Flow sorting of X and Y chromosome-bearing mammalian sperm: Activation and pronuclear development of sorted bull,boar, and ram sperm microinjected into hamster oocytes

Flow cytometric techniques were used to measure relative DNA content of X and Y chromosome-bearing bull, boar, and ram sperm populations and to separate the two sex-determining populations. Neat semen was prepared for flow cytometric analysis by washing, light sonication, and staining with 9 μM Hoechst 33342. Computer analysis of the bimodal histograms showed mean X-Y DNA differences of 3.9, 3.7, and 4.2% for bull, boar, and ram, respectively. Flow cytometric reanalysis of sorted bull, boar, and ram sperm showed purities greater than 90%. Bull, boar, and ram sperm nuclei were microinjected into hamster oocytes. Microinjected sperm were either unsorted, sorted, unsorted plus dithio-threitol (DTT) exposure, or sorted plus DTT exposure. Following microinjection, eggs were incubated 3 hr, fixed, and stained. A total of 579 eggs was observed for sperm activation (decondensation or formation of a male pronucleus). A lower percentage of sorted than unsorted (3 vs. 23%) boar sperm was activated (P <.05). However, sorted and unsorted DTT-exposed boar sperm or sorted and unsorted bull or ram sperm, regardless of DTT treatment, did not differ significantly. Sorted sperm nuclei of both rams and bulls exhibited higher activation rates than sorted boar sperm (P <.05). Treatment of sperm with DTT increased the activation rate (P < .05) for sorted boar sperm but not for bull or ram sperm. These data represent the first separation of bull, boar, and ram X and Y chromosome-bearing sperm populations and the first evidence that sperm of domestic animals sorted on the basis of DNA by flow cytometric procedures have the ability to decondense and to form pronuclei upon injection into a hamster egg.     

Influence of boar acrosin antibodies produced in rabbit and sheep on chymotrypsinogen activation catalyzed by acrosin from boar, bull, ram, rabbkt and human.

Activation of bovine chymotrypsinogen is catalyzed with increasing velocity by human, rabbit, boar, bull and ram acrosin. Antiboar-acrosin rabbit gamma-globulins cause a significant reduction in the proenzyme activation rate induced by boar and bull acrosin, but only a weak reduction or none if ram or rabbit acrosin is the activating agent. The antiboar-acrosin gamma-globulins from sheep strongly inhibit chymotrypsinogen activation by ram, bull and boar acrosin, and significantly inhibit the human acrosin-catalyzed reaction.     

The phospholipid-binding protein of the reproductive tract of the bull — Effect on the removal of spermatozoal cytoplasm droplets in other species and influence of antibodies on its reactivity

The phospholipid-binding protein (PBP) isolated from bull seminal vesicle fluid removed cytoplasm droplets not only from bull, but also from ram, boar and rabbit epididymal spermatozoa. However, the presence of a protein cross-reacting with anti-PBP antisera was demonstrated by immunofluorescent staining in ram seminal vesicles and ampullae. In contrast to PBP from bull, the ram PBP-like protein did not lyse bull or ram erythrocytes. Rabbit antiserum against PBP only negligibly reduced the ability of PBP to remove cytoplasm droplets from bull epididymal spermatozoa, but it inhibited the haemolytic effect of the protein.     

The occurrence of polyenoic fatty acids with greater than 22 carbon atoms in mammalian spermatozoa.

Fatty acids with carbon chain lengths greater than 22 (VLCFA) have been detected in boar, ram, bull and human spermatozoa. Saturated and mono-unsaturated fatty acids were present in all spermatozoa but, except for human spermatozoa, polyenoic fatty acids were quantitatively the most important components. Marked differences in polyenoic fatty acid composition were observed. Whereas human spermatozoa contain predominantly di-, tri- and tetraenoic fatty acids with up to 32 carbon atoms, boar, ram and bull spermatozoa also contain pentaenoic and/or hexaenoic acids with up to 34 carbon atoms. Human and boar spermatozoa differ markedly from those of the ram and bull in that only n-6 series acids are present.     

Testosterone and dihydrotestosterone concentrations in elephant serum and temporal gland secretions

Serum and temporal gland secretions (TGS) were obtained from mature wild African (Loxodonta africana) and captive Asian elephants (Elephas maximus). Samples were obtained from five cows and eight bulls culled for management purposes in Kruger National Park, South Africa, and from four females and two males residing at the Washington Park Zoo, Portland, Oregon. Our purpose was to describe the levels of the androgens, testosterone (T), and dihydrotestosterone (DHT), and to correlate these observations with sex, species and behavioral status. Male-female differences in serum T were pronounced in the Asian species, whereas male and female concentrations overlapped in the African elephant serum. Serum T concentrations in African females were greater than in Asian females. Serum DHT reflected T levels, except that the striking elevation of testosterone in Asian bulls during musth was not paralleled by equal increases in DHT levels. A species difference observed among males was higher serum T levels in nonmusth Asian bulls (1.84-5.35 ng/ml) compared to the levels in African bulls (0.38-0.68 ng/ml), except for one dominant African bull (6.64 ng/ml). This single African value was still considerably lower than the serum T values of the Asian males during musth. These musth values were the highest serum androgen concentrations: T was between 19 and 40 ng/ml (average 26.10 ng/ml). The TSG values of T and DHT were much higher than serum levels except in the Asian female. T/DHT ratios in TGS were more similar than in serum. One dominant African bull had a T TGS value of 78 ng/ml, which was much higher than the rest of the African males or females, but considerably lower than as Asian bull in musth (547 ng/ml). It seems apparent that a change in androgen status as reflected in serum and TGS levels of T and DHT precedes or is concomitant with overt alteration in behavior in the Asian male. The temporal gland appears to actively concentrate androgens in both African males and females, but in the Asian male the gland secretes only during musth when the greatest concentration of both T and DHT were observed. The apparent difference in the degree of temporal gland secretory activity between the two species suggests a more specific communicative function within the Asian male.     

Measurement and manipulation of cytoplasmic free calcium of ram and boar spermatozoa using quin 2

The highly selective fluorescent Ca2+ indicator 'quin 2' has been loaded into ram and boar spermatozoa as the acetoxymethyl ester, 'quin 2/AM', which is hydrolysed and trapped in the cytoplasm. Loadings of several mM were not toxic to spermatozoa as judged by motility. Fluorescence measurements (mean +/- S.E.M.) indicated a normal cytoplasmic free-calcium concentration, [Ca2+]i, of 193 nM +/- 0.2 (n = 10) for ejaculated ram sperm, 175 nM +/- 3.9 (n = 10) for cauda epididymal boar sperm and 105 nM +/- 10 (n = 10) for the caput sperm. After cold shock ejaculated ram and cauda epididymal boar sperm did not retain quin 2, due presumably to structural damage. However, cold shocked caput boar sperm could be readily loaded with quin 2 and had a [Ca2+]i similar to control sperm. Sodium azide, propranolol and caffeine did not affect the [Ca2+]i of ram and boar sperm, however theophylline, dibutyryl c-AMP and La3+ significantly reduced it. The inhibitors rotenone and antimycin A, and the uncouplers 2,4-DNP and CCCP caused a transient elevation of [Ca2+]i, most likely resulting from release of mitochondrial calcium. The increased [Ca2+]i following addition of the ionophore A23187, was highly pH dependent in ram spermatozoa and it was critical to increase the pH of the medium above 7.5; the increase in [Ca2+]i was apparently not dependent on the oxidative metabolism of the sperm as addition of the uncouplers 2,4-DNP and CCCP had no effect on [Ca2+ )i. Addition of filipin to ram and boar sperm resulted in a large increase in [Ca2+]i but addition of filipin to ionophore-treated sperm caused [Ca2+]i to fall well below control levels.     

Percentage binding of testosterone and dihydrotestosterone and unbound testosterone and dihydrotestosterone in rabbit maternal and fetal plasma during sexual organogenesis.

The percentages of bound testosterone (17 beta-hydroxy-4-androsten-3-one; T) and dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one; DHT) and their unbound concentrations were determined in pregnant rabbits and their fetuses from the 18th day of gestation to birth. T and DHT were also measured in fetal testes. In the testis, the total T/total DHT ratio, very high at 22 days (73.7 +/- 15.2), decreased until birth (6.7 +/- 0.8). In male fetuses the concentrations of total and unbound circulating T and DHT were always low and did not show any peak during sexual organogenesis. The percent binding of T (from 73.0 +/- 0.5 to 77.6 +/- 0.6) and DHT (from 76.5 to 83.7 +/- 1.1) in fetuses were similar in both sexes and significantly lower than those measured in mothers (T: from 87.2 +/- 0.6 to 91.6 +/- 0.9; DHT: from 87.3 +/- 0.9 to 93.8 +/- 0.9).     

Lipid dynamics in the plasma membrane of ram and bull spermatozoa after washing and exposure to macromolecules BSA and PVP.

Seminal plasma proteins and macromolecules in the external medium have a major influence on the functionality of sperm plasma membranes. In this investigation we have examined their effects on lipid diffusion in the surface membrane of ram and bull spermatozoa as measured by fluorescence recovery after photobleaching (FRAP). Results show that progressive removal of seminal plasma from ram spermatozoa by repeated centrifugation and resuspension in media +/- 4% bovine serum albumin (BSA) or 0.4% polyvinlypyrrolidone (PVP) causes a reduction in lipid diffusion in all regions of the membrane. By contrast, bull sperm membranes respond with an increase in diffusion in all regions. Repeated washing of bull spermatozoa whose membranes were previously immobile (i.e., showed no recovery after FRAP) restored lipid diffusion suggesting an inhibitory effect of seminal plasma proteins. Further analysis by atomic force microscopy revealed a close association between BSA and the plasma membrane. It is concluded that diffusion of lipids in the plasma membrane of ejaculated ram and bull spermatozoa is influenced by seminal plasma proteins and the composition of the suspending medium. Mol. Reprod. Dev. 59:306-313, 2001.     

Relation between the oxidation state of nicotinamide–adenine dinucleotide and the metabolism of spermatozoa

1. The existing procedures for extraction of oxidized and reduced nicotinamide coenzymes were adapted to spermatozoa to overcome the coenzyme-degrading activity of seminal plasma. 2. The content of total NAD(+) and NADH was determined in the spermatozoa of ram, bull, boar, stallion and cock. NADP(+) and NADPH were not detected in ram spermatozoa. 3. The oxidation state of sperm NAD depended on the seminal plasma, the removal of which produced a change in the percentage oxidation state of the coenzyme, 100x[NAD(+)/(NAD(+)+NADH)], without altering the total content of NAD(+)+NADH. 4. In suspensions of washed ram spermatozoa, incubated anaerobically at 25 degrees C, the percentage oxidation state of NAD declined with increasing spermatozoa concentration. 5. When ram or boar spermatozoa that had been previously washed and resuspended in Ringer phosphate medium, were incubated anaerobically at 25 degrees C with various substances, pronounced effects on the percentage oxidation state of NAD could be observed with l-lactate, pyruvate, oxaloacetate, dihydroxyacetone, formaldehyde and glyceraldehyde; sorbitol and acetoacetate acted only on ram spermatozoa; fructose, glucose, mannose and acetaldehyde acted predominantly on boar spermatozoa. Formaldehyde lowered the (NAD(+)+NADH) content of ram spermatozoa, but none of the other substances had a comparable effect. 6. The percentage oxidation state of sperm NAD was not influenced by exogenous cysteine, cystine, ergothioneine or ascorbate. 7. A highly active sorbitol dehydrogenase could be prepared from ram, but not from boar, spermatozoa. 8. Sorbitol, acetoacetate and 3-hydroxybutyrate effectively supported the respiration of ram, but not boar, spermatozoa. 9. ;Cold shock', resulting from sudden cooling of spermatozoa, abolished motility completely and irreversibly but produced only a slow and partial decrease in the total NAD content. Slight over-heating, sufficient to produce loss of motility, had no adverse effect on the total NAD content. 10. Storage of ram sperm at 14 degrees C produced only a small decrease of NAD after 2 days, but subsequently the loss became greater.     

Multiple forms of ram and bull sperm hyaluronidase revealed by using monoclonal antibodies

Two monoclonal anti-sperm hyaluronidase-producing cell lines were isolated following inoculation of mice with ram sperm hyaluronidase monomer. Both lines produced antibodies of the IgG1 class; these bound to ram hyaluronidase after 'Western blotting' but did not recognize the native enzyme. Whereas the 1A4 antibody was specific for ram hyaluronidase, and did not react with 'blotted' bull, boar or rabbit hyaluronidase, the 1D6 antibody recognized bull as well as ram hyaluronidase. The antibodies could be used for immunocytochemical localization of hyaluronidase in fixed spermatozoa. However, although some form of denaturation was required to unmask or form the epitopes with which the antibodies reacted, the degree and type of fixation required was critical, for the epitopes were readily destroyed; in particular, they were very sensitive to chemical modification such as glutaraldehyde treatment. It could be demonstrated that, like ram, bull spermatozoa contained an extended oligomeric family of hyaluronidase forms, apparently the result of intermolecular disulphide cross-linking of monomers. In spermatozoa of both species, the enzyme was confined to the anterior acrosomal region of the head.     

Glycolytic enzymes in mammalian spermatozoa. Activities and stabilities of hexokinase and phosphofructokinase in various fractions from sperm homogenates

1. Methods of homogenizing suspensions of washed mammalian spermatozoa were studied. The most useful methods were those using sonication and those using a French press. 2. Hexokinase, phosphofructokinase, glucose phosphate isomerase and adenosine triphosphatase activities in ram, bull and boar spermatozoa were investigated by using these two homogenization methods. Glucose phosphate isomerase, representative of soluble cytoplasmic material, was very readily extracted and remained entirely in the supernatant after centrifugation at 145000g for 60min. In contrast, the other three activities were less easily extracted and were sedimented in various proportions under the described conditions of centrifugation. 3. Attempts to obtain subcellular fractions from sperm homogenates by ;classical' methods failed, owing apparently to the inhomogeneity of subcellular particles in the homogenates. It is concluded that, after removal of sperm heads, the only meaningful fractionation is a separation of spermatozoal material which sediments at 145000g during 60min from that which does not. 4. The stabilities of hexokinase and phosphofructokinase activities in bull, boar and ram sperm homogenates were investigated. Hexokinases showed very little dependence on the various environments tested, whereas the optimum conditions for phosphofructokinase stability were: a minimum of sonication, the presence of phosphate ions and of a thiol-group protectant, and a pH7.5. Activities of hexokinase, phosphofructokinase and glucose phosphate isomerase per sperm cell were compared with published data on rates of fructolysis by spermatozoa; the potential catalytic activities were shown to be considerably in excess of these rates. However, phosphofructokinase may be the rate-limiting enzyme of glycolysis in vivo in bull and ram spermatozoa.     

Penetration of zona-free hamster eggs by liposome-treated sperm from the bull, ram, stallion, and boar

Spermatozoa from each of four rams, four stallions, and three boars (six semen samples) were treated with dilauroylphosphatidylcholine (PC12) liposomes and compared with control bull sperm to induce the acrosome reaction (AR) and study possible penetration of the sperm into zona-free hamster eggs. Diluted sperm were incubated with several concentrations of PC12 for 7 min at 39 degrees C prior to insemination of the hamster eggs in vitro. The sperm from the bull were diluted to 10(6) cells/ml, as previously studied. Sperm from the ram, stallion, and boar were diluted to 6 X 10(6) and 20 X 10(6) cells/ml. After addition to the eggs, the sperm concentration was reduced by 75 percent. Inseminated eggs were incubated with sperm for 3 h at 39 degrees C prior to being fixed, stained, and observed for sperm penetration. At an initial concentration of 6 X 10(6) cells/ml, bull sperm treated with 36.7 microM PC12 achieved an egg penetration rate of 92%, whereas under nearly identical conditions stallion spermatozoa achieved only 54% egg penetration. Under similar conditions, ram spermatozoa failed to penetrate eggs, but when the initial sperm concentration was increased to 20 X 10(6) cells/ml, sperm incubated with 51.1 microM PC12 achieved 52% egg penetration. Boar spermatozoa treated with PC12 at either sperm concentration failed to exhibit an AR or penetrate hamster eggs. In general, as PC12 concentration increased the percentage of sperm with an AR increased and sperm motility decreased. It is concluded that 1) PC12 liposomes are effective in inducing the AR in sperm from the bull, ram, and stallion, but under conditions tested are ineffective with boar sperm;(ABSTRACT TRUNCATED AT 250 WORDS)     

Self-administration of estrogen and dihydrotestosterone in male hamsters

Anabolic-androgenic steroids (AAS) are drugs of abuse. Previous studies have shown that male and female hamsters self-administer testosterone (T) and other AAS, suggesting that androgens are reinforcing in a context where athletic performance is irrelevant. AAS are synthetic derivatives of T, which may be aromatizable to estrogen and/or reducible to dihydrotestosterone (DHT). However, we do not know which metabolites of T are reinforcing. To determine if DHT, estradiol (E(2)), or DHT + E(2) are reinforcing, we tested intracerebroventricular (icv) self-administration in male hamsters. The hypothesis was that androgen reinforcement is sensitive to both androgenic and estrogenic T metabolites. If so, hamsters would self-administer DHT, E(2), and DHT + E(2). Twenty four castrated male hamsters (n = 8/group) received icv cannulas and sc T implants for physiologic androgen replacement. One week later, hamsters self-administered DHT (0.1, 1.0, 2.0 microg/microl), E(2) (0.001, 0.01, 0.02 microg/microl), or DHT + E(2), each for 8 days in increasing concentration (4 h/day). Operant chambers were equipped with an active and inactive nose-poke. At the medium concentration, hamsters self-administered DHT (active nose-poke: 47.9 +/- 13.9 responses/4 h vs. inactive: 18.7 +/- 4.8), E(2) (active: 44.8 +/- 14.9 vs. inactive: 16.6 +/- 2.6), and DHT + E(2) (active: 19.1 +/- 2.4 vs. inactive: 10.4 +/- 2.4, P < 0.05). At the highest concentration, males self-administered DHT (active: 28.3 +/- 7.7 vs. inactive: 15.0 +/- 3.5, P < 0.05) and DHT + E(2) (active: 22.6 +/- 3.8 vs. inactive: 11.6 +/- 2.5, P < 0.05), but not E(2). Hamsters did not self-administer the lowest concentrations of DHT, E(2), or DHT + E(2). These results support our hypothesis that both androgenic and estrogenic T metabolites are reinforcing. Together, they do not exert synergistic effects.     

Radiorespirometric studies of carbohydrate metabolism by washed spermatozoa of various species.

1. The patterns of 14CO2 evolution from specifically labeled glucose substrates by washed bull, ram, boar, rabbit, dog, rooster and turkey spermatozoa were similar and indicated the Embden-Meyerhof and Kreb's cycle pathways as the major route of energy metabolism. 2. Honey bee spermatozoa metabolized glucose-3,4-[14C], glucose-[U-14C] or fructose-[U-14C], but not glucose-1-[14C], glucose-2-[14C]or glucose-6-[14C], indicating the presence of the glycolytic pathway, but the absence of respiration via the Kreb's cycle. 3. The rate of glycolysis exceeded the rate of respiration in the spermatozoa of all the species studied. 4. A preferential utilization of glucose-1-[14C] over glucose-6-[14C] was evident in some sperm samples, but no consistent indication of pentose cycle metabolism was observed, due to considerable variability between samples within each group. 5. Fructose metabolism was greater than glucose metabolism in the rooster, less in the dog, boar and turkey, and similar in the spermatozoa from the other species examined. 6. Only ram and bull spermatozoa metabolized acetate-1-[14C] to any extent.     

Determination of testosterone and its tissue metabolites (DHT and 3 alpha-diol) in human plasma and prostatic tissue by isotopic dilution mass spectrometry

5 alpha-Dihydrotestosterone has been widely measured in human prostatic tissue using RIA since it is involved in the pathogenesis of human prostatic hyperplasia and seems to be the best index for the follow-up of patients affected by prostatic cancer under endocrine treatment. A GC-MS method for the simultaneous determination of testosterone (T), 5 alpha-dihydrotestosterone (DHT) and 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha-diol) in prostatic tissue based on the isotopic dilution technique was developed. Tri-deuterated internal standards of each compound were previously synthetized in our laboratory. After extraction and purification on Sep-Pak C18 and Sephadex LH-20, T and its metabolites were measured as heptafluorobutyric ester (HFB) derivatives. Quantitative analysis was performed on a VG 7070 EQ mass spectromer equipped with a fused silica capillary column using the Selected Ion Monitoring technique. Steroid values (mean +/- SD; ng/g tissue) found in nine human hypertrophic prostates were: T: 0.71 +/- 0.43; DHT: 4.46 +/- 1.41; 3 alpha-diol: 0.34 +/- 0.23. Preliminary results obtained from the detection of the three androgens in human prostatic hyperplasia treated for 3 months with GnRH before surgery seem to indicate that DHT concentration decreases more than 10 times. Values obtained (n = 1; ng/g tissue) were: T: 0.194; DHT: 0.255; 3 alpha-diol: 0.015.     

Proacrosin and the differentiation of the spermatozoa

Using the indirect immunofluorescence staining technique, the occurrence and localization of proacrosin, the zymogen form of acrosin, was studied during spermatogenesis in the bull, ram, boar and rabbit. Proacrosin staining was demonstrable for the first time in the early haploid spermatid and increased with the differentiation of the spermatid to spermatozoon. The spermatozoon is covered by a cap-like structure of uniform fluorescence corresponding to the acrosomal compartment of the male gamete. No fluorescence could be found in diploid spermatogenic cells, i.e., in spermatogonia and spermatocytes. An identical developmental pattern of proacrosin was observed with the indirect immunoperoxidase staining technique. However, with this staining technique a distinct distribution of proacrosin staining was observed in the acrosome of epididymal and ejaculated spermatozoa of the bull, ram, boar, rabbit and man. Proacrosin seems to be distributed in the acrosome in granules rather than in the homogeneous form, as was indicated by the results of indirect immunofluorescence staining.     

Immunofluorescent localization of acrosin in mammalian spermatozoa (1).

Acrosin was detected by immunofluorescence in the spermatozoan acrosomes of artiodactyla (bull, ram and boar), perissodactyla (horse), carnivora (dog and cat), lagomorpha (rabbit) and primates (human) using anti-bovine acrosin immunoglobulins. The results indicate that the acrosin molecules of several mammalian species possess antigenic similarities.     

Effect of ionic strength, serum albumin and other macromolecules on the maintenance of motility and the surface of mammalian spermatozoa in a simple medium

The seminal plasma in sperm suspensions from boar, bull, rabbit, ram and stallion was replaced with simple defined media as completely as possible by a combination of centrifugation through Ficoll and dilution. After this process, motility declined and the cells showed a tendency to agglutinate and/or stick to glass. Varying the ionic strength of the medium had little effect upon these parameters but sperm motility was preserved better in the presence of serum albumin. When a number of purified proteins and other macromolecules were tested individually in this way for their motility-preserving ability, bovine or human serum albumin was consistently the most effective. Defatting the albumin or altering its nature by mild reduction, oxidation or alkylation had little detectable effect on its motility-preserving ability; the protein did not appear to be acting as a chelator of metal ions, for it could not be replaced by EDTA. The response of the spermatozoa to replacemrnt of seminal plasma varied between species: bull spermatozoa were particularly sensitive and serum albumin had little effect upon their subsequent motility.     

Estimation by gas chromatography-mass spectrometry with selected ion monitoring of urinary excretion rates of 3 alpha-androstanediol during/after i.v. administration of 13C-labelled testosterone in man

To study in vivo the conversion of testosterone (T) into its metabolites, dihydro-testosterone (DHT) and 5 alpha-androstane-3 alpha, 17 beta diol (3 alpha-Diol) the urinary excretion rates of these steroids were determined by mass spectrometry in 6 healthy men during/after the i.v. infusion (t = 4 h) of 20 mg [13C]testosterone. In addition, plasma concentrations of T, DHT and 3 alpha-Diol were determined by radioimmunoassay. During steady state conditions at the end of the 4-h infusion of [13C]T the increase in the plasma concentrations of T from, basal, 405 +/- 140 ng/dl to 4205 +/- 804 ng/dl was paralleled by an increase in the plasma concentrations of DHT to 106.4 +/- 62.5 ng/dl) (basal: 30.8 +/- 21.8 ng/dl), and of 3 alpha-Diol to 32.2 +/- 12.5 ng/dl (basal: 12.5 +/- 13.9 ng/dl). Plasma concentrations of T, DHT and 3 alpha-Diol then returned to basal concentrations within 24 hours. Using mass-spectrometry we found a cumulative renal excretion of 13C-labelled T of 15.6 +/- 9.6 micrograms/24 h, equivalent to 0.08 +/- 0.05% of the infused amount (20 mg) of [13C]T. Whereas urinary excretion of [13C]DHT was below the level of detection by mass-spectrometry the cumulative excretion of [13C]3 alpha-Diol was 67.7 +/- 19.9 micrograms/24 hours which is equivalent to 0.3 +/- 0.1% of the infused dose of 13C-labelled testosterone. These data suggest that the determination of urinary 3 alpha-Diol by mass-spectrometry during/after the infusion of stable-labelled testosterone represents an alternative to the use of radioactive label for turnover studies.