weaner pigs, and features of these animals were compared to a control group. Porcine focal symmetrical poliomyelomalacia was produced in all experimental groups between 3 and 20 days after initiation of the treatment. Analysis of blood and several tissues revealed an elevated selenium content for all pigs. Histological lesions in the brain and the cervical lumbar/sacral spinal cord enlargements included endothelial proliferation, neuronal degeneration, microcavitation and glial cell reaction.

Porcine focal symmetrical poliomyelomalacia: test for an interaction between dietary selenium and niacin.

Experiments were conducted to test the hypothesis that dietary supplementation with nicotinamide would retard or eliminate the signs of selenium induced porcine focal symmetrical poliomyelomalacia (PFSP). Mixed-sex feeder pigs, approximately five weeks old, were divided into four groups and daily received, by oral capsule, the following treatments: no supplementation (control); 2.86 mg sodium selenite per kg body wt (selenium only); 44 mg nicotinamide per kg body wt (niacin only); or both the niacin and selenium (niacin + selenium). Over the ten day treatment body weights and behavior scores were recorded, as well as collection of fluid (blood, serum, urine) samples. Upon death, tissue samples (kidney, liver, brain, spinal cord and muscle) were obtained. All of these samples were analyzed for total selenium and bioactive niacin compounds. After gross pathological analysis, 11 samples from specific brain and spinal cord regions were taken for fixation and processing for histological analysis by light microscopy. The selenium only group showed behavior signs related to PFSP after two days of treatment with the average time of death at 6.5 days. Tissue levels of selenium were elevated and histological analyses established the expected lesions of PFSP. No disorders were noted in the control and niacin only groups. The niacin + selenium groups had slightly retarded changes in behavior scores (first differences from controls on day 4) but their mean day of death (7.5 days of treatment) did not differ from that of the selenium only groups. Histological analyses of these tissues revealed similar lesions to the selenium only group, but they may have been of lesser magnitude. The data were consistent with, but only partially supportive of, the above hypothesis.

Symposium summary and challenges for the future.

The poultry industry has grown and prospered over the past 50 yr by a repeated pattern of careful analysis of factors limiting production, followed by replacement of biological functions with management practices. Examples include assisted incubation, selection of sires, and survival via novel housing. Each resulted in a period of enhanced product output. Trends developing over the past decade raise the potential for consideration of another intervention, that of assisted reproduction. Examples illustrating the need to consider, and adopt at several levels, assisted reproduction are provided. Three critical aspects of poultry production should be monitored by careful documentation of: 1) genetic throughput from pedigree to product, best assessed by monitoring number of chicks produced per male; 2) product cost, best assessed by optimizing rate of lay and fertility of laid eggs for each hen; and 3) product quality, reflected in the homogeneity of progeny for desired traits. Each segment of the industry (turkey, egg or broiler; breeder or producer) will find unique solutions to these interacting factors. Presentations within the symposium are reviewed and integrated, and comments are provided relative to challenges facing the industry in the 21st century.