Showing papers in "Journal of Andrology in 1992"

Reactive oxygen species and human spermatozoa. I. Effects on the motility of intact spermatozoa and on sperm axonemes.

Abstract: Mammalian spermatozoa are sensitive to oxygen-induced damages mediated by lipid peroxidation of the cell membrane. The aim of this study was to evaluate whether reactive oxygen species (ROS) could also induce axonemal damage. When Percoll-separated spermatozoa were treated with hydrogen peroxide, or the combination xanthine and xanthine oxidase (X + XO), there was a progressive decrease, leading to a complete arrest, in sperm flagellar beat frequency. Once demembranated in a medium containing magnesium adenosine triphosphate (Mg.ATP), ROS-immobilized spermatozoa still reactivated motility; however, the percentage and duration of motility obtained in these tests gradually decreased to zero in the next hour. In 50% of the cases, motility of intact spermatozoa spontaneously reinitiated after 6 to 24 hours of immobilization due to ROS treatment, although with percentages and beat frequencies lower than those of untreated spermatozoa. Studies using ROS scavengers (such as catalase, superoxide dismutase, and dimethylsulfoxide) indicated that hydrogen peroxide was the most toxic of the ROS involved, but that .O2- and .OH probably also played a role in immobilization of spermatozoa by ROS. The data suggest that ROS induce a chain of events leading to sperm immobilization, that axonemes are affected, and that limited endogenous repair mechanisms exist to reverse these damages.

Evidence for increased lipid peroxidative damage and loss of superoxide dismutase activity as a mode of sublethal cryodamage to human sperm during cryopreservation.

Abstract: Cryopreservation of human sperm, now generally required in donor insemination programs, adversely affects the sperm in terms of standard sperm evaluation parameters and fertilizing ability. The freeze-thaw process appears to produce sublethal damage that appears only after a delay. The authors hypothesized that cryopreservation enhanced peroxidation of sperm membrane lipids, based on previous studies of sperm lipid peroxidation, which showed that the effects of peroxidative damage became evident only after a delay, depending on the peroxidation rate. The effect of cryopreservation on the phospholipid content, the composition of the acyl moieties of the phospholipids, and the activities of the peroxidation protective enzymes, superoxide dismutase (SOD) and glutathione peroxidase plus reductase, in human sperm were examined to test the hypothesis. Parallel determinations were made of the percent motility, the average path velocity of the motile cells, and the time to loss of motility under specified aerobic incubation conditions, which gives a good estimate of the lipid peroxidation rate. The phospholipid content decreases after cryopreservation, with loss of phosphatidylcholine and phosphatidylethanolamine being the more pronounced. Polyunsaturated acyl moieties were also preferentially lost. This loss pattern is observed also from lipid peroxidation. The activities of glutathione peroxidase plus reductase remained unchanged. The sperm SOD activities varied widely between samples before cryopreservation. In all samples there was a decline in SOD activity after freeze-thaw, but the extent of the decline was also widely variable. The time to loss of motility declined in parallel with SOD activity, and a strong correlation (R2 greater than 0.9) between SOD activity and time to loss of motility was found for all samples, before and after freeze-thaw. The authors conclude that cryopreservation does enhance lipid peroxidation in human sperm, as hypothesized, and that this enhancement is mediated at least in part by the loss of SOD activity occurring during the process.

Reactive oxygen species and human spermatozoa. II. Depletion of adenosine triphosphate plays an important role in the inhibition of sperm motility.

Abstract: Under moderate conditions, reactive oxygen species (ROS) have been shown to inhibit sperm motility after several hours of incubation The rapid decrease in flagellar beat frequency observed within the first hour of contact between ROS and spermatozoa was associated with a rapid loss of intracellular adenosine triphosphate (ATP) Motility of intact spermatozoa ceased when their ATP concentration was reduced by 85 +/- 5% Axonemal damage was confirmed when ROS-treated spermatozoa could not reactivate motility after demembranation in a medium containing magnesium adenosine triphosphate (MgATP) However, in conditions allowing rephosphorylation of the axonemes (addition of cyclic adenosine monophosphate, or cAMP, and protein kinase or sperm extracts to the demembranation medium), the motility could reactivate Three lines of evidence suggested that ATP depletion induced by ROS treatment was responsible for the effects observed in spermatozoa First, the rapid decrease in intracellular ATP observed after ROS treatment was closely followed by a decrease in beat frequency, loss of intact sperm motility, and axonemal damage due to insufficient phosphorylation Second, incubation of spermatozoa with the combination pyruvate-lactate allowed maintenance of sperm ATP at a normal level and prevented the effects of ROS; furthermore, spermatozoa immobilized after ROS treatment, then supplemented with pyruvate-lactate, were able to reinitiate motility in parallel with an increase in their ATP level Third, treatment of spermatozoa with rotenone, an ATP depleting agent, produced effects similar to ROS treatment and could also be reversed by the addition of pyruvate-lactate These data are consistent with the conclusion that ROS treatment produced axonemal damage mostly as a result of ATP depletion(ABSTRACT TRUNCATED AT 250 WORDS)

Normal and abnormal development of the male urogenital tract. Role of androgens, mesenchymal-epithelial interactions, and growth factors.

Abstract: Androgen-dependent male urogenital development occurs via mesenchymal-epithelial interactions in which mesenchyme induces epithelial morphogenesis, regulates epithelial proliferation, and evokes expression of tissue-specific secretory proteins. Mesenchymal-epithelial interactions continue to be important into adulthood. For example, mesenchyme of the urogenital sinus (UGM) and seminal vesicle (SVM) induce dramatic morphologic and functional changes in various adult epithelia. Since adult epithelial cells are unquestionably responsive to mesenchymes that can elicit expression of alternative morphologic and functional phenotypes, established carcinomas might also be influenced by their connective tissue environment. In this regard, Dunning prostatic tumor has been induced by UGM or SVM to differentiate into tall columnar secretory epithelial cells. This change in cytodifferentiation is associated with a reduction in growth rate and loss of tumorigenesis. The role of soluble growth factors in the mechanism of mesenchymal-epithelial interactions is discussed.

A long-term, prospective study of the physiologic and behavioral effects of hormone replacement in untreated hypogonadal men.

Abstract: This study describes sexual activity, nocturnal penile erections, and mood states as a function of serum levels of androgens in previously untreated hypogonadal men before and during hormone replacement, selected infertile men (elevated serum follicle-stimulating hormone levels), and normal men. Nocturnal penile tumescence and rigidity were measured with a portable monitor, and sexual activity and mood were assessed by prospective, self-reported written forms. Nocturnal erections were absent or of very low amplitude and duration in the untreated hypogonadal men compared to the infertile and normal men. Nocturnal erections increased steadily during hormone replacement and were in the normal range within 6 to 12 months of treatment. In contrast, serum testosterone concentration rapidly reached the upper range of normal. During treatment, the hypogonadal men reported increases in several aspects of sexual activity, including sexual interest and the number of spontaneous erections. On mood inventories, the untreated hypogonadal men scored significantly higher in ratings of depression, anger, fatigue, and confusion than did infertile and normal men. During hormonal replacement therapy these scores decreased, although the hypogonadal men continued to score higher in "depression" than did infertile and normal men. In most instances, the men with infertility and the normal men were statistically indistinguishable in nocturnal penile tumescence and rigidity parameters, self-reported sexual activity, and mood state. These data support the hypothesis that androgen treatment increases nocturnal and spontaneous erections, and sexual interest, and has some capacity to improve mood.

Testosterone and spermatogenesis. Identification of stage-specific, androgen-regulated proteins secreted by adult rat seminiferous tubules.

Abstract: The aim of this study was to identify potential androgen-regulated proteins (ARP) that might mediate the supportive effects of testosterone on spermatogenesis. Adult rats were injected with ethane dimethane sulphonate (EDS) to destroy Leydig cells and thus induce complete testosterone withdrawal. Other EDS-treated rats were injected with 25 mg testosterone esters (TE) every 3 days to maintain quantitatively normal spermatogenesis. A timeframe for the study of androgen action on spermatogenesis was deduced from enumeration of degenerating germ cells at stage VII of the spermatogenic cycle in perfusion-fixed testes from rats in the early stages (4 to 8 days) after EDS treatment. Based on this data and changes in testicular interstitial fluid volume, long seminiferous tubule segments were isolated from control rats and from EDS-treated rats (+/- TE-supplementation) at stages II-V, VI-VIII, or IX-XII, 2 days to 6 days after EDS treatment. Seminiferous tubule segments were incubated for 22 hours with 60 microCi 35S-labelled methionine. Incorporation into newly synthesized proteins in the seminiferous tubule culture medium (= secreted proteins) or in seminiferous tubule lysates (= intracellular proteins) was determined by trichloroacetic acid-precipitation followed by analysis using two-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis. In control rats, incorporation of 35S-methionine into proteins secreted by isolated seminiferous tubules was more than twice as great at stages VI-VIII than at stages II-V or IX-XII. This doubling in methionine incorporation into stages VI-VIII secreted proteins was abolished, however, 4 days after EDS treatment (when germ cell degeneration at stage VII was only just evident). A similar change occurred 4 days after testosterone withdrawal induced by immunoneutralization of luteinizing hormone. In the latter case and after EDS treatment, TE-supplementation of rats from day 0 maintained the normal control pattern of methionine incorporation into seminiferous tubule secreted proteins, although 6 days after EDS and TE treatment, incorporation into stages VI-VIII secreted proteins was 19% lower (P less than 0.05) than in the control group. In contrast, incorporation of methionine into proteins secreted by seminiferous tubules at stages II-V and IX-XII was unaffected by EDS and TE pretreatment, as was incorporation into intracellular proteins at all stages.(ABSTRACT TRUNCATED AT 400 WORDS)

Antioxidant Enzyme Activity in the Maturing Rat Testis

Abstract: Developmental profiles of the activity of the antioxidant enzymes superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px), glutathione transferase (GSH-Tr), and hexose monophosphate shunt (HMS) were measured in the rat testis and liver. The level of SOD in the testis was high at the age of 6 to 10 days, after which it dropped to approximately one third of that level by 20 days of age, and remained there up to 8 months of age. In the liver, SOD activity steadily increased from the neonatal to adult stage of life, reaching the same level as detected in the testis. The testicular activity of catalase was only 2% to 7% of that found in liver at all ages. It increased in both organs up to 6 weeks of age, whereafter the hepatic activity gradually decreased and no further changes were seen in the testis. The GSH-Px activity was low in the testis and declined slightly with age, whereas activity in the liver increased four-fold between birth and adulthood. The activity of GSH-Tr was similar in both organs studied: it increased after birth, showing a maximum in the liver at 1.5 months (ten-fold increase) and in the testis at 5 months of age (four-fold increase). The HMS activity was two to three times higher in the liver than in the testis, and decreased slightly with age in both organs. Thus, the basal levels and developmental profiles of antioxidant enzymes in the testis differ greatly from those in the liver. The very high ratio of SOD to catalase plus GSH-Px in testis (0.92 to 3.48), compared with that found in the liver (0.009 to 0.083), makes the former especially vulnerable to the harmful effects of reactive oxygen species. The peak in SOD activity at 6 to 10 days of age might be related to the physiologic events leading to cell differentiation at this time.

Thiol-disulfide status and acridine orange fluorescence of mammalian sperm nuclei.

Abstract: The relationship between thiol-disulfide status and acridine orange fluorescence of testicular, epididymal, and ejaculated spermatozoa in several mammalian species was investigated. Spermatozoa were fixed with acetic alcohol, stained with acridine orange, and examined with a fluorescence microscope. The majority of the nuclei of testicular spermatozoa of the hamster, mouse, and rabbit exhibited red acridine orange fluorescence. The proportion of sperm nuclei with red acridine orange fluorescence decreased as the spermatozoa descended the epididymis. Red acridine orange fluorescence was replaced by green acridine orange fluorescence. The site in the epididymis where 100% of the nuclei exhibited green fluorescence was the distal caput in the mouse, the corpus in the rabbit, and the proximal cauda in the hamster. In semen samples from men with proven fertility, normal semen parameters, or both, about 60% to 90% of the nuclei exhibited green acridine orange fluorescence. The proportion of sperm nuclei exhibiting green acridine orange fluorescence was higher in the spermatozoa pellet (containing highly motile spermatozoa) obtained by centrifugation through a Percoll gradient. From experiments using disulfide-reducing, thiol-oxidizing and thiol-detecting agents, we concluded that sperm nuclei fluoresce red when they are treated with acid while their DNA-associated protamines are poor in disulfides. Under such conditions, DNA is vulnerable to denaturation. Acridine orange binds to denatured (single-stranded) DNA as aggregates and emits red fluorescence. In contrast, when sperm nuclei are treated with acid while their DNA-associated protamines are rich in disulfides, DNA is resistant to denaturation. Acridine orange binds to native (double-stranded) DNA as a monomer and emits green fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgen regulation of programmed death of normal and malignant prostatic cells.

Abstract: Androgen-dependent normal prostatic glandular cells and androgen-dependent prostatic cancer cells can be induced to undergo cell death after androgen ablation. This death does not require the cells to proliferate and occurs as an energydependent process collectively referred to as «programmed cell death» in which the cells actively commit «suicide.» Associated with this programmed cell death pathway is the enhanced expression of a series of genes and the fragmentation of the genomic DNA into nucleosomal oligomers

The free hormone hypothesis. Distinction from the free hormone transport hypothesis.

Abstract: I. The free hormone transport hypothesis: distinction from the free hormone hypothesis. II. The free hormone hypothesis: importance of the rate-limiting step in tissue uptake. III. Explicit consideration of multiple plasma hormone-binding proteins in transport models. IV. Diffusion barriers in transport models. V. Functions of plasma hormone-binding proteins. VI. Conclusions and areas for future investigation.

Acute and long-term effects of a single dose of the fungicide carbendazim (methyl 2-benzimidazole carbamate) on the male reproductive system in the rat.

Abstract: The effects of carbendazim (methyl 2-benzimidazole carbamate) on the testis, efferent ductules, and sperm were determined in the adult rat after a single oral dose. Two experimental trials were performed: a time response between 2 hours and 32 days after exposure using 0 and 400 mg/kg, and a dose response at 2 and 70 days after exposure using 0 to 800 mg/kg doses. In experiment 1, effects were seen throughout the 32-day period, beginning 8 hours after exposure; the effects included first an increase in testis weight, then decreases in testicular spermatid numbers and in the percentage of morphologically normal cauda sperm. In experiment 2, significant testicular and efferent ductal alterations occurred in animals treated with doses of 100 mg/kg or greater. A dose-dependent increase in testicular weight 2 days after treatment was accompanied by increases in seminiferous tubular diameter and excessive loss of immature germ cells in a stage-dependent manner. There was also a dose-dependent increased incidence of occlusions in the efferent ductules. The occluded ductules were characterized by severe inflammation and exhibited disorganization of the epithelium. At 70 days, there were dose-dependent decreases in mean testis weight and mean seminiferous tubular diameter; however, only minimal long-term effects were seen at 50 mg/kg. In testes exhibiting seminiferous tubular atrophy of greater than 25% (100 mg/kg or greater doses), all of the testes were associated with efferent ductules containing occlusions. Caput sperm numbers were significantly reduced in these testes. Occlusions, abnormal ductules, fibrosis, spermatic granulomas, and mineralization were observed in the ductuli efferents. Long-term effects of carbendazim on the testis were induced primarily by ductal occlusions. Results show that carbendazim produces more severe short- and long-term effects on the male reproductive system than the fungicide benomyl.

Standardization and comparability of CASA instruments.

Abstract: Thirty human semen specimens were analyzed using a standard manual method, then videotaped and reanalyzed using two different computer-aided sperm analysis (CASA) instruments (the HTM system, Hamilton-Thorn Research, Danvers, MA, and the CTS system, Motion Analysis Corp., Santa Rosa, CA). Videotaped specimens were analyzed by CASA for 5 frames for sperm concentration (CON) and percent motility (MOT), and for 15 frames for kinematic variables (straight-line velocity, VSL; curvilinear velocity, VCL; linearity, LIN; and amplitude of lateral head displacement, ALH). Machine parameter settings for the two instruments were matched as closely as possible. CASA values were compared with each other for all measures and with manual results for sperm count and the percentage of motile sperm. Results show: 1) HTM and CTS average values for CON are not different from manual measures for the 5- or 15-frame analysis, but slight differences are seen between CTS and HTM; 2) average values for MOT for the 5-frame analysis are higher than the 15-frame analysis for both instruments, but the average manual measurement for percent motility is much higher than any CASA value; 3) average VSL and LIN are slightly higher for HTM than CTS, but pair-wise comparison shows a high degree of concordance between the instruments; and 4) the mean values for VCL and ALH are equal for the two instruments, and there is a close concordance for the pair-wise comparison for VCL; however, pair-wise comparison of ALH reveals significant differences between the instruments. Overall, the differences seen between these instruments are slight, and are probably not biologically or clinically significant.

Cryopreservation of human semen. Comparison of cryopreservatives, sources of variability, and prediction of post-thaw survival.

Abstract: Human semen was cryopreserved using Human Sperm Preservation Medium, TEST-Yolk buffer, or glycerol alone. Sperm characteristics for each specimen were measured before and after freezing to determine which cryopreservative resulted in better cryosurvival and recovery of motile sperm. Sperm frozen in Human Sperm Preservation Medium had a sig- nificantly better recovery of all semen parameters (motility, ye- locity, and recovery) than either TEST-Yolk or glycerol alone. Statistical analyses also were done to examine the variability between and within donor semen specimens. Differences be- tween donors, between specimens, and measurements within donors all contributed to variability of sperm characteristics. Specimen-to-specimen variability for a given donor represented 12% to 47% of the total variability, whereas processing and mea- surementvariability represented 12% to 41%. Donors also varied in the ability of their sperm to tolerate freezing. There was a relationship between motile count after dilution with cryopreser- vative and post-thaw motile count. This relationship allows the prediction of poor-thaw survival before freezing a specimen.

The Taurine and Hypotaurine Content of Human Semen

Abstract: Taurine and hypotaurine levels were measured in human sperm and seminal fluid. Sperm taurine ranged from 17 nmol/mg DNA to 348 nmol/mg DNA, and hypotaurine from 0 nmol/mg DNA to 251 nmol/mg DNA. Seminal fluid contained 319 mumol/L to 1590 mumol/L of taurine, but no detectable hypotaurine. The coefficient of variation in multiple ejaculates from a single man for these components ranged from 12% for hypotaurine to 24% for seminal fluid taurine, indicating a relative constancy in their concentrations. Sperm hypotaurine content was significantly correlated with sperm morphology, sperm relative forward progression, the percentage of motile sperm, and the total number of sperm in the ejaculate. By contrast, sperm taurine content was negatively correlated with these parameters. The mean hypotaurine content of sperm from 8 fertile men was 149 +/- 92 nmol/mg DNA, four times higher than that of sperm from 9 infertile men, which was 35 +/- 19 nmol/mg DNA (P = 0.011). In contrast, the mean sperm taurine content of the fertile men was lower than that of the infertile men (83 +/- 33 nmol/mg DNA versus 168 +/- 119 nmol/mg DNA, respectively; P = 0.07). Seminal fluid taurine concentrations, however, were similar for both groups. Hypotaurine, an antioxidant, may play an important role in protecting sperm from reactive oxygen species. Higher concentrations of taurine in the sperm of infertile men suggest that accelerated oxidation of hypotaurine to taurine may accompany the observed decline in other sperm parameters.

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

Abstract: 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.

Movement characteristics of boar sperm obtained from the oviduct or hyperactivated in vitro.

Abstract: The objectives of this study were to describe hyperactivated motility in boar sperm and to determine the incidence of hyperactivation among boar sperm flushed from the oviduct. Oviducts were surgically removed from 13 gilts 32 hours after mating them to fertile boars. The majority of the sperm flushed from the oviducts was immotile, weakly motile, or stuck to mucus or cellular debris. The mucus could not be penetrated by the sperm. The remaining 3% to 19% of the flushed sperm was free-swimming. Only five hyperactivated sperm were recovered, all from the ampulla of the oviduct. The remainder of the free-swimming sperm travelled in linear trajectories and possessed significantly higher flagellar curvature ratios (the flagella were less bent) than boar sperm measured in diluted semen. Hyperactivated motility was induced in washed ejaculated boar sperm, using a 1-minute pulse of 4 mumol/L calcium ionophore A23187. The ionophore-treated sperm had significantly lower straight-line velocities, linearities, and flagellar curvature ratios than controls, as would be expected for hyperactivated sperm. They were vigorous and swam in circles. It was concluded that, although few hyperactivated boar sperm could be recovered from the oviduct, boar sperm are capable of undergoing hyperactivation.

Sperm Surface Fibronectin Expression Following Capacitation

Abstract: The Arg-Gly-Asp (RGD) amino acid sequence plays a role in many cell-to-cell and cell-to-matrix adhesion systems, as a recognition sequence for cell membrane receptors termed integrins. Receptors of the VLA subfamily of integrins recognize fibronectin, laminin, and collagen. Given the authors' findings that fibronectin-derived, RDG-containing peptides competitively inhibit sperm-oolemmal adhesion and penetration in both heterologous (human-hamster) and homologous (hamster-hamster) gamete interactions, the expression of fibronectin on the surface of fresh, capacitated, and acrosome-reacted human spermatozoa was studied. The majority of fresh spermatozoa did not display fibronectin on their plasma membrane (0 to 16% positive), as demonstrated by the lack of binding of both monoclonal and polyclonal anti-fibronectin antibodies. In contrast, a significantly greater proportion of spermatozoa (varying between 18% to 100% for different donors) incubated overnight under capacitating conditions reacted with anti-fibronectin antibodies. The induction of an acrosome reaction with progesterone did not alter the proportion of sperm displaying fibronectin or its distribution on the sperm surface. A physiologic role of fibronectin in sperm-oolemmal interaction was suggested by the effects of anti-fibronectin antibodies on sperm oolemmal adhesion and penetration of hamster eggs by human spermatozoa, which were both significantly reduced (P less than 0.001).

Sperm penetration into a hyaluronic acid polymer as a means of monitoring functional competence.

Abstract: The diagnostic significance of sperm penetration assays based on a commercially available hyaluronate preparation (Sperm Select) has been investigated in the male partners of 77 couples characterized by a normal female partner. Sperm penetration into hyaluronate was highly correlated with the ability of the same sperm populations to penetrate bovine cervical mucus and, moreover, depended on the same attributes of semen quality, including the morphology of the spermatozoa, their number, and their motility as reflected by their mean path velocity. Stepwise multiple regression analyses employing these independent variables generated r values of 0.821 to 0.931, depending on the criterion of hyaluronate penetration used; path velocity was consistently the most informative variable according to the standardized regression coefficients. The relationship between hyaluronate penetration and sperm movement was so close that multiple regression equations could be generated that were capable of accounting for up to 76% of the variance in sperm velocity measurements obtained with a computerized image analysis system. Regression equations could also be generated using the hyaluronate penetration data that could account for 65% of the variance observed in an A23187-enhanced zona-free hamster oocyte penetration test, including the successful identification of the subpopulation of patients in whom 0% oocyte penetration had been recorded. Within the same data set, independent variables based on bovine cervical mucus penetration could only account for 43.5% of the variance in sperm-oocyte fusion. Hyaluronate penetration therefore appears to offer a simple, objective means of generating information on the functional competence of human spermatozoa that should find a role in routine diagnostic services where the more specialized tests are not available.

Androgen-induced prevention of the outgrowth of cranial gonadal suspensory ligaments in fetal rats.

Abstract: Normal and disturbed testicular descent is frequently approached exclusively through a consideration of the caudal testicular suspensory apparatus. This is surprising, because embryonal gonads develop with both cranial and caudal suspensory ligaments, and the sexes differ with respect to the persistence and development of both the cranial and the caudal ligaments. The current study examined the possible role of fetal testicular androgens in male-specific failure of the development of the cranial gonadal suspensory apparatus in rats. Normal male fetuses were studied, as well as fetuses exposed to the anti-androgen flutamide from day 10 after conception. Females were given daily injections of methyl-testosterone alone or in combination with the anti-androgen cyproterone acetate from day 15 after conception, and were studied on day 22. The cranial ligaments remained of minor extension in normal males but developed considerably in females. They developed in female fashion in males exposed to flutamide, and persisted throughout postnatal life. Cranial ligaments did not develop in females that had been exposed to methyl-testosterone. Simultaneous treatment with a large dose of cyproterone acetate effectively counteracted this effect. Fetal testicular testosterone thus appears to play a key role in the prevention of the outgrowth of the cranial gonadal/genital ligament in rats. The supposed function of this suspensory apparatus makes it likely that its persistence in males, as the consequence of inappropriate androgen action during fetal life, facilitates disturbance of testicular descent. This finding may contribute to understanding developmental disorders underlying disturbed testis descent in humans.

Testicular function in hyperthyroidism.

Abstract: Testicular function was assessed in nine men aged 17 to 35 years and seven men aged 36 to 46 years with Graves' disease. Levels of total testosterone, estradiol, sex hormone binding globulin, luteinizing hormone, and follicle stimulating hormone, and gonadotropin responses to gonadotropin releasing hormone were significantly greater than these levels and responses in age-matched controls. Although the percentage of free testosterone was lower than control values in the hyperthyroid men, calculated levels of free testosterone were not different from normal. Lower than normal percentages of free estradiol values resulted in higher than normal calculated free estradiol levels. As a result, the free testosterone/free estradiol ratio in the men with Graves' disease was lower than normal. Although mean sperm densities in the hyperthyroid men were not different from control values, the percent forward progressive motility of sperm from these men was significantly lower than control values. The hormonal and seminal abnormalities corrected when the patients became euthyroid after radioiodine therapy. The results of this study indicate that hyperthyroid men may have abnormalities in their hypothalamic-pituitary-testicular axes.

Sex hormone-binding globulin. Binding to cell membranes and generation of a second messenger.

Abstract: I n plasma, most steroid hormones are largely bound and transported by two specific steroid-hormone-binding proteins, corticosteroid-binding globulin (CBG) and sex hormone-binding globulin (SHBG) (Siiteri et al, 1982; Englebienne, 1984; Westphal, 1986; Rosner, 1986; Brien, 1981; Mercier-Bodard et al, 1981). Although it is an oversimplification (see Mendel, 1989), it is fair to state that the prevailing view of the function of these proteins is to bind steroids and thus trap them in the plasma compartment. Only a small fraction of free steroids are available to diffuse passively out of the capillary bed and across cell membranes to initiate hormonal effects. The best evidence supports this notion of how steroid hormones exit the vascular space and enter cells. In the past few years, however, there is undeniable evidence of additional functions for the steroidbinding proteins. The major lines of evidence that led to this conclusion are reviewed here.

Seasonal Effects on Seminal Quality, Plasma Hormone Concentrations, and GnRH-Induced LH Response in Fertile and Subfertile Stallions

Abstract: Seasonal effects on hormonal and seminal parameters in subfertile stallions have not been well documented and could provide information that is needed to understand the underlying endocrine mechanisms associated with testicular dysfunction. Such information may be useful in developing diagnostic tools to identify those stallions who are candidates for treatment. This investigation characterizes and compares the effects of season on endocrine function and seminal quality in fertile and subfertile stallions. Eight fertile and six subfertile stallions between the ages of 5 and 18 years were injected intravenously once every hour for 3 hours with either 1 mL saline on the first experimental day or 5 micrograms gonadotropin-releasing hormone in 1 mL saline on the second experimental day during the nonbreeding and breeding season. Heparinized blood samples were collected periodically through a jugular catheter before and after treatment for analysis of luteinizing hormone, follicle-stimulating hormone, testosterone, and estrogen conjugates by radioimmunoassay. Semen samples were collected twice, 1 hour apart, from all stallions in both seasons for analysis of volume, concentration, motility, pH, and morphology. A series of low intravenous doses (5 micrograms) of gonadotropin-releasing hormone induced a significant luteinizing hormone response (P less than 0.05) compared with saline treatment in both fertile and subfertile stallions. Fertile stallions had a twofold higher (P less than 0.05) net increase in plasma luteinizing hormone levels (peak levels minus baseline levels) in the breeding seasons than in the nonbreeding season. The magnitude of the luteinizing hormone response relative to baseline levels in fertile stallions, however, was one-and-one-half times greater (P less than 0.05) in the nonbreeding season than in the breeding season. In contrast, season did not have an effect on the net increase in plasma luteinizing hormone or the magnitude of the luteinizing hormone response relative to baseline levels in subfertile stallions. The net increase in plasma luteinizing hormone was similar between the two groups of stallions in both seasons. The magnitude of luteinizing hormone response relative to baseline levels, however, was lower (P less than 0.05) in subfertile stallions (141 +/- 14%) than in fertile stallions (235 +/- 46%) in the nonbreeding season; the two groups exhibited similar responses in the breeding season. Compared with fertile stallions, subfertile stallions had twofold to fourfold higher (P less than 0.05) plasma levels of gonadotropins and similar testosterone levels. The number of total progressively motile sperm was lower (P less than 0.05) in subfertile stallions in both seasons.(ABSTRACT TRUNCATED AT 400 WORDS)

FSH isoforms, radioimmunoassays, bioassays, and their significance.

Abstract: Follicle-stimulating hormone (FSH) plays a central role in steroidogenesis and gametogenesis In recent years, a great deal has been learned about the microheterogeneity of FSH using newly developed assay techniques This review article will attempt to discuss the advantages and the limitations of recently developed and commonly used assay systems

Dihydrotestosterone is a peripheral paracrine hormone.

Abstract: Androgen action in sexual tissues, especially skin and the prostate, is expressed by dihydrotestosterone (DHT) acting at the nuclear level. Dihydrotestosterone in the circulation and target tissues is almost solely derived from the peripheral conversion of secreted testosterone (T) in men and androstenedione in women. The general pathway is testosterone----DHT in equilibrium with androstanediol (3 alpha diol). However, a number of studies suggest that blood DHT or 3 alpha diol are not reliable indicators of peripheral DHT formation. This is particularly suggested by discrepancies in the specific activity of DHT in blood and urine following infusion of labeled DHT, suggesting that total body DHT formation is not reflected by blood levels. Thus, DHT should be thought of as a paracrine hormone formed and acting primarily in target tissues. 3 alpha androstanediol glucuronide (3 alpha diol G) is a major metabolite of DHT. An important site of its formation is the skin. Levels in blood and urine are increased in hirsutism and acne, and blood levels closely parallel pubertal development. 3 alpha diol G levels are especially increased in adrenal disorders of androgenicity such as andrenogenital syndrome; it is also a good marker of response to therapy. Levels are reduced in various forms of male pseudohermaphroditism. 3 alpha androstanediol glucuronide appears to be the best marker available of DHT formation in target tissues such as skin.

Simultaneous proliferation and differentiation of mast cells and Leydig cells in the rat testis. Are common regulatory factors involved

Abstract: The proliferation and differentiation of mast cells and Leydig cells were studied in adult sham operated or hypophysectomized rats after the administration of ethylene dimethane sulphonate (EDS) and in prepubertal rats after neonatal treatment with a gonadotropin-releasing hormone (GnRH) antagonist (Organon 30276; Oss, The Netherlands). After treatment with EDS, two proliferative waves were found. On day 3, several interstitial cell types proliferated, whereas mitotic cells corresponded to differentiating Leydig cells and mast cells around day 20. Differentiating Leydig cells showed a higher mitotic index than that of differentiating mast cells. Hypophysectomized animals showed high mitotic activity 3 days after treatment, but 21 days after treatment differentiating Leydig cells were absent and proliferative activity was reduced. The number of mast cells increased from day 15 to day 30 in EDS-treated rats and from day 15 to day 50 in hypophysectomized, EDS-treated rats. GnRH antagonist-treated rats showed poorly differentiated Leydig cells and abundant mitotic figures on day 23. Proliferation and differentiation of Leydig cells occurred concomitantly with the proliferation and differentiation of mast cells between 23 and 30 days of age. These results suggest that Leydig cells and mast cells in the rat testis share some common regulatory factors.

Direct effects of ethane dimethanesulphonate on epididymal function in adult rats. An in vitro demonstration.

Abstract: It was recently demonstrated that the Leydig cell toxicant ethane dimethanesulphonate (EDS) produces multiple effects on the epididymis after a single in vivo exposure. To determine whether any of the perturbations were mediated by a direct action of the compound, we used a novel system for the coculture of epididymal epithelial cells and sperm from the caput epididymidis. This system maintains the morphologic integrity and cell polarity of the epididymal epithelial cells before and during coculture, and the sperm recovered after coculture have intact plasma and acrosomal membranes. In addition, several functions required for epididymal sperm maturation are expressed, including the secretion of protein by the epididymal epithelium, the association of secreted protein with the plasma membrane of cocultured sperm, and the acquisition of progressive motility by cocultured sperm. In vitro exposure of epididymal epithelial cells and sperm to EDS results in a significant decline in protein secretion by the epithelial cells during coculture, and in particular, a dose-dependent decline in a 36- to 38-kd protein (PI 4.0 to 4.5) and a 34- to 36-kd protein (PI 4.5 to 5.0). Moreover, these and other proteins are not recovered from the sperm membrane of cocultured sperm after EDS treatment. Finally, EDSmore » results in a dose-dependent decline in the percentage of both motile and progressively motile sperm recovered after coculture compared with that of sperm from untreated cocultures.« less

Testicular regulation of epididymal protein secretion.

Abstract: The effects of different androgen and testicular fluid levels on the protein synthesis and secretion of the epididymis of mice and rats were examined. In mice, the in vitro protein synthesis and secretion of five epididymal segments were measured from 3 days up to 8 weeks after efferent duct ligation. During the entire period, alterations in the rate of protein secretion per milligram tissue were small. At 8 weeks, the mean rate of protein synthesis per milligram ligated epididymal tissue was 80% of that of the control side. As a consequence of the weight loss of the ligated epididymis, however, the protein secretion per organ can be estimated to be reduced by 50%. Changes in the protein profile were only found in the proximal segment, where a 40-kd protein appeared and a 29-kd protein disappeared. In rats, the effects of efferent duct ligation were studied in vivo for up to 8 months. Structural changes were present both in the proximal and in the distal epididymis. The most conspicuous change in the protein profile of secretory proteins was the disappearance of a 27-kd protein from the proximal segment. In the distal epididymis, a 32-kd protein was no longer secreted. In mice, the effects of castration on the profile of secreted proteins demonstrated that, without androgen stimulation, some proteins are still secreted 6 weeks after castration. Administering low or high doses of testosterone propionate to castrated mice resulted in almost similar profiles of secretory proteins. However one protein secreted in the proximal epididymis was preferentially stimulated by the high dose of testosterone propionate.

Sperm-zona pellucida interaction in cynomolgus and rhesus macaques.

Abstract: These experiments were carried out to establish and validate an in vitro system for studying macaque sperm-zona pellucida interaction. Sperm of rhesus and cynomolgus macaques were capacitated in vitro and incubated with cryopreserved zonae pellucidae. Homologous gamete incubations were tested, as well as cross-species combinations. Approximately 25% of macaque sperm bound to the zonae acrosome reacted within 1 minute of gamete coincubation, although the percentage of acrosome reactions in the sperm suspension was less than 1%. There was a small but consistent increase in the percent of acrosome reactions of zona sperm after an additional hour of incubation in sperm-free media. Similar results were obtained in the cross-species experiments, suggesting that zonae from the two macaque species can be used interchangeably in sperm-zona binding assays. Differences in the physiologic characteristics of the sperm of the macaque species were demonstrated. Cynomolgus sperm required activation with caffeine and dibutyryl cyclic adenosine monophosphate (dbcAMP) in order to bind to the zonae. Rhesus sperm were able to bind to the zonae and acrosome react in the absence of activators, although both sperm binding and percentage of acrosome reactions increased with the addition of activators. Large numbers of sperm from both macaque species bound to the zonae of hamster oocytes after treatment with activators, but the bound sperm did not acrosome react. These experiments demonstrate the importance of evaluating the acrosomal status of sperm when sperm-zona binding assays are performed with macaque gametes.

Sperm surface proteins after capacitation. Expression of vitronectin on the spermatozoan head and laminin on the sperm tail.

Abstract: Several integrins recognize as ligands proteins containing the Arg-Gly-Asp (RGD) sequence, such as fibronectin, vitronectin, and laminin. It has been previously demonstrated that oligopeptides containing the RGD sequence competitively inhibit both the adhesion of hamster and human sperm to zona-free hamster eggs and their subsequent penetration. In addition, the appearance of fibronectin on the surface of living human spermatozoa after capacitation has been demonstrated. In this work, it is shown that spermatozoa incubated overnight under capacitating conditions, but not fresh spermatozoa, also display the RGD-containing proteins vitronectin and laminin. Whereas the expression of fibronectin does not appear to be localized to any specific region of the sperm surface, laminin is present solely on the sperm tail, and vitronectin was detected mostly as an equatorial band on the sperm head. The percent of capacitated spermatozoa within each ejaculate reacting with antivitronectin antibodies (51% to 94%) was similar to that observed with antifibronectin antibodies (72% to 100%) in a series of fertile donors, and in a series of infertile men (7% to 98% for vitronectin versus 5% to 100% for fibronectin). In contrast, the percent of spermatozoa displaying laminin was lower, ranging from 2% to 42% for fertile donors and from 5% to 34% for infertile donors, and was unrelated to the expression of fibronectin or vitronectin. The time of appearance of both fibronectin and vitronectin when spermatozoa were incubated under capacitating conditions varied for different sperm donors, suggesting a difference in the process of their expression between different men. The specificity of antivitronectin antibody binding to human spermatozoa was demonstrated by competitive inhibition with purified human vitronectin. That there was no immunologic reaction between the antivitronectin antibodies used and fibronectin was demonstrated both by the failure of free fibronectin to inhibit antivitronectin antibody binding to spermatozoa, and by Dot blot analysis. A partial cross-reaction of the polyclonal antivitronectin antibody with fibronectin was shown by Western blot analysis, but this phenomenon was not present when the monoclonal antivitronectin antibody was used. In addition, both fibronectin and vitronectin could be extracted from capacitated spermatozoa solubilized in Chaps buffer, as shown by Dot blot and Western blot analysis. These observations suggest that vitronectin and fibronectin expressed on the surface of capacitated human spermatozoa could act as a ligand for specific receptors on the egg, and play a role in sperm-oolemmal adhesion.

Effects of insulin-like growth factor-I on androgen production by highly purified pubertal and adult rat Leydig cells.

Abstract: Leydig cells were isolated and purified from adult and midpubertal rats to study the effects of insulin-like growth factor-I (IGF-I) on steroidogenesis. Androgen production, as measured in Leydig cell conditioned culture media, from four different treatment groups (1 = no hormone; 2 = 70 ng/ml IGF-I; 3 = 0.1 ng/ml LH; 4 = 70 ng/ml IGF-I + 0.1 ng/ml LH) were compared daily. After 3 days in culture, the cells were treated with a maximally stimulating dose of luteinizing hormone (LH) (100 ng/ml) for 3 hours. Androgen production was highest in the cells treated with both IGF-I and low concentrations of LH. In the presence of IGF-I, regardless of LH, cells derived from pubertal animals had a greater increase in steroidogenesis during the culture period than did cells from adult animals. Pretreatment with IGF-I prior to maximal LH stimulation induced a greater increase in androgen production in cells from pubertal rats than in cells from adult animals. It is concluded that IGF-I has a direct effect on Leydig cells and may act synergistically with LH to promote androgen synthesis. The greater response in pubertal cells raises the possibility that IGF-I is important in the maturing process of the testis.