bluuebunny/crossref_metadata_2025 huggingface dataset:
Transport of Inositol into the Distal Cauda Epididymidis of the Rat
Authors: COOPER TREVOR G. , GUDERMANN THOMAS W. , YEUNG CHING‐HEI
Published: December 2015

The transport of [3H]myo‐inositol into the lumen of the rat distal cauda epididymidis was studied by luminal perfusion in vitro. Entry was time and tubule length‐dependent and saturable transport could be demonstrated with Vmax of 237 pmol/(30 min. cm) and K+ of 1 mM. Secretion of unlabeled inositol into the epididymal lumen was maintained for 5 hours at a concentration 6 times greater than that of the bathing solution. The turnover time of the epithelial pool of inositol was 4.5 hours, from which the intracellular concentration was estimated to be 26.6 mM. Transport was not reduced by metabolic inhibitors, and it was demonstrated that exchange diffusion across the basolateral membranes could drive the uptake in this region.
Effects of Prolactin on Luteinizing Hormone‐Stimulated Testosterone Secretion in Isolated Perfused Rat Testis
Authors: FUNG MAN‐CHEONG , WAH GEORGE C. , ODELL WILLIAM D.
Published: December 2015

The direct peripheral effect of prolactin on LH‐stimulated testosterone secretion was re‐evaluated using the intact, isolated, perfused rat testis. In paired experiments, one testis was infused with the hormones being studied; the other testis of the same rat was used as the control. A dose‐response curve of LH‐stimulated testosterone secretion was established first. A dose of 300 ng of LH, which was on the ascending portion of the dose‐response curve, was selected so that both stimulatory and inhibitory effects of prolactin could be observed. Prolactin doses ranging from 0 ng to 3000 ng were then tested to determine alterations in LH‐stimulated testosterone secretion. Prolactin inhibited LH‐stimulated testosterone secretion at low doses (< 300 ng), but augmented LH action at high doses (> 1000 ng). These results showed that prolactin and LH interact with each other in a biphasic dose‐dependent manner.
24‐Hour Pulsatile and Orcadian Patterns of Cortisol Secretion in Alcoholic Men
Authors: IRANMANESH ALI , VELDHUIS JOHANNES D. , JOHNSON MICHAEL L. , LIZARRALDE GERMAN
Published: December 2015

Pulsatile and circadian patterns of Cortisol secretion during acute (3 to 16 days) and chronic (29 to 39 days) abstinence were examined in alcoholic men with no clinical or laboratory evidence of hepatic dysfunction or nutritional deficiencies. Mean and integrated 24‐hour serum concentrations of Cortisol determined by sampling the blood every 20 minutes over a 24‐hour period were increased in six out of 10 alcoholic subjects during acute abstinence when compared with normal controls. Sustained abstinence in seven subjects with follow‐up studies caused significant decreases in the mean maximal Cortisol peak amplitude (13 ± 1.0 SEM acutely vs. 10.3 ± 0.52 μg/dl follow‐up; P = 0.01), mean 24‐hour serum Cortisol concentrations (10.9 μg/dl ± 1.2 vs. 8.5 μg/dl ± 0.26; P = 0.047), interpulse valley mean (9.3 μg/dl ± 0.88 vs. 6.5 μg/dl ± 0.34; P = 0.007), and valley nadir (7.9 μg/dl ± 0.69 vs. 5.4 μg/dl ± 0.30; P = 0.0036) concentrations. Cortisol pulse frequency was normal. Although circadian Cortisol rhythmicity was maintained in alcoholics, the timing of the circadian acrophase was delayed significantly (P = 0.006) during acute abstinence (1022 [clocktime] ± 34 min) as compared with normal controls (0743 [clocktime] ± 34 min), and the amplitude of circadian Cortisol rhythms exceeded normal in five of 10 alcoholics. Analysis of data in one alcoholic subject by a new multiparameter deconvolution method demonstrated increases in secretory burst amplitude (0.64 μg/dl ± 0.08 SD), mass of Cortisol released per burst (9.8 μg/dl ± 1.2 SD), and daily endogenous Cortisol production rate (22 mg ± 2.4 SD) during acute abstinence. These values were statistically different when compared with seven normal controls and the subjects' values during sustained abstinence (P < 0.02). In conclusion, the results of the present study suggest increased daily production of Cortisol as a possible mechanism underlying the elevated serum Cortisol concentrations in chronic alcoholics during acute abstinence. This abnormality is shown to be reversible with sustained abstinence from alcohol.
Molecular Basis of Signalling in the Spermatozoon*
Authors: GARBERS DAVID L.
Published: December 2015

The spermatozoon contains cell surface receptors for various egg‐associated molecules, one of which has now been identified as the enzyme guanylate cyclase. A single membrane‐spanning region divides the enzyme such that about one‐half is extracellular and one‐half is intracellular. A new paradigm for signal/transduction is established by these observations, in that a hormone or effector molecule binding to an extracellular site activates the catalytic domain of the same protein, resulting in the increased formation of a low molecular weight second messenger. Research on signalling mechanisms in the spermatozoon clearly apply to a diversity of cellular processes as well as to fertilization.
Pituitary Response to LHRH During Chronic Renal Insufficiency in the Male Rat
Authors: NAZIAN STANLEY J. , DIETZ JOHN R. , ROWLAND MELISA D.
Published: December 2015

Chronic renal insufficiency was induced in young adult male rats by a 2‐step 5/6 nephrectomy. Four weeks after surgery the rats were anesthetized with ketamine and injected with saline or 1, 5, 10, 50, or 100 ng of LHRH/100 g BW and blood samples were collected at various times and assayed for LH. A second group of rats was injected with saline or 5, 10, 50, 100, or 200 ng of LHRH/100 g BW and serum samples assayed for FSH. A third experiment tested for the presence of a self‐priming effect of LHRH on LH secretion. Rats were primed with three iv injections of 10 ng of LHRH/100 g BW or saline at 30‐min intervals and challenged with 50 ng of LHRH/100 g BW 30 min after the last priming injection. Ten min after the challenge injection, the LH increment was determined. The data indicate that male rats suffering from chronic renal insufficiency have an impaired LH response to low doses of LHRH, esentially normal FSH responses to most doses of LHRH examined, and lack a self‐priming effect of LHRH on LH secretion. These results suggest that part of the reproductive abnormalities found in chronic renal failure may be due to alterations in pituitary LH responses.
Nature of Alpha Subunit Secretion in Men: Circadian Rhythms, Pulsatile Release and Secretory Profiles
Authors: VELDHUIS JOHANNES D. , WINTERS STEPHEN J.
Published: December 2015

Alpha subunit complements LH as a marker of the activity of the hypothalamic GnRH pulse generator. To characterize episodes of ä subunit release and to determine if a circadian pattern of α subunit secretion is present in man, spontaneous α subunit pulsatility was analyzed in six healthy young men by blood sampling every 5 min for 24 h. The resulting α subunit concentration time series were analyzed by two statistically based independent peak detection methods, and subjected to Fourier transformation to assess underlying circadian rhythms. Cross‐correlation analyses and multiple parameter deconvolution were used to estimate the concordance of spontaneous and exogenous GnRH‐stimulated LH and α subunit secretion. These analyses revealed that two independent discrete peak detection algorithms yielded similar estimates of spontaneous α subunit pulse frequency, namely, 21 ± 1.1 (Cluster) and 21 ± 1.5 (Detect) α subunit peaks/24 h. Sampling intensity markedly influenced the estimate of endogenous α subunit pulse frequency, inasmuch as estimates from 5‐min sampling were significantly greater than those of 10‐min or 20‐min sampling. Fourier transformation unmasked a significant circadian α subunit rhythm in all six men, with maximal concentrations at 0836 h and an average amplitude of 28% of the 24‐hr mean hormone concentration. Cross‐correlation analysis of spontaneous glycoprotein release revealed that serum LH and α subunit concentrations were highly cross‐correlated when considered simultaneously, but not at various lags. Finally, deconvolution analysis of exogenous GnRH‐stimulated glycoprotein release disclosed distinct halftimes of α subunit and LH clearance with virtually simultaneous underlying secretory bursts. These data indicate that human α subunit is secreted in both a circadian and a discrete pulsatile fashion at a pulse frequency that is significantly underestimated at conventional sampling rates. The approximately hourly α subunit interpulse interval (68 ± 4.6 min) is similar to that reported earlier for LH in peripheral blood and for testosterone in gonadal vein blood in healthy men. Moreover, cross‐correlation analysis of endogenous GnRH‐driven α subunit and deconvolution analysis of exogenous GnRH‐stimulated α subunit and LH secretion suggest that these glycoproteins are secreted virtually simultaneously, but have significantly different endogenous clearance properties. The remarkably similar in vivo pulse frequencies for α subunit, LH, and testosterone in man suggest that the release of these three hormones is coordinately regulated.
The Effect of Tumor Necrosis Factor on Human Sperm Motility In Vitro
Authors: EISERMANN JUERGEN , REGISTER KAREN B. , STRICKLER RONALD C. , COLLINS JOHN LESLIE
Published: December 2015

Tumor necrosis factor (TNFα) is present in elevated levels in peritoneal fluid from infertile women with endometriosis. The effect of TNFα on human sperm motility in vitro was evaluated utilizing peritoneal fluid from infertile women with minimal endometriosis containing 0, 100, 400, or 800 U of TNFα/ml as well as similar concentrations of recombinant human TNFα. No reduction in progressive and total motility was found at recombinant TNFα concentrations of 100 U ml. However, 500 and 1000 U of recombinant TNFα/ml caused a significant reduction in progressive and total sperm motility after 4 and 21 hours of incubation when compared with controls. Similarly, peritoneal fluid containing 100 U of TNFα/ml did not significantly reduce progressive and total sperm motility after either 4 or 21 hours of incubation; but peritoneal fluid containing 400 U of TNFα/ml reduced progressive sperm motility after 4 and 21 hours and total sperm motility after 21 hours of incubation. Peritoneal fluid with a TNFα concentration of 800 U/ml caused a significant reduction in both progressive and total sperm motility after 4 and 21 hours when compared with controls of TNFα‐negative peritoneal fluid. The addition of polyclonal rabbit anti‐TNFα antibody or 30‐min heat inactivation at 56 C of TNFα‐positive peritoneal fluid reversed the inhibitory effect on sperm motility. The ability of TNFα to cause a significant reduction of sperm motility in vitro suggests that this may be a mechanism for the infertility observed in women with minimal endometriosis.
Cyclophosphamide in the Male Rat: New Pattern of Anomalies in the Third Generation
Authors: DULIOUST EMMANUEL J.B. , NAWAR NABIL Y. , YACOUB SAMEH G. , EBEL ALIX B. , KEMPF ELIANE H. , AUROUX MAURICE R.
Published: December 2015

Several abnormalities, such as postnatal deaths and behavioral impairments, have been previously reported in the progeny of male rats exposed to the cytostatic drug cyclophosphamide 60 days prior to mating. The anomalies were transmitted to the second generation (F2). The present results concern the third generation. Two experimental groups have been studied: a hybrid group, resulting from crosses between control subjects and either experimental F2 males or females, and a nonhybrid group, obtained by mating experimental F2 subjects together. Significant abnormalities were found in all experimental groups, whether the F2 subjects were male or female. F2 females had smaller litters whether they were mated with control or experimental males. Body weight was significantly increased in both hybrid and nonhybrid males. Increased postnatal mortality and learning deficit were also observed in the hybrid group. Such complex phenotypic changes confirm that frequent mutations probably have been inherited from the treated males but also suggest that genetic rearrangements have occurred from one generation to the next.
Quality Control During the Conventional Analysis of Semen, An Essential Exercise
Authors: DUNPHY BRUCE C. , KAY RICHARD , BARRATT CHRISTOPHER L.R. , COOKE IAN D.
Published: December 2015

Intra‐ and inter‐technician variability in assessing sperm motility by the methods recently advocated by the World Health Organization (WHO) were studied. The intra‐ and inter‐technician variability in estimating sperm concentration and the intra‐technician variability in assessing sperm morphology were also examined. Intra‐technician variability in assessing sperm motility appeared to be related to the natural ability and/or training of the observer. Although in general the intra‐technician variability was low, there were marked and clinically significant differences between observers when assessing the same semen sample. There was no significant difference between observers in the assessment of sperm concentration, and intra‐technician variability was low. When assessing sperm morphology, the intra‐technician variability was potentially large (above a level of 20% morphologically ideal spermatozoa). Technicians should be recruited who have natural ability as observers. Quality control appears to be an essential exercise for any center that plans to relate semen parameters to fertility outcome.
Seasonal Variation and Age‐Related Changes in Human Semen Parameters
Authors: Chen Zuying , Toth Thomas , Godfrey‐Bailey Linda , Mercedat Nelta , Schiff Isaac , Hauser Russ
Published: December 2015

ABSTRACT: Although semen quality has been discussed extensively with regard to age and season in the andrology literature, the results vary and firm conclusions are still outstanding. To investigate seasonal and age‐related variations in human semen parameters, we analyzed data that were collected from an andrology clinic population. We performed a retrospective review of 551 semen analysis records collected from 1989 to 2000 from the Vincent Memorial Andrology Laboratory at Massachusetts General Hospital. Semen volume, sperm concentration, total sperm count, motility, total motile sperm, and morphology significantly decreased as age increased. In addition, as age increased, the percentage of sperm with tail defects increased. Sperm concentration was significantly higher in winter (mean 157.9 million/mL) than in fall (mean 119.1 million/mL) (P < .05). The mean percentage of sperm with normal morphology was significantly higher in winter (9.2%) than in summer and spring (7.0% and 7.5%, respectively; P < .05). The mean percentage of sperm with head defects was significantly higher in fall and summer (74.0% and 72.3%, respectively) than in winter (68.6%; P < .05). Seasonal variations were found in sperm concentration and morphology, with higher sperm concentrations in winter than in fall, and a greater percentage of sperm with normal morphology in winter than in spring and summer. Sperm concentration was lowest in the fall, whereas the percentage of sperm with normal morphology was lowest in summer. Semen volume, sperm concentration, total sperm count, motility, total motile sperm, and morphology decreased as age increased.
Spontaneous DNA Fragmentation in Swim‐Up Selected Human Spermatozoa During Long Term Incubation
Authors: Muratori Monica , Maggi Mario , Spinelli Silvia , Filimberti Erminio , Forti Gianni , Baldi Elisabetta
Published: December 2015

ABSTRACT: The origin and the meaning of DNA fragmentation in ejaculated human spermatozoa are not yet clear, although some hypotheses have been proposed. In the present study, we used investigated sperm DNA fragmentation by terminal deoxynucleotidyl transferase‐mediated fluorescein‐dUTP nick end labeling (TUNEL)‐coupled flow cytometry to investigate DNA fragmentation in spermatozoa that were selected by the swim‐up procedure and incubated long‐term. In addition, using flow cytometry we detected annexin V binding assay and propidium iodide staining, and we also studied membrane phosphatidylserine translocation and the loss of membrane integrity in the same sperm populations that we used in the TUNEL investigation. We found that in vitro sperm DNA fragmentation 1) occurs after ejaculation under experimental conditions without the involvement of any external factor, 2) is not affected by treatment with the nuclease inhibitor aurintricarboxylic acid, 3) is increased by treatment with the glutathione peroxidase inhibitor mercaptosuccinate, 4) correlates with basal values (ie, just after swim‐up selection) of DNA fragmentation in teratozoospermic but not in normospermic semen samples, 5) develops in a sharply associated manner with the in vitro occurrence of sperm necrosis, and 6) is predicted by the basal value of annexin V binding in viable spermatozoa. These findings suggest an involvement of endogenously produced reactive oxygen species as the possible cause of in vitro sperm DNA fragmentation.
New Semen Quality Scores Developed by Principal Component Analysis of Semen Characteristics
Authors: Agarwal Ashok , Sharma Rakesh K. , Nelson David R.
Published: December 2015

ABSTRACT:The purpose of this study was to determine whether semen characteristics can be reduced to 2 semen quality (SQ) scores and whether these new scores can help the clinician in assessing the reproductive outcome. A cross‐sectional sample of 250 patients seeking infertility treatment were analyzed for semen characteristics. In addition, 177 male‐factor patients (prostatitis with infection, n = 40; varicocele, n = 77; varicocele with infections, n = 11; and vasectomy reversal, n = 43) were also assessed. Sperm motion kinetics were measured by computer‐assisted semen analysis (CASA) (concentration, percent motility, curvilinear velocity [VCL], straight‐line velocity [VSL], average path velocity [VAP], linearity [LIN], and amplitude of lateral head displacement [ALH]). Sperm morphology was assessed by both World Health Organization (WHO) guidelines and Tygerberg strict criteria. The principal component analysis model was used to construct an SQ score and a relative semen quality (RQ) score. A separate set of 25 normal donors was included as controls to determine normal ranges of the semen scores. Among the patient samples, SQ and RQ scores (median and 25% and 75% interquartile values) were 89.9, 25.1, and 130.4 and 106.1, 45.2, and 165.9, respectively. The SQ score for the varicocele and varicocele with infection groups was comparable (78.6 ± 17.4 and 84.8 ± 20.6) but significantly different from the control (100 ± 10, P < .001 and .03). Vasectomy reversal patients had an SQ score of 78.2 plus or minus 16.8 that was significantly lower than controls (P < .001). The correlation among semen characteristics allows for the efficient combining of semen measures. The composite scores can summarize overall SQ and quantity. Both SQ and RQ scores provide meaningful information on the quality of semen specimens for the clinician.
Regulation of Sulfated Glycoprotein‐1 and Cathepsin D Expression in Adult Rat Epididymis
Authors: Hermo Louis , Andonian Sero
Published: December 2015

ABSTRACT:Endocytosis, whereby proteins are internalized from the epididymal lumen to be eventually degraded in lysosomes, is one of the major functions of the epididymal epithelial cells in maintaining a proper luminal milieu conducive for sperm maturation. In the present study, using light microscope immunocytochemical methods, we examined the regulation of 2 lysosomal enzymes, sulfated glycoprotein‐1 (SGP‐1) and cathepsin D, in adult rat epididymides fixed in Bouin fixative and embedded in paraffin. After orchidectomy (O) with or without testosterone (T) supplementation, efferent duct ligation (EDL), or hypophysectomy (H), lysosomes of principal cells were intensely reactive with the anti‐SGP‐1 antibody, as were narrow, clear, and basal cells, with staining patterns similar to that of control animals. These experimental procedures also had no effect on cathepsin D expression in all cell types, except for clear cells of the corpus and cauda epididymidis, which after orchiedectomy and hypophysectomy, became intensely reactive, unlike their completely unreactive state in control animals. In O+T animals, as well as in EDL animals, clear cells remained unreactive. These data taken together suggest that expression of SGP‐1 is not under the control of testicular or pituitary factors, as is also the case for cathepsin D expression by principal, narrow, and basal cells. However, specific inhibition of cathepsin D expression by testosterone or one of its metabolites appears to occur in clear cells of the corpus and cauda epididymidis. Furthermore, in addition to small, typical lysosomes, principal cells also revealed large supranuclear and infranuclear spherical structures that were immunoreactive with both anti‐SGP‐1 and anti‐cathepsin D antibodies, suggesting their lysosomal nature. With electron microscopy, these structures appeared electron‐lucent and contained membranous profiles embedded in an electron‐dense, granular background. Such images suggest that the various experimental procedures adversely affect the expression of several other lysosomal enzymes in principal cells, leading to a lysosomal phenotype similar to that observed in various lysosomal storage diseases.
Specific Order in the Appearance of Protein Tyrosine Phosphorylation Patterns Is Functionally Coordinated With Dog Sperm Hyperactivation and Capacitation
Authors: Petrunkina Anna M. , Simon Katrin , Günzel‐Apel Anne‐Rose , Töpfer‐Petersen Edda
Published: December 2015

ABSTRACT: The aims of the present study were to characterize a slow capacitation system that records initial changes in the sperm membrane state, and, using a canine model, to order the specific protein tyrosine phosphorylation signaling in the sequence of capacitational events and to associate them with hyperactivated motility. Dog sperm washed through Percoll were incubated in complete bicarbonate Tyrode medium for 6 hours in 5% CO2. Capacitation was evaluated using chlortetracycline staining. Tyrosine phosphorylation patterns were assessed by immunocytochemistry. Parallel to this, a computer‐assisted motility analysis was performed. Significant changes in the percentage of capacitated and acrosome‐reacted cells were first observed after 90 minutes, increasing in a linear manner during further incubation (P < .05). Changes in the percentage of capacitated cells were accompanied by motility changes. During incubation, a strictly sequential phosphorylation of sperm tail (midpiece, principal piece, and end piece) and head proteins was observed. According to an analysis of kinetics, phosphorylation of head proteins occurred after the tail became completely phosphorylated. Changes in head phosphorylation progressed at the same rates as capacitation and acrosome reaction. Sperm motility, curvilinear velocity, average path velocity, straight line velocity, and lateral head displacement were correlated positively or negatively with phosphorylation of midpiece or end piece proteins, respectively. The bicarbonate‐stimulated increases in cyclic adenosine monophosphate levels and changes in protein phosphatase activity may be involved in the signaling system that controls membrane changes and motility in dog sperm. Phosphorylation kinetics of sperm proteins are potentially useful for diagnostic purposes to characterize the response of individual males to fertilizing conditions.
Male Genital Tract Inflammation: The Role of Selected Interleukins in Regulation of Pro‐Oxidant and Antioxidant Enzymatic Substances in Seminal Plasma
Authors: Sanocka Dorota , Jędrzejczak Piotr , Szumała‐Kaękol Anna , Frączek Monika , Kurpisz Maciej
Published: December 2015

ABSTRACT:Human semen contains spermatozoa as well as populations of round nonspermatozoal cells primarily consisting of leukocytes. Activation of white blood cells present in the seminal plasma during genital tract inflammation and cellular reactions against microbial agents may provoke a release of a variety of products such as cytokines and reactive oxygen species. The aim of this study was to evaluate whether a panel of selected cytokines (interleukin [IL]‐1β, IL‐6, IL‐8, and tumor necrosis factor‐α [TNFα]) detectable in seminal plasma during male genital tract inflammation could be considered as mediators between altered semen parameters and changed levels of pro‐oxidant and antioxidant substances. Studies using chemiluminometric, spectrophotometric, and enzyme‐linked immunosorbent assay methods indicate that proinflammatory cytokines such as IL‐1β, IL‐6, IL‐8, and TNFα may modulate pro‐oxidant and antioxidant activities in the male genital tract. The data also suggest that the function of pro‐oxidant and antioxidant systems in semen may directly influence basic semen parameters. The elevated numbers of leukocytes present in semen during male genital tract inflammation without an associated contribution of cytokines and semen antioxidant capacity appear to be of little prognostic value in evaluating male fertilization potential.
Long‐Term Culture and Transplantation of Murine Testicular Germ Cells
Authors: Jeong Dongkee , McLean Derek J. , Griswold Michael D.
Published: December 2015

ABSTRACT: The objectives of this study were to develop an in vitro culture system to optimize germ cell proliferation and to measure the potential of the cultured germ cells to produce mature spermatozoa after transplantation into a recipient. Donor germ cells isolated from ROSA26 male mice were cultured with a STO feeder cell layer in Dulbecco's minimal essential medium (DMEM) supplemented with fetal bovine serum (FBS), stem cell factor, leukemia inhibitory factor, basic fibroblast growth factor, insulin‐like growth factor 1, interleukin‐11, l‐glutamine, sodium pyruvate, 2‐mercaptoethanol, murine oncostatin M, and platelet‐derived growth factor. Donor germ cells formed colonies in the primary cultures after 8–21 days. These cultured colonies were maintained for 4 weeks or longer without subculture and proliferated for up to 8 passages over a period of 3 months. These colonies had alkaline phosphatase activity and incorporated 5‐bromo‐2′‐deoxyuridine. These colonies were positive partially when screened with antibody for germ cell nuclear antigen and c‐kit. Germ cells cultured with this supplemented medium showed enhanced colonization vs controls cultured with DMEM and FBS. Cultured germ cells from Rosa26 donors were transplanted into testes and were identified by X‐gal staining and histological screening. The cells cultured in the supplemented medium colonized the tubules and initiated spermatogenesis in the recipient mice. This is an improved method for culturing germ cells and may be useful in gene therapy and the production of transgenic animals.
Intracytoplasmic Spermatid Injection Can Result in the Delivery of Normal Offspring
Authors: Mansour Ragaa T. , Fahmy Ibrahim M. , Taha Ahmed K. , Tawab Nevine A. , Serour Gamal I. , Aboulghar Mohamed A.
Published: December 2015

ABSTRACT: Almost one‐third of all patients with nonobstructive azoospermia undergoing testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI) have cancelled cycles due to failure to find spermatozoa. For these patients, every attempt should be made to rescue the cycles by searching for spermatids. In this retrospective study, we report our experience in using elongating (stage Sb2) and elongated (stage Sc and Sd1) spermatids for ICSI. The study included 488 consecutive ICSI and TESE cycles performed for 452 patients with nonobstructive azoospermia. In 179 (36.7%) cycles, neither spermatozoa nor mature spermatids (stage Sd2) suitable for injection were found. After an extensive search only Sb2, Sc, and Sd1 spermatids were found in 22 of these 179 cycles (12.3%). These spermatids were used for injection of retrieved oocytes. The fertilization rate was 33.2%, and 19 patients (86.4%) reached the embryo transfer stage. In 6 cycles a chemical pregnancy occurred, and 3 clinical pregnancies were established, resulting in the delivery of 3 healthy boys with normal karyotypes. When normal living spermatozoa or mature spermatids (stage Sd2) cannot be found during TESE, late spermatids (stage Sb2, Sc, and Sd1) can be used successfully and result in the delivery of healthy offspring.
Identification of Differentially Expressed Genes in Mouse Spermatogenesis
Authors: Pang Alan L. Y. , Taylor H. Claire , Johnson Warren , Alexander Stefanie , Chen Yali , Su Yan A. , Li Xiaoquan , Ravindranath Neelakanta , Dym Martin , Rennert Owen M. , Chan Wai‐Yee
Published: December 2015

ABSTRACT: Complementary DNA microarray and quantitative polymerase chain reaction were used as tools for discovering genes that are differentially expressed in the mouse under normal physiological conditions at distinctive stages of male germ cell development, that is, type A spermatogonia, pachytene spermatocytes, and round spermatids. By using this strategy, we identified a set of genes exhibiting differential expression patterns in spermatogenesis, suggesting that specific functions of the encoded products occurred during the developmental process. Among them were several genes previously not known to be active in testis, which signified undiscovered functional roles of these genes during spermatogenesis. Many of the genes identified were not previously characterized. This study highlights new targets for manipulation to unravel the molecular mechanism of spermatogenesis.
Seminal Antioxidant Capacity in Pre‐ and Postoperative Varicocele
Authors: Mancini Antonio , Meucci Elisabetta , Milardi Domenico , Giacchi Elena , Bianchi Antonio , Pantano Angelo Lauria , Mordente Alvaro , Martorana Guiseppe Ettore , de Marinis Laura
Published: December 2015

ABSTRACT:In order to explore the impact of surgical treatment on antioxidant defense system in varicocele (VAR), we evaluated seminal total antioxidant capacity (TAC) in 25 patients affected by VAR, in 14 patients studied 10–24 months after varicocelectomy (post‐VAR) and separated into normo‐ and oligospermic groups, and in 24 non‐VAR control patients with seminal parameters matched to patients with VAR in the oligo‐ and normospermic groups (7 subjects with idiopathic oligospermia and 17 normal fertile subjects). TAC was measured in seminal plasma with the system H2O2‐metamyoglobin as a source of radicals, which interact with a chromogen 2,2′,‐azinobis (3‐ethylbenzothiazoline‐6‐sulphonate) (ABTS), generating a radical cation spectroscopically detectable. The presence of antioxidants induces a lag time in the production of ABTS cation proportional to the concentration of antioxidant compounds. When whole groups of patients were analyzed, lag values were significantly higher in VAR vs non‐VAR controls (mean ± SEM, 106.6 ± 8.8 seconds vs 78.7 ± 8.8 seconds) but were not modified by surgery (mean ± SEM, 105.8 ± 8.6 seconds). In groups separated according to seminal parameters, oligospermic VAR presented significantly higher lag values than oligospermic controls. Finally, when exploring a possible association of TAC with seminal parameters, we found a significant correlation between lag and sperm motility only in patients with VAR who were in the normospermic group (r = 0.65, P < .01). This correlation was not yet manifest post‐VAR. In conclusion, surgical treatment does not seem to modify absolute values of TAC but influences its fine regulation and relationships with sperm motility.
Postnatal Development and Regulation of β‐Hexosaminidase in Epithelial Cells of the Rat Epididymis
Authors: Hermo Louis , Adamali Huzaifa I. , Trasler Jacquetta M.
Published: December 2015

ABSTRACT: β‐Hexosaminidase (Hex) catalyzes the hydrolysis of terminal sugar residues from a number of substrates such as GM2 gangliosides, glycoproteins, glycolipids, and glycosaminoglycans. As an enzyme present in lysosomes of epithelial cells of the adult rat epididymis, it serves to degrade substances endocytosed from the epididymal lumen. In this way, it modifies and creates a luminal environment where sperm can undergo their maturational modifications. In this study, the postnatal developmental pattern of expression of Hex was examined in animals from days 7–56. In addition, the role of testicular factors on Hex expression in the different cell types and regions of the epididymis of adult rats was examined in orchidectomized and efferent duct‐ligated rats. Both parameters were examined on Bouin‐fixed epididymides in conjunction with light microscope immunocytochemistry. At postnatal day 7, the epithelium of the entire epididymis was unreactive for anti‐Hex antibody. By day 21, narrow and clear cells of their respective regions became reactive, whereas basal cells became reactive only by day 29. Principal cells displayed only an occasional reactive lysosome at day 21, several by day 29, and numerous reactive lysosomes by day 39, comparable to the region‐specific distribution noted for 90‐day‐old animals, and at an age when high androgen levels are attained. Thus, postnatal onset of Hex expression varies according to the different cell types of the epididymis, suggesting different regulatory factors. This finding was confirmed from studies employing adult orchidectomized and efferent duct‐ligated adult rats. Indeed, in all experimental animals, Hex immunostaining in narrow, clear, and basal cells was intense and comparable to control animals. In contrast, there was a notable absence of lysosomal staining in principal cells at all time points after orchidectomy, which was restored, however, following testosterone replacement. No effect on Hex expression was observed in efferent duct‐ligated animals. Taken together, the data suggest that Hex expression in lysosomes of principal cells is regulated by testosterone or one of its metabolites. However, the expression of Hex being independent of testicular factors in narrow, clear, and basal cells of adult animals, but occurring at different time points during postnatal development, suggests that different regulatory factors are responsible for onset of Hex expression in these cell types during development.
Preservation of Spermatogenesis in Spinal Cord Injured Rats With Exogenous Testosterone. Relationship With Serum Testosterone Levels and Cellular Localization of cAMP Responsive Element Modulator
Authors: Huang Hosea F. S. , Wang Shulun , Molina Carlos A. , Ottenweller John E.
Published: December 2015

ABSTRACT: The current experiment examined the effects of exogenous testosterone (T) on spermatogenesis in rats with spinal cord injury (SCI) and their relationship with the cellular distribution of a cyclic AMP‐responsive element modulator (CREM) in testicular cells. Implantation of T‐filled Silastic capsules (TCs, 1–20 cm) resulted in dose‐dependent, biphasic changes in testicular T levels and spermatogenesis in SCI rats. However, dose responsiveness of spermatogenesis to exogenous T in SCI rats differed from that in sham control rats. Specifically, implantation of 2‐cm TCs enhanced the effects of SCI on spermatogenesis, resulting in total regression of the seminiferous epithelium. Although 3‐cm TCs maintained complete spermatogenesis in sham control rats, this regimen failed to support complete spermatogenesis in SCI rats. Although complete spermatogenesis was maintained in SCI rats given 5‐20‐cm TC implants, various abnormalities persisted. Cellular distribution of CREM remained normal in SCI rats but was altered in those SCI rats that received 3‐ or 5‐cm TC implants. Such effects were associated with reduced CREM proteins in testicular tissues. These results were consistent with altered cAMP signaling and its regulation in testicular cells after SCI and provided possible mechanistic explanations for the effects of SCI on spermatogenesis. Conclusion: SCI resulted in changes in the responsiveness of spermatogenesis to exogenous T. These effects were associated with altered cAMP/CREM signaling in testicular cells. Further studies, including a study of the relationship between serum T levels and normalcy of sperm functions and the role of neural‐endocrine interactions in mediating the effects of SCI on spermatogenesis and sperm function, are needed so that therapeutic regimens can be designed for clinical use.
Inflammatory Cytokine Concentrations Are Elevated in Seminal Plasma of Men With Spinal Cord Injuries
Authors: Basu Sarmistha , Aballa Teodoro C. , Ferrell Sean M. , Lynne Charles M. , Brackett Nancy L.
Published: December 2015

ABSTRACT: The semen of most men with spinal cord injury (SCI) contains sperm with abnormally low motility. Studies suggest that the seminal plasma is the source of this condition. The seminal plasma of men with SCI contains an abnormally high number of white blood cells (WBC), specifically, activated T cells. It is known that activated T cells secrete cytokines and elevated concentrations of cytokines can be harmful to sperm. It is not known if the seminal plasma of men with SCI contains elevated concentrations of cytokines. The purpose of this study was to determine if the seminal plasma of men with SCI contained elevated concentrations of cytokines. Using the method of enzyme‐linked immunosorbant assay (ELISA), ten cytokines were measured in the seminal plasma of men with SCI as well as healthy non‐SCI control subjects. The cytokines of interest were grouped according to Th1 effector functions: interleukin 1 beta, interleukin 2, interleukin 12, tumor necrosis factor alpha, tumor necrosis factor beta, interferon gamma (IL1β, IL2, IL12, TNFα, TNFβ, INFγ, respectively) and Th2 effector functions: interleukin 4, interleukin 6, interleukin 10, transforming growth factor beta 1 (IL4, IL6, IL10, TGFβ1, respectively). The results showed a predominance of Th1 versus Th2 cytokine production in the seminal plasma of men with SCI compared with that of control subjects. This finding suggests an immunologic basis for infertility as a possible avenue of investigation in these men.
Immunolocalization and Regulation of Cystic Fibrosis Transmembrane Conductance Regulator in the Adult Rat Epididymis
Authors: Ruz Ricardo , Andonian Sero , Hermo Louis
Published: December 2015

ABSTRACT: Cystic fibrosis is the most common serious autosomal recessive condition in whites, and more than 95% of men with cystic fibrosis are infertile. The cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic adenosine monophosphate (cAMP)‐regulated chloride channel, has been localized in the efferent ducts; however, to our knowledge, its expression and regulation in the epididymis by testicular factors have not been examined. In the present study, these parameters were examined immunocytochemically by the light microscope with an anti‐CFTR antibody in Bouin‐fixed, paraffin‐embedded control adult rat epididymides and both orchidectomized adult rats with or without testosterone supplementation and efferent duct—ligated rats sacrificed at different time points. In control animals, a thick dense band of immunoperoxidase reaction product was visualized over the apical plasma membrane of the principal cells but not their microvilli. The apical band was prominent only in the corpus and cauda regions. While there was no CFTR expression in basal cells, clear cells of the corpus and cauda regions showed a weak‐to‐moderate band of apical plasma membrane staining. An examination of orchidectomized, orchidectomized and testosterone, and efferent duct—ligated rats revealed that CFTR was no longer expressed as an intense band on the apical plasma membrane of the principal cells of the corpus and cauda regions. However, under these conditions, an intense apical/supranuclear reaction was noted in the form of small vesicular structures. Clear cells were unaffected by the different experimental treatments. Together, these data indicate that CFTR is expressed in a cell‐ and region‐specific manner and that, while its synthesis in principal cells is not under the control of testicular factors, targeting to the apical plasma membrane is regulated by a testicular luminal factor.
Involvement of A1 Adenosine Receptors in the Acquisition of Fertilizing Capacity
Authors: Minelli Alba , Liguori Lavinia , Bellazza Ilaria , Mannucci Roberta , Johansson Björn , Fredholm Bertil B.
Published: December 2015

ABSTRACT: Ejaculated mammalian spermatozoa acquire competence to fertilize oocytes by a two‐step process: capacitation followed by acrosome reaction. The biochemical and biophysical modifications occurring in vivo in the female reproductive tract can be reproduced in vitro, and previous studies have suggested a capacitative role for adenosine A1 receptor (A1R). Mice with a targeted disruption of the Adora 1 gene (A1R–/– mice) provide a useful model for better understanding the role of the A1R in fertility. Murine spermatozoa express A1R in the head, neck, midpiece region, and tail. The number of capacitated spermatozoa incubated in human tubal fluid was significantly reduced in A1R–/– compared with A1R+/+ and A1R+/– spermatozoa. The difference between A1 R+/+ and A1R−/− mouse spermatozoa was mainly in the time necessary to reach the maximum percentage of capacitation. A1R+/+ murine sperm obtained the full state of capacitation within 90 minutes whereas A1R−/− sperm required 240 minutes. Caffeine, a known antagonist of A1 and A2A adenosine receptors, lowered the number of capacitated sperm and affected the time of capacitation in a dose‐dependent manner, mimicking the effects of the lack of A1 receptors. Although number, motility, and viability of A1R−/− murine sperm was not significantly different from A1R+/+ mouse spermatozoa, a significant reduction of the number of pups produced by A1R−/− male mice suggests that A1 receptors must be fully operative to accomplish the optimal degree of capacitation and thereby fertilization.
The Relationship Between Environmental Exposure to Phthalates and Computer‐Aided Sperm Analysis Motion Parameters
Authors: Duty Susan M. , Calafat Antonia M. , Silva Manori J. , Brock John W. , Ryan Louise , Chen Zuying , Overstreet James , Hauser Russ
Published: December 2015

ABSTRACT: The general population is exposed to phthalates through consumer products, diet, and medical devices. The present study explored whether phthalates, reproductive toxins in laboratory animals, were associated with altered sperm movement characteristics in men. Two‐hundred twenty subjects provided a semen sample for computer‐aided sperm analysis (CASA) and a urine sample for measurement of phthalate monoesters, monoethyl (MEP), monobenzyl (MBzP), mono‐n‐butyl (MBP), mono‐2‐ethylhexyl (MEHP), and monomethyl (MMP). Three CASA parameters, straight‐line velocity (VSL), curvilinear velocity (VCL), and linearity (LIN), were used as measures of sperm progression, sperm vigor, and swimming pattern, respectively. There were suggestive dose‐response relationships (shown as the predicted change in mean sperm motion parameter for the second and third tertiles compared with the first tertile; P value for trend) for MBzP with VSL (−2.36 μm/s, −2.81 μm/s; P = .09) and VCL (−1.67 μm/s, −2.45 μm/s; P = .4). There were suggestive negative associations between MBP and VSL (−3.07 μm/s, −2.87 μm/s; P = .08) and VCL (−3.25 μm/s, −3.46 μm/s; P = .2), and between MEHP with VSL (−1.09 μm/s, −2.73 μm/s; P = .1) and VCL (−0.29 μm/s, −2.93 μm/s; P = .3). In contrast to the other phthalates, MEP was positively associated with VSL and VCL but negatively associated with LIN. No consistent relationship was found for MMP and any sperm motion parameter. Although we did not find statistically significant associations, trends between CASA parameters, sperm velocity, and forward progression, and increased urinary levels of MBP, MBzP, and MEHP warrant further follow‐up.
Survival and Fertility of Boar Spermatozoa After Freeze‐Thawing in Extender Supplemented With Butylated Hydroxytoluene
Authors: Roca Jordi , Gil Maria A. , Hernandez Marta , Parrilla Inma , Vazquez Juan M. , Martinez Emilio A.
Published: December 2015

ABSTRACT:This study evaluated the protective effect of butylated hydroxytoluene (BHT), a lipid‐soluble antioxidant, against cryopreservation injuries to boar spermatozoa. In experiment 1, the lowest BHT concentrations able to reduce lipid peroxidation in boar spermatozoa were determined. Nine BHT concentrations (ranging from 0.025 to 3.2 mM) were evaluated, and the lowest (P < .05) production of malondialdehyde (MDA), as an indicator of lipid peroxidation, was obtained when BHT ranged from 0.2 to 1.6 mM. In experiment 2, sperm survivability was evaluated when BHT was added to a postthaw freezing extender by measuring the degree of sperm lipid peroxidation (using MDA production) and by measuring parameter such as motility, plasma membrane and acrosome integrity, and cell apoptosis. The ability of thawed spermatozoa to fertilize in vitro—matured oocytes and of embryos to develop to the blastocyst stage in vitro was also assessed. Pooled sperm‐rich fractions collected from 3 mature Pietrain boars were frozen in 0.5‐mL straws after dilution with lactose‐egg yolk‐glycerol‐Orvus ES Paste extender supplemented with 0, 0.2, 0.4, 0.8, and 1.6 mM BHT. Postthaw sperm survival, evaluated 30 and 150 minutes after thawing, was higher in BHT‐treated spermatozoa, being significant (P < .05) when the freezing extender was supplemented with 0.2, 0.4, and 0.8 mM BHT. The addition of BHT to the freezing extender resulted in a significant (P < .05) decrease in the MDA concentration in thawed spermatozoa, irrespective of the level of BHT used. BHT had no effect on oocyte cleavage rates, but the development to blastocyst was improved for embryos derived from spermatozoa frozen in extender supplemented with 0.4 mM BHT (16% vs 29% of blastocysts per total oocytes; P < .05). In conclusion, under the conditions tested in the present study, the addition of BHT to the freezing extender improved the overall efficiency of thawed boar spermatozoa.
Anatomy of the Human Penis: The Relationship of the Architecture Between Skeletal and Smooth Muscles
Authors: Hsu Geng‐Long , Hsieh Cheng‐Hsing , Wen Hsien‐Sheng , Hsu Wen‐Long , Wu Chih‐Hsiung , Fong Tsorng‐Harn , Chen Shyh‐Chyan , Tseng Guo‐Fang
Published: December 2015

ABSTRACT: To investigate the anatomy of the ischiocavernosus muscle, bulbospongiosus muscle, and tunica albuginea and to determine their relationships to smooth muscle, which is a key element of penile sinusoids, we performed cadaveric dissection and histologic examinations of 35 adult human male cadavers. The tunica of the corpora cavernosa is a bilayered structure that can be divided into an inner circular layer and an outer longitudinal layer. The outer longitudinal layer is an incomplete coat that is absent between the 5‐o'clock and 7‐o'clock positions where 2 triangular ligamentous structures form. These structures, termed the ventral thickening, are a continuation of the anterior fibers of the left and right bulbospongiosus muscles. On the dorsal aspect, between the 1‐o'clock and 11‐o'clock positions, is a region called the dorsal thickening, a radiating aspect of the bilateral ischiocavernosus muscles. In the corpora cavernosa, skeletal muscle contains and supports smooth muscle, which is an essential element in the sinusoids. This relationship plays an important part in the blood vessels' ability to supply the blood to meet the requirements for erection, whereas in the corpus spongiosum, skeletal muscle partially entraps the smooth muscle to allow ejaculation when erect. In the glans penis, however, the distal ligament, a continuation of the outer longitudinal layer of the tunica, is arranged centrally and acts as a trunk of the glans penis. Without this strong ligament, the glans would be too weak to bear the buckling pressure generated during coitus. A significant difference exists in the thickness of the dorsal thickening, the ventral thickening, and the distal ligament between the potent and impotent groups (P ≤ .01). Together, the anatomic relationships between skeletal muscle and smooth muscle within the human penis explain many physiologic phenomena, such as erection, ejaculation, the intracavernous pressure surge during ejaculation, and the pull‐back force against the glans penis during anal constriction. This improvement in the modeling of the anatomic‐physiologic relationship between these structures has clinical implications for penile surgeries.