bluuebunny/crossref_metadata_2025 huggingface dataset:
Relationship of Human Sperm Acrosin and Proacrosin to Semen Parameters II. Correlations
Authors: GOODPASTURE JESSIE C. , ZAVOS PANAYIOTIS M. , ZANEVELD LOURENS J.D.
Published: December 2015

Previous results, simultaneously confirmed by others, suggest that a relationship exists between sperm acrosin levels and fertility in man. The assessment of sperm acrosin may therefore be a useful addition to the semen analysis, but only if the more standard semen parameter measurements cannot predict acrosin levels. Ejaculates from 102 men were analyzed and the relationship of the sperm acrosin system (acrosin, proacrosin, and acrosin inhibitor) to other seminal characteristics was determined. Very little correlation was observed between enzymatic and nonenzymatic parameters. Only five non‐enzymatic parameters, all of which were morphologic, showed correlation coefficients of ≥ 0.35 with acrosin and proacrosin, but none had an r‐value above 0.48. The total acrosin and proacrosin levels were highly correlated to each other (r = 0.93). It is concluded that sperm acrosin/proacrosin levels cannot be predicted by other seminal parameters; thus, measurement of sperm acrosin/proacrosin may be clinically useful as a diagnostic parameter.
Influence of Photoperiod, Ambient Temperature and Melatonin on Testosterone Synthesis and Release During Reproductive Maturation in Male Deer Mice
Authors: FAIL PATRICIA A. , WHITSETT J. MAL
Published: December 2015

Four experiments were designed to investigate the influence of photoperiod and other environmental factors on androgen production and reproductive maturation in deer mice. Male prairie deer mice (Peromyscus maniculatus), born in a light/dark cycle of 6L:18D, either remained in this short photoperiod or were switched to a long day regimen of 16L:8D at weaning. In a cross‐sectional experiment, the deer mice were killed between 3 and 8 weeks of age for measurement of serum testosterone concentration and reproductive organ weights. In a second experiment, blood was collected from each mouse at weekly intervals between 3 and 9 weeks of age. This repeated measures design was used to reduce the high variability in testosterone values observed in the first experiment. Reproductive organs were weighed at the termination of the experiment. Testosterone concentrations and reproductive organ weights were greater in males reared in the long photoperiod in both experiments. In a third experiment, the animals were housed under five different conditions to test the influence of high ambient temperature and melatonin as well as photoperiod. At 7 weeks of age, they received an injection of hCG or saline. More testosterone was released in deer mice reared in 16L:8D and 27 C than in those reared in short days (6L:18D) or those reared in high ambient temperature (35 C) or those treated with exogenous melatonin. One week later, animals were sacrificed. The single hCG treatment caused significant reversal of the suppression of accessory sex organ weights following melatonin, short days or 35 C temperature. In a fourth experiment, the additive influence of melatonin and 35 C temperature was tested. Animals treated with 35 C or both melatonin and 35 C had lower serum testosterone at 7 weeks of age, released less testosterone after hCG, and had smaller organ weights with or without hCG than long day controls. The influence of melatonin treatment and 35 C temperature appears to be additive for testicular weight and testosterone release after hCG. Thus, the attenuation of reproductive development that accompanied short days, melatonin treatment and high ambient temperature occurred via diminished testosterone secretion, which can be overcome at least in part by gonadotropin treatment.
Relationships Between Computerized Measurements of Motion of Frozen‐Thawed Bull Spermatozoa and Fertility
Authors: BUDWORTH PAUL R. , AMANN RUPERT P. , CHAPMAN PHILIP L.
Published: December 2015

A computerized system (CellSoft, CRYO Resources, Ltd.) was validated using video tapes of frozen‐thawed bull spermatozoa diluted in filtered (0.2 μm) egg yolk‐citrate extender (8 × 106 spermatozoa/ml) and analyzed at 30 frames/sec for the percentage of motile spermatozoa (≥ 20 μm/sec) and linear velocity of motile spermatozoa. Virtually all motile spermatozoa were detected and debris rarely were classified as immotile spermatozoa if the extender had been filtered. Variation about the mean for percent motile cells was similar when only 12 rather than 20 or 30 frames/field were analyzed. Use of 20 frames/‐ field was adequate to determine the percentage of motile bull spermatozoa. Five mixtures of live and killed spermatozoa were analyzed (four bulls) to evaluate accuracy. Percent motile spermatozoa was correlated (r = 0.97) with the ratio of live:killed spermatozoa. Mean linear velocity of motile spermatozoa was similar for each mixture (P > 0.05). To further evaluate accuracy, percent motile spermatozoa was determined by computer and by “track motility” (20 samples; 0 to 63% motile spermatozoa); values were correlated (r = 0.95). The system was precise (CV of 6% based on triplicate analyses of the same samples) and reasonably accurate for evaluating bull sperm motility if the extender had been filtered and 20 to 25 fields (≥ 200 spermatozoa) were evaluated. Correlations between measurements of sperm motion and fertility were studied using cryopreserved semen from two fertility trials. For the first, 75‐day nonreturn rate data for 20 samples of bull semen (10 bulls) were not significantly correlated with evaluations made by CellSoft. For the second fertility trial, the competitive fertility index (a measure of relative fertility) for nine bulls was correlated (r ≥ 0.68; P < 0.05) with percent motile spermatozoa, linear velocity and straight‐line velocity. Multiple correlations based on six characteristics evaluated by CellSoft, at 0 or 1.5 hours, and the competitive fertility index were ≥0.94. Based on the latter data, the system may facilitate prediction of the relative fertility of bull spermatozoa.
Effect of Germ Cells on Vectorial Secretion of Androgen Binding Protein and Transferrin by Immature Rat Sertoli Cells In Vitro
Authors: JANECKI ANDRZEJ , JAKUBOWIAK ANDRZEJ , STEINBERGER ANNA
Published: December 2015

The influence of germ cells (> 85% pachytene spermatocytes) on vectorial secretion of androgen binding protein (ABP) and transferrin by immature rat Sertoli cells was investigated using two‐compartment culture chambers. The ratio of ABP secreted into the outer and inner compartment in control cultures of Sertoli cells alone was 1.9, and was not influenced by either FSH or testosterone. Co‐culture of Sertoli cells in direct contact with germ cells in the presence of FSH decreased this ratio, the decrease being most pronounced (0.7) after 2 days of co‐culture. This effect was not observed if the germ cells were not in direct contact with Sertoli cell monolayers. The outer to inner compartment ratio of transferrin in Sertoli cell‐alone cultures was 1.6 and, in contrast to ABP, was not significantly influenced by the addition of germ cells, even in the presence of FSH. It is concluded that in immature rat Sertoli cells the polarity of ABP secretion, but not that of transferrin, may be regulated by pachytene spermatocytes (and possibly other germ cells), and that this process is FSH‐dependent.
Simultaneous Determination of Testosterone, Dihydrotestosterone and 5α‐Androstan‐3α,‐17β‐Diol by Isotopic Dilution Mass Spectrometry in Plasma and Prostatic Tissue of Patients Affected by Benign Prostatic Hyperplasia Effects of 3‐Month Treatment with a GnRH Analog
Authors: SALERNO ROBERTO , MONETI GLORIANA , FORTI GIANNI , MAGINI ANGELA , NATALI ALESSANDRO , SALTUTTI CARLO , Di CELLO VALERIO , COSTANTINI ALFIERO , SERIO MARIO
Published: December 2015

This article reports an isotope dilution mass spectrometric method for the simultaneous measurement of testosterone (T), dihydrotestosterone (DHT), and 5α‐androstan‐3α, 17β‐diol (3α‐diol) in human plasma and prostatic tissue. After addition of tri‐deuterated steroids as internal standards to prostatic tissue homogenates or plasma samples, extraction was performed with diethylether. The extracts were purified by two chromatographic steps (Sep‐Pak C 18 cartridge and Sephadex LH‐20) and injected into a gas chromatograph coupled with a mass spectrometer after derivatization with heptafluorobutyric acid. This method was highly specific and showed good precision, accuracy, reproducibility and sensitivity. T, DHT, and 3α‐diol were measured in human plasma and in prostatic tissue of seven patients with benign prostatic hyperplasia (BPH) treated for 3 months with a long acting GnRH analog before surgery. Plasma levels of T, DHT, and 3α‐diol were reduced by GnRH analog treatment to castrate levels. The tissue concentrations of the same steroids, compared with those obtained from 19 untreated patients, showed a mean reduction of about 90% for DHT and 3α‐diol, and about 75% for T. These results suggest that about 90% of prostatic DHT and 3α‐diol depend on testicular activity because they are dramatically reduced after pharmacologic castration.
Doppler Examination in Varicocele A Standard Method of Evaluation
Authors: ANNONI FEDERICO , COLPI GIOVANNI M. , MARINCOLA FRANCESCO M. , NEGRI LUCIANO
Published: December 2015

Doppler sonography is considered a reliable method for detecting reflux due to venous valvular incompetence, which occurs in varicocele. It is, however, a matter of debate whether the characteristics of this reflux can be correlated quantitatively in a clinical setting.

In this study, a two‐step method was utilized to identify reflux in basal conditions with the patient standing and breathing spontaneously and to determine the time of centrifugal secondary venous reflux of the distal spermatic cord during the squeezing and relaxing maneuver. This method is closely related to that used by the vascular surgeon to detect valvular incontinence of the saphenous vein. Since Doppler sonography is much more reproducible than Valsalva's maneuver, it is therefore much more reliable.

In a 12‐month period, 625 patients with pathologic findings in at least two spermiograms were studied. Thirty percent showed constant basal reflux not influenced by respiratory exhilation. The squeezing and relaxing maneuver induced secondary reflux longer than 1.6 seconds in 17%, between 0.8 and 1.6 seconds in 17% and less than 0.8 seconds in 36% of the patients.

The higher sensitivity and specificity of Doppler examination compared with thermography and angiography, as well as its low cost and noninvasiveness, make this the procedure of choice in the diagnosis of venous reflux in varicocele.
Significance of Reactive Oxygen Species and Antioxidants in Defining the Efficacy of Sperm Preparation Techniques
Authors: AITKEN R. JOHN , CLARKSON JANE S.
Published: December 2015

The mechanisms responsible for mediating the influence of sperm preparation protocols on human sperm function have been investigated. Techniques that involved the separation of motile spermatozoa prior to centrifugation were found to yield sperm suspensions of highest quality. If the spermatozoa were centrifuged prior to isolation of the motile cells, sperm function was impaired. The detrimental effects of centrifugation were associated with a sudden burst of reactive oxygen species production by a discrete subpopulation of cells (characterized by significantly diminished motility and fertilizing capacity) that could be separated from normal functional spermatozoa on Percoll gradients. If unfractionated sperm suspensions were subjected to centrifugation, the reactive oxygen species generated by this subpopulation impaired the functional competence of normal spermatozoa in the same suspension. Assessment of the ability of the antioxidants, butylated hydroxytoluene, and vitamin E, to curtail the peroxidative damage inflicted by such cells in response to centrifugation revealed a significant improvement of sperm function in the presence of vitamin E.
Can the Fertility Potential of a Seminal Sample Be Predicted Accurately?*
Authors: AMANN RUPERT P.
Published: December 2015

This paper highlights the most critical aspects of the problem of predicting fertility. To determine if a laboratory test(s) is highly correlated with fertility it is essential to have: a) specific, precise and accurate laboratory tests, and b) precise and accurate fertility data. Acquisition of precise and accurate data for laboratory tests and fertility of spermatozoa in the same sample is not easy. Data derived from in vitro fertilization are not tests of fertility, because only a subset of the attributes important for fertilization in vivo are tested. Because of deficiencies in fertility data, there probably is no valid report for human spermatozoa correlating results of laboratory tests and fertility, and very few valid studies for laboratory or domesticated animals. There is little doubt that objective measures of sperm motion, acrosomal status, or other characteristics are significantly correlated with fertility. However, establishment of the correlations between a group of attributes and fertility is not the question of interest. The goal is prediction of fertility. There has been no recent effort to develop a prediction of fertility or fecundity based on sperm characteristics, and achievement of this goal may be elusive.
Relationship Between the Results of Sperm Analysis and GIFT
Authors: RODRIGUEZ‐RIGAU LUIS J. , AYALA CARMA , GRUNERT GEORGE M. , WOODWARD RELL M. , LOTZE EBERHARD C. , FESTE JOSEPH R. , GIBBONS WILLIAM , SMITH KEITH D. , STEINBERGER EMIL
Published: December 2015

In vitro fertilization (IVF) and GIFT have been proposed as therapeutic approaches in infertile couples where a significant male factor is present. To date, few published data are available relating the success rate of GIFT to the severity of the male factor. In this report the results of the first 172 GIFT cases were analyzed. The overall pregnancy rate was 18.0%. The relationship between the occurrence of pregnancy and sperm count (millions/ml), total sperm count (millions/ejaculate), % motility, motile sperm count (millions/ml) and total motile sperm count (millions/ejaculate) were examined. Significant direct correlations were observed between the clinical pregnancy rate and sperm count, total sperm count, motile sperm count and total motile sperm count. Motile sperm count and total motile sperm count had the best correlations with clinical pregnancy rates, which were over 24.0% in groups with motile sperm counts exceeding 40 × 106 cells/ml or total motile sperm counts greater than 100 × 106/ejaculate. The clinical pregnancy rates were 12.5% and 7.7%, respectively, for groups with motile sperm counts under 10 × 106/ml and total motile sperm counts below 25 × 106/ejaculate. No correlation was found between percent motile cells and pregnancy rate. Results of the sperm penetration assay using zona‐free hamster eggs were available in a subpopulation of 27 patients. No significant correlation between this sperm penetration assay and pregnancy rate could be demonstrated. The incidence of chemical pregnancy showed a significant negative correlation with the total motile sperm count, indicating a higher incidence of early pregnancy wastage in cases of oligozoospermia. Using an objective method for measuring sperm motility and quality, a significant correlation between forward sperm motility and the incidence of clinical pregnancy after GIFT was demonstrated.
Estradiol and Plasminogen Activator Secretion by Cultured Rat Sertoli Cells in Response to Melanocyte‐Stimulating Hormones
Authors: BOITANI CARLA , FARINI DONATELLA , CANIPARI RITA , BARDIN C. WAYNE
Published: December 2015

Pro‐opiomelanocortin‐derived peptides, α‐MSH and β‐endorphin, are synthesized and secreted by Leydig cells, and are believed to have paracrine effects on Sertoli cells in the testis. Peptides with MSH activity stimulate adenylate cyclase and cAMP accumulation in Sertoli cell‐enriched cultures. The purpose of the present study was to determine whether such peptides would affect Sertoli cell parameters, such as aromatase and plasminogen activator activities, that are known to be regulated by cAMP, α‐MSH stimulated aromatase activity in Sertoli cell‐enriched cultures prepared from 10‐day‐old rats and this effect was potentiated by methyl isobutylxanthine (MIX). The combination of α‐MSH plus MIX was not as potent as FSH. α‐MSH, des‐acetyl‐α‐MSH, β‐MSH, ACTH(1–13), and ACTH(1–24) stimulated aromatase activity to a similar extent, suggesting that Sertoli cells do not distinguish between the activities of these peptides. α‐MSH potentiated the action of dbcAMP and forskolin on Sertoli cell aromatase, but unexpectedly had no effect on the action of either half‐maximal or maximal doses of FSH. The regulation of plasminogen activator was examined next; urokinase was markedly suppressed by FSH in 10‐day‐old Sertoli cells. Although neither α‐MSH nor MIX alone had an effect on urokinase secretion, in combination they were as effective as FSH. In 10‐day‐old Sertoli cells each of these peptides had little or no effect on tissue plasminogen activator. It was concluded that molecules such as the MSH/ACTH peptides modulate Sertoli cell function via cAMP, that there is a differential response depending more upon the Sertoli cell products examined rather than the peptide tested, and that the magnitude of the responses to α‐MSH and MIX examined to date do not exceed those produced by FSH.
Effect of MK‐906, a Specific 5α‐reductase Inhibitor, on Serum Androgens and Androgen Conjugates in Normal Men
Authors: RITTMASTER ROGER S. , STONER ELIZABETH , THOMPSON DAVID L. , NANCE DWIGHT , LASSETER KENNETH C.
Published: December 2015

To determine the hormonal effects of MK‐906, an orally active 5α‐reductase inhibitor, on serum androgens and androgen conjugates, 12 healthy men were given 10, 20, 50, and 100 mg MK‐906 2 weeks apart in randomized order in a 4‐period crossover design. Serum testosterone (T), dihydrotestosterone (DHT), androstanediol glucuronide, and androsterone glucuronide were measured before and 24 hours after each dose. The effect of MK‐906 on glucuronyl transferase activity, the enzyme responsible for androstanediol glucuronide and androsterone glucuronide formation, was assessed in vitro using rat prostate tissue. Serum T levels were unchanged after all doses. Serum DHT, androstanediol glucuronide, and androsterone glucuronide were suppressed by 70, 40, and 56%, respectively, after the 10‐mg dose, and by 82, 52, and 66% after the 100‐mg dose (P < 0.02 for the comparison between the 10 and 100‐mg doses for all three steroids), respectively. Baseline serum T and DHT levels were strongly correlated (R = 0.89, P = 0.0002), as were androstanediol glucuronide and androsterone glucuronide levels (R = 0.78, P = 0.003), but there was no correlation between DHT levels and the levels of either conjugated steroid. MK‐906 had no effect on glucuronyl transferase activity in vitro. It was concluded that single doses of MK‐906 suppress both conjugated and unconjugated 5 α‐reduced androgens. While all three steroids appeared to be good markers of systemic 5 α‐reductase inhibition, further research will be needed to determine which steroid best reflects tissue DHT levels in patients receiving these inhibitors.
Cytochalasin D Inhibits Penetration of Hamster Eggs by Guinea Pig and Human Spermatozoa
Authors: ROGERS B. JANE , BASTIAS CRISTINA , COULSON RICHARD L. , RUSSELL LONNIE D.
Published: December 2015

Fertilization experiments using zona‐free hamster eggs and spermatozoa from both guinea pig and human were conducted in the presence of cytochalasin D to evaluate the possible role of actin filaments in fertilization processes. When the actin filament inhibitor, cytochalasin D, was added to fertilization media at concentrations of 10 to 30 μM, penetration of eggs was significantly inhibited. Preincubation of the eggs with cytochalasin D and washing prior to addition of spermatozoa had no effect on penetration as quantitated by the number of swollen heads in the egg cytoplasm. However, spermatozoa preincubated with cytochalasin D and subsequently washed prior to egg addition showed reduced penetration of the same magnitude as when spermatozoa and eggs were coincubated with cytochalasin D. Both the percentage of zona‐free eggs showing decondensed sperm heads and the penetration indices (total decondensed spermatozoa/total eggs) were significantly affected when spermatozoa were exposed to cytochalasin D. The DMSO vehicle used to dissolve cytochalasin D had little effect on the number of decondensed heads. When the concentration of cytochalasin D was increased (DMSO remaining constant) in human sperm experiments, percent penetration decreased and progressively fewer decondensed spermatozoa were recorded, indicating dose‐responsiveness to cytochalasin D. Motility parameters of human spermatozoa were not altered at any of the concentrations of cytochalasin D tested. Neither guinea pig sperm motility nor acrosome reaction was altered significantly by cytochalasin D or the DMSO vehicle. These experiments suggest that cytochalasin D may be an inhibitor of some fertilization processes such as sperm penetration or sperm head decondensation.
The Actions of Calcitonin on the TM3 Leydig Cell Line and on Rat Leydig Cell‐Enriched Cultures
Authors: NAKHLA ATIF M. , BARDIN C. WAYNE , SALOMON YORAM , MATHER JENNIE P. , JÄNNE OLLI A.
Published: December 2015

Studies demonstrating calcitonin receptors on Leydig cells have suggested that these cells may be one of the many sites affected by this peptide. To investigate this possibility, the effect of synthetic salmon calcitonin on the TM3 Leydig cell line (derived from immature mouse Leydig cells) and on primary Leydig cell‐enriched preparations was examined. Synthetic salmon calcitonin stimulated the conversion of [3H]adenine to [3H]cyclic AMP in TM3 cells. In addition, the hormone stimulated the basal secretion of testosterone in both TM3 cell‐ and Leydig cell‐enriched cultures and potentiated the action of hCG on Leydig cell‐enriched cultures. Synthetic salmon calcitonin also increased the concentration of androgen and estrogen receptors in cultured TM3 Leydig cells by 2‐ and 4‐fold, respectively, when added to the culture medium (1 μg/ml). The fact that 8‐bromo‐cyclic AMP decreased both androgen and estrogen receptor concentrations suggested that the effect of calcitonin on sex steroid receptors is not mediated by its effect on cyclic AMP in these cells. The possibility that the action of calcitonin on steroid receptors might be mediated by another messenger such as calcium (Ca2+) was therefore considered. Progressively lowering the concentration of Ca2+ in the culture medium of the cells from 1.5 mM to less than 0.01 mM decreased the concentration of both androgen and estrogen receptors. Returning the Ca2+ concentration to normal levels (1.5 mM) restored steroid receptor levels. Receptor levels were also decreased when the extracellular Ca2+ concentration was lowered to 0.5 mM, and treatment with the Ca2+ ionophore, A23187 (1 μM), restored receptor levels to normal. The calcium channel blocker, verapamil, decreased the androgen receptor concentration but unexpectedly increased the concentration of estrogen receptors. It was concluded that calcitonin stimulates cAMP formation and testosterone secretion, and increases the concentration of sex steroid receptors. These observations provide evidence that the previously demonstrated calcitonin receptors on Leydig cells may be coupled to several biologic responses in this cell type.
Demonstration of the Role of Prostate‐Specific Antigen in Semen Liquefaction by Two‐Dimensional Electrophoresis
Authors: LEE CHUNG , KEEFER MARTIN , ZHAO ZHONG WEN , KROES ROGER , BERG LORI , LIU XIANXI , SENSIBAR JULIA
Published: December 2015

Two‐dimensional protein profiles of human semen, prostatic fluid, and seminal vesicle fluid were compared to demonstrate changes in the protein composition of human semen before and after liquefaction. Semen specimens were obtained from a volunteer. Prostatic fluid specimens were collected by rectal massage from patients visiting a urology clinic. Samples of seminal vesicle fluid were collected by needle aspiration from isolated seminal vesicles, which were removed at surgery. All specimens were prepared and processed according to the ISO‐DALT system for separation of proteins in two‐dimensional gels. Following electrophoresis, protein spots in the gels were visualized by silver staining. Prostatic fluid and seminal vesicle fluid showed their characteristic protein profiles. The protein profile of human semen contained specific proteins of both prostatic fluid and seminal vesicle fluid. One major group of proteins in seminal vesicle fluid (Mw 28,000–68,000 daltons), designated as seminal vesicle‐specific antigen, was observed in freshly ejaculated human semen, but disappeared from the two‐dimensional profile when the ejaculate was allowed to stand at room temperature for 30 min. When prostatic fluid or prostate‐specific antigen was mixed with seminal vesicle fluid and incubated at 37C for 30 min, the seminal vesicle‐specific antigen also disappeared from the two‐dimensional profiles. The findings indicate that seminal vesicle‐specific antigen, a group of predominant proteins in seminal vesicle fluid, is the structural component of seminal coagulum, and that prostate‐specific antigen is the enzyme which digests seminal vesicle‐specific antigen and liquifies semen coagulum.
Human Sperm Mitochondrial Function Related to Motility: A Flow And Image Cytometric Assessment
Authors: AUGER JACQUES , RONOT XAVIER , DADOUNE JEAN P.
Published: December 2015

Current evaluation of male fertility, routinely estimated by sperm count, motility, and morphology, provides only crude information about the fertility state of individuals. Both flow and image cytometry were applied to mitochondrial activity and sperm motility respectively. Sperm samples from fertile donors were concomitantly measured for Rhodamine 123 (Rh123) uptake (an estimation of mitochondrial activity), percentage of dead cells, and motility characteristics, such as percentage of motility, curvilinear velocity, and amplitude of lateral head displacement. These measurements were done under experimental conditions known to modulate sperm motility (temperature and time course survival in a capacitating medium). Bimodal distributions were found for Rh123 uptake. Flow cytometry‐derived parameters were essentially time‐dependent whereas motility characteristics were primarily temperature‐dependent. Correlations were found between various flow cytometry‐derived parameters and motility characteristics. Most of the correlations were obtained after a 24 h incubation in a capacitating medium. The most significant correlation in every experimental condition concerned the percentage of motile spermatozoa and the Rh123 uptakes. The drop in motility observed after a 24 h incubation was paralleled by a markedly lower drop in mitochondrial activity. The data suggest that these two complementary techniques represent an improvement in basic and/or clinical assessment of the functional spermatozoa status.
Developmental and Stage‐Specific Expression of Smad2 and Smad3 in Rat Testis
Authors: Xu Jian , Beyer Amanda R. , Walker William H. , McGee Elizabeth A.
Published: December 2015

ABSTRACT: Members of the transforming growth factor beta type (TGFβ) superfamily and their receptors are expressed in the testis, and are believed to play important paracrine and autocrine roles during testicular development and spermatogenesis. The Smad proteins are downstream mediators for the family of TGFβ growth factors. Smad2 and Smad3 are associated with both TGFβ and activin signaling. However, very little is known about the expression and regulation of the Smad signaling proteins in the testis. In the present study, we have determined that Smad2 and Smad3 proteins are expressed in the postnatal testes of rats from 5 days to 60 days of age. Expression levels for both proteins are higher in young rats than in sexually mature rats. Smad2 and Smad3 messenger RNA levels parallel protein expression. Smad2 and Smad3 proteins are mainly localized in the cytoplasm of meiotic germ cells, Sertoli cells, and Leydig cells. Smad3 protein is localized to the nucleus of preleptotene to zygotene primary spermatocytes in young rats. Both proteins are expressed throughout all stages of the cycle of seminiferous tubules but are expressed at their lowest levels at stages VII—VIII in the seminiferous epithelium of adult rats. The presence of these downstream mediators in these cell types supports a role for TGFβ and activin during spermatogenesis. The difference between the expression of Smad2 and Smad3 suggests that they may have different functions within the testis.
Cavernous Neurotomy Causes Hypoxia and Fibrosis in Rat Corpus Cavernosum
Authors: Leungwattanakij Somboon , Bivalacqua Trinity J. , Usta Mustafa F. , Yang Dae‐Yul , Hyun Jae‐Seog , Champion Hunter C. , Abdel‐Mageed Asim B. , Hellstrom Wayne J.G.
Published: December 2015

ABSTRACT: The etiologies of erectile dysfunction (ED) after nerve‐sparing radical prostatectomy have not been clearly elucidated. The aim of this study was to evaluate the effects of cavernous nerve injury on cavernous fibrosis, and to consider measures to prevent irreversible damage to the cavernous tissues. Twenty male Sprague‐Dawley rats constituted the study population. The animals were divided into 2 groups; group 1 consisted of sham‐operated rats (n = 10), and group 2 consisted of rats that underwent incision of both cavernous nerves (n = 10). Three months later, all rats underwent intracavernous papaverine injection (300 and 600 mg), and intracorporal pressures were recorded. Transforming growth factor‐β1 (TGF‐β1) messenger RNA (mRNA) expression from rat penile tissue was measured using reverse transcriptase‐polymerase chain reaction. Hypoxia‐inducible factor‐1α (HIF‐1α), TGF‐β1, and collagen I and III protein expressions were determined by Western blot analysis and immunohistochemical staining. Erectile function as studied with intracavernosal papaverine injection and histological analysis of penile cross‐sections at 3 months was similar in both groups. TGF‐β1 mRNA expression, HIF‐1α, TGF‐β1, and collagen I and III protein expressions were significantly greater in the neurotomy group. Immunohistochemical staining for TGF‐β1, HIF‐1α, and collagen III were qualitatively more positive in the neurotomy group, whereas collagen I staining was similar. This study demonstrates an increase in TGF‐β1, HIF‐1α, and collagen III synthesis in rat cavernosal smooth musculature after cavernous neurotomies. In theory, cavernous fibrosis may be reduced by employing various vasoactive agents or interventions that increase oxygenation to the corporal tissues during the postoperative period.
Deterioration of Plasma Membrane Is Associated With Activated Caspases in Human Spermatozoa
Authors: Paasch Uwe , Grunewald Sonja , Fitzl Guenter , Glander Hans‐Juergen
Published: December 2015

ABSTRACT: Spermatozoa with deteriorated plasma membranes can be separated by magnetic‐activated cell sorting (MACS) after binding superparamagnetic annexin V‐conjugated microbeads (ANMBs) to membrane phosphatidylserine (PS). Semen samples from 15 donors and 25 infertile patients were divided into 2 spermatozoal fractions by annexin V‐MACS. Activated caspases (aCPs), which mediate degradations of cell quality, were determined by CaspaTag in the 2 subpopulations. Spermatozoa from donors showed lower levels of bound annexin V (3.6% ± 0.5% vs 11.9% ± 1.1%; P < .01) and aCPs (21.8% ± 2.6% vs 43.2% ± 2.1%; P < .01) than did spermatozoa from infertile patients. MACS resulted in a decrease of spermatozoa with aCPs from 21.8% ± 2.6% (before separation) to 9.2% ± 1.4% (in the ANMB‐negative fraction) in donors and from 43.2% ± 2.1% to 18.8% ± 2.6% in infertile patients (mean ± SEM; P < .01). Separation effects of the MACS technique were confirmed with flow cytometry using anti‐annexin V antibodies and with electron microscopy. ANMB‐MACS removes spermatozoa with PS‐bound annexin V and produces a higher quality spermatozoal fraction. Spermatozoa with a deteriorated membrane are characterized by an increase in aCPs. A higher percentage of spermatozoa with ANMBs bound to PS and with aCPs were found in infertile patients.
Neoplastic Potential of Germ Cells in Relation to Disturbances of Gonadal Organogenesis and Changes in Karyotype
Authors: Słowikowska‐Hilczer Jolanta , Romer Tomasz E. , Kula Krzysztof
Published: December 2015

ABSTRACT: The study consisted of 46 intersexual patients who underwent gonadectomy at the age of 3 months to 19 years because of gonadal dysgenesis (GD; 40 cases) or true hermaphroditism (bisexual gonads; 6 cases). In patients with GD, the incidence of the 46,XY karyotype was 67.5%, whereas the remaining patients exhibited numerical and structural aberrations of sex chromosomes (NSASs), and all patients with bisexual gonads revealed NSAS. Seminoma was diagnosed in 1 patient with the 46,XY karyotype and pure GD (streak gonads). Intratubular carcinoma in situ (CIS) appeared as an exclusive lesion in 61.5% of 13 patients with mixed GD, in 54% of 11 patients with partial GD (bilateral testes), in 16.7% of 6 patients with bisexual gonads, and in none of 13 patients with pure GD. CIS also appeared in tubules in the vicinity of sex cord‐derived tumors (gonadoblastoma nests and unclassified mixed germ cell‐sex cord‐stromal tumor; MGCSCST) and within the tumors. In 3 patients, gonadoblastoma replaced the whole bilateral gonads and is referred to as gonadoblastoma‐only GD. The incidence of neoplastic lesions (mostly bilateral) was 90.9% in patients with partial GD, 76.9% (mostly unilateral) in patients with mixed GD, 23.1% (unilateral) in patients with pure GD, and 16.7% (unilateral) in patients with bisexual gonads. Disregarding types of disturbances of gonadal organogenesis, the incidence of lesions was 71.4% in 28 patients with the 46,XY karyotype and 35.3% in 17 patients with NSAS. We conclude, first, that NSAS is not a prerequisite for the appearance of GD and GD is more frequently associated with the 46,XY karyotype. Second, the spectrum of germ cell neoplastic lesions in GD is wider than reported. Besides germ cell carcinoma, CIS, and gonadoblastoma nests, the spectrum also includes a tumor of gonadoblastoma‐only in cases of GD and MGCSCST. Third, the incidence of neoplastic lesions is related more to the severity of the disturbances of gonadal organogenesis than it is to aberrations in sex chromosomes. Fourth, less disturbed testicular organogenesis predisposes these patients more toward germ cell neoplastic lesions, which suggests that the testicular environment of a dysgenetic gonad plays an important role in germ cell neoplasia initiation, maintenance, or both.
An Increased CAG Repeat Length in the Androgen Receptor Gene in Azoospermic ICSI Candidates
Authors: Mengual Lourdes , Oriola Josep , Ascaso Carlos , Ballescà José L. , Oliva Rafael
Published: December 2015

ABSTRACT:The androgen receptor gene has a polymorphic trinucleotide repeat that encodes a polyglutamine tract in its N‐terminal transactivation domain. We started this study in order to find out whether a correlation existed between the length of this polymorphic tract and the presence of azoospermia in candidates for intracytoplasmic sperm injection (ICSI). The CAG repeat length in exon 1 of the androgen receptor (AR) gene was directly sequenced in 102 patients with azoospermia and in 96 fertile controls. Hormone levels were also measured in patients with azoospermia. The mean AR gene CAG repeat length was significantly larger in azoospermic subjects than it was in control fertile men (23.25 ± 2.7 versus 22.42 ± 2.8; P = .033). A receiver operating characteristic analysis evidenced a cutoff point at 22/23 CAG repeats at which the probability of being azoospermic increased 2.2 times. Subsequent logistic regression analysis of the data showed that the odds for azoospermia increased with the number of CAG repeats. Men with more than 26 CAG repeats have a 4.09 greater risk of being azoospermic. Therefore, in our candidates for ICSI, a direct correlation exists between the CAG repeat length in the exon 1 of the AR gene and the risk of being azoospermic.
Evidence for a Role of Glycogen Synthase Kinase‐3β in Rodent Spermatogenesis
Authors: Guo Taylor B. , Chan Kam C. , Hakovirta Harri , Xiao Yang , Toppari Jorma , Mitchell Aaron P. , Salameh Wael A.
Published: December 2015

ABSTRACT: Glycogen synthase kinase‐3β (GSK‐3β) regulates cell metabolism, cell cycle, and cell fate through the phosphorylation of a diverse array of substrates. Herein, we provide evidence that supports a role for GSK‐3 in mammalian meiosis and spermatogenesis. Immunostaining of testis sections showed that while GSK‐3α was ubiquitous in the seminiferous tubules, GSK‐3β was expressed in premeiotic type B spermatogonia, in both meiotic preleptotene and leptotene spermatocytes, as well as in Sertoli cells in both the mouse and rat. Thus, GSK‐3β is expressed in germ cells entering meiosis. In addition, intense immunoreactivity was detected in rat step 6 though 11 spermatids. In situ hybridization (ISH) in rat testis confirmed the immunostaining pattern in leptotene and spermatids and showed a GSK‐3β messenger RNA (mRNA) signal in some pachytene spermatocytes. The restricted pattern of expression suggests cell‐specific regulation of Gsk‐3β mRNA. To determine whether GSK‐3 is required for meiosis entry, rat stage VIIa seminiferous tubule segments were cultured with selective small‐molecule GSK‐3 inhibitors. These compounds markedly and dose‐dependently suppressed meiotic synthesis (S)‐phase DNA. Since a yeast GSK‐3 homolog, Rim11p (regulator of inducer of meiosis), is pivotal to meiosis entry, we tested whether GSK‐3β complements Rim11p function in meiosis. Rim11p phosphorylates transcription factors Ume6p (unscheduled meiotic gene expression) and Ime1p (inducer of meiosis) to induce meiosis entry. Overexpression of murine GSK‐3β in a rim11 mutant yeast failed to rescue the sporulation defect. Our finding that GSK‐3β interacted only with Ume6p but not with IME1 in a yeast 2‐hybrid assay suggests that noncomplementation reflects partial divergence in substrate specificity. This work provides the basis for future studies of GSK‐3β signaling in mammalian meiosis and spermatogenesis.
The Use of Novel Semen Quality Scores to Predict Pregnancy in Couples With Male‐Factor Infertility Undergoing Intrauterine Insemination
Authors: Bedaiwy Mohamed A. , Sharma Rakesh K. , Alhussaini Tarek K. , Mohamed Maher S. , Aleem Ali M. Abdel , Nelson David R. , Thomas Anthony J. , Agarwal Ashok
Published: December 2015

ABSTRACT: The purpose of this study was to determine whether 2 new semen quality (SQ) scores could predict pregnancy in patients undergoing intrauterine insemination (IUI) for male‐factor infertility and whether an overall score could help in counseling these couples with assisted reproductive technique (ART) options. Ninety‐three couples with male‐factor infertility were examined for semen analysis. Samples were prepared by density gradient separation (47% and 90%), and IUI was performed. On the basis of the 2 semen scores (SQ and relative quality [RQ]), the IUI‐semen pregnancy score (IUI‐SPS) was calculated. Of the 192 IUI cycles, 14% (27 of 192) resulted in pregnancy. Both prewash SQ and RQ scores were significantly related to pregnancy (P = .02 and P < .001), as was the postwash RQ score (P < .001). Of the IUI cycles in which the postwash RQ score was greater than 125, 40% (13 of 32) resulted in pregnancy compared to 9% of cycles (14 of 160) in which the postwash SQ score was less than 125. The prewash IUI‐SPS score was significantly related to IUI‐induced pregnancy (P < .001). Both the pre‐ and postwash SQ and RQ scores can predict pregnancy in male infertility patients undergoing IUI. Patients with an IUI‐SPS less than 150 may be advised to seek in vitro fertilization (IVF), whereas those with an IUI‐SPS greater than 150 may be advised to seek IUI.
Relationship Between the Characteristics of Epididymal Red Deer Spermatozoa and Penetrability Into Zona‐Free Hamster Ova
Authors: Soler Ana J. , Garde José J.
Published: December 2015

ABSTRACT: A heterologous (zona‐free hamster oocytes) in vitro fertilization (IVF) system was used to evaluate the relationship between sperm factors and penetration capacity of epididymal red deer spermatozoa. The sperm parameters evaluated in 36 sperm samples obtained postmortem from stags selectively shot during the rutting season were sperm motility, functional integrity of plasma membrane by means of the hypo‐osmotic swelling test (HOST), and, simultaneously, viability and acrosomal status via a triplestain technique. Zona‐free hamster oocytes were used to evaluate the capacity of the different sperm assays to predict in vitro penetration. In order to increase the variability in sperm quality, we recovered samples from stags at different intervals between the death of the male and the collection of the genitalia. All measures of sperm quality declined progressively (P < .001) with increasing intervals between death and sample collection. In addition, many sperm parameters were related to penetration ability in vitro. Subsequently, sperm samples were rearranged in 2 categories according to the interval that had elapsed between death and the collection of the genitalia (group 1, short interval = 0–12 h; group 2, large interval = 18–40 hours). When samples were grouped, less correlation achieved significance, especially for group 1, than when samples were not divided. Also, correlation between the number of sperm per oocyte and sperm parameters for group 1, which had the highest values of sperm quality, failed to reach significance. It is concluded that the classical parameters accepted in assessing the viability of deer spermatozoa can be good predictors of the penetrating ability of the spermatozoa when satisfactory in vitro conditions are used for the development of the IVF system. Also, this study demonstrates that compatible heterologous gamete interaction allows thorough assessment of sperm function in a wild deer.
Hormonal Regulation of Bovine Secretory Proteins Derived From Caput and Cauda Epididymal Epithelial Cell Cultures
Authors: de Pauw Ingrid M. C. , Goff Alan K. , van Soom Ann , Verberckmoes Steven , de Kruif Aart
Published: December 2015

ABSTRACT: The goal of this study was to investigate the effect of hormones (testosterone, dihydrotestosterone [DHT], and hydrocortisone) on the protein secretion of caput and cauda epididymal epithelial cells cultured in principal cell medium (PCM). A confluent monolayer of caput and cauda epididymal epithelial cells was obtained from serum‐containing PCM in the presence or absence of hormones after 7 days of culture at 38.5°C (5% CO2 in air). The protein secretion of epididymal epithelial monolayers incubated in serum‐free PCM for 3 days was examined. The secreted proteins were separated by 2‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (2D SDS‐PAGE). A comparison of the different protein patterns showed 61 spots, of which 11 were secreted only in the presence of hormones, 3 appeared to show hormone‐related changes, and 25 were region‐specific. Most of these secreted proteins were low‐molecular‐weight acidic proteins. To obtain evidence of the epididymal origin of the secreted proteins, proteins present in caput and cauda epididymal plasma were analyzed. In conclusion, our data indicate that hormones influence the synthesis of a number of caput and cauda epididymal proteins. Some of these proteins could be important for improving our understanding of spermatozoa maturation and storage and their acquisition of fertilizing ability.
A Putative 12‐Transmembrane Domain Cotransporter Associated With Apical Membranes of the Epididymal Duct
Authors: Obermann Heike , Wingbermühle Anke , Münz Sibylle , Kirchhoff Christiane
Published: December 2015

ABSTRACT: The epithelial cells lining the epididymal duct play an important role in establishing and maintaining the luminal fluid microenvironment. A cDNA, canine epididymal cDNA 11 (CE11), was cloned from the dog epididymis predicting a transport protein involved in this process. The full‐length sequence encoded a 12‐transmembrane domain protein of 481 amino acids; a splice variant predicted a shorter isoform. Northern blot analysis and in situ transcript hybridization revealed that the CE11 mRNA is highly expressed by the epididymal duct epithelium. By reverse transcription‐polymerase chain reaction, however, lower levels of both splice variants were detected in other tissues as well. Database searches suggested that CE11 is homologous to the previously described so‐called thymic stromal cotransporter (TSCOT) with weak similarity to sugar (and other) cotransporters. We show here that the mouse and human TSCOT mRNAs, although enriched in immune tissues, have a broader distribution than previously assumed. Employing Western blot analysis, we identified the endogenous CE11 protein in membrane preparations of the canine epididymis. In accordance with the occurrence of 2 splice variants, 2 immunopositive bands were detected, most probably representing the predicted CE11 isoforms. Immunoperoxidase staining and indirect immunofluorescence localized the antigen in the apical membrane compartment of the adult organ, suggesting that CE11 functions as an apical transport protein involved in the establishment and/or maintenance of the specific luminal microenvironment of the epididymal duct.
Neonatal Coadministration of Testosterone With Diethylstilbestrol Prevents Diethylstilbestrol Induction of Most Reproductive Tract Abnormalities in Male Rats
Authors: Rivas Ana , McKinnell Chris , Fisher Jane S. , Atanassova Nina , Williams Karin , Sharpe Richard M.
Published: December 2015

ABSTRACT: The primary purpose of this study was to evaluate whether the coadministration of testosterone (TE; 200 μg) with 10 μg of diethylstilbestrol (DES) between days 2 and 12 postnatally could prevent the adverse gross reproductive tract changes and associated loss of androgen receptor (AR) expression induced by DES treatment alone. Various endpoints (rete testis area, efferent duct lumen area, epithelial cell height of efferent ducts, and vas deferens) were quantified to check for the abnormal changes that have been shown to occur after neonatal treatment with a high dose (10 μg) of DES. Additionally, DES induction of an aberrant pattern of estrogen receptor alpha (ER‐α) immunoexpression in the vas deferens and seminal vesicles was evaluated. The coadministration of DES with TE prevented the induction of all but one of the abnormalities induced by DES treatment on its own, coincident with the restoration of normal/supranormal TE levels and normal immunoexpression of the AR and ER‐α in the tissues studied. The exception was DES‐induced lumenal distension of the efferent ducts, which was only partially prevented by the coadministration of DES with TE. These evaluations were made on day 18, but the described abnormalities were already somewhat evident by day 8 in DES‐treated animals. It was therefore tested whether a delay of TE replacement until days 8–12 was still able to reverse the abnormalities already induced by DES treatment alone. A delayed treatment with TE reversed the adverse changes in epithelial cell height and in ER‐α and AR immunoexpression in the same tissues by day 18; however, rete testis overgrowth was only partially prevented, and efferent duct distension was not prevented at all. These results provide further evidence that DES‐induced disorders of reproductive tract development in the male result from a disturbance of the androgen‐estrogen balance rather than from estrogen action alone.