Showing papers in "Journal of Andrology in 2005"
TL;DR: Data show that sperm DNA damage can be efficiently treated with oral antioxidants administered during a relatively short time period.
Abstract: Sperm DNA fragmentation is known to compromise male fertility. Previous findings have suggested the implication of oxidative stress in the etiology of this pathological condition. The present study was conducted to find out if the pathologically increased incidence of DNA fragmentation in ejaculated spermatozoa can be reduced by oral treatment with two antioxidants, vitamins C and E. Sixty-four men with unexplained infertility and an elevated (> or = 15%) percentage of DNA-fragmented spermatozoa in the ejaculate were randomized between an antioxidant treatment (1 g vitamin C and 1 g vitamin E daily for 2 months) group and a placebo group. Sperm DNA fragmentation was evaluated by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling assay before and after treatment. No differences in basic sperm parameters were found between the antioxidant treatment and the placebo group before or after treatment. However, the percentage of DNA-fragmented spermatozoa was markedly reduced (P < .001) in the antioxidant treatment group after the treatment (9.1 +/- 7.2) as compared with the pretreatment values (22.1 +/- 7.7). No difference in the pretreatment and posttreatment incidence of sperm DNA fragmentation was observed in the placebo group. These data show that sperm DNA damage can be efficiently treated with oral antioxidants administered during a relatively short time period.
434 citations
TL;DR: Among these properties, pH and buffering capacity, osmolarity, ionic strength, and rheological properties play dominant roles in the physico-chemical processes that govern drug release kinetics and delivery vehicle distribution.
Abstract: A fluid medium was developed to simulate the salient physical and chemical properties of human semen. The composition of the medium was based upon an extensive review of the literature on constituents of human semen. In choosing the ingredients for this medium, the goal was to emphasize properties that influence interactions of human semen with topical contraceptive, prophylactic, or therapeutic products. Among these properties, pH and buffering capacity, osmolarity, ionic strength, and rheological properties play dominant roles in the physico-chemical processes that govern drug release kinetics and delivery vehicle distribution.
408 citations
TL;DR: Spermatozoa are sensitive to OS because they lack cytoplasmic defenses, and the sperm plasma membrane contains lipids in the form of polyunsaturated fatty acids, which are vulnerable to attack by ROS.
Abstract: The greatest paradox of aerobic respiration is that oxygen, which is essential for energy production, may also be detrimental because it leads to the production of reactive oxygen species (ROS) (Saleh and Agarwal, 2002). When levels of reactive oxygen species (ROS) overwhelm the body’s antioxidant defense system, oxidative stress (OS) occurs. OS is a condition in which the elevated levels of ROS damage cells, tissues, or organs (Moller et al, 1996; Sharma and Agarwal, 1996; Saleh et al, 2003). ROS are free radicals that play a significant role in many of the sperm physiological processes such as capacitation, hyperactivation, and sperm-oocyte fusion (Aitken et al, 2004; Allamaneni et al, 2004; de Lamirande et al, 1998). However, they also trigger many pathological processes in the male reproductive system, and these processes have been implicated in cancers of the bladder and prostate, as well as in male infertility (Bankson et al, 1993; Hietanen et al, 1994; Agarwal and Saleh, 2002). Spermatozoa are sensitive to OS because they lack cytoplasmic defenses (Donnelly et al, 1999; Saleh and Agarwal, 2002). Moreover, the sperm plasma membrane contains lipids in the form of polyunsaturated fatty acids, which are vulnerable to attack by ROS. ROS, in the presence of polyunsaturated fatty acids, triggers a chain of chemical reactions called lipid peroxidation (Agarwal et al, 1994; Kobayashi et al, 2001; Zalata et al, 2004). ROS can also damage DNA by causing deletions, mutations, and other lethal genetic effects (Moustafa et al, 2004; Tominaga et al, 2004). It is difficult to block the OS-
303 citations
TL;DR: Sperm P1 and P2 concentrations inversely correlate with DNA fragmentation, indicating a protective role of the protamines against sperm DNA damage, and a possible clinical significance for human sperm protamine levels.
Abstract: The objective of this study was to examine the relationship between DNA integrity and protamines in human sperm. One hundred forty-nine male infertility patients were included in an Institutional Review Board-approved study. Sperm were evaluated for DNA fragmentation using the DNA Integrity Assay, a test equivalent to the sperm chromatin structure assay (SCSA). Additionally, nuclear proteins were extracted and the protamine-1/protamine-2 ratio (P1/P2), protamine-1 (P1), protamine-2 (P2), and total protamine concentrations were evaluated. We identified 37 patients with abnormally low P1/P2 ratios, 99 patients with normal P1/P2 ratios, and 13 patients with abnormally high P1/P2 ratios. DNA fragmentation was significantly elevated in patients with low P1/P2 ratios (37.1 +/- 6.02) vs those with normal and high P1/P2 ratios (26.7 +/- 1.9 and 23.8 +/- 3.2, respectively; P < .05) and was inversely correlated with the P1/P2 ratio (R(s) -0.18, P < .05), P1 concentration (R(s) -0.29, P < .001), P2 concentration (R(s) - 0.24, P < .005), and total protamine concentration (R(s) -0.28, P < .001). Furthermore, chi2 analysis revealed a significant increase in the incidence of marked DNA fragmentation in patients with diminished levels of either P1 or P2. The present study is the first to report that human sperm protamine content is significantly related to DNA fragmentation. In particular, sperm P1 and P2 concentrations inversely correlate with DNA fragmentation, indicating a protective role of the protamines against sperm DNA damage. In light of recent studies highlighting the negative effect of sperm DNA damage on ART outcomes, these findings indicate a possible clinical significance for human sperm protamine levels.
237 citations
TL;DR: A unique microarray system capable of directly identifying antisense RNAs and predicted transcripts was utilized, and 68 shared RNAs were revealed, some of which were similar to those previously defined as micro-RNAs, whereas others were the antisense of previously in silico-predicted transcripts.
Abstract: We recently described a complex population of spermatozoal coding RNAs that are delivered to the oocyte on fertilization. These are derived throughout spermatogenesis, representing a record of past events. Recently, evidence has been provided that micro-RNAs are present in testes, suggesting that they might also be carried in ejaculate spermatozoa. To directly test this hypothesis, a unique microarray system capable of directly identifying antisense RNAs and predicted transcripts was utilized. RNA isolated from the ejaculate spermatozoa of 6 normal fertile men was directly hybridized to sense oligonucleotide arrays containing 10 000 elements. This revealed 68 shared RNAs, some of which are similar to those previously defined as micro-RNAs, whereas others were the antisense of previously in silico-predicted transcripts. The results and implications of this study are described in this communication.
190 citations
TL;DR: Irrespective of the concentration used, SOD and CAT significantly improved postthaw sperm survival, in terms of total sperm motility and viability, and improved the ability of frozen-thawed spermatozoa to produce embryos in vitro.
Abstract: In the present study the potential benefit of reactive oxygen species (ROS)-scavenging enzymes superoxide dismutase (SOD) and catalase (CAT) when cryopreserving boar spermatozoa was evaluated. Pooled ejaculate sperm-rich fractions collected from 3 fertile boars were frozen in a split design, after being extended in a conventional freezing extender (control) or the same extender supplemented with SOD (150 or 300 IU/mL, experiment 1), CAT (200 or 400 IU/mL, experiment 2), or SOD + CAT in combination (150 + 200 or 300 + 400 IU/mL, experiment 3). Irrespective of the concentration used, SOD and CAT, alone or in combination, significantly improved postthaw sperm survival, in terms of total sperm motility (assessed with CASA) and viability (assessed with a triple stain; propidium iodide/R123/fluorescein isothiocyanate-labeled peanut agglutinin). Moreover, CAT alone, at a concentration of 400 IU/mL, or in combination with SOD, at concentrations of 200 and 400 UI/mL, improved the ability of frozen-thawed spermatozoa to produce embryos in vitro (zygote cleavage and blastocyst formation as end points). Additional data of ROS generation (luminol- and lucigenin-dependent chemiluminescence) and membrane lipid peroxidation (malondialdehyde [MDA] production) indicated that SOD and CAT reduced postthaw ROS generation by boar spermatozoa, without any influence on MDA production.
180 citations
TL;DR: It is concluded that adiponectin levels increase within days of experimental T deficiency in normal men, and the increase in adip onectin is prevented by T replacement, which supports the hypothesis that T, its metabolites, or both directly suppress adipocyte production of adiponECTin.
Abstract: Testosterone (T) administration to men increases lean body mass and decreases fat mass. Adiponectin is produced by adipocytes and is thought to influence insulin sensitivity. In this study, we sought to determine whether experimental alterations in serum T change adiponectin levels in normal men. We measured adiponectin levels in 28 healthy men ages 18-35 years before and during treatment with a potent gonadotropin-releasing-hormone (GnRH) antagonist, acyline. Decreased T levels led to increased serum adiponectin within 7 days; maximal adiponectin levels were reached on day 21 (baseline 8.6 +/- 0.9 compared with 12.2 +/- 1.0 microg/mL on day 21, P <.05) and persisted through day 30, despite no significant changes in body mass index (BMI) and an increase in leptin. The addition of T to acyline, maintaining serum T levels within the normal range, prevented the increase in adiponectin following acyline alone. In a second study, 25 men aged 55-85 years were treated with 3 weeks of high-dose T (testosterone enanthate [TE], 600 mg/wk intramuscularly). With high serum T levels, adiponectin levels decreased significantly by day 21 of treatment (baseline 14.3 +/- 1.9 compared with 10.8 +/- 1.5 microg/mL, P <.05 vs baseline and placebo), BMI slightly increased, and leptin levels were decreased. We conclude that adiponectin levels increase within days of experimental T deficiency in normal men, and the increase in adiponectin is prevented by T replacement. Furthermore, supraphysiologic T administration results in decreased adiponectin levels. Our data support the hypothesis that T, its metabolites, or both directly suppress adipocyte production of adiponectin.
159 citations
TL;DR: It is hypothesize that androgen deprivation promotes differentiation of progenitor stroma cells into an adipogenic lineage producing fat-containing cells, thus altering erectile function.
Abstract: Androgens are deemed to be critical for the development, growth, and maintenance of penile tissue as well as for erectile function Androgens are also reported to inhibit differentiation of stroma progenitor cells into adipocytes and promote differentiation into smooth muscle The objective of this study was to investigate whether androgen deprivation results in accumulation of adipocytes in the corpus cavernosum Mature, New Zealand white male rabbits were subjected to sham surgery (control) or orchiectomy Two weeks after surgery, erectile function was assessed by monitoring changes in intracavernosal blood pressure (ICP) in response to pelvic nerve stimulation All ICP measurements were normalized to the mean systemic arterial blood pressure In parallel studies, penile cross sections from control and orchiectomized rabbits were fixed and stained with either Masson's trichrome or hematoxylin and eosin to assess smooth muscle and connective tissue content Alternatively, tissue sections were stained with Toluidine blue to assess accumulation of fat-containing cells Orchiectomy resulted in loss of erectile function and penile atrophy, associated with reduced trabecular smooth muscle and increased connective tissue content Most strikingly, tissue from orchiectomized animals exhibited accumulation of fat-containing cells (adipocytes) in the subtunical region of the corpus cavernosum We hypothesize that androgen deprivation promotes differentiation of progenitor stroma cells into an adipogenic lineage producing fat-containing cells, thus altering erectile function
143 citations
TL;DR: It is demonstrated that this sperm nuclease is present in the sperm nucleus and that it is activated by the presence of both calcium and magnesium much more efficiently than with either ion alone, resulting in DNA degradation in 30 minutes.
Abstract: Recent work from our laboratory provided evidence for the existence of a nuclease in hamster spermatozoa. This endogenous nuclease cleaves sperm chromatin at the bases of DNA loop domains into large fragments with an average size of roughly 50 kb. Here, we demonstrate that this sperm nuclease is present in the sperm nucleus and that it is activated by the presence of both calcium and magnesium much more efficiently than with either ion alone, resulting in DNA degradation in 30 minutes. We also show that similar nucleases are present in mouse and human spermatozoa. The human nuclease can be activated by freeze-thawing spermatozoa in noncryoprotective media. The activity of the sperm nuclease in all 3 species resembles that of a group of somatic cell DNAses that also require both calcium and magnesium and that digest the chromatin into loop-sized fragments during apoptosis.
132 citations
TL;DR: The ASMA protocol used in this study was useful to detect subtle morphometric differences between spermatozoa, and the combination of this analysis with a multivariate statistical procedure gave new information on the biological characteristics of boar ejaculates that is not given by conventional sperm analysis.
Abstract: A statistical approach using sequentially principal component analysis (PCA), clustering, and discriminant analyses was developed to identify sperm morphometric subpopulations in well-defined portions of the fresh boar ejaculate. Semen was obtained as 2 portions (the first 10 mL of the sperm-rich fraction and the rest of the ejaculate, respectively) and frozen using a conventional protocol. Before freezing, an aliquot was used for computer-assisted sperm morphometry analysis (ASMA). Postthaw quality was evaluated using computer-assisted sperm analysis (CASA), and an annexin-V/PI assay evaluated sperm membranes. The PCA revealed that 3 variables represented more than 78% of the cumulative variance in sperm subpopulations. The clustering and discriminant analyses, based on 5780 individual spermatozoa, revealed the existence of 4 sperm subpopulations. The relative percentage of these subpopulations varied between boar and ejaculate portions. Linear regression models based on measured morphometric characteristics could account for up to 36% of the percentage of intact sperm membranes postthaw. The ASMA protocol used in our study was useful to detect subtle morphometric differences between spermatozoa, and the combination of this analysis with a multivariate statistical procedure gave new information on the biological characteristics of boar ejaculates that is not given by conventional sperm analysis.
127 citations
TL;DR: The present study confirms the heterogeneity of sperm movement patterns in boar semen, patterns that vary through the cryopreservation procedure, especially after removal of the seminal plasma by centrifugation and subsequent extension at 17 degrees C and after the slow cooling at 5 degrees C, when obvious increases in hyperactivated movement appeared.
Abstract: The present study evaluates the effect that various steps of a conventional cycle of cryopreservation have on the patterns of movement exhibited by boar spermatozoa. Sperm-rich ejaculate fractions collected from 24 mature fertile boars (1 ejaculate per boar) were cryopreserved following a standard freeze-thaw procedure with 0.5-mL plastic straws. Overall sperm motility and the individual kinematic parameters of motile spermatozoa (assessed by the computer-aided sperm analysis system Sperm Class Analyzer [SCA]) were recorded in 5 steps of the cryopreservation procedure. These steps were as follows: 1) at the time that the fresh semen was extended, 2) at 17 degrees C, after sperm concentration by centrifugation and re-extension of the pellet with lactose-egg yolk extender; 3) at 5 degrees C, after added freezing extender; 4) at the time that thawed semen was held in a water bath at 37 degrees C for 30 minutes; and 5) at the time that thawed semen was held in a water bath at 37 degrees C for 150 minutes. Data from individual motile spermatozoa, defined by 7 kinematic parameters (curvilinear velocity [VCL], straight-line velocity [VSL], average path velocity [VAP], linearity [LIN], straightness [STR], mean amplitude of lateral head displacement [ALH], and beat cross frequency [BCF]), were analyzed using a pattern analysis technique (PATN) to identify and quantify populations and subpopulations of motile sperm within the semen samples. After the first cluster analysis, 3 motile sperm populations (P) were identified (P1: progressive and/or vigorous cells [90.4%], P2: poorly progressive cells [8.3%], and P3: nonprogressive cells [1.3%]). These populations remained constant (P greater than .05) throughout the 5-step cryopreservation procedure. A second PATN was carried out within the P1 sperm population, which identified 3 sperm subpopulations (sP) (eg, sP1: cells with progressive and vigorous movement [58.7%], sP2: progressive cells only [24.6%], and sP3: vigorous cells only, hyperactive-like [16.7%]). Although the relative frequency of these 3 subpopulations varied among ejaculates (boars), there was no interaction with any cryopreservation step we examined. Whereas sP1 remained constant (P greater than .05), sP2 and sP3 varied significantly (P less than .05) through the cryopreservation procedure, with the increase in sP3 after centrifugation at 17 degrees C and during cooling at 5 degrees C considered particularly relevant. In conclusion, the present study confirms the heterogeneity of sperm movement patterns in boar semen, patterns that vary through the cryopreservation procedure, especially after removal of the seminal plasma by centrifugation and subsequent extension at 17 degrees C and after the slow cooling at 5 degrees C, when obvious increases in hyperactivated movement appeared. The vast majority of spermatozoa, those exhibiting progressive and vigorous movement, remained constant during the cryopreservation procedure, although the proportion differed among boars.
TL;DR: It is proved that 2 protein bands of approximately 14 (P14) and 20 (P20) kd isolated from seminal plasma are responsible for this protective effect and suggest that both proteins are involved in sperm CA and gamete interaction, first by stabilizing the sperm membrane and then by participating in CA in the female reproductive tract.
Abstract: We have already shown that seminal plasma proteins in the ram can repair cold-shock sperm membrane damage and that the addition of seminal plasma proteins before cold-shock treatment prevents sperm membrane injury. In this study, we prove that 2 protein bands of approximately 14 (P14) and 20 (P20) kd isolated from seminal plasma are responsible for this protective effect. In vitro capacitation (CA) and acrosome reaction (AR) modified the content of both proteins on the sperm surface. P20 release began at the beginning of CA, and the induction of the AR accounted for an additional release of both proteins; not more than 35% of these proteins remained on acrosome-reacted sperm. Cytochemical analysis detected that there are several binding sites for P14 and P20 on the sperm surface and that membrane alterations induced by CA and the AR accounted for the loss and redistribution of both proteins to the equatorial and postequatorial regions. The P14-sequenced fragment showed a high homology with several seminal plasma proteins of different species and contained the FN 2 domain like bovine PDC-109. However, the sequence of the P20 fragment was not homologous with any reported protein. By immunochemical analysis, we obtained evidence that P14 is phosphorylated in serine and threonine residues and that P20 is a glycosylated protein. These results suggest that both proteins are involved in sperm CA and gamete interaction, first by stabilizing the sperm membrane and then by participating in CA in the female reproductive tract. The protective effect of P14 and P20 could be related to their decapacitating role.
TL;DR: It is concluded that clomiphene citrate administration may result in sperm in the ejaculate of patients with nonobstructive azoospermia or the simplification of testis sperm retrieval.
Abstract: Clomiphene citrate is a well-established agent thathas been empirically used in cases of idiopathic oligospermia. Clo-miphene increases endogenous gonadotropin-releasing hormonesecretion from the hypothalamus and gonadotropin hormone secre-tion directly from the pituitary and, thus, increases intratesticulartes-tosterone concentration. Using intracytoplasmic sperm injection(ICSI), very few sperm may be required for fertilization.Theobjectiveof this study was to determine if the application of clomiphenecitratein males with nonobstructive azoospermia might produce sufficientsperm for ICSI, either by resulting in sperm identified in the ejaculateor by potentially improving outcomes of surgical testicular sperm ex-traction. Forty-two patients with nonobstructive azoospermia (agerange, 25–39 years) from 3 international centers were evaluatedwithroutine history, physical examination, and hormonal assessment.Ini-tial testicular biopsy demonstrated maturation arrest in 42.9% andhypospermatogenesis in 57.1% of patients. Clomiphene citrate wasadministered, with the dose titrated to achieve serum testosteronelevels between 600 ng/dL and 800 ng/dL, and semen analyses wereperformed at periodic intervals. In patients remaining azoospermicon semen analysis, surgical testicular biopsy and sperm extractionwere performed. After clomiphene citrate therapy, 64.3% of the pa-tients demonstrated sperm in their semen analyses ranging from 1to 16 million sperm/mL, with a mean sperm density of 3.8 million/mL. Sufficient sperm for ICSI was retrieved by testicular sperm ex-traction in all patients, even though 35.7% remained azoospermic.Additionally, clomiphene citrate administration resulted in a statisti-cally significant increase in testis biopsy patterns associated withgreater likelihood of sperm obtained by surgical extraction (P, .05).We conclude that clomiphene citrate administration may result insperm in the ejaculate of patients with nonobstructive azoospermiaor the simplification of testis sperm retrieval. Surgeons may considera course of clomiphene citrate administration prior to surgical spermretrieval in patients with nonobstructive azoospermia.Key words: Hypospermatogenesis, maturation arrest, ICSI,TESE.J Androl 2005;26:787–791
TL;DR: The main findings emerging from this study were that the addition of GSH to the freezing media resulted in an improvement in percent motility and motion parameters of thawed spermatozoa, as measured by both microscopic analysis and computer-assisted semen analysis (CASA).
Abstract: In this study, we evaluated the effects of glutathione (L-gamma-glutamyl-L-cysteinylglycine; GSH) supplementation of the freezing extender on semen parameters during the cooling (2 hours at 5 degrees C) and freezing phases of the cryopreservation process to compensate for the decrease in GSH content observed during sperm freezing. To fully address these questions, we incorporated a new set of functional sperm tests. These included tests of mitochondrial function, inducibility of the acrosome reaction, in vitro penetration (IVP) of oocytes, changes in sulfhydryl group content in membrane proteins, and capacitation status. The main findings emerging from this study were that the addition of GSH to the freezing media resulted in 1) an improvement in percent motility (%MOT) and motion parameters of thawed spermatozoa, as measured by both microscopic analysis and computer-assisted semen analysis (CASA); 2) a higher number of total viable spermatozoa; 3) a higher number of noncapacitated viable spermatozoa; and 4) a decrease in the number of spermatozoa with changes in the sulfhydryl groups in membrane proteins. This protective effect on sperm function was more pronounced with 1 mM of GSH than with 5 mM of GSH.
TL;DR: It is demonstrated that a sustained disruption of sperMatogenesis occurs despite the continued presence of stem spermatogonia in the seminiferous epithelium, suggesting that cisplatin-induced germ cell loss may occur, in part, as a result of Sertoli cell injury-dependent alterations in germ cell microenvironment.
Abstract: Exposure to cisplatin results in impaired spermatogenesis, azoospermia, and, sometimes, permanent infertility in male patients. The mechanism(s) by which cisplatin induces damage to testicular cells is poorly understood. We previously reported that acute exposure to cisplatin results in elevated germ cell apoptotic rates and that this indicates long-term damage to the seminiferous epithelium. Here, we present data that implicate an injury to Sertoli cells as a possible mechanism to explain an elevated rate of germ cell apoptosis and consequent infertility. Normal adult C57/Bl/6J mice were exposed to 1, 2, or 4 rounds of 1, 2.5, or 5 mg/kg cisplatin in a regimen designed to resemble clinical chemotherapeutic exposure (1 injection daily for 5 days with a recovery phase of 16 days between cycles). A dose-dependent reduction in testicular weight due to germ cell loss was observed. While exposure to 1 mg/kg caused only temporary germ cell depletion, higher doses (2.5 and 5 mg/kg) revealed widespread testicular atrophy as evidenced by gaps in the epithelium due to cytoplasmic vacuolization and loss of differentiating germ cells. Although the acute loss of germ cells by apoptosis can result in temporary infertility, the testis has the ability to repopulate itself with mature cells, provided the stem germ cell population remains unharmed. Here, we demonstrate that a sustained disruption of spermatogenesis occurs despite the continued presence of stem spermatogonia in the seminiferous epithelium. These results suggest that cisplatin-induced germ cell loss may occur, in part, as a result of Sertoli cell injury-dependent alterations in germ cell microenvironment.
TL;DR: If antioxidant intake in the range studied is indeed beneficial for fertility in healthy men, it does not appear to be mediated through the integrity of sperm chromatin, and the results of this study do not preclude possible beneficial effects of high antioxidant intake on sperm Chromatin integrity for men with fertility problems.
Abstract: Oxidative stress is detrimental to sperm function and a significant factor in the etiology of male infertility. This report examines the association between dietary and supplementary intake of the antioxidants vitamin C, vitamin E, and beta-carotene and sperm chromatin integrity. Eighty-seven healthy male volunteers donated semen samples, completed food-frequency questionnaires, and provided information about their sociodemographic characteristics, medical and reproductive histories, and lifestyle habits. Sperm chromatin integrity was measured using the DNA fragmentation index (DFI) and related parameters, obtained from the sperm chromatin structure assay (SCSA). SCSA measures the susceptibility of sperm DNA to acid-induced denaturation in situ. After adjusting for age and duration of abstinence, there was no dose-response association between any DFI outcome and any antioxidant intake measure. Non-dose-related associations were found between beta-carotene intake and both the standard deviation of DFI (SD DFI) and the percent of immature sperm. Participants with moderate, but not high, beta-carotene intake had an increase in SD DFI compared with participants with low intake (adjusted means 206.7 and 180.5, respectively; P = .03), as well as an increase in the percentage of immature sperm (adjusted means 6.9% and 5.0%, respectively; P = .04). If antioxidant intake in the range studied is indeed beneficial for fertility in healthy men, it does not appear to be mediated through the integrity of sperm chromatin. The results of this study do not preclude possible beneficial effects of high antioxidant intake on sperm chromatin integrity for men with fertility problems.
TL;DR: Both chronic pelvic pain syndrome and premature ejaculation are common disorders, but their ethiopathogeneses are not well understood and the incidence of psychogenic sexual problems was higher than in the normal population.
Abstract: Chronic pelvic pain syndrome is a common and se- rious health problem affecting the quality of life in men. Limited stud- ies exist on the relation of this condition to premature ejaculation. We evaluated prevalence rates of premature ejaculation in Turkish male patients with chronic pelvic pain syndrome and compared them with healthy control subjects. Sixty-six men with chronic pelvic pain syndrome were included in the study (group 1). A questionnaire con- sisting of 2 parts—demographic data and a Turkish version of the National Institutes of Health Chronic Prostatitis Symptom index— was administered to all patients. Premature ejaculation was defined as intravaginal ejaculation latency of less than 2 minutes with the same partner for at least 6 months. All patients were evaluated with physical examinations and routine laboratory tests. If erectile dys- function was noted from the medical history, penile Doppler ultra- sonography also was performed. The results were compared with the results of 30 healthy men without urinary symptoms (group 2). The x2 test was used for statistical analyses. Of 66 patients with chronic pelvic pain syndrome, 51 had premature ejaculation (77.3%), and in 10 (15.2%) patients, premature ejaculation and erectile dys- function were found together. Penile Doppler ultrasonography showed no vascular pathology in patients with erectile dysfunction. The rate of premature ejaculation was higher in patients in the study group than it was in patients in the control group, and this difference was statistically significant (P , .05). Both chronic pelvic pain syn- drome and premature ejaculation are common disorders, but their ethiopathogeneses are not well understood. In Turkish men with chronic pelvic pain syndrome, the incidence of psychogenic sexual problems was higher than in the normal population.
TL;DR: Late gestation in utero treatment of pigs with low doses of busulfan and testicular irradiation of goats at 1 week of age will result in a reduction in the endogenous germ cell population that could facilitate donor cell colonization after germ cell transplantation.
Abstract: The efficiency of germ cell transplantation, the procedure of transferring germ cells from a donor male into the testes of recipient males, can be greatly increased by reduction of endogenous germ cells in recipient animals. To develop effective methods for suppression of endogenous spermatogenesis in potential pig and goat recipients, we either administered busulfan to pregnant sows or irradiated the testes of immature goats. Piglets from sows treated twice with busulfan (7.5 mg/kg) at days 98 and 108 of gestation showed reduced gonocyte numbers at 2, 4, and 8 weeks of age and reduced initiation of spermatogenesis at 16 weeks of age. For goats, groups of 3 kids at 1, 5, or 9.5 weeks of age received fractionated irradiation of the testes with 3 doses of 2 Gy on 3 consecutive days. At 2 months after irradiation, 5%-10% of seminiferous tubule cross sections contained pachytene spermatocytes, compared with 50%-100% in controls. At 3 months after irradiation, spermatozoa appeared in 20% of tubule cross sections in all treated goats and in 100% of tubules in control goats. By 6 months after irradiation, spermatogenesis had recovered in 60% of tubules in goats treated at 5 or 9.5 weeks of age but in only 29% of tubules after treatment at 1 week of age. Therefore, late gestation in utero treatment of pigs with low doses of busulfan and testicular irradiation of goats at 1 week of age will result in a reduction in the endogenous germ cell population that could facilitate donor cell colonization after germ cell transplantation.
TL;DR: It is shown that human CRISP-3 is a quantitatively minor seminal plasma protein not associated with prostasomes, and expression was found in the secretory epithelium throughout the male genital tract, with particularly high expression in the cauda epididymis and ampulla vas deferens.
Abstract: Mammalian members of the cysteine-rich secretory protein (CRISP) family are expressed predominantly in the male reproductive tract and are implicated in the process of reproduction from spermiogenesis, posttesticular sperm maturation, and capacitation to oocyte-sperm fusion, and possibly also penetration of the zona pellucida. Rodents express only 2 CRISPs (CRISP-1 and CRISP-2) in their male reproductive system, whereas humans and horses express an additional third member named CRISP-3. We have previously demonstrated that this protein is present in human seminal plasma as well as in other exocrine secretions, in blood plasma, and in neutrophilic granulocytes. To characterize the protein in seminal plasma and localize the production of CRISP-3 in the human male reproductive tract, we performed immunoblotting and enzyme-linked immunosorbent assay measurements of seminal plasma and immunohistochemistry and in situ hybridization of tissue specimens. We were able to show that human CRISP-3 is a quantitatively minor seminal plasma protein not associated with prostasomes. Furthermore, CRISP-3 expression was found in the secretory epithelium throughout the male genital tract, with particularly high expression in the cauda epididymis and ampulla vas deferens. Examination of seminal plasma from vasectomized males indicates that organs downstream of the epididymis are probably the major sources of seminal plasma CRISP-3.
TL;DR: Addition of GSH to the thawing extender could be of significant benefit in improving the function and fertilizing capacity of frozen boar spermatozoa.
Abstract: In this study, we evaluated the effects of glutathione (L- g-glutamyl-L-cysteinylglycine; GSH) supplementation of the thawing extender on semen parameters to compensate for the decrease in GSH content observed during sperm freezing. To fully address these questions, we used a set of functional sperm tests. These included tests of motility and motion parameters, changes in sulfhydryl group content in membrane proteins, capacitation status, measures of intra- cellular reactive oxygen species generation, sperm chromatin conden- sation, and in vitro penetration of immature oocytes. The main findings emerging from this study were that addition of GSH to the thawing media resulted in a lower number of capacitated viable spermatozoa, a decrease in the number of spermatozoa with changes in the sulf- hydryl groups in membrane proteins, a reduction of the reactive oxy- gen species generation, a lower chromatin condensation, and a higher penetration ability of oocytes in vitro and a higher proportion of de- condensated sperm heads. GSH appears to play an important role in sperm antioxidant defense strategy. Addition of GSH to the thawing extender could be of significant benefit in improving the function and fertilizing capacity of frozen boar spermatozoa.
TL;DR: This study shows specific variability in the transition nuclear protein genes (TNPs) of sterile male patients and analysis of the human TNP1 and TNP2 gene sequences revealed 5 amino acid substitution-causing single nucleotide polymorphisms (SNPs) in the open-reading frame of the T NP2 gene.
Abstract: Previously, we examined the relationship between protamine gene variations and human male infertility. In this study, we show specific variability in the transition nuclear protein genes (TNPs) of sterile male patients. Transition nuclear proteins (TPs) are major nuclear proteins that replace nuclear histones, leading to eventual substitution by protamines during human spermiogenesis. Analysis of the human TNP1 and TNP2 gene sequences in 282 sterile male patients and 270 (TNP1) and 266 (TNP2) proven-fertile male volunteers revealed 5 amino acid substitution-causing single nucleotide polymorphisms (SNPs) in the open-reading frame of the TNP2 gene. On the other hand, a deletion of 15 nucleotides, which encompassed the recognition site for the cAMP response element (CRE) transcription factor, was found in the 5'-promoter region of the TNP1 gene in infertile men. This deletion reduces TNP1 expression and may cause human male infertility.
TL;DR: The FSHR haplotype does not associate with different serum FSH levels but it is differently distributed in normal and azoospermic men, which might represent genetic factors contributing to phenotypic expression of severe spermatogenetic impairment.
Abstract: The human follicle-stimulating hormone (FSH) receptor (FSHR) gene possesses single nucleotide polymorphisms (SNP) in exon 10, which influence serum FSH levels in women, but not in men. In the present study we extend our previous investigation and for the first time analyze a novel, common SNP at position -29 of the FSHR core promoter in men. The SNP in codon 680 was analyzed in 438 men with nonobstructive azoospermia and in 304 controls. The SNP in codon 307 and at position -29 was analyzed in 345 men with nonobstructive azoospermia and 186 controls. SNPs were determined by allelic discrimination. No significant difference in the frequency of the polymorphism at position 680 and serum FSH levels was found. At position -29 (A/G) the A(-29) allele was less frequent than the G(-29) allele both in controls (25% vs 75%) and in patients (30% vs 70%) (P not significant). Together the three SNPs form four discrete haplotypes (A-Thr-Asn, G-Thr-Asn, A-Ala-Ser, and G-Ala-Ser) occurring in 10 combinations. A statistically significant difference in the allelic distribution between controls and azoospermic men was found (P < .05 by chi2 test). The A-Ala-Ser allele was more frequent in patients (9.1%) than in controls (5.4%), whereas the G-Thr-Asn allele was less frequent in patients (33.1%) than in controls (40.6%) (P < .01 by Fisher's exact test). No significant correlation between serum FSH levels and FSHR allele was found. We conclude that the FSHR haplotype does not associate with different serum FSH levels but it is differently distributed in normal and azoospermic men. The A-Ala-Ser and the G-Thr-Asn allele might represent genetic factors contributing to phenotypic expression of severe spermatogenetic impairment.
TL;DR: It is demonstrated that sp32, a (pro)acrosin binding protein, is the p 32, a tyrosine phosphorylated protein related to capacitation, a protein implicated in proacros in maturation.
Abstract: Mammalian sperm must undergo capacitation, a preparation period in the female reproductive tract or in vitro, in order to fertilize. We have previously described a Mr 32 000 tyrosine phosphorylated protein, "p32," that appears in pig sperm during capacitation. The identity of p32 remains unknown; if and how it is involved during capacitation is not understood. The objective of the present study was to identify p32 by proteomic techniques. Western blotting of proteins separated successively under nonreducing and then reducing conditions showed the appearance of the tyrosine phosphorylated p32 only when sperm were incubated in capacitating conditions. The spot was sequenced by mass spectrometry/mass spectrometry and identified as "sp32," a protein implicated in proacrosin maturation. The same membranes probed with anti-sp32 antibody demonstrated that sp32 is present in both noncapacitating and capacitating conditions and revealed exactly the same spot as p32. Immunoprecipitation with either anti-phosphotyrosine or anti-sp32 antibody corroborated these results. Indirect immunofluorescence with anti-phosphotyrosine antibody or anti-sp32 antibody show similar labeling of capacitated sperm, supporting the hypothesis that p32 is a tyrosine phosphorylated form of sp32. After ionophore treatment to induce the acrosome reaction, anti-sp32 and anti-phosphotyrosine labeling on the acrosome disappeared. These results demonstrate that sp32, a (pro)acrosin binding protein, is the p32, a tyrosine phosphorylated protein related to capacitation. We will now focus on the significance of tyrosine phosphorylation on sp32 function during fertilization-related events.
TL;DR: The data indicate that aging leads to impairment in the neurogenic erectile response in BN rats involving a possible derangement in penile hemodynamic mechanisms of the erectile tissue.
Abstract: Physiological aging is a significant risk factor in the on-set of male erectile dysfunction (ED) and an imbalance in factors that modulate cavernosal smooth-muscle tone may play a role in these altered penile hemodynamic mechanisms. To evaluate the association between aging and male erectile function, we monitored neurogenic erectile response and its correlation to systemic arterial pressure changes in old (21-23 months of age) vs young (6-9 months of age) Brown-Norway (BN) rats. We tested the hypothesis that age-associated ED is due to unregulated vasoconstrictive tone, contributed in part by an increased Rho-kinase activity, and that antagonism of Rho-kinase activity attenuates the age-related decline in male erectile function. We also examined the hypothesis that a combination of Rho-kinase antagonism and phosphodiesterase-5 (PDE-5) inhibition has a synergistic effect in improving the erectile response in these aging animals. Erectile function in old BN rats was evaluated before and after intracavernosal injection of a specific inhibitor of Rho-kinase (Y-27632) alone or in combination with zaprinast, a PDE-5 inhibitor. Erectile capabilities of the young and old BN rat groups were significantly different in corpus cavernosum pressure response after electrical-field stimulation of the major pelvic ganglion. Y-27632 administration attenuated the aging-related changes in male erectile function seen in BN rats. Rho-kinase antagonism and PDE-5 inhibition had a synergistic effect in improving erectile function in old rats. Our data indicate that aging leads to impairment in the neurogenic erectile response in BN rats involving a possible derangement in penile hemodynamic mechanisms of the erectile tissue. Rho-kinase inhibition may be of value in treating age-related ED.
TL;DR: While T production in rat Leydig cells is highly sensitive to NO and an endogenous NO-generating system is not present in these cells, NOS activity is more likely to reside in activated testicular macrophages.
Abstract: The free radical nitric oxide (NO), generated through the oxidation of L-arginine to L-citrulline by NO synthases (NOSs), has been shown to inhibit steroidogenic pathways. NOS isoforms are known to be present in rat and human testes. Our study examined the sensitivity of Leydig cells to NO and determined whether NOS activity resides in Leydig cells or in another cell type such as the testicular macrophage. The results showed a low level of L-[14C]arginine conversion in purified rat Leydig cell homogenates. Administration of the NOS inhibitor L-N(G)-nitro-arginine methyl ester (L-NAME), or the calcium chelator ethylenebis (oxyethylenenitrilo)tetraacetic acid (EGTA), had no effect on L-[14C]citrulline accumulation. Increased intracellular Ca2+ concentrations that were induced by a calcium ionophore, or the addition of luteinizing hormone (LH), failed to affect NO formation in intact cells that were cultured in vitro. Introduction of a high concentration of the NO precursor L-arginine did not decrease testosterone (T) production, and NOS inhibitors did not increase T biosynthesis. However, exposing Leydig cells to low concentrations of the NO donor S-nitrosoglutathione (GSNO) induced a dramatic blockade of T production under basal and LH-stimulated conditions. DNA array assays showed a low level of expression of endothelial NOS (eNOS), while the neuronal and inducible isoforms of NOS (nNOS and iNOS) were below detection levels. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses confirmed these findings and demonstrated the presence of high iNOS messenger RNA (mRNA) levels in activated testicular macrophages that produced large amounts of NO. These data suggest that, while T production in rat Leydig cells is highly sensitive to NO and an endogenous NO-generating system is not present in these cells, NOS activity is more likely to reside in activated testicular macrophages.
TL;DR: Assessment of chamber flow dynamics, flow uniformity, inflow cell velocity, and results of concentration measurements under different flow conditions for latex bead and porcine and human sperm suspensions provided further support that hemacytometry, when performed properly, remains the gold standard.
Abstract: Capillary loaded chambers are frequently used for semen analysis. Poiseuille flow of specimen into these chambers causes migration of suspended particles or cells in a direction transverse to the flow, which results in their preferential accumulation in the Segre-Silberberg (SS) planes. This SS effect depends on the transverse velocity gradient in the laminar flow. For semen analysis in thin capillary-loaded slides, the SS effect can lead to erroneous estimation of sample sperm-cell concentration. To better understand chamber flow dynamics and SS effect significance, we assessed flow uniformity, inflow cell velocity, and results of concentration measurements under different flow conditions for latex bead and porcine and human sperm suspensions. Overall, a concentration peak was present at the meniscus, which continued through chamber loading. High-velocity SS preferred planes, which channeled particles toward the meniscus, were located at the fractional positions of beta = .27 and beta = .73, where beta is the distance from wall to plane normalized to the chamber depth. In computer-automated semen analysis, a standard 20-microm x 18-mm x 6-mm chamber is commonly used, and these studies supported our previously published fluid-flow theory for this type of chamber. Conversely, the SS effect does not appear to have time to develop in the 100-microm-depth hemacytometer, which is deeper than the standard slide, has lower transverse velocity gradient, and consequently does not exhibit concentration variation due to the SS effect. These findings provide further support that hemacytometry, when performed properly, remains the gold standard. Applicability of our findings to routine semen analyses was then tested in 2 studies performed with independent boar studs. These studies compared diluted boar semen concentrations estimated by standard hemacytometry and in capillary-loaded 20-microm slides, using a computer-automated semen-analysis system designed to compensate for the SS effect. Good numerical agreement for sperm concentration with a high degree of correlation (r(2) = .936) was found between the 2 techniques. These findings reaffirm the need to critically assess new technologies for accuracy, repeatability, and precision.
TL;DR: It is suggested that age-related reductions in intracellular cAMP may lead to the reduced testosterone production that characterizes aged Leydig cells, supported by recent studies from the lab demonstrating that long-term culture of old Leydigs cells with dbcAMP restored testosterone production to levels approximating those of young cells.
Abstract: Previous studies have shown that the capacity of Leydig cells from aged (21-24-month-old) Brown Norway rats to produce testosterone is reduced from young (4-month-old) levels, and that this is correlated with reductions in steroidogenic acute regulatory protein (StAR), peripheral benzodiazapine receptor (PBR), and the levels and activities of the steroidogenic enzymes. The age(s) at which particular changes in the steroidogenic pathway occur, and the relationship of particular changes to reduced testosterone production, are not known. We examined 3 critical components of the steroidogenic pathway, cyclic adenosine monophosphate (cAMP) production, StAR, and P450 side-chain cleavage enzyme (P450scc) in relationship to age-related decreases in testosterone production. Leydig cells isolated from Brown Norway rats of increasing ages (4, 9, 15, and 20 months) were evaluated. The ability of Leydig cells to produce testosterone was reduced at 9 months, although not significantly. Significant reductions in testosterone production were first seen in cells isolated from rats of 15 months of age, and further reductions occurred thereafter. Reduced testosterone was correlated with reductions in StAR, P450scc mRNA, and protein. Significant decline in luteinizing hormone-stimulated intracellular cAMP levels was seen by 9 months, before significant reductions in testosterone, StAR, and P450scc. Further declines in cAMP levels were seen at 15 and 20 months. These studies suggest that age-related reductions in intracellular cAMP may lead to the reduced testosterone production that characterizes aged Leydig cells. This suggestion is supported by recent studies from our lab demonstrating that long-term (3 days) culture of old Leydig cells with dbcAMP restored testosterone production to levels approximating those of young cells.
TL;DR: Differences between CASA results and hemacytometry are established, in large part, due to the Segre-Silberberg effect, which occurs during Poiseuille flow in high-gradient fluid flow in thin capillary-loaded slides.
Abstract: Accurate determination of sperm concentration in fluid suspension is a critical component in a semen analysis. Inaccurate estimations can lead to misinterpretation of the spermiogram and, in the case of livestock production, can lead to faulty insemination doses, which can adversely affect stud power, fertility, fecundity, and cost effectiveness of breeding programs. Capillary-loaded slides, like the hemacytometer, have been the standard for calibration of other concentration estimation modalities such as photometry, Coulter counter, flow cytometry, and computer-automated semen analysis (CASA). Single-use capillary-loaded slides, much smaller than the hemacytometer, are frequently used by many of the current CASA systems. As the use of CASA increases, more field reports are suggesting differences between CASA results and hemacytometry. In this article, we establish that these differences are, in large part, due to the Segre-Silberberg effect, which occurs during Poiseuille flow in high-gradient fluid flow in thin capillary-loaded slides. We develop the theory of this phenomenon and derive the scaling and significance of the effect. Finally, we graphically provide a means for predicting the necessary compensation factor when using capillary-loaded slides to determine sperm concentration.
TL;DR: Correlations between sperm viability as assessed by flow cytometry and NRR56 was slightly lower than observed for microscopic assessment of sperm motility, suggesting poorer sperm survival during postthaw incubation compared with Triladyl extender with whole and with clarified egg yolk.
Abstract: A newly developed flow cytometric method for determination of sperm concentration and viability was tested in an insemination trial with cryopreserved bull sperm to establish the relationship between sperm viability and nonreturn rates. Semen for experimental inseminations was produced from 157 young sires (114 Holstein and 43 Jersey), each contributing 4 experimental semen collections. Straws containing approximately 15 × 106 motile sperm before freezing were used in 118 680 experimental inseminations performed by 254 artificial insemination technicians in 6352 Danish herds. Statistical analysis based on 44 946 experimental first inseminations showed that the major part (95.4%) of variation in the 56-day nonreturn rate (NRR56) was residual. Only 0.38% of the total variation in NRR56 was due to bulls and differences between ejaculate within bull. However, bulls were preselected, and a relatively high insemination dose was used. Correlations between sperm viability as assessed by flow cytometry and NRR56 was slightly lower than observed for microscopic assessment of sperm motility. However, flow cytometry makes it possible to achieve an objective and precise determination of sperm viability. It was therefore possible to calculate the effect on NRR56 provided selection of semen is based on the flow cytometric method. Three freezing extenders were used in this experiment, but a significant difference in NRR56 was not observed. Flow cytometric results for 1 extender (Biociphos Plus) indicated poorer sperm survival during postthaw incubation compared with Triladyl extender with whole and with clarified egg yolk.