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Showing papers in "Journal of Andrology in 1995"


Journal ArticleDOI
TL;DR: Clinicians need to be aware of exaggerated claims of antioxidant benefits by various commercial supplements for fertility purposes until proper multicenter clinical trial have been completed.
Abstract: Oxygen toxicity is an inherent challenge to aerobic life, including spermatozoa, the cells responsible for propagation of the species. How this toxicity affects the spermatozoan in its interactions with the ovum is still unknown. An increase in oxidative damage to sperm membranes, proteins, and DNA is associated with alterations in signal transduction mechanisms that affect fertility. Recent evidence suggests that spermatozoa and oocytes possess an inherent but limited capacity to generate ROS to aid in the fertilization process. Though a variety of defense mechanisms encompassing antioxidant enzymes (SOD, catalase, and GSH peroxidase and reductase), vitamins (E, C, and carotenoids), and biomolecules (GSH and ubiquinol) are available, a balance of the benefits and risks from ROS and antioxidants appears to be necessary for the survival and functioning of spermatozoa. An assay system for the evaluation of OSS needs to be developed. Such an assay will assist the clinician in the assessment of fertility status of both male and female partners. The determination of this OSS value will also theoretically identify the subgroups of responders and nonresponders to any putative antioxidant therapy. Though the therapeutic use of antioxidants appears attractive, clinicians need to be aware of exaggerated claims of antioxidant benefits by various commercial supplements for fertility purposes until proper multicenter clinical trial have been completed.

378 citations


Journal ArticleDOI
TL;DR: It is hypothesized that the DNA strand breaks produced in the normal transition from a somatic cell histone complex to a protamine complex are not ligated properly, resulting in residualDNA strand breaks and altered chromatin structure, which could lead to the accessibility of the endogenous DNA strands.
Abstract: Sperm from four mammalian species were analyzed by the sperm chromatin structure assay (SCSA) and the terminal deoxynucleotidyl transferase assay (TdTA) using flow cytometry. The SCSA quantitates the susceptibility of sperm nuclear DNA to in situ acid denaturation, while the TdTA quantitates the presence of endogenous DNA strand breaks in sperm nuclear chromatin. Correlations were seen between the percentage of sperm cells showing susceptibility to in situ acid denaturation and the percentage of cells showing the presence of DNA strand breaks for humans (r = 0.56, P = 0.004), rams (r = 0.84, P < 0.001), bulls (r = 0.78, P < 0.001), and stallions (r = 0.65, P < 0.001). No significant differences were seen when using fresh or frozen samples for either assay. These results suggest that sperm cells that are more susceptible to in situ DNA denaturation may have a greater number of accessible endogenous DNA strand breaks. We hypothesize that the DNA strand breaks produced in the normal transition from a somatic cell histone complex to a protamine complex are not ligated properly, resulting in residual DNA strand breaks and altered chromatin structure. Alternatively, altered chromatin structure could lead to the accessibility of the endogenous DNA strand breaks.

270 citations


Journal ArticleDOI
TL;DR: The present review initially focuses on the human epididymis, and it seems likely that much of this sperm storage capacity is in what some would call the proximal convoluted vas deferens in the human.
Abstract: A variety of mammalian species have been used in studies of epididymal function. The knowledge gained from these laboratory and domestic species has been applied to the human as well, but the anatomy and biology of the human epididymis is not commonly investigated directly. Nonpathological specimens from men within their reproductive years are understandably difficult to obtain, and even when such epididymides are obtained, they often lack the proper preservation for good biological studies. Because the human epididymis is not often addressed directly, the present review initially focuses on that organ before going on to more general comments. In nature, the distal portion of the human epididymis disappears into the epididymal fat, making it appear that the epididymis has a minimal cauda region. In fact, the still-coiled human epididymis is 10-12 cm long before becoming the convoluted vas, and the convoluted vas extends for approximately another 7-8 cm. Nevertheless, the human epididymis does not have a bulbous cauda for sperm storage, as do the caudae of most other species, including the rat (Fig. 1A). Whereas the cauda of the rat epididymis contains approximately 60% of the sperm in that epididymis (Amann, 1981) the ‘tail’ of the human epididymis (Fig. 1B) contains at least 50% of the sperm present in that epididymis (Amann and Howards, 1980; Johnson and Varner, 1988). Because the demarcation of the tail or cauda of the epididymis has been unclear in most human studies, it seems likely that much of this sperm storage capacity is in what some would call the proximal convoluted vas deferens (Fig. 1B). In fact, it may be that the bulbous cauda seen in most species is simply extended into what is called the proximal convoluted vas in the human. This makes the human cauda

189 citations


Journal ArticleDOI
TL;DR: It is concluded that, although spermatozoa do not possess detectable nitric oxide synthase activity, low levels of Nitric oxide induce human sperm capacitation, and this action likely involves hydrogen peroxide.
Abstract: The influence of nitric oxide on human sperm hyperactivation and capacitation, as well as its mechanism of action and its possible origin from spermatozoa were studied. Percoll-washed spermatozoa from healthy volunteers were incubated in Ham's F-10 medium supplemented or not with the nitric oxide-releasing agents, diethylamine-NONOate or spermine-NONOate, in combination or not with superoxide dismutase or catalase (scavengers for the superoxide anion and for hydrogen peroxide, respectively), or with sodium nitrate, sodium nitrite, or preincubated NONOates. Sperm hyperactivation, capacitation, and nitric oxide synthase activity were determined. High concentrations (0.3 to 1 mM) of NONOates reduced sperm motility. However, a lower concentration (0.1 mM) of the two NONOates had no effect on the percentage of sperm motility or of hyperactivation but resulted in a significant increase in sperm capacitation (24% +/- 4%) when compared to that of control spermatozoa (Ham's F-10 alone, 12% +/- 2%). Nitric oxide released by the NONOates appeared responsible for this effect because sodium nitrate or nitrite or preincubated NONOates (to exhaust the formation of nitric oxide) had no influence on sperm capacitation. Catalase, but not superoxide dismutase, abolished the capacitating action of the NONOates. No nitric oxide synthase activity was detected in spermatozoa, whether they were in their basal state or already capacitated. Furthermore, the nitric oxide synthetase inhibitor L-NG nitroarginine methyl ester did not block sperm capacitation induced by fetal cord serum ultrafiltrate. It is therefore concluded that, although spermatozoa do not possess detectable nitric oxide synthase activity, low levels of nitric oxide induce human sperm capacitation, and this action likely involves hydrogen peroxide.

184 citations


Journal ArticleDOI
TL;DR: This review will examine several aspects of these epididymal genes including tissue and regional-specific expression, differential regulation by androgens and/or testicular factors, mechanisms by which these genes may be controlled, and the putative functional significance of these genes in the epididsymis.
Abstract: The metamorphosis of an immature and nonfunctional sperm into a mature sperm capable of progressive motility and fertility is thought to be the result of a highly regulated and complex series of events in the epididymis. It is well established that sperm maturation is not intrinsic to sperm cells themselves but requires the interaction of spermatozoa with proteins that are synthesized and secreted by the epididymal epithelium in a highly regionalized manner. This regional expression of epididymal proteins results in the sperm encountering a unique and ever-changing luminal fluid environment as they progress through the epididymis, which ultimately results in sperm maturation. These maturational events also require the presence of androgens. Although epididymal secretory proteins have been studied for some time, the identity and function of these proteins in sperm maturation and the mechanisms by which their genes are controlled have not been elucidated. The identity and analysis of genes expressed only in discrete regions of the epididymis provide valuable information in the quest to determine the regional-specific functions necessary for sperm maturation. This review will examine several aspects of these epididymal genes including tissue and regional-specific expression, differential regulation by androgens and/or testicular factors, mechanisms by which these genes may be controlled, and the putative functional significance of these genes in the epididymis. Minireview

136 citations


Journal ArticleDOI
TL;DR: It is concluded that TALP-TEST medium supports stallion sperm capacitation in vitro, progesterone-induced acrosome reactions are physiological, and sperm from fertile stallions may be more responsive to progester one- induced acrosomes than those of subfertile stallions.
Abstract: Mammalian sperm that have completed capacitation are capable of undergoing the acrosome reaction in response to a number of biological and chemical stimuli. In the present report, we have investigated the ability of progesterone to stimulate acrosome reactions of stallion sperm capacitated in vitro. Motile sperm were selected by a two-layer Percoll gradient centrifugation and were incubated in TALP medium modified by the 1:1 (v/v) addition of TEST-yolk medium for 5 hours at 39 degrees C, under 5% CO2 in humidified air. Sperm incubated in vitro in TALP-TEST medium had a higher percentage of acrosome reactions following the addition of progesterone (3.18 mumol/L) compared to controls (P < 0.05). Furthermore, sperm from stallions classified as fertile on the basis of breeding history had higher percentages of progesterone-induced acrosome reactions in comparison with stallions classified as subfertile (P < 0.05). Acrosome reactions were assessed routinely by fluoresceinated lectin binding, but the physiological appearance of induced acrosome reactions was confirmed at the ultrastructural level by transmission electron microscopy. We conclude that 1) TALP-TEST medium supports stallion sperm capacitation in vitro, 2) progesterone-induced acrosome reactions are physiological, and 3) sperm from fertile stallions may be more responsive to progesterone-induced acrosome reactions than those of subfertile stallions. This is the first report in a nonhuman species that differences exist between the sperm of fertile and subfertile males in the ability to capacitate and acrosome react in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

116 citations


Journal ArticleDOI
TL;DR: It is ascertain that the supplementation of subfertile men with yeast-rich Se showed a more pronounced effect on Se concentrations and GSH-Px activities in blood components and seminal fluid than selenite did.
Abstract: The objective of this study was to evaluate the effect of selenium (Se) supplementation on Se concentration and glutathione peroxidase (GSH-Px) activity in blood components and seminal fluid and on spermatozoal quality characteristics in subfertile men. Thirty-three men were supplemented for 12 weeks with 200 micrograms Se/day in the form of yeast-rich Se (group I, n = 16) or sodium selenite (group II, n = 17). Blood samples and sperm were collected at the start of the study and after 2, 4, 8, and 12 weeks following Se supplementation. Se concentration in whole blood and plasma and GSH-Px activity in red cells and plasma increased significantly during the study, but in the group supplemented with yeast-Se the effect was more pronounced. Se concentration in seminal fluid also increased in both groups, but the effect of yeast-Se was markedly higher than that of selenite. In both groups statistically significant correlations were found between Se concentration in plasma and seminal fluid. GSH-Px activity in seminal fluid in the yeast-Se group increased significantly and reached a plateau after 2 weeks, whereas in the selenite group the activity did not change throughout the whole study period. Weak correlations between Se concentrations and GSH-Px activities in seminal fluid were seen, but only in the yeast-Se group were the relations statistically significant. The subjects in both groups showed no response in sperm count, motility, and morphology. In conclusion, we can ascertain that the supplementation of subfertile men with yeast-rich Se showed a more pronounced effect on Se concentrations and GSH-Px activities in blood components and seminal fluid than selenite did. Se supplementation did not improve the spermatozoal quality characteristics of sperm count, motility and, morphology.

87 citations


Journal ArticleDOI
TL;DR: It is suggested that spinal cord injury will result in a temporary, but profound, effect on the pituitary-testicular hormone axis and Sertoli cell function, and changes may impair certain aspects of Sertolis cell function that could render these cells incapable of supporting normal spermatogenesis.
Abstract: The present study investigated the time course of the onset of the abnormalities in spermatogenesis following spinal cord injury, and their relationship to changes in the pituitary testicular hormonal axis and Sertoli cell function. Spinal cord injury (SCI) was induced in adult male rats by surgical transection of the spinal cord at the level of T9 and L1 vertebrae. Animals were killed 3, 7, and 14 days after the operation. As early as 3 days following SCI, abnormalities in spermatogenesis, including delayed spermiation and vacuolization of the nucleus of spermatids, were noted in both the T9 and L1 animals. By 14 days, other lesions, including phagocytosis of mature spermatids, incomplete cellular associations, and total regression of seminiferous epithelium, became apparent. Concurrently a transient but significant (P 0.10). Northern blot analysis of testicular poly(A)+ RNA revealed a transient but significant reduction in the steady-state level of the 2.7-kilobase (kb) Sertoli cell transferrin mRNA transcript in both the T9 and the L1 animals 3 days after the operation (P < 0.05). On the other hand, the 1.7-kb androgen binding protein (ABP) mRNA remained unaffected during the 2-week study period. The steady-state level of mRNA transcripts for spermatogenic cell-specific hemiferrin and spermatid specific transition protein 2 and protamine 1 also remained unchanged. These results suggest that spinal cord injury will result in a temporary, but profound, effect on the pituitary-testicular hormone axis. These changes may impair certain aspects of Sertoli cell function that could render these cells incapable of supporting normal spermatogenesis. However, the severity of spermatogenic lesions and the disparate responses of the two major Sertoli cell proteins make it unlikely that hormone deficiency is the only mechanism responsible for the impaired spermatogenesis following spinal cord injury.

82 citations


Journal ArticleDOI
TL;DR: The existence of a temporal correlation between the appearance of the gap junction protein Cx43 and both the germ cell differentiation and the modulation in the junctional barrier between Sertoli cells is documented.
Abstract: To test whether the gap junction protein connexin 43 (Cx43) is associated with germ cell differentiation and with the Sertoli cell junctional blood barrier, we recorded the temporal changes in its distribution before birth, through the neonatal period, puberty, and adulthood in guinea pig, and throughout the annual seasonal reproductive cycle in the mink. We used the immunoperoxidase labeling technique on Bouin's perfused-fixed testes and with site-specific polyclonal affinity-purified antibodies against Cx43. Cx43 was localized between Leydig cells in fetal guinea pig testis. In this species, after birth, the appearance of Cx43 concurred with the onset of spermatogenesis. In the seminiferous epithelium, the distribution of Cx43 coincided with the gap junctions of the Sertoli cell junctional blood barrier. In guinea pig and mink, the distribution of the protein in the tubules changed in accordance with the germ cell differentiation in a stage-dependent manner and with the modulation, i.e., the assembly and disassembly of the junctional barrier accompanying the translocation of spermatocytes into the lumenal compartment. In the mink, the reaction product persisted during testicular regression but showed a similar distribution from one tubule to the next. In this paper, we documented the existence of a temporal correlation between the appearance of the gap junction protein Cx43 and both the germ cell differentiation and the modulation in the junctional barrier between Sertoli cells. The paper also discusses the possibility that cell-to-cell communications, generally attributed to gap junctions, may help changes in the barrier to take place in coordination with spermatogenesis.

82 citations


Journal ArticleDOI
TL;DR: The results suggest that SLPI has a local protective function against proteolytic degradation of the male reproductive tract tissues during inflammation.
Abstract: Secretory leucocyte protease inhibitor, SLPI, is a low-molecular-weight, acid-stable protein present in the liquid part of fresh human ejaculate but not demonstrable in the gel structure. No fragmentation of SLPI occurred during gel dissolution, but a slow proteolytic cleavage of SLPI was seen on incubation of the liquified semen at 37°C. The same pattern of degradation products was seen after incubation of SLPI with prostatic secretion and also with purified prostate-specific antigen, PSA. We could identify Arg 20-Tyr 21 and Met 73-Leu 74 to be the primary cleavage sites upon proteolytic modification of SLPI by purified PSA. However, we did not find any inhibition of the enzymatic activity of PSA by SLPI, even at a 100-fold molar excess of the inhibitor. The slow degradation of SLPI facilitated sampling and the reliable determination of the normal level of SLPI in seminal plasma, which was about 20 mg/L. We investigated the glandular origion of SLPI in the genital tract by immunocytochemistry. A strong immunostaining for SLPI was demonstrated in epithelial cells within the glandular lumina of the prostate gland, seminal vesicles, and epididymis but not in the stromal parts of these glands. In addition the immunostaining was also detected in the deferent ducts and the germinal epithelium of the testes. Taking into account that SLPI is a strong inhibitor of several proteases, including leukocyte elastase and cathepsin G, the results suggest that SLPI has a local protective function against proteolytic degradation of the male reproductive tract tissues during inflammation.

80 citations


Journal ArticleDOI
TL;DR: Because GnRH antagonist-antiandrogen treatment can protect stem spermatogonial survival and/or function in the rat from cyclophosphamide-induced damage, hormonal pretreatment should be useful for preventing the prolonged azoospermia caused by chemotherapy with cycloph phosphate-containing protocols.
Abstract: Studies of protection of testicular function from cyclophosphamide with hormonal pretreatment have been limited by the lack of a convenient model for cyclophosphamide-induced inactivation of stem spermatogonia In the rat, the mortality from cyclophosphamide had prevented the administration of sufficient dosages to produce detectible damage to stem spermatogonia To overcome this problem, we used bone marrow transplantation and sodium 2-mercaptoethanesulfonate (Mesna) treatment to raise the lethal dose for 50% of the animals (LD50) for cyclophosphamide from 275 to > 400 mg/kg body weight In addition we used irradiation, 2 weeks prior to injection of cyclophosphamide, to greatly enhance the measured toxicity of cyclophosphamide towards stem spermatogonia Whereas sperm counts at 9 weeks after a 300 mg/kg cyclophosphamide dose were reduced by only a factor of 16 without prior irradiation, they were reduced by a factor of 60 when 25 Gy of irradiation had been given Dramatic protection against this toxicity was produced by hormone treatment with a gonadotropin-releasing hormone (GnRH) antagonist (Nal-Glu) and an antiandrogen (flutamide) following the radiation but prior to cyclophosphamide This hormone treatment did not modify the stem cell toxicity of the radiation and it therefore must be protecting stem cells against cyclophosphamide-induced damage Because GnRH antagonist-antiandrogen treatment can protect stem spermatogonial survival and/or function in the rat from cyclophosphamide-induced damage, if the same principles are applicable in human, hormonal pretreatment should be useful for preventing the prolonged azoospermia caused by chemotherapy with cyclophosphamide-containing protocols

Journal ArticleDOI
TL;DR: A formula considering all statistical possibilities for defects of the examined sperm to be present in a sperm cell, the total number of affected spermatozoa, and, as consequence, that of sperm devoid of defects is developed, also considering the probability of association characteristics of some of them.
Abstract: This paper concerns the mathematical evaluation of sperm quality as examined by electron microscopy. Proceeding with a Bayesian technique, we have developed a formula considering all statistical possibilities for defects of the examined sperm to be present in a sperm cell, the total number of affected spermatozoa, and, as consequence, that of sperm devoid of defects, also considering the probability of association characteristics of some of them. The formula has been studied in three applications. The first concerns the number of healthy spermatozoa present in ejaculates of fertile men. We have found an enormous variability, but we have observed that the minimal number of spermatozoa free of defects assuring a normal fertility seems to be a little higher than 2 x 10(6). The second and third examples concern varicocele and assisted fertilization. In both cases the formula allows a precise simultaneous evaluation of the totality of studied characters and a determination of the number of spermatozoa free from defects. This comparative analysis shows that the formula is sufficiently sensitive to distinguish the different degrees of the varicocele condition and the various possibilities of fertility power in cases of natural or artificial insemination. In this way we will easily control the level of improvement of sperm quality in cases of varicocele treated pharmacologically or surgically, or we will better predict the success of artificial insemination.

Journal ArticleDOI
TL;DR: It is hypothesized that androgens under control of an intact fetal hypothalamic-pituitary axis alter the viscoelastic properties of the gubernaculum and push the testicle into the scrotum.
Abstract: Descent of the testes is a complex event mediated by hormonal and mechanical factors. At present we hypothesize that testicular descent occurs as the result of the secretion of descendin from a normal testicle. Descendin secretion results in selective growth of the gubernacular cells. Gubernacular outgrowth results in masculinization of the inguinal canal. At the beginning of testicular descent, the patent processus migrates into the inguinal canal, transmitting intraabdominal pressure to the gubernaculum. The gubernaculum in turn applies traction to the testicle to introduce the testicle into the inguinal canal. Descent of the testes into and through the inguinal canal is an interplay between intraabdominal pressure transmitted by a patent processus vaginalis and androgen-induced gubernacular regression. Specifically, we hypothesize that androgens under control of an intact fetal hypothalamic-pituitary axis alter the viscoelastic properties of the gubernaculum. Reductions in the turgidity of the gubernaculum allow intraabdominal pressure to push the testicle into the scrotum. Functional abnormalities in any of the above factors will result in cryptorchidism.

Journal ArticleDOI
TL;DR: Results indicated that cell proliferation and cell death in rat prostatic epithelial cells in culture could be modulated by EGF and TGF-beta 1.
Abstract: A tissue culture system for rat prostatic epithelial cells was developed, and the effect of epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and transforming growth factor beta 1 (TGF-beta 1) on these cells was evaluated. The primary culture was prepared by DNAse/collagenase dissociation of minced ventral prostates. Cells were initially plated in RPMI-1640 medium containing 10% fetal bovine serum to allow the preferential attachment of stromal cells. Twenty-four hours later, the unattached epithelial cells were replated in WAJC-404 medium supplemented with insulin (5 micrograms/ml), transferrin (5 micrograms/ml), and selenious acid (5 ng/ml). Bovine pituitary extract (BPE) (30 micrograms/ml), EGF (10 ng/ml), and TGF-beta 1 (0, 0.1, and 1.0 ng/ml) were added either alone or in combination according to experimental requirements. The rate of cell proliferation was assessed by counting the total cell number and by [3H]thymidine incorporation. Prostatic epithelial cells exhibited a bell-shaped growth curve in a span of 7-8 days, with a growth peak at day 3 or 4 of culture. Treatment of cells with EGF or TGF-alpha resulted in a concentration-dependent increase in cell growth, whereas addition of TGF-beta 1 into the culture resulted in an inhibition of cell proliferation that could be reversed with increasing concentrations of EGF. Cell death was assessed using the terminal deoxynucleotidyl transferase (TdT)-mediated immunoperoxidase-digoxigenin nick end labeling technique and the trypan blue exclusion test. Epithelial cells cultured in media containing EGF had the lowest incidence of cell death. Cells cultured in the absence of EGF demonstrated a marked increase in cells undergoing cell death. The addition of TGF-beta 1 into the EGF-depleted medium caused a further increase of cell death. These results indicated that cell proliferation and cell death in rat prostatic epithelial cells in culture could be modulated by EGF and TGF-beta 1. The former stimulated cell proliferation and prevented cell death, whereas the latter inhibited proliferation in the presence or absence of EGF and induced cell death.


Journal ArticleDOI
TL;DR: In vitro studies suggest that the protease activity of PSA might play a functional role to facilitate the growth and spreading of cancerous prostatic cells and hK2 may have similar properties.
Abstract: In vitro studies suggest that the protease activity of PSA might play a functional role to facilitate the growth and spreading of cancerous prostatic cells. hK2 may have similar properties. Further investigation to prove their in vivo effects is required. Regulation of PSA and hKLK2 gene expression is mediated not only by androgens, but also by a number of autocrine and paracrine factors, suggesting that the control mechanisms for expression of these genes are complex and multifaceted. Such factors may also be integral for the growth and differentiation of prostate cells. Thus PSA and hKLK2 may serve as useful markers to study the regulation of gene expression during cell growth and differentiation of the prostate.

Journal ArticleDOI
TL;DR: The presence in the ejaculate of one or more gamete forms belonging to the epididymal caput, corpus, or cauda will allow workers to better establish the intensity of stress produced by a high frequency of semen collection.
Abstract: Sperm quality in the caput, corpus, and cauda regions of the epididymis of healthy and sexually mature Landrace boars was studied. Epididymal sperm characteristics were examined by light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Sperm vitality decreased very slightly although progressively with the transport of sperm through the epididymis. Osmotic resistance of acrosomes was very low in the sperm from the caput and approximately 100% in the corpus and cauda. The incidence of spermatozoa with the head detached from the tail remained stable in the first two regions of the epididymis, increasing notably in the cauda. Sperm agglutination increased progressively as sperm progressed along the epididymal duct. The percent of mature spermatozoa and aberrant spermatozoa increased from the caput to the cauda, whereas the percent of immature spermatozoa decreased. In the caput and corpus the percent of immature spermatozoa was similar, although in the caput they were characterized by the presence of a proximal cytoplasmic droplet; in the corpus the cytoplasmic droplet was distal. The acrosomal protuberance was highly developed in spermatozoa from the epididymal caput, but its volume was considerably reduced in those from the epididymal cauda. The electron density of the acrosomal content was lower in spermatozoa from the caput than in those from the epididymal cauda. The mitochondrial sheath of spermatozoa from the caput was made of voluminous mitochondria of unequal size, with a low electron-dense matrix. In the cauda region, the mitochondria were smaller in diameter, homogeneous in size, and with greater matrix electron density. This last fact is related to the loss of the capacity of spermatozoa to fold their tail by the midpiece as they progress along the epididymal duct. The complex epididymal maturation process of the sperm results in quantitative and qualitative changes that can be characterized in each of the three epididymal regions. The presence in the ejaculate of one or more gamete forms belonging to the epididymal caput, corpus, or cauda will allow workers to better establish the intensity of stress produced by a high frequency of semen collection.

Journal ArticleDOI
TL;DR: Significant steroidogenic reserve was still present in testes after torsion, which in previous studies had been shown to have permanent loss of spermatogenesis.
Abstract: Little is known about specific testicular cell responses to periods of testicular torsion. In particular, the steroidogenic capacity of Leydig cells in the post-torsion testis is unknown. Male Sprague-Dawley rats (450-550 g) underwent no torsion (control) or a 720 degrees unilateral testicular torsion for either 0 (sham), or 1 or 2 Such torsions have previously been shown to cause progressive damage to the rat testis. One, 15, or 30 days after torsion repair all animals (n = 5-10/group) were prepared for testicular venipuncture and intravenous infusion of ovine luteinizing hormone (LH) via the femoral vein. Testicular venous blood was collected directly from the surface of the testis both 5 minutes prior to and 90 minutes after infusion of predetermined ED50 (0.1 microgram) or ED100 (0.5 microgram) doses of LH. Testicular venous serum (TVS) was assayed for testosterone (T) by radioimmunoassay. Control animal TVS T concentrations before LH infusion and 90 minutes after ED50 and ED100 LH stimulation were 103 +/- 25, 621 +/- 103, and 1,055 +/- 140 ng/ml, respectively. Testes having experienced a 1-hour torsion did not have a significantly (P < 0.05) reduced capacity to respond to ED50 and ED100 stimulation at either 15 and 30 days after the torsion. Testes having experienced a 2-hour torsion did have significantly reduced (P < 0.05) ED50 responses at both 1 hour and 30 days after torsion repair. More remarkably, significant steroidogenic reserve was still present in testes after torsion, which in previous studies had been shown to have permanent loss of spermatogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Journal ArticleDOI
TL;DR: The results indicate that the ability of cryopreserved equine spermatozoa to attach to equine OEC or ZP in vitro is reduced compared to fresh extended spermatozosa due to changes other than a reduction in post-thaw motility or membrane integrity.
Abstract: Two bioassays were used to evaluate the interaction of fresh and cryopreserved equine semen with oviductal epithelial cells (OEC) and with the zona pelludda (ZP). Split ejaculates were either stored at room temperature or frozen and thawed. In experiment 1, progressive motility and membrane integrity were evaluated for each treatment. Fluorescent labeled spermatozoa were cocultured with monolayers of OEC for 30 minutes, and the number of sperm attached to OEC was counted by fluorescence microscopy and analysis of digitized images. Motility of spermatozoa attached to OEC was observed at 0.5, 3, 6, 18, 24, and 48 hours after insemination. In experiment 2, progressive motility, membrane integrity, and acrosomal integrity were determined. Differential labeling with the fluorochromes fluorescein isothiocyanate (FITC) or tetra-methylrhodamine isothiocyanate (TRITC) was used to distinguish fresh and frozen-thawed spermatozoa. Equal numbers of motile spermatozoa from each treatment were incubated with salt-stored equine oocytes for 4 hours, and the number of spermatozoa firmly bound to the ZP was counted using dual-wavelength epifluorescence microscopy. Fewer (P < 0.001) cryopreserved spermatozoa attached to OEC compared to spermatozoa stored at room temperature. The motility of spermatozoa attached to OEC decreased over time within each treatment group (P < 0.001), but this decrease was not different between treatments. The mean number of spermatozoa bound per ZP and percentage of acrosome-intact spermatozoa were lower (P < 0.05) for frozen-thawed than for fresh spermatozoa. There was no effect of stallion on acrosomal status of frozen-thawed spermatozoa; however, the number of spermatozoa bound per ZP was different between stallions within treatments (P < 0.05). These results indicate that the ability of cryopreserved equine spermatozoa to attach to equine OEC or ZP in vitro is reduced compared to fresh extended spermatozoa due to changes other than a reduction in post-thaw motility or membrane integrity. The decreased ability of frozen-thawed spermatozoa to attach to OEC or to ZP could explain, in part, the reduced fertility of cryopreserved compared to fresh spermatozoa in the horse.

Journal ArticleDOI
TL;DR: Washing and resuspending sperm to a standard concentration increased the number of sperm per microscopic field in low-concentration samples and reduced the field-to-field variation in all samples and decreased thenumber of sperm recognition errors by the ASMA instrument.
Abstract: New methods of specimen preparation were developed and a new method of objective, automated sperm morphometry analysis (ASMA) was performed to reduce the technical variation in the rabbit sperm morphology assay. The optimal staining procedure was a modified GZIN stain, which allowed the ASMA instrument to accurately recognize the distal end of the sperm head and to achieve the highest sperm recognition rate (94%). Washing and resuspending sperm to a standard concentration increased the number of sperm per microscopic field in low-concentration samples and reduced the field-to-field variation in all samples. Washing also decreased the number of sperm recognition errors by the ASMA instrument. Mean metric measurements for all sperm were: length, 7.38 microns; width, 3.91 microns; width/length, 0.53; area, 22.10 microns; and perimeter, 19.20 microns. Within-specimen coefficients of variation (CVs) ranged from 0.8% to 5.5% and between-animal CVs ranged from 2.7% to 7.5%. The use of standard specimen preparation techniques and ASMA technology can significantly reduce the technical variation in the rabbit sperm morphology assay.

Journal ArticleDOI
TL;DR: Future studies will be directed toward understanding the coordinate functional domains of AR, AR binding to specific androgen response elements, AR dimerization, AR phosphorylation, and AR interaction with accessory proteins that direct cell- and temporal-specific regulation of gene transactivation.
Abstract: The clinical and pathophysiologic features of AIS provide a human model for understanding the role of androgen and its receptor in the induction and maintenance of male sex differentiation and function Upon inspection, one is immediately impressed by the diverse nature of the mutations involved in the spectrum of AIS and the heterogeneous distribution of these mutations throughout the coding region of the AR gene Because of the large number and diverse array of these naturally occurring mutations and their associated clinical phenotypes, there is great potential for understanding the structure-function relationships of AR from the in vitro expression of the mutant receptors in various cell lines Future studies will be directed toward understanding the coordinate functional domains of AR, AR binding to specific androgen response elements, AR dimerization, AR phosphorylation, and AR interaction with accessory proteins that direct cell- and temporal-specific regulation of gene transactivation

Journal ArticleDOI
TL;DR: The results suggest the secretory contribution of major prostatic proteins and zinc per ejaculate to be significantly decreased in oligo-asthenozoospermic men, and the importance of this finding in relation to poor sperm count and motility as indicators of impaired gonadal function requires further investigation.
Abstract: The secretory function of the human prostate and the seminal vesicles is a prerequisite for gel formation and liquefaction of semen, but the relation to poor sperm motility and low sperm count in infertile men remains to be clarifyed. Our aim was to evaluate the secretory function of the prostate and the seminal vesicles in normozoospermic men (n = 35) and in asthenozoospermic men, who were all also oligozoospermic (n = 27). All 62 subjects belonged to couples undergoing routine infertility evaluation. In liquefied seminal fluid we measured the concentrations of fructose and protein C inhibitor (PCI) contributed by the seminal vesicles, PCI complexed to prostate-specific antigen (PSA), and the prostatic contribution of zinc, PSA, acid phosphatase (PAP), beta-microseminoprotein (beta-MSP), and Zn alpha 2-glycoprotein (Zn alpha 2-GP). The concentration of each prostatic secretory protein correlated significantly with that of zinc (P < 0.01) in both the normozoospermic (NZS) and oligo-asthenozoospermic (OAZS) subgroups, but the PCI concentration did not correlate significantly with that of fructose. There was no significant difference between the NZS and OAZS subgroups in ejaculate volume or secretory contribution from the seminal vesicles, whereas the OAZS subgroup was characterized by significantly lower secretory contributions of Zn alpha 2-GP (P = 0.001), Zn, PSA, PAP (P < 0.01), and beta-MSP (P < 0.05). The two subgroups did not differ significantly in the serum concentration of luteinizing hormone (LH), testosterone, or sex hormone-binding globulin (SHBG). The results thus suggest the secretory contribution of major prostatic proteins and zinc per ejaculate to be significantly decreased in oligo-asthenozoospermic men. The importance of this finding in relation to poor sperm count and motility as indicators of impaired gonadal function requires further investigation.

Journal ArticleDOI
TL;DR: A convergent mechanism of crosstalk between the PKA and PKC pathways leading to the human sperm AR is suggested, with an additive AR response of ED50 concentrations but not for ARmax doses.
Abstract: The human sperm acrosome reaction (AR) occurs via the activation of at least two signal transduction pathways. The purpose of this investigation was to characterize two of the pathways, the protein kinase A (PKA) and C (PKC) pathways, and determine whether pathway "crosstalk" occurs between them in eliciting the AR in capacitated spermatozoa. Stimulators of each pathway were tested in a dose-dependent manner. ARmax, ED50, and delta ARmax (%ARmax-%ARcontrol) values were calculated. The PKA pathway stimulators forskolin and dibutyryl cyclic AMP (dbcAMP) induced an ARmax at 1.0 microM and 1.0 mM, respectively. The ED50 and delta ARmax values were: 0.01 microM and 17% for forskolin and 0.069 mM and 13% for dbcAMP. Two stimulator types of the PKC pathway were tested: synthetic diacylglycerols (DG) and a phorbol diester. 1,2-dioleoyl-sn-glycerol and 1,2-dioctanoyl-sn-glycerol, analogues of the PKC-activating second messenger DG, each induced an ARmax at 50 microM. The ED50 and delta AR max values were: 33 microM and 24% for 1,2-dioleoyl and 34.8 microM and 34% for 1,2-dioctanoyl. 4 beta-Phorbol-12,13-didecanoate, a PKC stimulator, induced an ARmax at 0.1 microM. The ED50 and delta ARmax were 0.021 microM and 26%. An inhibitor of each kinase was added at the end of the capacitation period and prior to stimulation by inducers at their ARmax dose. KT5720, a PKA inhibitor, caused a dose-dependent reduction of the forskolin and dbcAMP-induced AR. Calphostin C, a PKC inhibitor, prevented stimulation of the AR by 1,2-dioleoyl and 4 beta-phorbol-12,13-didecanoate. To investigate pathway "crosstalk," the following experiments were conducted: (1) stimulators of each pathway were combined and tested at the ARmax and ED50 concentrations for each; (2) spermatozoa were pretreated with a kinase inhibitor and then stimulated using an alternative pathway stimulator; and (3) a PKA or PKC inhibitor and a combination of PKA and PKC stimulators, at ED50 concentrations, were tested. The results for (1) indicate an additive AR response of ED50 concentrations but not for ARmax doses. The results for (2) demonstrate that a kinase inhibitor for one pathway prevents induction of the AR by a stimulator of the alternative pathway. Finally, the results for (3) show that a kinase inhibitor for one pathway prevents induction of the AR by the combined use of separate pathway stimulators. When taken collectively, the present results suggest a convergent mechanism of crosstalk between the PKA and PKC pathways leading to the human sperm AR.

Journal ArticleDOI
TL;DR: Results show that iNOS can be expressed in RPSMC in a cell passage-dependent fashion that has so far not been reported for other cell lines, and that the induction reaches much higher levels than in rat or human vascular smooth muscle cells.
Abstract: Nitric oxide (NO), the main mediator of penile erection, is assumed to be synthesized in the penis by the neuronal constitutive nitric oxide synthase (nNOS). However, nNOS has not been identified in the penile smooth muscle, the target of NO action. The other NOS isozymes, the inducible NOS (iNOS) and the endothelial NOS (eNOS) have not been reported in any penile tissue. The smooth muscle vascular and trabecular tissue from rat corpora cavernosa is represented in vitro by cell cultures designated RPSMC. To determine whether iNOS can be expressed in penile smooth muscle, RPSMC were treated with different lymphokines and/or bacterial lipopolysaccharide (LPS). The selected inducer, LPS/interferon, elicited at 48 hours up to a 50-fold increase in nitrites in the medium ; the increase continued by 72 hours. The induction was inhibited by L-Nω-nitroarginine methyl ester (L-NAME), aminoguanidine, actinomycin D, cycloheximide, transforming growth factor-β1 (TGF-β1), and dexamethasone, but was resistant to nifedipine and platelet-derived growth factor-AB (PDGF-AB). iNOS induction increased with cell passage. The [ 3 H] L-arginine/citrulline measurement of NO synthesis with intact cells confirmed these results. Incubations of soluble and particulate fractions showed that the cytosol contained most of the activity (Km = 43 μM), which was partially inhibited by ethyleneglycal-bis-tetraacetic acid (EGTA). The 4.4-kb iNOS mRNA peaked at a late period (24-30 hours) and remained high for up to 72 hours. iNOS mRNA induction was strongly inhibited by actinomycin D and dexamethasone, partially inhibited by TGF-β1, inhibited slightly by PDGF-AB, and unaffected by nifedipine. These results show that iNOS can be expressed in RPSMC in a cell passage-dependent fashion that has so far not been reported for other cell lines, and that the induction reaches much higher levels than in rat or human vascular smooth muscle cells. The expression pattern is also distinctive for the penile cells in time course of induction, Ca 2+ dependence, response to certain agents, and mRNA stability.

Journal ArticleDOI
TL;DR: DMA is a suitable probe to identify human sperm mannose-binding sites crucially involved in sperm-zona interaction that appear to require free calcium concentrations to operate, and their expression changes with capacitation and acrosome reaction.
Abstract: A D-mannosylated albumin (DMA) neoglycoprotein was assessed to validate experimentally a probe capable of detecting mannose-binding sperm receptors involved in human sperm-egg interaction. DMA specifically blocked zona binding of swim-up human spermatozoa in a concentration-dependent manner. While no considerable effect was observed on sperm-zona initial contact, almost 50% of spermatozoa bound to the zona during a 2-hour period detached from it when DMA was introduced in the incubation medium. DMA inhibition was evident when 10% fetal bovine serum, but not 3.5% human serum albumin (HSA), was used as Ham's F10 medium supplementation. This may be due to the amount of free calcium in the medium since addition of 40 mM CaCl2 to F10-HSA restored DMA inhibition. Furthermore, the higher the calcium concentration in the incubation buffer, the greater the DMA blockage of sperm-zona binding. Unfixed sperm presented fluorescent DMA label over the entire acrosomal area (cap pattern), or concentrated at the equatorial segment (bar pattern). These patterns increased during capacitation, appearing on an average of 20% of the sperm after overnight incubation. They also increased, especially the bar pattern, following calcium ionophore treatment. Nearly all of methanol-fixed spermatozoa displayed the fluorescent label at the head level. Concomitant assessment of sperm membrane integrity and DMA fluorescent patterns revealed that DMA fluorescence coincided mostly with permeabilized or altered sperm plasma membrane. In conclusion, DMA is a suitable probe to identify human sperm mannose-binding sites crucially involved in sperm-zona interaction. These sites appear to require free calcium concentrations to operate, and their expression changes with capacitation and acrosome reaction. Precise location on human spermatozoa, however, warrants further investigation.

Journal ArticleDOI
TL;DR: The results suggest that the prepubertal testis may be more refractory to the effects of short periods of torsion than the adult, and demonstrate that ipsilateral torsions does not result in contralateral testicular damage in either adult or prepuberal animals.
Abstract: Previous studies of experimental torsion have suggested that there may be important differences between the adult testis and the prepubertal testis in their responses to torsion, especially with regard to the potential for contralateral damage following unilateral testicular torsion. In the present study, adult Sprague-Dawley rats and prepubertal Sprague-Dawley rats (35 days of age) were subjected to unilateral 1-, 2-, and 4-hour periods of 720 degrees or 360 degrees testicular torsion. Ipsilateral and contralateral testes were examined 30 and 60 days after torsion repair for effects on testis weight, histology, and daily sperm production (DSP). Other animals were subjected to 1-/or 4-hour 720 degrees torsion, and testicular vein testosterone concentrations were determined 30 days later. In adult animals, 1-, 2-, and 4-hour 720 degrees torsion resulted in a significant decline in ipsilateral testis weight within 30 days. Endocrine and exocrine function were also clearly disrupted. In prepubertal animals, 1-hour 720 degrees torsion significantly reduced testis weight 60 days after surgery. Also, prepubertal DSP values and testicular vein testosterone concentrations were not significantly reduced from control values by 1-hour 720 degrees torsion when examined 30 days after surgery. Longer periods of 720 degrees torsion resulted in damage similar to that seen in the adult animals. Neither adult nor prepubertal animals regained any lost function when examined 60 days after insult. Torsion of 360 degrees induced no testicular injury in adult or prepubertal animals. Contralateral testes were not affected by degree or duration of torsion in either adult or prepubertal animals. These results suggest that the prepubertal testis may be more refractory to the effects of short periods of torsion than the adult. More importantly, these results demonstrate that ipsilateral torsion does not result in contralateral testicular damage in either adult or prepubertal animals.

Journal ArticleDOI
TL;DR: Observations done after different surgical denervation procedures demonstrated that the superior spermatic nerve was the source of fibers for testicular vessels and for the nerve network of the upper pole, and fibers from the inferior speratic nerve were restricted to the nervenetwork of the lower pole.
Abstract: Sections of the rat testis and whole-mounts of the testicular capsule were studied microscopically using the glyoxylic acid-induced fluorescence method, to detect monoamines, and immunohistochemical procedures for the detection of immunoreactivities to protein gene-product 9.5 (PGP 9.5), the C-terminal accompanying peptide of neuropeptide Y (CPON), and vasoactive intestinal polypeptide (VIP). Monoaminergic nerves were only observed around the intracapsular blood vessels: the initial segment of the testicular artery and the superior venous plexus, and in the anterior aspect of the upper and lower testicular poles. These capsular nerve networks were associated with the superior and inferior ligaments of the testis. Nerves displaying PGP 9.5 and CPON immunoreactivity appeared in the same sites and followed the same distribution as monoaminergic nerves. By contrast, VIP-immunoreactive fibers were only found in the nerve network of the lower pole. Observations done after different surgical denervation procedures demonstrated that the superior spermatic nerve was the source of fibers for testicular vessels and for the nerve network of the upper pole. On the other hand, fibers from the inferior spermatic nerve were restricted to the nerve network of the lower pole.

Journal ArticleDOI
TL;DR: The way in which gonocytes adhere to Sertoli cells appears to change during the immediate postnatal period, as reflected by the observed change in phopholipase sensitivity, perhaps indicating production of a phospholipases C-resistant NCAM isoform by several days after birth.
Abstract: During neonatal development of the rat testis, gonocytes resume mitosis and display renewed motility to migrate toward the basal lamina, two events that occur in vitro when these cells are cocultured with Sertoli cells. However, although substantial evidence suggests that development of gonocytes depends on Sertoli cells, little is known of how these cell types interact beyond our previous observations that they communicate via gap junctions and adhere avidly to each other. In the present study, we utilized several approaches to examine the mechanism by which gonocytes adhere to Sertoli cells in vitro. First, we characterized this attachment in general by (1) determining its susceptibility to brief trypsinization in decreasing concentrations of Ca2+, (2) assessing the ability of gonocytes to adhere to Sertoli cells at reduced temperature, and (3) examining the effect of phospholipase C treatment on the number of gonocytes attached to a Sertoli cell monolayer. Because the findings suggested that a non-cadherin mechanism is involved, we used immunofluorescence to identify the presence of neural cell adhesion molecule (NCAM) at virtually all gonocyte-Sertoli cell (and Sertoli cell-Sertoli cell) boundaries and found that incubation of cocultures in the continuous presence of NCAM antibodies caused release of essentially all gonocytes (but not Sertoli cells) from the monolayer. We also found, in (3) above, that gonocyte-Sertoli cell adhesion was very susceptible to phospholipase C in cocultures isolated from newborns and maintained in vitro for 2 hours or 1 day but not in cultures maintained for 3 days. Moreover, cells isolated from pups 5 days old were as resistant to enzyme treatment at 2 hours postplating as were cultures from newborns after 3 days in vitro. Thus, the way in which gonocytes adhere to Sertoli cells appears to change during the immediate postnatal period, as reflected by the observed change in phopholipase sensitivity, perhaps indicating production of a phospholipase C-resistant NCAM isoform by several days after birth. These data constitute new information on the way in which postnatal gonocytes adhere to Sertoli cells and provide a basis for future work in our ongoing exploration of germ cell development in the neonatal rat testis.

Journal ArticleDOI
TL;DR: The data indicate that ACE release from human spermatozoa during capacitation is independent of acrosome reaction, and measurement of ACE release may be a clinically useful assay for human sperm capacitation.
Abstract: The present study examines the release of angiotensin-converting enzyme (ACE) from human spermatozoa during capacitation conditions and in correlation to acrosome reaction and cell death. The ACE content of spermatozoa was measured by treating the cells with different detergents. Glass wool-filtered and washed human spermatozoa were incubated for 3 hours at 37 degrees C. Percentages of acrosome-reacted and dead spermatozoa did not change significantly, but the ACE release increased from 0 to 2.93 +/- 0.44 mU/100 x 10(6) spermatozoa after 180 minutes (P < 0.001). In order to study the influence of acrosome reaction on ACE release, the acrosome reaction of noncapacitated spermatozoa was induced by 10 microM calcium ionophore A23187. The percentages of acrosome reaction and viability in noncapacitated spermatozoa as well as the ACE release were compared to corresponding data from experiments using capacitated spermatozoa (3 hours, 37 degrees C) from the same donors. Although the number of living acrosome-reacted spermatozoa after ionophore treatment (30.5 +/- 4.0%) was significantly higher than after capacitation (13.3 +/- 2.8%, P < 0.001), ACE release from ionophore-treated, noncapacitated spermatozoa was lower (P < 0.05). The data indicate that ACE release from human spermatozoa during capacitation is independent of acrosome reaction. Measurement of ACE release may be a clinically useful assay for human sperm capacitation.

Journal ArticleDOI
TL;DR: The seminal concentration of ETs are produced in different parts of the male genital tract and that they do not represent an useful tool for the diagnosis of male reproductive diseases, according to reverse-phase high-performance liquid chromatography analysis.
Abstract: We have previously found the presence of endothelin (ET) receptor and ET-like immunoreactivity in rat testis We now extend our studies from rat to human testis We found expression of a specific transcript for ET-1 and ET-1-like immunoreactivity in human testis Positive staining was confined to the Sertoli cells of the tubular compartment, although few peritubular and interstitial cells were also stained We also identified specific ET A and ET B receptor transcripts in human testis ; ET A expression was more abundant than the ET B expression Mathematical analysis of multiple self-and cross-competition studies among [ 125 I]ET-1, [ 125 I]ET-3, and analogues confirmed the presence of the ET A and ET B isoreceptors In testicular homogenates, the ET A receptor was sevenfold more concentrated than the ET B receptor In order to localize the receptors, we performed [ 125 I]ET-1 autoradiography Binding sites were mostly concentrated into the seminiferous tubules, although interstitial and peritubular myoid cells were also positive Within the seminiferous tubules, [ 125 I]ET-1 binding sites were confined to primary and secondary spermatocytes and early spermatids, whereas Sertoli cells were negative We were unable to demonstrate the presence of functional ET receptors in ejaculated spermatozoa Because ET-like immunoreactivity was present in Sertoli cells, we next asked whether authentic ET-1 is present in human seminal fluid and represents a good index for Sertoli cell function Reverse-phase high-performance liquid chromatography analysis of ET-like immunoreactivity in seminal fluid indicated that most of the detected peptides correspond to the ET-1 precursor, big-ET-1 The seminal concentration of ET-like immunoreactivity was similar in normospermic, oligospermic, azoospermic, and vasectomized men, indicating that ETs are produced in different parts of the male genital tract and that they do not represent an useful tool for the diagnosis of male reproductive diseases In conclusion, this study demonstrated, for the first time, the presence of ET-1 and its receptors in human testis