Showing papers in "Journal of Andrology in 1984"

Analysis of human sperm function following exposure to the ionophore A23187. Comparison of normospermic and oligozoospermic men.

Abstract: Time exposure photomicrography and interspecific in vitro fertilization techniques have been used to compare the responses to the divalent cation ionophore A23187 of spermatozoa from normal fertile and oligozoospermic men. The fertilizing capacity of spermatozoa from the fertile controls produced a bell-shaped dose response curve when assessed in the presence of ionophore. The optimal responses occurred in the presence of 50 and 100 microM A23187. At this concentration, a mean penetration rate of about 75%, in association with multiple polyspermy, was observed without significant changes in motility patterns. At higher doses of A23187, there was a decline in fertilization rates, an independent reduction in sperm motility, and a significant decrease in the amplitude of lateral sperm head displacement. In contrast to the fertile controls, spermatozoa recovered from patients with oligozoospermia failed to exhibit a significant change in their fertilizing potential following exposure to A23187. Calculations based on the Poisson distribution theory indicated that this lack of responsiveness was not related to any differences in the motility of the spermatozoa from the oligozoospermic patients compared to the controls. These results suggest that calcium ionophores may be of value in providing a rapid and sensitive indicator of the functional competence of human spermatozoa, which circumvents problems concerning the rate and efficiency of sperm capacitation encountered with conventional protocols.

Secretion of androgen binding protein by Sertoli cells is influenced by contact with germ cells.

Abstract: Sertoli cells were cultured alone or with germ cells to evaluate the effect of the association with germ cells on the secretory activity of Sertoli cells. Secretion of androgen-binding protein, which is specifically secreted by Sertoli cells, was measured under several experimental conditions. The following experimental models were utilized: 1) cultures of explants of seminiferous epithelium from prepubertal animals in which germ cells adherent to Sertoli cells are present (Sertoli cell enriched cultures); 2) monolayers formed only by Sertoli cells, obtained by removing germ cells from Sertoli cell enriched cultures, and 3) cocultures of Sertoli cell only cultures and germ cell populations at defined stages of differentiation. The results obtained indicated that FSH-induced ABP secretion was greatly reduced in Sertoli cell only cultures as compared to enriched Sertoli cell cultures, and that this difference was stable throughout the first eight days of culture. In addition, cocultures of Sertoli cell only cultures with germ cells induced an increase of ABP when cocultured germ cells were at differentiation stages, such as pachytene spermatocytes, which are able to recognize and firmly adhere to the Sertoli cell monolayers. Cocultures with round spermatids, which do not adhere to Sertoli cells, did not increase the amount of FSH-induced ABP production. The addition of nongerminal cells such as lymphocytes and fibroblasts were also not effective in stimulating ABP secretion. Surface interaction between Sertoli cells and cocultured germ cells seemed to be necessary for this FSH-induced ABP production.(ABSTRACT TRUNCATED AT 250 WORDS)

A Prospective Study of the Relationship Between Semen Quality and Fertility in Cases of Unexplained Infertility

Abstract: The male partners of 68 couples exhibiting 5.1 +/- 0.3 (SEM) years of unexplained infertility were assessed using the conventional criteria of semen quality, the movement characteristics of the spermatozoa and the outcome of the zona-free hamster egg penetration test. After a follow-up period of 2.3 +/- 0.06 (SEM) years, 25 (37%) of these patients were found to have initiated a pregnancy, thereby permitting an analysis of those aspects of semen quality which most accurately predicted their subsequent fertility. A multivariate discriminant analysis revealed that the conventional semen profile, per se, was not of significant value in discriminating the incidence of pregnancy. However, significant discrimination (P = 0.0173) was obtained when the postcapacitation movement characteristics of the spermatozoa were incorporated into the analysis. The accuracy of this prognosis was further increased if either the duration of infertility or the outcome of the zona-free hamster egg penetration test was taken into consideration. Overall classifications of fertility were then 76.3% and 76.5% accurate, respectively. These results suggest that in vitro assessments of human sperm function are of significant value in evaluating male fertility.

The Meaning of Sperm Capacitation A Historical Perspective

Abstract: It should be recalled that sperm capacitation was originally defined in 1952 as some physiological changes of the spermatozoa in the female genital tract before they are capable of penetrating and fertilizing the eggs. It was found further that capacitation can be achieved outside the female tract, first in the presence of biological fluids, and then in the absence of biological fluids. Later on it was found that capacitated rabbit uterine spermatozoa still have acrosome and that the acrosome reaction of rabbit spermatozoa occurred in contact with eggs in the oviduct. Thus, several authors separated acrosome reaction from capacitation and considered capacitation as a preparation for the acrosome reaction, even though the titles of their articles still implied that capacitation included acrosome reaction. During the past 30 years we have found many membrane changes on the molecular and immunological level in spermatozoa that prepare them for physiological changes such as "hyperactivation," and morphological changes such as "the acrosome reaction." These events lead to more vigorous motility and to the release of various enzymes for the penetration of the egg. Undoubtedly, further study will reveal more molecular, physiological, and morphological changes in the mammalian spermatozoa before they are capable of fertilization. There are definite changes before hyperactivation and acrosome reaction, but these changes are parts of capacitation, if we prefer to keep its original meaning. It is proposed here that in order to save further confusion, capacitation of spermatozoa should be defined as originally proposed, that is, to include all the events that lead to the development of the capacity of mammalian spermatozoa to penetrate eggs.(ABSTRACT TRUNCATED AT 250 WORDS)

The Effects of Ethylene Glycol Monomethyl Ether on Testicular Histology in F344 Rats

Abstract: Ethylene glycol monomethyl ether (EGME) has been found to produce testicular atrophy in experimental rodents. The studies that follow were designed to determine the testicular cell type(s) most susceptible to EGME administration. For histologic studies, F344 rats were gavaged with 150 mg/kg/day of EGME 5 days per week, and serially sacrificed. In sections from perfusion-fixed tissue, necrotic changes were observed in some meiotic and premeiotic spermatocytes 24 hours after a single dose. Also, nuclear condensation was seen in occasional early pachytene spermatocytes. These effects were magnified after two doses; there were more necrotic pachytene and meiotic spermatocytes than necrotic stage I pachytene spermatocytes. By day 4, testes from all treated animals were affected; there was a pronounced maturation-depletion effect, seen as the absence of round spermatids from tubules in stages I to III. These effects continued to develop at days 7 and 10, leaving only Sertoli cells, spermatogonia, and late stage spermatids populating the epithelium. Other animals were treated similarly, but subject to efferent duct ligation 16 hours prior to sacrifice. Fluid production, as judged by weight gain in the testes after efferent duct ligation, was unaffected by EGME treatment. Analysis of the fluid collected at the rete testis indicated that there was no treatment-related change in the relative amounts of androgen binding protein. The data indicate that the spermatocyte is the primary target cell for the histologic effects of EGME in the testis of F344 rats.

Specific binding of the glycosaminoglycan 3H-heparin to bull, monkey, and rabbit spermatozoa in vitro.

Abstract: In Vitro binding and some binding parameters of the glycosaminoglycan heparin to viable epididymal or ejaculated bull spermatozoa, ejaculated rabbit spermatozoa, and frozen-thawed rhesus monkey spermatozoa were investigated. Nonspecific binding was affected only by the concentration of 3H-heparin, whereas specific binding was saturable, reversible, and dependent on the pH, temperature, and calcium concentration of the incubation medium. Magnesium concentration dependence was observed in the presence of calcium but could not be detected in the absence of calcium. Bound 3H-heparin was displaced by several orders of magnitude greater concentrations of chondroitin sulfate. Scatchard plot analysis suggested multiple binding affinities for 3H-heparin to spermatozoa. 3H-heparin was shown to bind to sperm heads and flagella. Fluorescein-labeled heparin bound to acrosomal, postacrosomal, and flagellar membranes. It was concluded that the specific binding of heparin involved a proteinaceous component on, or intercalated with, spermatozoal membranes. Thus, glycosaminoglycans present in the female reproductive tract may contribute to sperm capacitation and enhance the likelihood of successful fertilization in mammals.

Identification of Arginine Esterase as the Major Androgen‐dependent Protein Secreted by Dog Prostate and Preliminary Molecular Characterization in Seminal Plasma

Abstract: This work was undertaken to determine the identity of the major androgen-dependent 15,000 molecular weight protein previously observed on SDS polyacrylamide gel electrophoresis of both dog prostate cytosol and dog seminal plasma. The protein was identified as one of the two chains of arginine esterase on the basis of its ability to bind 3H-diisopropylphosphofluoridate (DFP), an active site titrant of serine proteases. Furthermore, since the other polypeptide chain was heterogeneous, at least five distinct peaks of arginine esterase activity could be separated by chromatofocusing under nonreducing conditions. The molecular weight of the seminal plasma protein was estimated at 29,500 by Sephadex G-100 gel filtration, and at 25,000 by SDS polyacrylamide gel electrophoresis in the absence of mercaptoethanol. In the presence of mercaptoethanol, two major peaks were observed with molecular weights of 15,000 and 14,000. These results show that arginine esterase of dog seminal plasma is a serine protease composed of two different chains linked by disulfide bridges. One of the chains contains the reactive serine group. The other one is probably glycosylated since it presents several isoelectric points.

Effects of an LHRH agonist analog upon sexual function in male dogs. Suppression, reversibility, and effect of testosterone replacement.

Abstract: Male beagle dogs were injected once daily with 10 micrograms/kg of [6-D-(2-naphthyl)alanine]-LHRH (D-Nal(2)6-LHRH), a potent LHRH agonist, for periods up to 42 days, with recovery periods up to 172 days. Blood samples collected at regular intervals were assayed for LH, FSH, and testosterone; total ejaculates were collected and analyzed weekly, and animals were sacrificed at various intervals for sex organ weights and histology. The first injection of D-Nal(2)6-LHRH caused an acute elevation in plasma levels of LH, FSH, and testosterone, measured at 2 and 4 hours after the injection. This acute response to injection was attenuated with each successive injection and by two weeks no elevation was seen, suggesting a down-regulation of pituitary response. Basal levels of LH and testosterone were maximally depressed by four days of treatment. Testis volume, duration of erection, ejaculate volume, sperm count, sperm motility and testis volume all declined during treatment, with sperm count significantly lowered by two weeks and ejaculation volume becoming zero by five weeks of treatment. Spermatogenesis, assessed histologically, was partially suppressed at ten days and completely suppressed by 38 days of treatment. All parameters returned to normal following cessation of treatment. Recovery time was longer for the dogs treated for 42 days than for those treated for ten days. When testosterone was supplemented during 42 days of agonist treatment, basal plasma testosterone levels were maintained at the low end of the normal range. Testosterone supplementation did not prevent pituitary down-regulation, suppression of spermatogenesis, or the decrease in testis and epididymis weights, but prevented the decline in duration of erection. Ejaculate volume and sperm count declined more slowly with combination treatment than with agonist alone. During the decline in sperm count sperm motility was maintained with combination treatment. Injection of hCG into control and agonist treated dogs resulted in similar percentage increases in plasma levels of testosterone, although peak levels were greater in control than in treated animals. The data suggest a pituitary desensitization with this LHRH agonist in the dog but only a minor role for testicular desensitization.

Comparison of glycerol and a zwitter ion buffer system as cryoprotective media for human spermatozoa. Effect on motility, penetration of zona-free hamster oocytes, and acrosin/proacrosin

Abstract: This study compared the cryoprotective effect of glycerol with that of a zwitter ion buffer system (TESTCY). Spermatozoa that are cryopreserved in the presence of glycerol possess a somewhat higher progressive motility immediately after thawing than those preserved in the presence of TESTCY. However, after a 1-hour incubation in glycerol-free medium, the progressive motilities of the glycerol- and TESTCY-treated spermatozoa become essentially identical. After 2 hours in culture medium, TESTCY-treated spermatozoa possess a higher motility than glycerol-treated spermatozoa, indicating that TESTCY is a better preservative than glycerol for the long-term motility of human spermatozoa. The fertilizing potential of the cryopreserved spermatozoa was assessed by their ability to penetrate zona-free hamster oocytes in vitro. Spermatozoa that are cryopreserved in the presence of TESTCY produce three- to four-fold higher penetration rates than glycerol-treated, cryopreserved spermatozoa. Cryopreservation in the presence of TESTCY also results in a higher stability of the acrosin/proacrosin system than when the spermatozoa are preserved in glycerol, since about two- to three-fold higher levels of proacrosin are retained. These results indicate that TESTCY is a better cryopreservative for human spermatozoa than glycerol.

Origin of a motility inhibitor within the male reproductive tract.

Abstract: Seminal plasma contains a motility inhibitor of demembranated reactivated spermatozoa. We investigated its origin within the reproductive tract. The highest level of inhibitor was detected in seminal vesicle fluids from the three species investigated (bull, rat, rabbit). Significant levels of inhibitor were also observed in prostatic fluids. Testes and epididymal fluids, as well as bulbo-urethral and coagulating gland homogenates were essentially devoid of inhibitor. On a mg protein basis, the inhibitor in seminal vesicle fluid was about four times less active than the inhibitor of seminal plasma. The high level of inhibitor in seminal plasma can not be explained by the synergistic effect of the combination of seminal vesicle, prostatic and epididymal fluids. Dialysis experiments suggested that the high level of inhibitor in seminal plasma was mainly due to the presence of a dialysable activator. This activator is capable of potentiating up to four-fold the inhibitor present in seminal vesicle fluid.

The Effect of Prepubertal Spermatic Cord Torsion on Subsequent Fertility in Rats

Abstract: This study was undertaken to determine the effects of various durations of testicular torsion in prepubertal rats on their subsequent fertility, and to determine whether these effects could be altered by removal of the torsioned testis. Sixty rats (35 days old) were subjected to 720 degrees unilateral spermatic cord torsion for 0, 1, 3, 5, 9, or 12 hours. The torsioned testis was then either detorsioned or removed. At 65 days of age each male was housed with two females for three weeks. Rats undergoing detorsion of the spermatic cord demonstrated a linear decrease in fertility with respect to the duration of torsion (r = -0.904). However, all of the animals undergoing unilateral torsion with subsequent orchiectomy were fertile, regardless of the duration of torsion. In addition, the percentage of females impregnated, the number of embryos produced, and the mean embryo size decreased with increasing intervals of torsion (r = -0.834 to r = -0.979); the sharpest decline occurred between 5 and 9 hours of torsion. All of these parameters were significantly lower (P less than 0.001 to P less than 0.05) in the detorsioned group as compared to the orchiectomized group. There was a decrease in seminiferous tubule diameter in the contralateral testis with respect to the duration of torsion (P less than 0.01). These data indicate that unilateral spermatic cord torsion in young rats significantly reduced their subsequent fertility with respect to duration of the torsion, and that this detrimental effect may be minimized if the damaged testis is removed rather than untwisted and replaced into the scrotum.

Pulsatile Secretion of Gonadotropins and Prolactin in Male Marathon Runners Relation to the Endogenous Opiate System

Abstract: We tested the hypothesis that sustained, strenuous physical training alters the neuroendocrine regulation of pulsatile gonadotropin and/or prolactin secretion in men. Blood was sampled at 20-minute intervals over 8 hours in five endurance-trained men after a 10-15 mile run in the middle of the active training season, and in 11 nonendurance trained normal controls. In these two groups, basal patterns of physiologically pulsatile secretion of LH, FSH, and prolactin (PRL) were not significantly different in relation to the following parameters: mean serum concentration of each of the three hormones (N = 25 samples); areas under the hormone concentration vs. time curves; fractional, incremental, and absolute pulse amplitudes; and pulse frequency, or periodicity. To test for enhanced suppressive effects of endogenous opiates in trained male marathon runners, subjects were administered the potent opiate-receptor antagonist, naltrexone (1 mg/kg). This antagonist significantly stimulated pulsatile LH secretion by increasing mean serum LH values from 10.94 to 13.58 mIU/ml (P = 0.007); area under the LH concentration vs. time curve increased from 5370 to 6510 mIU/ml X 8 hours (P = 0.05) and, pulse frequency rose from 2.8 to 4.9 pulses/8 hours (P = 0.006). Naltrexone also enhanced pulse frequency of FSH secretion from 3.4 to 5.4 pulses/8 hours (P = 0.009), but did not alter serum prolactin concentrations. None of these responses differed significantly from those in normal sedentary controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of rat caput epididymidis contractility by prostaglandins.

Abstract: Mechanical activity of the rat caput epididymidis in vitro was recorded using a videomicrography system. The effects of prostaglandin (PG)F2 alpha, PGE2, and aspirin on caput epididymidis contractility were determined by measuring the frequency of contraction, luminal diameter, and amplitude of contraction at various concentrations of each test compound in vitro. PGF2 alpha stimulated contractility of the tubules at physiological concentrations, while PGE2 reduced contractility. Aspirin strongly inhibited contractility at concentrations of 10(-3) and 10(-2)M. Endogenous levels of PGF2 alpha and PGE were determined for rat testes, caput, corpus, and cauda epididymidis and vas deferens. While the concentrations of PGE were consistently higher than those of PGF2 alpha, both compounds were relatively low in the testes, high in the vas deferens, and intermediate throughout the epididymis. Results from these experiments strongly suggest that PGs are important regulators of proximal epididymidis contractions and thus may regulate sperm transport through that organ.

Organ culture of rat caput epididymal tubules in a perifusion chamber.

Abstract: We have been able to culture caput epididymal tubules from rats by modifying an organ culture system (Orgebin-Crist et al, 1980) which was used to culture rabbit corpus epididymal tubule segments. The morphology of the epithelium was consistently good throughout seven days in culture, although sloughed epithelium was commonly seen during the first 24 hours. Evidence of this sloughing was much less frequent thereafter. Throughout seven days of culture, autoradiography of SDS-PAGE of luminal fluid obtained from tubules cultured in medium containing 14C-L-leucine showed incorporation into bands identical to those stained by Coomassie Blue. Rat epididymal alpha-lactalbumin was consistently localized on the luminal surface of the epithelium and the middle piece of the spermatozoa. Spermatozoa appeared to have normal morphology throughout the first three days in culture; thereafter, decapitated spermatozoa were frequently seen. Caput spermatozoa only quiver in place prior to culture, but after three days in culture, 53% of the spermatozoa from distal caput tubules are progressively motile upon dilution in a balanced salt solution. Since the transit time for spermatozoa in the caput epididymidis of the rat is approximately three days, it should be possible with this culture system to study maturational events involving interactions between spermatozoa and the epididymal epithelium.

Physiopathologic aspects of Leydig cell function in varicocele patients

Abstract: Leydig cell function was studied in 108 varicocele (V) patients with a mean age of 30.9 years, and a control group (C) of 46 men with a mean age of 30 years. Plasma gonadotropin levels were determined before and after GNRH stimulation. Testosterone (T), 17-OH-progesterone (17-OH-P), dihydrotestosterone (DHT) and estradiol (E2) were also assayed. Mean plasma T levels were significantly decreased in varicocele patients (V = 416 +/- 12.9, n = 106; C = 487 +/- 19.9, n = 40; P less than 0.01), while the basal 17-OH-P/T ratio was significantly increased (V = 0.38 +/- 0.02, n = 56; C = 0.28 +/- 0.02, n = 40; 0.02 greater than P greater than 0.01) and remained higher after hCG stimulation (P less than 0.01). No significant differences in mean sex steroid levels were observed when comparing varicocele patients with normal sperm counts (VN) and those who had oligozoospermia (VO). There was a significant negative linear correlation between age and 17-OH-P (n = 56; r = -0.47; P less than 0.01) and T values (n = 106; r = 0.27; P less than 0.01) in varicocele patients, which contrasted with the absence of any significant correlation with age in the controls. These data suggest that the duration of idiopathic varicocele influences testicular hormone secretion.

Do endocrines play an etiological role in diabetic and nondiabetic sexual dysfunctions

Abstract: Sexually dysfunctional diabetic and nondiabetic males were compared with a group of normal controls using different endocrinological, psychophysiological, and psychological parameters. One hundred male subjects participated in this study: 47 diabetics with sexual dysfunction (DD), 31 nondiabetics with sexual dysfunction (NDD), and 22 normal controls (C). They were evaluated by an internist (physical examination and medical history), a psychologist (psychological and sexual functioning tests), a psychiatrist (psychiatric history and mental status examination), a urologist (genitourinary physical examination), and an endocrine biochemist (evaluation of endocrine factors). Additionally, subjects were evaluated for nocturnal penile tumescence (NPT) during three nights in the sleep laboratory to obtain a differential diagnosis of impotence, that is, psychogenic vs. organic. Both sexually dysfunctional groups showed significant differences on several measures in the psychological and psychophysiological evaluations. There were also significant differences between these two groups and the control group. Plasma levels of total testosterone and serum levels of prolactin, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) showed no significant differences among the three groups, but there were some significant correlations between the endocrine and psychological measures. No significant correlations were found between the endocrine and psychophysiological measures.

Similar biochemical properties of human seminal plasma and epididymal alpha-1,4-glucosidase.

Abstract: We have studied some characteristics of alpha-1,4-glucosidases in human male reproductive organs in order to obtain information on the origin of the enzyme in seminal plasma. Acid and neutral enzymes could be distinguished on the basis of their selective inhibition either by SDS (acid enzyme) or MTT (neutral enzyme). Only the epididymis contained a significant amount of SDS resistant neutral alpha-1,4-glucosidase which was comparable to what has been isolated in seminal plasma. The similarity of epididymal and seminal plasma neutral enzymes was further confirmed by ultracentrifugation on sucrose density gradients, which permitted a complete separation of neutral (11S) and acid (4S) iso-enzymes. The 11S form was present in epididymis and in seminal plasma, but was totally absent in seminal vesicles, prostates and testis. The epididymal enzyme also had some of the unique characteristics found in the seminal plasma enzyme: it precipitated upon dialysis against distilled water, and its mobility on SDS polyacrylamide gel electrophoresis was identical to that of form 1 in seminal plasma. These results, although they do not constitute absolute proof of the identity of epididymal and seminal plasma alpha-glucosidase, certainly provide strong support for this hypothesis.

Effects of Electroejaculation and Ketamine‐HCl on Serum Cortisol, Progesterone, and Testosterone in the Male Cat

Abstract: The influence of manual restraint, ketamine-hydrochloride anesthesia and electroejaculation under anesthesia on circulating levels of cortisol, progesterone and testosterone was examined in male domestic cats. In the first experiment, cats were anesthetized with ketamine-HCl (17.5 mg/kg of body weight) and serially bled (controls) or serially bled and electroejaculated. These animals showed signs of recovering from anesthesia within 45 to 60 minutes of ketamine-HCl injection. Average serum cortisol concentrations increased (P < 0.01) over the 84-minute sampling interval in both the electroejaculated and control groups. Cortisol levels reached their maximum concentration in the electrically stimulated males immediately postelectroejaculation (95.1 ng/ml) and were significantly greater (P < 0.01) than in the controls (36.1 ng/ml) at a comparable time. Maximal mean cortisol concentrations in the control group (62.8 ng/ml) occurred 54 minutes after the first blood sample and occurred together with the onset of anesthesia recovery. Mean testosterone levels did not differ between electroejaculated and control cats, but did decrease (P < 0.05) between the first and last blood sampling in both groups. In the second experiment, cats were bled on the same time schedule as in Experiment 1, but were bled while awake and manually restrained, or else during a deeper plane of anesthesia induced and maintained with higher doses of ketamine-HCl (initial dose, 23 mg/kg). Mean serum cortisol levels were greater (P < 0.05) during manual restraint (range, 36.3–41.1 ng/ml) compared to deep anesthesia (range, 16.7–25.8 ng/ml), but did not change over the 84 minute sampling interval in either group. Although the decline in mean concentrations was not significant, testosterone decreased by 31–94% in four of six individual males during both manual restraint and deep anesthesia. Serum progesterone levels in both Experiments 1 and 2 averaged less than 0.5 ng/ml. Although progesterone increased (P < 0.05) between the first and last blood samples in Experiment 1, the absolute quantities detected were indistinguishable from possible progesterone antibody cross-reactivity with the hormone cortisol. The results of this research indicate that electroejaculation of the anesthetized cat produces acute stress as demonstrated by elevated serum cortisol levels measured immediately after electroejaculation. However, significant increases in cortisol also are detected in non-electroejaculated cats recovering from ketamine-HCl anesthesia. Such a rise is abolished if males are maintained under a more prolonged and deeper plane of anesthesia during blood sampling, or if no ketamine-HCl is given. Because serum testosterone declines markedly in most cats independent of a detectable rise in cortisol there is no clear, physiological coupling of these two hormones in this species.

Ultrastructural Lesions in Testes from Hyperprolactinemic Men

Abstract: Testicular tissue from eight men with prolactinomas and elevated serum prolactin were evaluated by light (LM) and transmission electron microscopy (TEM). A semiquantitative assessment of testicular morphology was employed to provide a morphology index for each tissue specimen. Although in each biopsy specimen germ cell exfoliation was evident, as was abnormal structural change in the seminiferous epithelium, there was no apparent correlation with the overall degree of tissue pathology (morphology index) and the serum level of prolactin. All of the tissue displayed variably thickened seminiferous tubule walls which, when viewed by TEM, were composed of thickened laminae propriae and redundant and involuted basal laminae. Likewise, all tubules contained Sertoli cells with overt cytoplasmic degeneration, principally in the apical (adluminal) region of the cell. This was visualized, in part, as a retraction of the apical cytoplasm from periluminal spermatids and degeneration or absence of Sertoli-germ cell junctional specializations. Sertoli-Sertoli cell junctional complexes appeared structurally intact. Leydig cell ultrastructure was typical of normal cells and contained a variable amount of lipid and smooth endoplasmic reticulum. This also was without positive correlation with the overall degree of tissue pathology or level of serum prolactin. Our results demonstrate the variable degree of testicular pathology associated with hyperprolactinemia in man, and suggest that abnormal tubule walls and altered Sertoli cell ultrastructure are consistent findings in this abnormal endocrine condition.

Reproductive History and Semen Analysis in Prevasectomy Fertile Men with and without Varicocele

Abstract: A group of 598 allegedly fertile men requesting vasectomy were investigated; varicocele was found in 97 (16.2%) of these men. The mean ages and age distributions of men with and without varicocele were not significantly different. Reproductive histories (number of pregnancies, living children and spontaneous abortions, as well as incidence of present pregnancy) were similar in both groups. The average seminal characteristics (semen volume, sperm count, total sperm count, percentage of motile spermatozoa, quality of motility, morphology) were not different for men with and without varicocele, except for a slight, but significantly higher incidence of oval-headed sperm in men without varicocele. However, the incidence of varicocele was significantly higher in men with sperm counts below 40 million/ml. Three important observations may be made from this study: 1) the incidence of varicocele in this prevasectomy population was similar to that reported for the general population, but lower than the incidence reported for male partners of infertile couples; 2) in this population of allegedly fertile men, the presence of a varicocele did not significantly affect reproductive performance; 3) even though the incidence of varicocele was higher in men with sperm counts below 40 million/ml, the average seminal characteristics were not different in men with and without varicocele.

Mechanism of action of gonadotropin-releasing hormone-stimulated Leydig cell steroidogenesis. I. The stimulatory effect is calcium dependent and not mediated by cyclic nucleotides.

Abstract: UNLABELLED The present study was designed to elucidate mechanisms responsible for gonadotropin-releasing hormone (GnRH)-stimulated testosterone formation. Purified Leydig cells from adult Sprague-Dawley rats were incubated with varying concentrations of GnRH agonist (des-Gly10, (D-Ala6) GnRH N-ethylamide), hCG, 8-bromo cAMP or pregnenolone; testosterone, cAMP, cyclic GMP (cGMP) and cAMP-dependent protein kinase activity were measured after various time periods. Basal testosterone levels were 2.54 +/- 0.13 ng/10(5) cells, increasing to 3.18 +/- 0.14, 4.32 +/- 0.08, and 4.63 +/- 0.12 ng within 1 hour after the addition of 10(-9), 10(-8), and 10(-7) M GnRH agonist, respectively. After a 3-hour incubation a 10(-7) M dose of GnRH agonist increased testosterone production four-fold above control. GnRH agonist potentiated hCG-stimulated testosterone formation, but had no significant effects on cGMP levels and cAMP-dependent protein kinase activity. Cyclic AMP levels in the incubation medium increased slightly. GnRH agonist also enhanced 8-bromo-cAMP and pregnenolone-induced testosterone formation. Furthermore, GnRH agonist increased testosterone formation both in the absence and presence of phosphodiesterase inhibitor. These results suggest that the major effect of GnRH agonist is probably beyond the cAMP step. When purified Leydig cells were incubated in a calcium-free medium, the stimulatory effects of GnRH agonist on testosterone formation were completely abolished, but could be restored by the addition of calcium to the incubation medium. GnRH agonist-induced testosterone formation was also blocked by the addition of nifedipine (a calcium channel blocking agent, 0.1 to 10 micrograms/ml). Finally, GnRH antagonist in a concentration of 10 micrograms/ml completely inhibited GnRH agonist-stimulated testosterone formation. IN CONCLUSION GnRH agonist stimulated Leydig cell testosterone formation in short-term incubations. The stimulatory effect is calcium dependent and not mediated by cyclic nucleotides.

Heterogeneity of sperm density profiles following 20-week therapy with high-dose LHRH analog plus testosterone.

Abstract: Eight normal male volunteers received an LHRH analog, 100 to 500 micrograms daily, for 20 weeks. Testosterone enanthate, 100 mg, was given by injection every second week. Sperm density fell to 5.5 X 10(6)/ml and to 0 in two of the subjects receiving 100 micrograms, but was unchanged in the third. Three of the subjects who received 500 micrograms displayed azoospermia, whereas the other two showed no significant change in sperm density. The reasons for the heterogeneity are not clear. Only one of the three nonresponders had testosterone values that were higher than the other subjects. Gonadotropin levels were similar in responders and nonresponders. It is possible that the response to LHRH analog in man is determined by the extent of the reduction in LH bioactivity.

Specific inhibition of the testicular mitochondrial respiratory chain in vitro by gossypol.

Abstract: Optically inactive gossypol is an effective male antifertility agent in several mammalian species, while optically active (+)-gossypol has no antifertility effect in the rat and hamster Recently, it was suggested that the mitochondria of spermatogenic cells may be a subcellular target of gossypol We are reporting the effects of optically inactive gossypol and (+)-gossypol on the respiratory chain of mitochondria isolated from the testes and liver of rats and hamsters The mitochondria were incubated with the test compounds and difference spectra were recorded Complete inhibition of the testicular mitochondrial respiratory chain was observed at a concentration of approximately 75 microM In contrast, no inhibition of the liver mitochondrial respiratory chain was observed with the test compounds at concentrations as high as 300 microM These results demonstrate selective inhibition of the testicular mitochondrial respiratory chain by gossypol isomers

Control of metastases in the Nb rat prostatic adenocarcinoma model.

Abstract: Chemotherapeutic agents, as well as acetyl-salicylic acid, heparin, and indocin, were evaluated in regard to their effect on the rate of metastasis in the androgen-insensitive Noble rat prostate adenocarcinoma system. Acetyl salicylic acid, heparin and indocin were similar to the chemotherapeutic agents cyclophosphamide and adriamycin in reducing the incidence of metastases, but had less effect on tumor volume. In the heparin and indocin groups, the rate of metastases was approximately one half that of the control group (25% vs. 59%). In all three experiments evaluating heparin and indocin, there was a decrease in the number of animals with metastasis compared with control groups.

The hormonal regulation of premeiotic steps of spermatogenesis in the newborn rat.

Abstract: The effect of high doses of testosterone propionate (TP) on the development of the first spermatogenic wave was systematically analyzed to determine the period of maximal susceptibility to testosterone. Two mg of TP were administered daily to groups of immature male rats, starting from birth, or days 5, 10, 15, and 20 until day 35 of life. Animals injected from birth or day 5 showed severe testicular atrophy, with reductions of more than 70% of testicular weight, diminished tubular diameters, and spermatogenic arrest during the meiotic prophase. Groups treated from days 10 or 15 showed increasing testicular weights with qualitatively normal spermatogenesis. When treatment started at 20 days, completely normal testicular development was achieved. To test the responsiveness of the neonatal hypothalamo-pituitary axis to TP administration, groups of 5-day-old male rats received daily injections of TP, and plasma FSH was determined at ten, 20, and 35 days. FSH levels were not detectable at ten and 20 days, and extremely depressed at 35. A group of 5-day-old male rats was injected simultaneously with 2 mg of TP and 14 IU of FSH (human menopausal gonadotropin: 71 IU of FSH and 80 IU of LH/ml) until day 35. Testicular weights and tubular diameters were increased compared to controls, and spermatid differentiation had proceeded to more mature steps than those seen in control animals. Inhibition of testicular development by neonatal TP administration was paralleled by a sharp decrease in circulating FSH levels and reversed by FSH replacement. We show here that a “critical period” of maximal susceptibility to TP administration and FSH deprivation exists during the first ten days of life, and that testosterone (T) is unable to stimulate spermatogenesis at that age.

Culture of principal cells from the ram epididymis. A comparison of the morphology of principal cells in culture and in situ.

Abstract: A procedure was developed to isolate and culture principal cells from the initial segment, central caput, distal caput, and proximal corpus epididymidis. The morphology of cultured cells and of cells in situ was compared. Over four to ten days, principal cells cultured in a floating collagen matrix with serum-free medium formed clusters that developed into either large sheets of columnar cells or tubular structures of cuboidal cells. Structural polarity was evident and junctional complexes reformed. The distribution and relative abundance of organelles in principal cells in situ differed depending on the region examined, and most of these regional characteristics were retained by principal cells in culture. Microvilli and membrane-bound vesicles were less conspicuous in cultured cells. Cultured principal cells were shorter than principal cells in situ, but were of similar volume. The high purity (90%) and viability (70%) of principal cells after seven to ten days in culture, their retention of morphologic characteristics unique to the region of origin, and the formation of function units are evidence that such cultures should be valuable for studying regional differences in the function of principal cells.

Periodic Exposure to a Brief Light Signal Stimulates Neuroendocrine‐Gonadal Activity in Golden Hamsters

Abstract: The photoperiodic effects of a periodic light pulse on neuroendocrine-gonadal activity in the male golden hamster was examined using a night interruption paradigm. Adult male hamsters that had been housed 3 to 5 per cage on LD 14:10 were placed either in individual cages, each equipped with a running wheel, or maintained in communal housing conditions, without access to a running wheel. Animals were then transferred to either LD 6:18 or to a LD 6:18 light cycle with a periodic 10-second night interruption (8 hours after lights off) occurring once every two, four, or seven days. As expected, exposure to LD 6:18 for 11 weeks induced complete regression of the testes and seminal vesicles, accompanied by low serum levels of LH and FSH, in both individually and communally-housed animals. However, in individually-housed animals receiving a 10-second night interruption every other day on LD 6:18, paired testis and seminal vesicle weights, as well as serum gonadotropin levels, were maintained at values comparable to those normally observed in hamsters exposed to photostimulatory long days. Furthermore, the presentation of a periodic 10-second night interruption once every four or seven days to individually-housed animals with access to a running wheel was sufficient to partially or totally block the inhibitory effects of short days on neuroendocrine-gonadal activity. Communally-housed hamsters receiving a 10-second light pulse once every two, four, or seven days also exhibited paired testis and seminal vesicle weights, as well as serum gonadotropin levels, that were consistently higher than the values obtained for animals exposed only to LD 6:18. However, the efficacy of each night interruption paradigm in maintaining testicular and seminal vesicle weight in communally-housed animals was considerably less than that observed for animals with access to a running wheel. These results demonstrate that the periodic presentation of a brief photic stimulus once every two, four, or seven days during the dark phase of an inhibitory light cycle can influence the neuroendocrine events underlying the photoperiodic regulation of gonadal function. This experimental approach, whereby a discrete stimulus is periodically administered at a specific arcadian time to induce a photoperiodic response, should provide a valuable model for examining the physiologic mechanisms by which light regulates the neuroendocrine-gonadal axis in mammals.

Effects of Long‐ and Short‐term Vasectomy on Structural and Functional Parameters of the Rat

Abstract: The effects of vasectomy were examined by comparing various parameters from sham operated and vasectomized rats that had undergone surgery at 90 days of age and were killed at 190 or 390 days of age Significant alterations in the vasectomized rats from sham rats included: testicular and epididymal hypertrophy, formation of pathologic vas deferens granulomas, decreased total serum protein, lowered alpha-globulin levels as shown by serum electrophoresis, and increased sperm agglutinin antibody titers For vasectomized rats, the differential white blood cell count showed increased numbers of neutrophils and large lymphocytes and decreased numbers of small lymphocytes and basophils Both the number and extent of many vasectomy-induced alterations were greater in long-term vasectomized than in short-term vasectomized rats

Mechanism of action of gonadotropin-releasing hormone-stimulated Leydig cell steroidogenesis. II. Gonadotropin-releasing hormone stimulates phospholipid labeling.

Abstract: To investigate mechanisms responsible for gonadotropin-releasing hormone (GnRH)-stimulated Leydig cell steroidogenesis, the effects of GnRH agonist [des-Gly10, (D-Ala6) GnRH] on phospholipid turnover were studied. GnRH agonist in concentrations of 10(-9) to 10(-7)M increased phosphatidic acid labeling 292 +/- 16% (mean +/- SE), and phosphatidylinositol labeling 258 +/- 13.2%. GnRH agonist-stimulated phospholipid labeling was detectable as early as 2 minutes. GnRH antagonist completely blocked GnRH agonist-induced testosterone formation and phosphatidic acid and phosphatidylinosital labeling. Nifedipine in concentrations of 1 and 10 micrograms/ml inhibited GnRH agonist-stimulated testosterone formation but had no effect on 32P incorporation into phospholipids. Our results suggest that GnRH agonist-stimulated Leydig cell steroidogenesis is calcium dependent and correlated with increased phospholipid turnover.

Fibroblast Androgen Receptors in Patients with Genitourinary Anomalies

Abstract: The etiology of certain disorders of sexual differentiation is unclear. The authors have examined the hypothesis that hypospadias and other disorders compatible with a defect in androgen action, such as cryptorchidism, micropenis, chordee/penile torsion, and ectopic testis, might be explained by androgen receptor abnormalities. Therefore, 25 subjects were studied who were selected only because they had one of these developmental defects, together with a predominantly male phenotype, and no readily ascertainable explanation for the defect. Four of these subjects had mixed gonadal dysgenesis with multiple genito-urinary anomalies. They were included for comparative purposes, since there is no evidence for androgen resistance in this disorder. Patients with testicular regression syndrome (gross testosterone deficiency), impaired testosterone biosynthesis (relative testosterone deficiency), 5α-reductase deficiency (altered T/DHT ratio), and a family history or endocrine profile suggestive of androgen resistance, were all excluded from evaluation. Androgen receptor content (Ro) and binding affinity (Kd) were measured in 26 genital or pubic skin fibroblast strains cultured from 25 affected patients using a dispersed, whole cell assay at 22 C. There was no difference in the mean androgen receptor content (approximately 10,000 sites/cell) or binding affinity (approximately 1 nM) between the patients' fibroblasts and those from 26 fibroblast strains established from 26 normal males. Moreover, there were no differences in the nuclear uptake of [3H]dihydrotestosterone into dispersed, intact fibroblasts incubated at 37 C when 11 patient and seven normal male fibroblast strains were compared. In conclusion: these disorders of sexual differentiation cannot be attributed to androgen resistance on the basis of decreased androgen receptor binding affinity or receptor number, as reflected in whole cells or nuclei of cultured skin fibroblasts.