Showing papers in "Journal of Andrology in 1988"

Significance of Reactive Oxygen Species and Antioxidants in Defining the Efficacy of Sperm Preparation Techniques

Abstract: The mechanisms responsible for mediating the influence of sperm preparation protocols on human sperm function have been investigated. Techniques that involved the separation of motile spermatozoa prior to centrifugation were found to yield sperm suspensions of highest quality. If the spermatozoa were centrifuged prior to isolation of the motile cells, sperm function was impaired. The detrimental effects of centrifugation were associated with a sudden burst of reactive oxygen species production by a discrete subpopulation of cells (characterized by significantly diminished motility and fertilizing capacity) that could be separated from normal functional spermatozoa on Percoll gradients. If unfractionated sperm suspensions were subjected to centrifugation, the reactive oxygen species generated by this subpopulation impaired the functional competence of normal spermatozoa in the same suspension. Assessment of the ability of the antioxidants, butylated hydroxytoluene, and vitamin E, to curtail the peroxidative damage inflicted by such cells in response to centrifugation revealed a significant improvement of sperm function in the presence of vitamin E.

Relationships between computerized measurements of motion of frozen-thawed bull spermatozoa and fertility.

Abstract: A computerized system (CellSoft, CRYO Resources, Ltd.) was validated using video tapes of frozen-thawed bull spermatozoa diluted in filtered (0.2 micron) egg yolk-citrate extender (8 X 10(6) spermatozoa/ml) and analyzed at 30 frames/sec for the percentage of motile spermatozoa (greater than or equal to 20 microns/sec) and linear velocity of motile spermatozoa. Virtually all motile spermatozoa were detected and debris rarely were classified as immotile spermatozoa if the extender had been filtered. Variation about the mean for percent motile cells was similar when only 12 rather than 20 or 30 frames/field were analyzed. Use of 20 frames/field was adequate to determine the percentage of motile bull spermatozoa. Five mixtures of live and killed spermatozoa were analyzed (four bulls) to evaluate accuracy. Percent motile spermatozoa was correlated (r = 0.97) with the ratio of live:killed spermatozoa. Mean linear velocity of motile spermatozoa was similar for each mixture (P greater than 0.05). To further evaluate accuracy, percent motile spermatozoa was determined by computer and by "track motility" (20 samples; 0 to 63% motile spermatozoa); values were correlated (r = 0.95). The system was precise (CV of 6% based on triplicate analyses of the same samples) and reasonably accurate for evaluating bull sperm motility if the extender had been filtered and 20 to 25 fields (greater than or equal to 200 spermatozoa) were evaluated. Correlations between measurements of sperm motion and fertility were studied using cryopreserved semen from two fertility trials. For the first, 75-day nonreturn rate data for 20 samples of bull semen (10 bulls) were not significantly correlated with evaluations made by CellSoft. For the second fertility trial, the competitive fertility index (a measure of relative fertility) for nine bulls was correlated (r greater than or equal to 0.68; P less than 0.05) with percent motile spermatozoa, linear velocity and straight-line velocity. Multiple correlations based on six characteristics evaluated by CellSoft, at 0 or 1.5 hours, and the competitive fertility index were greater than or equal to 0.94. Based on the latter data, the system may facilitate prediction of the relative fertility of bull spermatozoa.

The sperm chromatin structure assay. Relationship with alternate tests of semen quality and heterospermic performance of bulls.

Abstract: Data obtained by the sperm chromatin structure assay (SCSA) on spermatozoa from nine bulls were correlated with fertility, measured by heterospermic performance (-0.94, P less than 0.01) and by alternate tests of sperm quality, including motility, acrosome integrity, Sephadex filtration and morphology of spermatozoa (all significant at P less than 0.05 to P less than 0.01). The SCSA uses flow cytometry to determine the susceptibility of nuclear DNA to low pH-induced denaturation in situ as measured by the ratio of acridine orange binding to double- or single-stranded DNA. The error associated with multiple SCSA measurements was relatively low. The primary finding is that the assay of chromatin structure stability performed on killed spermatozoa was as highly correlated with the heterospermic performance of semen as the best of the classical tests for semen quality. The SCSA may therefore be a highly useful technique for evaluation of sperm quality.

Epididymal markers in human infertility.

Abstract: Alpha-glucosidase, glycerophosphocholine, and L-carnitine were measured in sperm-free seminal plasma to determine whether these markers reflected the epididymal function of men attending an infertility clinic. The putative markers correlated well with each other (r = 0.66 to 0.70) and in 92% of 283 cases were accurate in categorizing semen as containing normal or subnormal amounts of markers. Glucosidase was considered the best index of epididymal function and was used for a further 306 samples. The ejaculate content of epididymal markers was correlated with testicular volume and serum testosterone below values of 30 ml and 30 nmol/l, respectively. Markers were also correlated with the concentration and motility of spermatozoa in semen. Seventy-one of 425 patients (17%) displayed subnormal epididymal secretions, mainly in association with hypogonadism (Klinefelter syndrome, Kallman syndrome, idiopathic hypogonadotropic hypogonadism) but also in cases of obstructed ducts, maldescended testicles, and local irradiation following hemicastration. Because azoospermic patients had reduced epididymal markers with both high and low FSH levels and a large proportion of men with reduced glucosidase and normal FSH suffered from testicular failure, it is suggested that other indices of testicular function are required for correct interpretation of reduced epididymal markers. Thirteen patients (3%) had low markers for which no cause was apparent; these may be cases of infertility due to isolated epididymal dysfunction.

The regulation of the proliferation and differentiation of rat Leydig cell precursor cells after EDS administration or daily HCG treatment.

Abstract: The proliferation and differentiation of possible Leydig cell precursors in adult rats were studied after destruction of the existing Leydig cells with EDS or after daily treatment with hCG. After 2 days with either treatment, a 12- to 16-fold increase in the number of [3H]thymidine-incorporating interstitial cells was found. In the case of hCG treatment, this was probably due to the high plasma hCG levels. However, after EDS treatment, LH levels start to rise between days 1 and 3, suggesting a paracrine stimulation of the proliferation of interstitial cells. After hCG treatment, a substantial increase in the numbers of Leydig cells was already found at day 2. It was concluded that hCG induced a rapid differentiation, without cell division, of existing precursor cells into recognizable Leydig cells. In rats treated with both EDS and hCG, new Leydig cells were not formed during the first 10 days. This indicates that EDS destroys not only mature Leydig cells but also those Leydig cell precursors that are able to differentiate rapidly into recognizable Leydig cells.

Acute Effects and Long-Term Sequelae of 1,3-Dinitrobenzene on Male Reproduction in the Rat ·II. Quantitative and Qualitative Histopathology of the Testis

Abstract: This study determined the quantitative and qualitative histopathologic effects of a single oral dose of 1,3-dinitrobenzene (48 mg/kg) on the rat testis from 1 to 175 days postexposure. The testis was damaged severely by hour 24, as evidenced by increased numbers of regressive seminiferous tubules that exhibited degenerating pachytene spermatocytes, chromatin margination in spermatids, giant cells, deformed spermatid heads, retained spermatids, and reduced numbers of meiotic figures. The major effects during the first 48 hours posttreatment were degeneration or exfoliation of pachytene spermatocytes and round spermatids and the retention of step 19 spermatids. These regressive effects continued until 24 days, after which the tubules either recovered or became atrophic. At the end of the study (175 days), three males were normal, one had regressed testicles, and three males had atrophic tubules (15 to 45%). Several cellular abnormalities were common throughout the period. In addition, the frequency of the stages of spermatogenesis was altered, an indication of a disturbance in the kinetics of spermatogenesis. 1,3-Dinitrobenzene produced profound and specific lesions in the seminiferous tubules, and recovery was slow and incomplete. Atrophic tubules seemed to form if the normal cellular associations were not reestablished within 24 days, possibly due to the inability of Sertoli cells to reorganize the synchrony of germ cell development.

The selective removal of pachytene spermatocytes using methoxy acetic acid as an approach to the study in vivo of paracrine interactions in the testis.

Abstract: Testicular weight and morphology, serum gonadotropins, intratesticular levels of testosterone and ABP levels in testicular interstitial fluid were studied in adult rats at intervals of 1 to 70 days after a single oral dose of 650 mg/kg methoxy acetic acid. At 3 days, this treatment resulted in the selective loss or depletion of pachytene and later spermatocytes from seminiferous tubules at all stages other than VIII to XI of the spermatogenic cycle. At later times this lesion was expressed as an absence mainly of round (14 days) or elongated (21 days) spermatids from the majority of seminiferous tubules. Other than these changes, spermatogenesis did not appear to be affected by treatment and was qualitatively normal in all tubules at 70 days after treatment. As deduced from cell counts at 3 days posttreatment, the initial action of methoxy acetic acid was restricted to late zygotene spermatocytes (stage XII) and pachytene spermatocytes at all stages other than early- to mid-stage VII. Levels of FSH in serum and those of ABP in testicular interstitial fluid indicated that Sertoli cell function was altered in rats treated with methoxy acetic acid. Both were increased at 1 to 3 days posttreatment, returned to normal at 7 to 14 days but were increased again at 21 days before finally returning to control levels at 28 days. In contrast, the levels of testosterone in serum, isolated seminiferous tubules and testicular interstitial fluid were unaffected by treatment, as also were the serum levels of LH. The two periods of increase in FSH and ABP levels coincided with the times of greatest decrease (approximately 20%) in testicular weight, and may be related either to the type of germ cell missing from the affected tubules and/or to the stage of the cycle of the affected (or unaffected) tubules. These data suggest that chemicals such as methoxy acetic acid may prove useful in the study of paracrine interactions in vivo.

Diagnostic value of sperm function tests and routine semen analyses in fertile and infertile men

Abstract: The results of routine semen analyses, the zona-free hamster oocyte penetration test, the hypoosmotic swelling test, and semen adenosine triphosphate levels were studied in 66 fertile and 130 infertile men. Multivariate discriminant analysis demonstrated that routine semen parameters including semen volume, sperm count, percent sperm motility, and percent normal spermatozoa in combination could predict the fertility of these patients with 70.4% accuracy. Of the three sperm function tests evaluated, the zona-free hamster oocyte penetration test and the hypoosmotic swelling test were selected by the multivariate discriminant analysis as variables capable of providing significant information on the fertility status of the patients. However, the addition of the results of these two tests to the routine semen analysis did not significantly improve the predictability of fertility. The overall correct prediction rate was 77.6% after incorporation of the results of these two sperm function tests. In this group of subjects, the presently available sperm function tests did not predict the fertility status of a patient with a high degree of accuracy.

Purification and Characterization of a Sperm Motility Inhibitor in Human Seminal Plasma

Abstract: A sperm motility inhibitor from human seminal plasma was purified and characterized. The purification procedure includes dialysis, ion exchange chromatography on SP-Sephadex C-25 and adsorption chromatography on hydroxylapatite. With this procedure, the seminal plasma motility inhibitor was purified 290-fold with a 24% recovery in inhibitory activity. Its molecular weight has been estimated at 18,000 to 22,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, but at 13,000 to 15,000 according to molecular sieving under native conditions. The mobility inhibitor has an isoelectric point pH 9.1. It is stable over a wide range of pH (5 to 10) and at temperatures up to 60 C. The observation that the seminal plasma factor inhibited purified bull dynein ATPase in a concentration-dependent manner may suggest that it blocks the motility of demembranated spermatozoa by interfering with dynein arm function.

The relationship between the motility and morphology of spermatozoa in human semen.

Abstract: High-speed videomicrography was used to assess simultaneously the morphology and motility of seminal spermatozoa from 10 fertile donors and 10 patients being evaluated for infertility. In both donors and patients, morphologically normal spermatozoa were more likely to be motile and had significantly higher straight line velocity, greater rolling frequency and flagellar beat frequency than abnormally shaped cells. For donors and patients there were highly significant, linear correlations (R = 0.7 to R = 0.98) between the movement characteristics of morphologically normal and abnormal spermatozoa within an ejaculate. A greater percentage of normal donor spermatozoa were motile than were the normal spermatozoa from patients (56% vs. 28%, respectively, P less than 0.005) and normal donor spermatozoa also swam faster than normal patient spermatozoa (49.1 +/- 3.2 microns/sec vs. 37.4 +/- 4.3 microns/sec, mean +/- sem, respectively, P less than 0.05). Overall, a multivariate analysis of variance, including straight line velocity, rolling frequency, beat frequency, and flagellar beat amplitude, demonstrated that these movement characteristics were significantly greater for the normal cells from donors than for the normal spermatozoa from patients. These biologic distinctions notwithstanding, the discrimination between semen from donors and patients was not improved when only morphologically normal cells were analyzed for motility.

Acute effects and long-term sequelae of 1,3-dinitrobenzene on male reproduction in the rat. I. Sperm quality, quantity, and fertilizing ability.

Abstract: Groups of eight adult male rats were given a single oral dose of 0 or 48 mg/kg of 1,3-dinitrobenzene and sacrificed at 1, 2, 4, 8, 16, 24, 32, 72, and 175 days posttreatment. The groups killed at 175 days were bred to untreated females during weeks 3, 4, 6, 9, 13, and 24. Decreased testis weight and testicular sperm numbers were observed by day 4; decreased cauda sperm reserves and epididymis weight occurred by day 8 and day 16, respectively. Reduced numbers of motile spermatozoa and abnormal sperm morphology were seen in spermatozoa from the cauda epididymidis by day 16. Fertilizing ability, as indicated by the presence of two pronuclei and a sperm tail in eggs flushed from the oviducts of inseminated females, was slightly reduced by week 4 and declined to zero by week 6. Group means for reproductive organ weights, sperm production, and sperm reserves failed to return to control levels although some individual animals approached full recovery. Normal fertilizing ability was restored in most animals by week 13, but two of seven remained infertile. Occlusion of some efferent ductules was observed in three of seven animals at 175 days. This study indicates that 1,3-dinitrobenzene is a potent testicular toxicant in the rat, capable of producing marked testicular damage, infertility, and possibly sterility from a single exposure.

Electron Microscopic Study of the Secretion Process in Bovine Reproductive Organs

Abstract: The origin and mechanism of the secretion of membrane-bound particles in bovine seminal plasma were studied with transmission (TEM) and scanning (SEM) electron microscopy of the epididymis, vas deferens, ampulla, and seminal vesicle of adult bulls. In the SEM study, all these organs were found to contain apical protrusions in the lining of the epithelial cells. Eventually the protrusions became detached and formed secretory bodies within the lumina of these organs. In the epididymis, the TEM study disclosed a granular and rather homogeneous content in the protrusions and bodies, whereas in the vas deferens they contained dilated cisternae of smooth endoplasmic reticulum. In the ampulla and seminal vesicle, the formation of the apical protrusions was associated with an accumulation of membrane-bound vesicles. These vesicles were found to be released from the storage bodies into the secretory fluid of the lumen. Both could be harvested from isolated seminal vesicle secretions by Percoll gradient centrifugation. It was concluded that various parts of the bovine reproductive organs discharge their secretory products at least partly by an apocrine mechanism. The membrane-bound particles in the seminal plasma, however, appear to be mainly derived from the ampulla and seminal vesicle.

Computer-assisted human semen analysis. Sampling errors and reproducibility.

Abstract: Videomicrographic computer-automated semen analysis systems allow quantitative description of sperm motility, velocity, progression, and head movement amplitude and frequency with unprecedented ease. The minimum number of spermatozoa needed for stable results, the variability of measurements and optimum methods of sampling the ejaculate were determined for one such system (Cell-Soft, CRYO Resources, New York, NY). Sampling a minimum of 225 spermatozoa yields stable measurements, and analyzing four microscope fields in triplicate provides data with the lowest coefficient of variation. The variability attributable to the instrument itself was acceptable for all measurements (6.2% to 15.4%) except mean amplitude of lateral head displacement. Limitations of these results and the potential utility of videomicrographic sperm movement analysis are discussed.

Simultaneous Determination of Testosterone, Dihydrotestosterone and 5α-Androstan-3α,-17β-Diol by Isotopic Dilution Mass Spectrometry in Plasma and Prostatic Tissue of Patients Affected by Benign Prostatic Hyperplasia Effects of 3-Month Treatment with a GnRH Analog

Abstract: This article reports an isotope dilution mass spectrometric method for the simultaneous measurement of testosterone (T), dihydrotestosterone (DHT), and 5α-androstan-3α, 17β-diol (3α-diol) in human plasma and prostatic tissue. After addition of tri-deuterated steroids as internal standards to prostatic tissue homogenates or plasma samples, extraction was performed with diethylether. The extracts were purified by two chromatographic steps (Sep-Pak C 18 cartridge and Sephadex LH-20) and injected into a gas chromatograph coupled with a mass spectrometer after derivatization with heptafluorobutyric acid. This method was highly specific and showed good precision, accuracy, reproducibility and sensitivity. T, DHT, and 3α-diol were measured in human plasma and in prostatic tissue of seven patients with benign prostatic hyperplasia (BPH) treated for 3 months with a long acting GnRH analog before surgery. Plasma levels of T, DHT, and 3α-diol were reduced by GnRH analog treatment to castrate levels. The tissue concentrations of the same steroids, compared with those obtained from 19 untreated patients, showed a mean reduction of about 90% for DHT and 3α-diol, and about 75% for T. These results suggest that about 90% of prostatic DHT and 3α-diol depend on testicular activity because they are dramatically reduced after pharmacologic castration.

Effect of repeated washing on sperm-bound immunoglobulin G.

Abstract: The effect of sperm washing on the stability of sperm-bound immunoglobulin G (IgG) antibody derived from plasma from four patients and also IgG bound in vivo on the spermatozoa of four other men was quantitatively evaluated. In the first series of experiments, human spermatozoa were incubated with an IgG antibody-containing plasma and subjected to 18 cycles of sperm washing. In the second set of experiments, spermatozoa from men positive for sperm-bound IgG were subjected to four cycles of sperm washing. The amount of residual antibody bound to a constant number of spermatozoa was quantitated by a radiolabeled antiglobulin assay during and following the washing procedures. There was no significant loss of sperm-bound antibody due to the washing procedures. The results of these studies undermine the utility of sperm washing as an effective treatment of antibody-mediated infertility in men.

Effect of germ cells on vectorial secretion of androgen binding protein and transferrin by immature rat Sertoli cells in vitro.

Abstract: The influence of germ cells (greater than 85% pachytene spermatocytes) on vectorial secretion of androgen binding protein (ABP) and transferrin by immature rat Sertoli cells was investigated using two-compartment culture chambers. The ratio of ABP secreted into the outer and inner compartment in control cultures of Sertoli cells alone was 1.9, and was not influenced by either FSH or testosterone. Co-culture of Sertoli cells in direct contact with germ cells in the presence of FSH decreased this ratio, the decrease being most pronounced (0.7) after 2 days of co-culture. This effect was not observed if the germ cells were not in direct contact with Sertoli cell monolayers. The outer to inner compartment ratio of transferrin in Sertoli cell-alone cultures was 1.6 and, in contrast to ABP, was not significantly influenced by the addition of germ cells, even in the presence of FSH. It is concluded that in immature rat Sertoli cells the polarity of ABP secretion, but not that of transferrin, may be regulated by pachytene spermatocytes (and possibly other germ cells), and that this process is FSH-dependent.

Antimitotic drugs in the male rat. Behavioral abnormalities in the second generation.

Abstract: The second generation descended from rats treated either with cyclophosphamide alone or with both cyclophosphamide and vinblastine were investigated. As in the first generation, the offspring were evaluated for mean litter size, sex ratio, frequency of gross external malformations and, within the first 4 months of life, growth and mortality. When they reached adulthood, between 12 and 16 weeks of age, the offspring were also tested for spontaneous activity and learning capacity. At birth, the progeny of the treated grandfathers did not show malformations or any other obvious disorder. However, when compared with the control population, the experimental animals showed significantly decreased success rates in a learning task, whatever the learning performance of their parents. Furthermore, decreased spontaneous activity was observed in the male subjects from unsuccessful parents. The similarities between the anomalies found in the first and the second generations argue for the induction of mutations by antimitotic drugs. This hypothesis and the subtle differences between generations and between sexes are discussed.

Serum levels of total testosterone and sex hormone binding globulin in hypothyroid patients and normal subjects treated with incremental doses of L-T4 or L-T3.

Abstract: Plasma testosterone (T) and sex hormone binding globulin (SHBG) were assayed in normal controls (N = 9) and hypothyroid patients (N = 17) receiving increasing doses of L-T4 (0.2 mg, 0.4 mg for 30 days), followed first by 30 days without medication and then by 30 days each of 0.05 mg L-T3 and 0.2 mg L-T3. Normal male controls showed a significant increase in plasma T only at high doses of either L-T4 (0.4 mg) or L-T3 (0.2 mg). A small but significant increase in plasma T levels was observed in normal female subjects at 0.4 mg of T4. In both men and women, plasma SHBG increased in a dose-dependent manner with L-T4 or L-T3 and correlated positively and significantly with serum thyroid hormone levels. Hypothyroid male subjects had significantly lower levels of plasma T (mean +/- SD) of 279 +/- 131 ng/dl as compared with normal males (431 +/- 118 ng/dl), which reached the normal range only at a relatively high dose of either L-T4 (0.4 mg) or L-T3 (0.2 mg). No significant changes in plasma T were seen in the hypothyroid female group. Basal plasma SHBG levels were significantly lower in both hypothyroid men and women and increased towards normal levels during L-T4 and L-T3 therapy, although the response to thyroid hormones was significantly lower than that of normal controls. These results indicate that thyroid hormone therapy increases plasma SHBG levels in both normal and hypothyroid patients and that this increase precedes the expected elevation of plasma T in males.

Differential effects of (+) and (-) gossypol enantiomers on mitochondrial function and proliferation of cultured TM4 cells.

Abstract: The in vitro effects of (+) and (-)gossypol enantiomers on the mitochondrial functions of mouse transformed Sertoli, TM4 cells were investigated by monitoring mitochondrial rhodamine 123 accumulation. When TM4 cells were cultured in medium without fetal calf serum, 5 micrograms/ml of both enantiomers caused similar declines in mitochondrial rhodamine 123 staining. By contrast, (-)gossypol had a greater adverse action than did (+)gossypol on the mitochondrial of TM4 cells that were cultured in medium supplemented with 2% fetal calf serum. Construction of dose response curves for the effects of the two enantiomers on rhodamine 123 accumulation by TM4 cells after 5 hr of drug treatment gave an EC50 of 7.5 micrograms/ml for the (-)gossypol isomer compared with 18 micrograms/ml for the (+)gossypol isomer. Similarly, TM4 cell proliferation was also more disturbed by (-)gossypol in medium supplemented with other concentrations of fetal calf serum. The results suggest that the lower effect of (+)gossypol on TM4 cells may be attributed to its higher affinity to serum components, which may impede its entrance into TM4 cells.

Morphologic changes induced by short-term ischemia in the rat testis are not affected by treatment with superoxide dismutase and catalase.

Abstract: Short-term testicular ischemia was induced unilaterally by ligating the testicular artery for 60 or 100 minutes in adult rats. After 7 days, the rats were fixed by vascular perfusion. The effect of ischemia and reperfusion was quantified using morphometric techniques. Occlusion of the testicular artery for 60 and 100 minutes resulted in a mild and moderate damage, respectively, to the seminiferous tubules. The contralateral control testis was not affected. To study the role of oxygen radicals in ischemia-reperfusion-induced testicular damage, rats were treated with superoxide dismutase and catalase. These radical scavengers did not influence the extent of testicular damage.

Changes in Testicular Morphology in Boars Actively Immunized Against Gonadotropin Hormone‐Releasing Hormone

Abstract: Alterations in testicular morphology were studied in boars actively immunized against gonadotropin hormone releasing hormone (GnRH). Ten boars were divided equally into two experimental groups (five GnRH-immunized, and five controls). Antibody production was achieved by conjugating GnRH to human serum globulin (hSG). The GnRH-hSG conjugate was emulsified in complete Freund's adjuvant, and administered to boars at 12 weeks of age. Boars were given a booster in incomplete Freund's adjuvant on week 18 and 20. The presence of high antibody titers to GnRH caused luteinizing hormone and testosterone to decline to nondetectable levels. Morphometric examination showed a reduction in percentage volume in Leydig cells/unit testis, seminiferous tubule diameter and seminiferous epithelial height, and an increase in non-Leydig cell interstitial tissue in GnRH-immunized boars compared with controls. Histologic evaluation displayed severe damage of the seminiferous epithelium, absence of spermatids, incomplete cell associations, disruption of Sertoli cells, formation of multinucleated giant cells, and a striking reduction in size and cytoplasmic structures of Leydig cells in GnRH-immunized animals. These results demonstrate the potent inhibitory effects of GnRH immunoneutralization on the boar reproductive system.

Isolation and Characterization of the Plasma Membrane of Rat Cauda Epididymal Spermatozoa

Abstract: 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.

Localization of the human prostatic secretory protein PSP94 and its mRNA in the epithelial cells of the prostate.

Abstract: The histologic distribution of a prostatic secretory protein of 94 amino acids (PSP94) and its mRNA were examined simultaneously by conventional immunohistochemistry and in situ hybridization histochemistry. The results show localization of immunoreactive PSP94 and its mRNA in the epithelial cells of the prostate gland, thus providing strong evidence of PSP94 synthesis in these cells. Preliminary analysis of PSP94 by radioimmunoassay and of its mRNA by Northern blot analysis indicates that PSP94 biosynthesis in pathologic prostatic tissues is variable. The value of PSP94 as a marker of prostate gland secretory activity is discussed.

Effects of Ethanol Consumption on the Morphology of the Rat Seminiferous Epithelium

Abstract: The effects of ethanol consumption on the morphology of the seminiferous epithelium, with particular emphasis on Sertoli cell ultrastructure, were examined during and following pubertal development. Sprague-Dawley rats were maintained on chronically high levels of ethanol for 7 weeks beginning at 29 days of age. Animals in Group 1 were fed a liquid diet containing ethanol (36% ethanol-derived calories) ad libitum. Group 2 animals were paired with animals in Group 1 and fed a liquid control diet in the amount consumed by their ethanol partners (g/kg body wt/day). Animals in Group 3 were fed Purina rodent chow ad libitum. Blood samples were collected at 60 days for determination of plasma testosterone levels. On day 79, each epididymis, the adrenals and the right testis were removed from anesthetized animals and weighed; the left testis was removed and processed for light and electron microscopy. Blood alcohol levels were consistently high throughout the feeding period, averaging 272.6 ± 9.7 mg/100 ml at 1900 hours (1 hour after lights off) and 178.8 ± 20.8 mg/100 ml at 1330 hours. Testosterone levels were lower in ethanol-consuming animals than in pair-fed or control subjects. Testis weight was also somewhat reduced in ethanol-consuming animals; however, when adjusted for body weight, relative testis weights were found to be increased in ethanol and pair-fed animals. Epididymal weights were reduced in both ethanol and pair-fed animals. Relative adrenal weights were increased by ethanol. The most dramatic effect of ethanol consumption was on the morphology of the seminiferous tubules. Although intercellular junctions appeared intact in all groups, there was a dramatic increase in lipid at the base of Sertoli cells in alcohol-fed animals. Lipid was also observed in spermatogenic cells. Pair-fed animals were similar to controls. Lipid accumulation may be indicative of ethanol-induced damage to Sertoli cells and/or the associated germ cells.

Measurement of seminal LDH-X and transferrin in normal and infertile men.

Abstract: LDH-X, an isoenzyme of lactate dehydrogenase specific for germinal epithelium activity, has been measured in the seminal plasma of infertile subjects whose infertility had different origins. In the same samples, seminal transferrin, an index of Sertoli cell function, was also measured. In this investigation, seminal LDH-X was not detectable in the vasectomized subjects, in patients with azoospermia due to seminiferous tubular damage, nor in patients who showed a marked decrease in sperm concentration (less than 1 X 10(6)/ml). In oligozoospermic patients (sperm concentration less than 20 X 10(6)/ml) seminal LDH-X levels were reduced to about one-third of those found in normal controls. Seminal LDH-X levels correlated (r = 0.7237) with total sperm count better than seminal transferrin levels (r = 0.5511), while no correlation was found between these two biochemical parameters and sperm motility, viability and morphology. To study their spontaneous variations with time, LDH-X and transferrin were also measured in semen specimens collected monthly from five healthy men, over 1 year. In these samples (N = 60), sperm count variability (43.1%, calculated in terms of the coefficient of variation), was similar to that of LDH-X (40.4%) and higher than for transferrin (23.0%).

Origin and fate of autophagosomes in Leydig cells of normal adult rats

Abstract: Autophagosomes were observed frequently in electron microscope photographs of Leydig cells from normal adult rat testis. Their formation, evolution and fate were analyzed morphologically in preparations treated to show cytidine monophosphatase (CMPase) and glucose-6-phosphatase (G-6-Pase) activities and in animals sacrificed at various time intervals ranging from 5 min to 6 hrs after a single intratesticular injection of cationic ferritin. Analysis of the morphologic data led to the following interpretation and model. Preautophagosomal structures appeared as flattened, elongated membranous profiles. These expanded, took on a C-shape and fused at their edges to demarcate a small cytoplasmic territory containing normal-looking smooth endoplasmic reticulum (ER) and mitochondria. Such early autophagosomes were thus delimited by two membranes separated by a narrow lumen. Following fusion of these elements with secondary lysosomes, the space between the two membranes increased in size, the inner membrane disintegrated and the enclosed organelles no longer could be identified. The late autophagosomes then reached the cell surface and appeared to exocytose their residual content. In contrast to secondary lysosomes and trans-Golgi elements, which were CMPase-positive, the preautophagosomal flattened membranous elements and early autophagosomes were CMPase-negative. The late autophagosomes on the contrary were CMPase-positive. While ER cisternae were G-6-Pase-positive, the pre-, early and late autophagosomal structures were unreactive for this enzyme. Cationic ferritin tracer experiments showed that only late autophagosomes became labeled with cationic ferritin following their fusion with secondary lysosomes into which the tracer had accumulated following its endocytosis from the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

The pituitary-testicular axis at rest and during moderate exercise in males with diabetes mellitus and normal sexual function.

Abstract: Hormonal studies of pituitary-testicular function in insulin-dependent diabetes mellitus were examined at rest and during moderate exercise to assess whether diabetes per se caused abnormalities of nocturnal penile tumescence and androgen function in men with normal sexual function. The present study compared 10 healthy men and eight men with Type I diabetes mellitus in whom normal sexual function was determined by clinical history. Urinary gonadotropin excretion, semen analysis and diurnal variation of serum glucose, prolactin, testosterone and free testosterone were determined in both groups. In addition, the serum levels of testosterone, free testosterone, prolactin, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were measured at rest, during 45 minutes of exercise on a bicycle ergometer at 50% of the subjects previously determined maximal oxygen uptake (VO2 max) and during a 30-minute recovery period. Nocturnal penile tumescence and parameters of semen analysis were similar in both groups. Urinary FSH excretion and serum FSH were higher (P less than or equal to 0.01) in the diabetic subjects while urinary LH excretion was similar. Diurnal variation of serum prolactin, testosterone and free testosterone were similar in both groups. Exercise produced a significant (P less than or equal to 0.01) increase in maximal free and total testosterone in both groups without changes in serum FSH or LH. Prolactin increased significantly (P less than or equal to 0.01) during exercise in the diabetic group only. We conclude that, for the most part, the pituitary-testicular axis and nocturnal penile tumescence under basal conditions and the pituitary-testicular axis during moderate exercise are similar in healthy males and insulin-dependent diabetic males with normal sexual function.

HCG treatment increases intratesticular pressure in the abdominal testis of unilaterally cryptorchid rats.

Abstract: Adult, unilaterally cryptorchid rats were given a single subcutaneous injection of hCG. HCG treatment of 100 I.U. (but not 10 I.U.) resulted in a marked increase in intratesticular pressure (approximately 40 mm Hg) in the abdominal testis that was maximal 24 hours after treatment. This increase in pressure is caused by increased vascular permeability coupled with insufficient lymph drainage. In the scrotal testis, hCG treatment resulted in increased vascular permeability and lymph flow, but this did not result in a marked increase in testicular pressure. No morphologic signs of hCG-induced damage were observed in either the abdominal or scrotal testis 10 days after hCG treatment. Testicular microcirculation, as studied by laser doppler flowmetry, was abnormal in the abdominal testis, but hCG treatment inhibited vasomotion in both the abdominal and scrotal testis.

Doppler examination in varicocele. A standard method of evaluation.

Abstract: Doppler sonography is considered a reliable method for detecting reflux due to venous valvular incompetence, which occurs in varicocele. It is, however, a matter of debate whether the characteristics of this reflux can be correlated quantitatively in a clinical setting. In this study, a two-step method was utilized to identify reflux in basal conditions with the patient standing and breathing spontaneously and to determine the time of centrifugal secondary venous reflux of the distal spermatic cord during the squeezing and relaxing maneuver. This method is closely related to that used by the vascular surgeon to detect valvular incontinence of the saphenous vein. Since Doppler sonography is much more reproducible than Valsalva's maneuver, it is therefore much more reliable. In a 12-month period, 625 patients with pathologic findings in at least two spermiograms were studied. Thirty percent showed constant basal reflux not influenced by respiratory exhilation. The squeezing and relaxing maneuver induced secondary reflux longer than 1.6 seconds in 17%, between 0.8 and 1.6 seconds in 17% and less than 0.8 seconds in 36% of the patients. The higher sensitivity and specificity of Doppler examination compared with thermography and angiography, as well as its low cost and noninvasiveness, make this the procedure of choice in the diagnosis of venous reflux in varicocele.

Alterations in the pulsatile properties of gonadotropin secretion in alcoholic men.

Abstract: The functional characteristics of the hypothalamic-pituitary-testicular axis were examined quantitatively in 10 chronic alcoholic men without hepatic dysfunction or clinical nutritional deficiencies. Spontaneous gonadotropin pulsatility was analyzed in blood sampled every 20 minutes over a 24-hour period 3 to 16 days after abstinence from alcohol and again 29 to 39 days later. The numbers of LH and FSH pulses per 24 hours were normal in these alcoholic men compared with controls. However, we found increased mean 24-hour concentrations of immunoactive LH (P = 0.012) and FSH (P = 0.018), increased peak heights for LH (P = 0.035) and FSH (P = 0.004), decreased fractional LH (P = 0.002) and FSH (P = 0.044) pulse amplitudes and increased interpulse valley mean LH (P = 0.010) and FSH (P = 0.018) concentrations. Serum levels of total testosterone, total estradiol and estrone were normal, whereas concentrations of free testosterone and free estradiol were increased. Pituitary release of LH and FSH was normal in response to low (5-micrograms) and high (95-micrograms) doses of GnRH given intravenously. The present observations indicate that in chronically alcoholic men, acute abstinence from ethanol is associated with elevated circulating concentrations of immunoactive gonadotropins in the presence of intact spontaneous gonadotropin pulsatility, preserved pituitary responsiveness to exogenous GnRH, and increased concentrations of free testosterone and free estradiol. Such findings are consistent with alterations in the endogenous feedback actions of sex steroid hormones in this setting.