bluuebunny/crossref_metadata_2025 huggingface dataset:
Susceptibility of Glycolytic Enzyme Activity and Motility of Spermatozoa From Rat, Mouse, and Human to Inhibition by Proven and Putative Chlorinated Antifertility Compounds In Vitro
Authors: BONE WILHELM , JONES ALAN R. , MORIN CHRISTOPHE , NIESCHLAG EBERHARD , COOPER TREVOR G.
Published: November 2023

ABSTRACT: Nonhormonal contraceptives that act by blocking energy metabolism within sperm have the advantage over spermatogenic inhibitors by their fast onset of infertility and their almost immediate restoration of fertility after withdrawal of the contraceptive agent. This study was done to test new chlorinated compounds for their contraceptive potency on rodent and human sperm in vitro. Cells were incubated in a medium containing glucose as the sole energy source with 1‐chloro‐3‐hydroxypropanone (CHOP) and 1,6‐dichloro‐1,6‐dideoxy‐d‐fructose (DCDF), chlorinated analogues of glycolytic substrates, as well as racemic (R,S)‐α‐chlorohydrin (ACH). After incubation, enzymatic activity and kinematic parameters were estimated. A dose‐dependent inhibition of the glycolytic enzyme, glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), of rat and mouse distal cauda epididymidal and human ejaculated sperm by ACH, CHOP, and DCDF was demonstrated. Triosephosphate isomerase (TPI) was inhibited by ACH, but not by CHOP and DCDF, irrespective of species. All compounds inhibited sperm motility and kinematic parameters with increasing concentration. The results confirm that inhibition of glycolytic enzymes of sperm, including those of human, can be effectively brought about by a variety of chloro‐compounds that can be converted to (s)‐3‐chlorolactaldehyde, the stereospecific chloro‐derivative of the enzyme's natural substrate, (r)‐glyceraldehyde 3‐phosphate, and could be developed into contraceptive agents for men.
Differential Expression of CCAAT/Enhancer‐Binding Protein‐δ (C/EBPδ) in Rat Androgen‐Dependent Tissues and Human Prostate Cancer
Authors: YANG GENYAN , GREGORY CHRISTOPHER W. , SHANG QUAN , O'BRIEN DEBORAH A. , ZHANG YONG‐LIAN
Published: November 2023

ABSTRACT: CCAAT/enhancer‐binding protein δ (C/EBPδ) is a nuclear transcription factor that regulates cellular growth and differentiation. In this study we demonstrate that C/EBPδ gene expression is differentially regulated in rat androgen‐dependent tissues and human prostate cancer. C/EBPδ messenger RNA (mRNA) levels were very low in adult rat ventral prostate, epididymis, and testis. In ventral prostate and epididymis, expression of C/EBPδ mRNA increased more than sixfold when testicular testosterone was eliminated by surgical castration or treatment with ethane‐1, 2‐dimethanesulfonate (EDS). Testosterone replacement reduced C/EBPδ mRNA levels to near control values in both tissues. CWR22 is a human prostate cancer xenograft that mimics biological characteristics of androgen‐dependent and androgen‐independent human prostate cancer. In androgen‐dependent CWR22 tumors, expression of C/EBPδ mRNA declined in response to castration. Both C/EBPδ mRNA and protein levels increased following testosterone administration. However, C/EBPδ mRNA and protein levels were variable in recurrent CWR22 tumors growing in the absence of testicular androgen for ∼5 months. C/EBPδ expression was also variable in androgen‐independent human prostate carcinomas (n = 3), although mRNA levels were substantially lower than those in androgen‐dependent tumors (n = 3). These studies demonstrate that androgen down‐regulates C/EBPδ levels in androgen‐dependent rat tissues, but induces C/EBPδ expression in androgen‐dependent human prostate cancer. Deregulation of C/EBPδ occurs when prostate cancer progresses to the androgen‐independent state.
Evaluation of Nitric Oxide Synthase and Arginase in the Induction of a Peyronie's‐Like Condition in the Rat
Authors: BIVALACQUA TRINITY J. , CHAMPION HUNTER C. , LEUNGWATTANAKIJ SOMBOON , YANG DAE YUL , HYUN JAE‐SEOG , ABDEL‐MAGEED ASIM B. , SIKKA SURESH C. , KADOWITZ PHILIP J. , HELLSTROM WAYNE J. G.
Published: November 2023

ABSTRACT: Peyronie's disease is an idiopathic, localized connective tissue disorder of the penis, involving the tunica albuginea of the corpus cavernosum and adjacent areolar space. Current proposals as to the origin of Peyronie's disease suggest that fibrosis and collagen changes of the tunica are the result of an inflammatory process following vascular trauma. Our laboratory and other investigators have recently proposed an animal model for the study of Peyronie's disease. When transforming growth factor‐β1 (TGF‐β1) was injected into the rat tunica albuginea, tissue fibrosis was observed at 6 weeks. Therefore, our aim was to assess arginase II, endothelial and inducible nitric oxide synthase isoforms, and nitrotyrosine levels—all factors involved in inflammatory reactions—in the cavernosal tissue of saline‐injected and TGF‐β1‐injected rats after 6 weeks in order to evaluate the roles these enzymes may play in the induction of a Peyronie's‐like condition in the rat. To examine the expression of endothelial nitric oxide synthase (eNOS), iNOS, and arginase II protein, and mRNA in the corpus cavernosum, immunoblot analysis, and reverse transcriptase—polymerase chain reaction were performed. We also determined immunohistochemically the expression of nitrotyrosine, a marker of peroxynitrite formation, in the rat penis. After 6 weeks, iNOS protein and gene expression was up‐regulated and eNOS protein and gene expression was down‐regulated in the corpora cavernosa of the TGF‐β1‐injected penises. Furthermore, arginase II protein expression as well as immunohistochemical localization of nitrotyrosine was significantly higher in the TGF‐β1‐injected corpora cavernosa. These results suggest that iNOS is the key control element for peroxynitrite formation, arginase II expression, and eNOS down‐regulation in the induction of a Peyronie's‐like condition in the rat.
Androgen Receptor Regulation of G1 Cyclin and Cyclin‐Dependent Kinase Function in the CWR22 Human Prostate Cancer Xenograft
Authors: GREGORY CHRISTOPHER W. , JR RAYMOND T. JOHNSON , PRESNELL SHARON C. , MOHLER JAMES L. , FRENCH FRANK S.
Published: November 2023

ABSTRACT: Human prostate cancer is initially dependent on androgens for growth, and androgen‐dependent cells undergo apoptosis after castration. However, a subset of androgen‐responsive cells survives and eventually proliferates in the absence of testicular androgen. The high levels of androgen receptor in both androgen‐de‐pendent and recurrent tumors led us to investigate androgen regulation of cell cycle proteins in human prostate cancer using the CWR22 xenograft. Cellular proliferation decreased dramatically in CWR22 tumors after castration. Testosterone propionate (TP) treatment of castrated mice restored cellular proliferation after 24–48 hours. Growth of CWR22 tumors in the absence of testicular androgen recurred several months after castration. CDK1 and CDK2, and cyclin A and cyclin B1 messenger RNAs were decreased 6 days after castration, increased 6–12 hours after TP treatment, and were expressed at high levels in recurrent CWR22 tumors. Coimmunoprecipitated cyclin B1/CDK1 and cyclin D1/CDK4 protein complexes decreased after castration and increased after TP treatment of castrated mice. In addition, CDK1 and CDK2 kinase activities were up‐regulated by androgen in parallel with hyperphosphorylation of retinoblastoma (Rb) protein. Despite the absence of testicular androgen in recurrent CWR22, the levels of these androgen‐regulated cyclin/CDK protein complexes and hyperphosphorylation of Rb were equal to or greater than in tumors from intact mice. The results indicate that androgen receptor regulates cellular proliferation by control of CDK and cyclins at the transcriptional level and by post‐translational modifications that influence cell cycle protein activity.
Mouse Seminal Vesicle Secretory Protein of 99 Amino Acids (MSVSP99): Characterization and Hormonal and Developmental Regulation
Authors: MOREL LAURENT , BROCHARD DENIS , MANIN MICHÈLE , SIMON ANNE‐MARIE , JEAN CLAUDE , VEYSSIERE GEORGES
Published: November 2023

ABSTRACT: Polyclonal antibodies have been generated to investigate the localization, tissue and species distribution, androgen regulation, and ontogeny of a protein secreted by mouse seminal vesicle, designated as MSVSP99 (ie, mouse seminal vesicle secretory protein of 99 amino acids). MSVSP99 is a polymorphic compound with a molecular weight of around 14 kilodaltons and a positive immunoreactivity range of 5.23 to 5.70. Positive immunoreactivity was restricted to the epithelial cells of the seminal vesicle. Western blot analysis showed organ specificity for MSVSP99, which could not be detected in several organs in the mouse. Time course decrease of MSVSP99 after castration closely followed that of its mRNA. In contrast, the length of time required to restore control levels after testosterone treatment was higher for the protein than it was for its mRNA. Whereas the MSVSP99 gene is already active in 10‐day‐old males, MSVSP99 is first detected at 27 days. Then, we conclude that factors other than the accumulation of the mRNA regulate MSVSP99 expression.
Altered Protamine 2 Expression Is Uncommon In Donors of Known Fertility, but Common Among Men With Poor Fertilizing Capacity, and May Reflect Other Abnormalities of Spermiogenesis
Authors: CARRELL DOUGLAS T. , LIU LIHUA
Published: November 2023

ABSTRACT: During the spermatid elongation stage of spermiogenesis approximately 85% of sperm nuclear histones are replaced by protamines. Protamines increase the packing ratio of sperm chromatin, presumably facilitating sperm motility and function. In this study we evaluated the incidence of abnormal protamine expression in 75 patients undergoing in vitro fertilization (IVF) and 50 donors of known fertility by isolation of sperm nuclear proteins, quantitative gel electrophoresis, and Western blot analysis. In addition, we evaluated the relationship between abnormal protamine expression and semen quality, sperm penetration ability, chromatin stability, and IVF outcome. Seventeen percent (13/75) of IVF patients had no measurable protamine 2 (P2) versus 0% (0/50) of donors of known fertility (P < .005). Sperm penetration rates were decreased in 12 of 13 patients without P2, and mean penetration rates (4.6 ± 1.2 vs 32.8 ± 2.9, P < .005), normal morphology (22.4 ± 3.6 vs 48.7 ± 4.2, P < .05), and progressive motility (22.3 ± 2.5 vs 35.4 ± 2.1, P < .05) were all significantly decreased compared with patients with measurable P2. The mean sperm concentration was not significantly different. The presence of protamine precursor bands was also associated with a diminished penetration capacity (18.4 ± 2.8 vs 36.7 ± 3.0, P < .05). Sperm chromatin decondensation following exposure to heparin sulfate was significantly increased in patients without a measurable P2 band. Twelve patients with no measurable P2 underwent intracytoplasmic sperm injection (ICSI), with 6 patients (6/12, 50%) becoming pregnant. ICSI fertilization and subsequent embryo cleavage were not different in patients without P2 compared with other patients undergoing ICSI. These data indicate that abnormal sperm protamine levels are a common defect in infertility patients, but not in donors of known fertility. It appears that abnormal protamine levels may reflect defects of late spermiogenesis, including sperm penetration capacity.
Intracavernosal Sildenafil Facilitates Penile Erection Independent of the Nitric Oxide Pathway
Authors: MCAULEY IAIN W. , KIM NOEL N. , MIN KWEONSIK , GOLDSTEIN IRWIN , TRAISH ABDULMAGED M.
Published: November 2023

ABSTRACT: Sildenafil, in nanomolar serum levels, is an effective phosphodiesterase type 5 inhibitor, and facilitates penile erection only during sexual stimulation. However, there is limited information on the pharmacological activity of this agent when tissue levels approach millimolar concentrations following intracavernosal injection. The aim of this study was to investigate whether sildenafil causes penile erection in the absence of active neurogenic input. Organ bath preparations of rabbit penile cavernosal tissue strips were contracted with 1 μM phenylephrine and exposed to increasing concentrations of sildenafil in the absence or presence of the nitric oxide (NO) synthase inhibitor, NΩ‐nitro‐l‐arginine methyl ester (l‐NAME; 0.6 mM). Sildenafil caused dose‐dependent relaxation of rabbit cavernosal smooth muscle at high concentrations (>0.1 μM) with little or no effect at concentrations below 0.1 μM. The addition of l‐NAME did not affect this response. In a separate protocol, sildenafil dose response determinations were performed in the presence of the guanylyl cyclase inhibitor, 1H‐[1,2,4]‐oxadiazolo‐[4,3‐a]‐quinoxalin‐1‐one (ODQ; 3 μM) or vehicle (50% dimethyl sulfoxide [DMSO]). Relaxation to sildenafil in the presence of DMSO was significantly enhanced relative to sildenafil alone. ODQ treatment partially attenuated relaxation to sildenafil, but failed to completely inhibit the response. In cavernosal tissue strips, sildenafil elevated basal cyclic guanosine monophosphate (cGMP) levels twofold (0.54 vs. 1.10 pmol/mg prot). To further investigate these observations, anesthetized rabbits were injected intracavernosally with sildenafil (0.3–1.3 mg). In the absence of pelvic nerve stimulation, the magnitude and duration of the intracavernosal pressure increased in a dose‐dependent fashion in response to sildenafil, approaching the systemic arterial pressure at higher doses. Intracavernosal administration of l‐NAME, at doses that inhibited pelvic nerve stimulated penile erection, did not alter the response to intracavernosal sildenafil at 1.3 mg. Sildenafil, at the doses tested, did not significantly change the systemic arterial pressure. These data suggest that intracavernosal sildenafil, at tissue levels approaching millimolar concentrations, can cause relaxation of vascular smooth muscle and penile erection by a novel mechanism independent of the classical NO/cGMP pathway.
Purification of Rat Leydig Cells: Increased Yields After Unit‐Gravity Sedimentation of Collagenase‐Dispersed Interstitial Cells
Authors: SALVA ANTONIO , KLINEFELTER GARY R. , HARDY MATTHEW P.
Published: November 2023

ABSTRACT: Procedures for purification of Leydig cells have facilitated studies of their regulatory biology. A multistep procedure that includes a filtration with nylon mesh (100‐micron pore size) to separate interstitial cells from the seminiferous tubules, combining centrifugal elutriation and Percoll density gradient sedimentation, has been used to obtain a 95% enrichment of rat Leydig cells. However, the number of recovered Leydig cells by this procedure represents only a small fraction of the 25 million, on average, that exist in the adult rat testis. The objective of this study was to test whether the yield of purified Leydig cells might be enhanced by substitution of unit‐gravity sedimentation (S method) for the filter step (F method). We also asked whether a greater number of Leydig cell clusters, macrophages, or both would be recovered by this new method, and if the presence of Leydig cell clusters is associated with increased capacity for testosterone production in vitro. The number of purified Leydig cells was 1.9‐fold higher for the S method than for the F method, with no differences in purity assessed by 3β‐hydroxysteroid dehydrogenase histochemical staining. Leydig cell clusters were also found in greater numbers with the S method both after collagenase dispersion and at the end of the purification. No differences were seen in testosterone production or in the number of macrophages present in the Leydig cells that were prepared by the 2 methods. These results indicate that the new method recovers greater numbers of Leydig cells by collecting clustered Leydig cells that are systematically eliminated when a filtration step is used.
Does Sperm Morphology Play a Significant Role in Increased Sex Chromosomal Disomy? A Comparison Between Patients With Teratozoospermia and OAT by FISH
Authors: GOLE L. A. , WONG P. F. , NG P. L. , WANG X. Q. , NG S. C. , BONGSO A.
Published: November 2023

ABSTRACT: Eight semen samples from men with teratozoospermia (T), along with samples from 3 men with normal fertility and 5 samples from men with oligoasthenoteratozoospermia (OAT) were analyzed for X and Y chromosomal anomalies with the use of fluo‐rescently labeled centromeric probes. This study was undertaken to determine whether patients with abnormal sperm morphology (teratozoospermia) have increased or decreased incidence of a sex chromosomal anomaly. The sex chromosome disomy for the T samples was 0.36% and for the OAT group it was 0.61%, compared with baseline value for the normozoospermia group (0.09%).
Cordyceps sinensis and Its Fractions Stimulate MA‐10 Mouse Leydig Tumor Cell Steroidogenesis
Authors: HUANG BU‐MIIN , JU SY‐YEUAN , WU CHING‐SHYI , CHUANG WOEI‐JER , SHEU CHIA‐CHIN , LEU SEW‐FEN
Published: November 2023

ABSTRACT: The effects of Cordyceps sinensis (CS) and its extracted fractions on steroidogenesis in MA‐10 cells were determined. Different concentrations of CS and 3 fractions of CS (F1, a water‐soluble polysaccharide; F2, a water‐soluble protein; and F3, a poorly water‐soluble polysaccharide and protein) were added to MA‐10 mouse Leydig tumor cells with or without human chorionic gonadotropin (hCG), and the production of steroid and the expression of steroidogenic acute regulatory protein (StAR) were examined. The results showed that CS alone (2–10 mg/mL) stimulated MA‐10 cell progesterone production in a dose‐dependent relationship. Fractions F1 and F3 (2–10 mg/mL) also had significant (P < .05) stimulatory effects on MA‐10 cell steroidogenesis with a dose‐dependent relationship. However, fraction F2 did not have an effect on MA‐10 cells. CS and F3, but not F1, significantly induced more steroid production in hCG‐stimulated MA‐10 cells (P < .05). As a temporal relationship, F1 and F3 (2 mg/mL) maximally stimulated progesterone production between 1 and 3 hours after stimulation in MA‐10 cells. In addition, CS and F3 significantly enhanced MA‐10 cell StAR protein expression, which indicates that CS and F3 may use a cyclic adenosine monophosphate signal transduction pathway to activate MA‐10 Leydig cell steroidogenesis in a manner to that of luteinizing hormone.
Dibromoacetic Acid, a Prevalent By‐Product of Drinking Water Disinfection, Compromises the Synthesis of Specific Seminiferous Tubule Proteins Following Both In Vivo and In Vitro Exposures
Authors: HOLMES MICHAEL , SUAREZ JUAN D. , ROBERTS NAOMI L. , MOLE M. LEONARD , MURR ASHLEY S. , KLINEFELTER GARY R.
Published: November 2023

ABSTRACT: Dibromoacetic acid (DBA) is a by‐product of drinking water disinfection that alters spermatogenesis in adult male rats. To identify a mechanism by which DBA alters spermatogenesis, seminiferous tubules representing specific groups of spermatogenic stages were exposed either in vivo or in vitro, and structural and functional consequences were evaluated. Seminiferous tubules representing stages I–V, VI–VIII, and IX–XIV were isolated from testes of adult rats and cultured overnight in conditions of reduced oxygen and temperature. For in vivo exposures, seminiferous tubules were recovered from animals that had received 250 mg/kg DBA via gavage for 5 days. For in vitro exposures, 180 and 600 μM concentrations were tested; these concentrations bracket the concentration of DBA observed within the testis following in vivo exposure. Protein synthesis was evaluated by 35S‐methionine labeling overnight and quantitative analysis of radiolabeled proteins in mini, 2‐dimensional (2D) sodium dodecyl sulfate‐polyacrylamide gel electrophoresis gels. Radio‐inert cultures were processed for light and electron microscopy. Morphological evaluation indicated that all spermatogenic stages of the seminiferous tubules from control animals were well maintained during the isolation and culture period. Although no treatment‐related lesions were observed following in vivo exposure, histological alterations were observed at the lowest in vitro exposure. There was a significant diminution (P < .05) in the synthesis of 4 cytosolic proteins following both in vivo and in vitro exposures. Diminution in these proteins was restricted to stages I–V and IX–XIV of spermatogenesis, suggesting that proteins involved in the early stages of spermiogenesis are uniquely sensitive to DBA exposure. Because histology and protein synthesis were affected by relevant in vitro exposures, this indicates that DBA is capable of altering spermatogenesis directly.
Changes in Hormone Sensitivity in the Ventral Prostate of Aging Sprague‐Dawley Rats
Authors: MORRISSEY COLM , BUSER ADAM , SCOLARO JOHN , O'SULLIVAN JACINTHA , MOQUIN AMY , TENNISWOOD MARTIN
Published: November 2023

The adult rat ventral prostate, which has been used extensively as a model for hormone‐dependent prostate cancer, is composed of hormone‐dependent columnar secretory epithelial cells and a mixture of hormone‐independent cuboidal epithelial cells, basal epithelial cells, and stromal cells. Androgen ablation causes the gland to regress due to the selective loss of the secretory luminal epithelial cells that undergo apoptosis. Most, if not all, of the studies examining the induction of apoptosis and the mechanism of regression have used young adult males at around 3 months of age. Prostate cancer, however, is a disease of older males, and we have therefore investigated whether age‐related changes in hormone sensitivity and apoptosis occur in the ventral prostate of aged animals (12 months old) compared to young animals (3 months old). We have observed distinct differences in the morphology of the prostate between young and old rats prior to castration and a significant slowing in the rate of regression after castration in older animals. These changes are accompanied by changes in lipofuscin accumulation and levels of the antioxidant enzymes catalase and manganese (Mn) superoxide dismutase in the gland.
Expression and Regulation of Aquaporins 1, 8, and 9 in the Testis, Efferent Ducts, and Epididymis of Adult Rats and During Postnatal Development
Authors: BADRAN HAITHAM H. , HERMO LOUIS S.
Published: November 2023

Aquaporins (AQPs) are membrane protein channels that allow the rapid passage of water through an epithelium containing tight junctions. In the present study, light microscope immunocytochemistry was utilized to localize several members of the AQP family in the testis, efferent ducts, and epididymis of normal adult animals during postnatal development and after various experimental procedures on adult animals. In the testis of normal adult animals, AQP‐8 was expressed exclusively in Sertoli cells, while AQP−l9 outlined Leydig cells. In the efferent ducts, AQP‐1 was expressed on the microvilli, basolateral plasma membranes, and apical endosomes of the nonciliated cells and cilia of ciliated cells, while AQP−9 was present only on the microvilli of nonciliated cells. In the epididymis, AQP−9 was localized to the microvilli of the principal cells of all regions, with the most intense reaction being noted in the initial segment and cauda regions. The clear cells of the cauda region expressed only AQP−9. AQP‐1 was not expressed in the testis or the epididymal epithelium, but it was expressed over the endothelial cells of the vascular channels of the efferent ducts and epididymis. After efferent duct ligation or orchidectomy, there was no change in the expression of AQP‐1 or −9 over the microvilli or cilia of epithelial cells in the case of the efferent ducts, suggesting that testicular factors do not regulate their expression in this region. In contrast, AQP‐9 expression in the principal cells of the initial segment, but not of other regions, and also in the clear cells of the cauda region was dramatically reduced after both treatments. As the expression was not restored to control levels by testosterone replacement, the data suggest that a luminal factor(s) derived from the testis regulates AQP‐9 expression in the principal cells of the initial segment and in the clear cells of the cauda region. Postnatal studies revealed that the expression of AQP‐1 and −9 in the different cell types of the efferent ducts and epididymis occurred between days 7 and 29, eliminating sperm and high androgen levels as possible regulating factors. Taken together, these data suggest cell specificity with respect to the expression of AQP‐8 and −9 in the testis. In the efferent ducts and epididymis, specificity exists in cell, region, and tissue distribution with respect to the expression of AQP‐1 and −9, and their expression does not appear to be regulated by androgens.
Segment‐Specific Decrease of Both Catecholamine Concentration and Acetylcholinesterase Activity Are Accompanied by Nerve Refinement in the Rat Cauda Epididymis During Sexual Maturation
Authors: SILVA ADILSON M. , QUEIRÓZ DANIEL B.C. , NETO EDUARDO F. CASTRO , NAFFAH‐MAZZACORATTI MARIA DA GRAĈA , GODINHO ROSELY O. , PORTO CATARINA S. , GUTIÉRREZ‐OSPINA GABRIEL , AVELLAR MARIA CHRISTINA W.
Published: November 2023

In the present work, histochemical and biochemical studies were conducted to analyze changes in the pattern of autonomic innervation during sexual maturation, using the rat epididymis as a model. Glyoxylic acid histochemistry and immunohistochemical studies against dopamine β‐hydroxylase (DβH) and acetylcholinesterase (AChE) indicated a reduction in the amount of catecholaminergic and AChE‐positive neurons, fibers, and puncta detected in the cauda epididymis of adult rats (120 days old), when compared to immature (40 days) and young adult (60 days) animals. No obvious age‐related variations were detected in the few catecholaminergic and AChE‐positive fibers and puncta present in the caput region. AChE‐positive fibers were found sorting out among epithelial cells and ending free upon the epithelial surface or into the tubular lumen of the cauda region of adult rats. Furthermore, a positive staining for AChE in epithelial cells was also detected in the caput and cauda epididymis in all ages studied. Biochemical analysis confirmed a significant decrease in noradrenaline concentration as well as AChE activity in the cauda epididymis with sexual maturation. Immunohistochemical studies against microtubule‐associated protein 1B (MAP 1B), a neuronal cytoskeletal marker, further substantiated the quantitative changes observed in catecholaminergic and AChE‐positive neuronal elements in the cauda epididymis. Thus, our results documented segment‐specific variations in noradrenaline concentration and AChE activity during epididymal sexual maturation and suggest that such variations result, at least in part, from the refinement of the autonomic innervation pattern with age.
Lipid Diffusion in the Plasma Membrane of Mouse Spermatozoa: Changes During Epididymal Maturation, Effects of pH, Osmotic Pressure, and Knockout of the c‐ros Gene
Authors: CHRISTOVA YONKA , JAMES PETER S. , COOPER TREVOR G. , JONES ROY
Published: November 2023

It is well known that the plasma membranes of mammalian spermatozoa undergo extensive remodeling during maturation in the epididymal duct. In this investigation, we have used fluorescence recovery after photobleaching (FRAP) techniques to: 1) measure rates of lipid diffusion in the plasma membrane of mouse spermatozoa at different stages of maturation; 2) examine the effects of varying external conditions found in the epididymal duct (pH, temperature, and osmotic pressure) on lipid diffusion in mature sperm; and 3) investigate the effects of the c‐ros null mutation that causes tail angulation in cauda spermatozoa after ejaculation as a result of cell swelling due to altered membrane function. Our results show that lipid diffusion (as measured using reporter probes 5‐(N‐octadecanoyl)aminofluorescein [ODAF] and 2‐(6‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino)hexanoyl‐1‐hexadecanoyl‐sn‐glycero‐3‐phos‐phocholine [NBD‐C6‐PC]) is several times faster across the membrane on the sperm head than on the tail and that it increases significantly during passage from caput to cauda. Temperature variations between 20°C and 37°C have a substantial effect on diffusion coefficients, with the sperm head being more responsive than the tail. Changes in external pH (6.5–8.5) or osmotic pressure (202–389 mOsm/kg), however, have little relative effect on lipid diffusion on any region of the sperm. The rate of diffusion of 22‐(N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino)‐23,24‐bisnor‐5‐cholen‐3βol (NBD‐cholesterol) is 10‐fold higher across the head plasma membrane than across the tail and does not change significantly during epididymal maturation. Similarly, lipid diffusion in hairpin‐shaped cauda sperm from c‐ros (‐/‐) males is not significantly different from (+/+) controls. These results suggest that temperature and compositional changes are 2 of the important factors that regulate the dynamics of lipid molecules in the mouse sperm plasma membrane.
Preclinical Evaluation of Sodium Cellulose Sulfate (Ushercell) as a Contraceptive Antimicrobial Agent
Authors: ANDERSON ROBERT A. , FEATHERGILL KENNETH A. , DIAO XAIO‐HUI , COOPER MORRIS D. , KIRKPATRICK RISA , HEROLD BETSY C. , DONCEL GUSTAVO F. , CHANY CALVIN J. , WALLER DONALD P. , RENCHER WILLIAM F. , ZANEVELD LOURENS J.D.
Published: November 2023

The spread of sexually transmitted infections (STIs) and limited methods for control of pregnancies presents high risks to the reproductive health of women. Methods controlled by women and directed toward disease prevention and contraception are needed. We report on preclinical studies of the biological properties of sodium cellulose sulfate (Ushercell) currently being developed for use as a topical contraceptive antimicrobial agent. Ushercell was evaluated with tests designed to identify its contraceptive and antimicrobial properties. Ushercell inhibits hyaluronidase (reversible; IC50 = 1.7 mg/mL), impairs sperm penetration of cervical mucus (approximately 70% inhibition at 1 mg/mL), and acts as a stimulus for acrosomal loss (IC50 = 52 ng/mL). It prevents conception in rabbits when added to spermatozoa (approximately 95% inhibition at 1 mg/mL) or when vaginally applied (complete contraception by 45 mg) before insemination. However, up to 50 mg/mL, Ushercell does not irreversibly immobilize spermatozoa, suggesting that Ushercell is not cytotoxic. Ushercell has a broad spectrum of antimicrobial activity in vitro. Inhibited microbes include human immunodeficiency viruses (different laboratory strains and clinical isolates; IC50 values range from 3 to 78 μg/mL), herpes viruses, HSV‐1 (IC50 = 59 ng/mL) and HSV‐2 (IC50 = 24 ng/mL), Neisseria gonorrhoeae (IC50 = 2 μg/mL), and Chlamydia trachomatis (IC50 = 78 μg/mL). In contrast, Ushercell does not inhibit growth of beneficial vaginal bacteria, Lactobacillus gasseri, at 5 mg/mL. These results suggest that the antimicrobial effects of Ushercell are selective, and not likely mediated by nonspecific cytotoxic mechanisms. These data provide the basis for further clinical development of Ushercell as a vaginal agent to prevent unplanned pregnancy and STIs.
Cholesterol Transport, Peripheral Benzodiazepine Receptor, and Steroidogenesis in Aging Leydig Cells
Authors: CULTY MARTINE , LUO LINDI , YAO ZHI‐XING , CHEN HAOLIN , PAPADOPOULOS VASSILIOS , ZIRKIN BARRY R.
Published: November 2023

The cellular mechanisms responsible for age‐related decline in the ability of Leydig cells to produce testosterone are not yet fully understood. The decline in testosterone production could result from a reduction in the Leydig cell enzymatic activities mediating testosterone synthesis, the amount of substrate available for these enzymes, or both. In the present study, we examined the effect of age on a critical early step in the steroidogenic pathway, the transport of cholesterol into mitochondria. Leydig cells were isolated from the testes of young and old Brown Norway rats and incubated with human chorionic gonadotropin (hCG) and the side‐chain cleavage cytochrome P450scc inhibitor aminoglutethimide (AMG). Mitochondria were isolated from these cells in the presence of AMG. Upon removal of AMG, the mitochondria from old cells produced 80% less steroid than those from young cells, only a fraction of which could be accounted for by a decrease in P450scc activity. These results suggest that the accumulation of hormonally recruited cholesterol into mitochondria is defective in old Leydig cells. With this in mind, we turned our attention to peripheral benzodiazepine receptor (PBR), a mitochondrial cholesterol‐binding protein known to be involved in mediating cholesterol transport. PBR messenger RNA (mRNA) and protein expression were decreased in old cells. Moreover, both the dissociation constant (Kd) and the number of binding sites (Bmax) of the PBR were decreased in the old cells by 50% and 30%, respectively. Taken together, these results suggest that alterations in cholesterol transport and in PBR may play critical roles in age‐related decreases in testosterone production in Brown Norway rat Leydig cells.
Mild Testicular Hyperthermia Induces Profound Transitional Spermatogenic Suppression Through Increased Germ Cell Apoptosis in Adult Cynomolgus Monkeys (Macaca fascicularis)
Authors: LUE YAN‐HE , LASLEY BILL L. , LAUGHLIN LISA S. , SWERDLOFF RONALD S. , HIKIM AMIYA P. SINHA , LEUNG ANDREW , OVERSTREET JAMES W. , WANG CHRISTINA
Published: November 2023

ABSTRACT: We have previously demonstrated that mild testicular hyperthermia induces stage‐specific and germ cell‐specific apoptosis in rat and mouse testes. The objectives of this pilot study were to examine whether mild testicular hyperthermia induces azoospermia and oligozoospermia in non‐human primates, and to determine whether spermatogenesis suppression was due to acceleration of germ cell apoptosis. Three adult Cynomolgus monkeys (Macaca fascicularis) were used in this study. The scrota containing the testes were immersed in a water bath at 43°C for 30 minutes once daily for 6 consecutive days. Semen and blood samples were collected at 2 and 1 weeks before, and 2, 4, 6, 8, 10, and 12 weeks after the first heat treatment. Testicular biopsies were performed before and at 3 and 7 days, and 12 weeks after the first heat exposure. Apoptosis in testicular biopsy was assessed by TUNEL assay, by electron microscopy, and by detection of cleaved Poly(ADP‐ribose)polymerase with Western blotting. A transient decrease in serum testosterone levels was observed in 2 monkeys 2 weeks after heat treatment. Serum inhibin B levels declined in all 3 monkeys 2 weeks after testicular hyperthermia and remained at relatively low levels throughout the study in 2 of 3 monkeys. Two of 3 monkeys exhibited azoospermia by 6 or 8 weeks after the first heat treatment; the remaining monkey had marked oligozoospermia (8 × 106/ejaculate, 10.89% of pretreatment levels) 6 weeks after the first heat treatment. Increased germ cell apoptosis in testicular biopsy samples was found at 3 and 7 days after the first heat exposure. Using immunohistochemistry, we observed that the immunoactivity of proapoptotic Bax protein accumulated in heat‐induced apoptotic germ cells. Full recovery of spermatogenesis was noted 12 weeks after the first heat treatment. We conclude that, similar to rodents, mild testicular hyperthermia results in azoospermia and oligozoospermia in monkeys through increased germ cell apoptosis with minimal effect on the hormonal milieu.
High‐Level Expression of Zinc Transporter‐2 in the Rat Lateral and Dorsal Prostate
Authors: IGUCHI KAZUHIRO , USUI SHIGEYUKI , INOUE TAKAHIRO , SUGIMURA YOSHIKI , TATEMATSU MASAE , HIRANO KAZUYUKI
Published: November 2023

ABSTRACT: Zinc is present at high concentrations in the prostate gland, however, the zinc‐retention system in the prostate remains obscure. In this study, we investigated the expression of zinc transporters in the rat prostate and found that zinc transporter‐2 (ZnT2), which sequesters zinc to the lysosome‐like compartment, is expressed at high levels in the lateral prostate (LP) and dorsal prostate (DP), and that these areas contain higher levels of zinc than other tissues such as the ventral prostate (VP), liver, and kidney. Zinc levels in LP from castrated rats were lower than those in sham‐operated rats. However, expression of ZnT2 in LP and DP was unaffected by castration. Expression of other zinc transporters (ZnT1, ZnT4, and divalent cation transporter 1) did not correlate with zinc levels. These results suggest that factors that regulate zinc homeostasis other than zinc transporters are involved in lowering zinc content after castration in rat prostate.
Possible Relationship Between Seminal Plasma Inhibin B and Spermatogenesis in Patients With Azoospermia
Authors: GAREM YEHIA F. EL , ARINI ALI F. EL , BEHEIRY ADEL H. EL , ZEID SAMI A. ABOU , COMHAIRE FRANK H.
Published: November 2023

ABSTRACT: Inhibin B is bidirectionally secreted by Sertoli cells, basal secretion into the circulation exerts negative feedback on follicle‐stimulating hormone secretion, and serum inhibin B is considered a marker of spermatogenesis. The precise role of apical secretion is unknown. The objective of our work was to study the relationship between seminal inhibin B and spermatogenesis. Dimeric inhibin B was measured by immunoassay in seminal plasma of volunteers with normozoospermia (n = 10, group 1), in men after vasectomy (n = 10, group 2), and in men with azoospermia (n = 50, group 3). Testicular biopsy and testicular sperm extraction were performed in men with azoospermia. Seminal inhibin B levels were higher in men in group 1 than in men in groups 2 and 3 (P < .0001). In seminal plasma, inhibin B presents a positive correlation with alpha glucosidase activity (r = .37, P = .002). Seminal inhibin B is inversely related with serum FSH (r = ‐.58, P < .001), and presents a weak positive correlation with serum testosterone concentration (r = .29, P = .03). No difference was found between inhibin B levels in seminal plasma of patients with nonobstructive or obstructive azoospermia, and between positive or negative outcome of TESE. We conclude that inhibin B secretion by Sertoli cells is differentially regulated. The contribution of accessory sex glands limits the use of seminal plasma inhibin B as a marker of spermatogenesis.
Effect of High‐Dose Liothyronine on Semen Quality and Recovery Following Withdrawal in Rabbits
Authors: FOOTE ROBERT H.
Published: November 2023

ABSTRACT: Endocrine aspects of the thyroid in the pituitary‐thyroid‐gonadal axis have been studied extensively, but few controlled studies have been conducted on sperm output in males with hyperthyroidism. The rabbit was used as a model to study the effects of hyperthyroidism induced with supraphysiologic doses of triiodothyronine (T3). Semen was collected from 18 sexually mature Dutch rabbits for 9 weeks before the experiment to standardize semen collection procedures, and to provide semen data to equalize treatment groups. Controls were continued on the same regimen for 18 weeks, whereas the treatment group received daily T3 doses of 10 μ/kg per body weight for 5 weeks, followed by 13 weeks of recovery. Feed consumption was reduced precipitously during T3 treatment, but it recovered rapidly following termination of T3 administration. Gradual weight loss and recovery accompanied changes in feed consumption. Loss of libido was not detectable with treatment. Sperm output during 9 weeks following T3 treatment was 46% of that collected during the standardization period. During the last 4 weeks, sperm output had recovered to 88% of pretreatment values, and subsequently, treated males had normal fertility. Histologic sections showed that the thyroid was inhibited by T3 treatment and that spermatogenesis was moderately depressed. Both recovered by the termination of the experiment. These studies indicate that effects of induced hyperthyroidism on sperm production are transient.
Lignosulfonic Acid Blocks In Vitro Fertilization of Macaque Oocytes When Sperm Are Treated Either Before or After Capacitation
Authors: TOLLNER THEODORE L. , OVERSTREET JAMES W. , LI MING W. , MEYERS STUART A. , YUDIN ASHLEY I. , SALINAS EDWARD R. , CHERR‡ GARY N.
Published: November 2023

ABSTRACT: Lignin‐derived macromolecules (LDMs) are biologically active compounds that affect a variety of cell‐to‐cell interactions including the inhibition of fertilization and embryo development in a number of nonmammalian species. The effect of lignosulfonic acid (LSA), a highly sulfonated LDM, on cynomolgus macaque sperm‐oocyte interaction was evaluated with a zona pellucida binding assay and by in vitro fertilization (IVF). Sperm were treated with LSA (1.5 mg/mL) either before washing or after capacitation. Capacitation included centrifugation through 80% Percoll followed by 2 consecutive washes with medium, overnight incubation, and activation with dibutyryl cyclic adenosine monophosphate and caffeine. The zona binding assay was performed using immature oocytes that had adhered to the center of glass “binding chambers.” The number of capacitated sperm that attached to the zona over a 3‐minute period was recorded. Sperm attachment was significantly inhibited by LSA as compared to controls whether treatment occurred after capacitation (92.5%; P < .001) or before washing (82.5%; P < .001). When sperm were treated similarly with fucoidin, a sulfated polysaccharide known to inhibit sperm‐oocyte interaction, sperm‐zona binding was significantly inhibited by postcapacitation treatment but not by prewash treatment. Treatment of sperm with LSA consistently blocked fertilization over 4 IVF cycles both before washing and after capacitation. Fertilization rate for controls was 65% ± 17%. No LSA‐treated sperm were observed on the surface of lightly rinsed oocytes after 4 hours of coincubation. Localization of biotinylated LSA showed labeling over the entire sperm surface with the greatest intensity observed over the head and midpiece. LSA treatment had no effect on the percentage of motile sperm or quality of sperm motility. Due to the antifertility properties of this nontoxic molecule, LSA appears to have potential as a vaginal contraceptive.
The Kv2.2 α Subunit Contributes to Delayed Rectifier K+ Currents in Myocytes From Rabbit Corpus Cavernosum
Authors: MALYSZ JOHN , FARRUGIA GIANRICO , OU YIJUN , SZURSZEWSKI JOSEPH H. , NEHRA AJAY , GIBBONS SIMON J.
Published: November 2023

ABSTRACT: K+ currents are known to regulate the excitability of corpus cavernosum myocytes and therefore to play a role in the control of penile erection and detumescence. We used electrophysiology and molecular cloning techniques to identify ion channel proteins that contribute to K+ currents in rabbit cavernosal myocytes. Currents were recorded from freshly isolated myocytes using whole‐cell patch clamp techniques. Cavernosal myocytes expressed a delayed rectifier voltage‐gated K+ current that appeared to contribute to the resting membrane potential. This voltage‐gated K+ (Kv) current was inhibited by the nonselective compounds 4‐aminopyridine (1–10 mM), (+)‐fenfluramine (10 μM‐1 mM), and Grammostola spatulata venom (1:100) in a dose‐dependent and reversible fashion. Hanatoxin‐1 (1 μM), a selective Kv2 channel inhibitor, partially inhibited the current, but α‐dendrotoxin (200 nM), a Kv1 channel blocker, had no effect. The nucleotide sequence of K+ channel subunits was determined by polymerase chain reaction‐based cloning techniques using RNA derived from cavernosal muscle strips and single identified myocytes. Molecular cloning techniques identified the full‐length sequence of the rabbit ortholog of the Kv2.2 α subunit. This sequence contains 911 amino acid residues and is 92% identical to the recently revised human Kv2.2 sequence. Identified cavernosal myocytes of the type used in physiological recordings expressed Kv2.2 messenger RNA. We conclude that Kv2.2 α subunits contribute to whole‐cell currents in rabbit cavernosal myocytes. Further, Kv currents play a role in regulating membrane potential and hence excitability in rabbit cavernosal myocytes.
Functional Role of Hepatocyte Growth Factor Receptor During Sperm Maturation
Authors: CATIZONE A. , RICCI G. , GALDIERI M.
Published: November 2023

ABSTRACT: Mammalian spermatozoa acquire motility and fertilizing capacity during their transit through the epididymis. Hepatocyte growth factor (HGF) is a pleiotropic cytokine with potent motogenic capacities that has been identified in different organs, including the mammalian male genital tract. In mice, HGF is present in the testis and, in large amounts, in the distal part of the epididymis. In prepuberal rats, we have demonstrated that HGF is synthesized by the peritubular myoid cells and in men, HGF is present in significant quantities in seminal plasma. It has been suggested that in mice, HGF has a role in initiating sperm motility, whereas in men, no significant correlations between HGF concentration and sperm motility have been found. In the present paper we report that in rats, HGF receptor, c‐met, is expressed in testicular and epididymal spermatozoa. Through immunocytochemistry, we have found that c‐met is exclusively localized on the head in testicular sperm. A different localization of c‐met has been found in sperm isolated from caput and cauda epididymidis. Cells isolated from epididymal caput show a c‐met localization exclusively restricted to the head in most cells. In a minority of caput epididymis spermatozoa the receptor is localized both in the cell head and along the flagellum. Spermatozoa isolated from the epididymal cauda were quite homogeneous, showing the receptor localized along the entire cell surface. We also report that HGF is synthesized and secreted by the rat epididymis as indicated by the scatter effect of epididymal cell homogenate and culture medium on MDCK cells. To clarify whether HGF is involved in the acquisition of sperm motility in the epididymis, its maintenance, or both, spermatozoa isolated from caput epididymidis have been cultured in medium alone or supplemented with HGF. The results obtained indicated that HGF has a positive effect on the maintenance of sperm motility which, in the absence of HGF, significantly decreases during the first hour of culture, whereas it is maintained for at least 3 hours when HGF is present in the culture medium. We also report that HGF does not influence spermatozoa viability as indicated by the cytometrical analysis of propidium iodide‐labeled sperm; an equal number of dead cells appeared in control and in HGF‐treated preparations. In conclusion, our data strongly support the hypothesis that HGF positively influences sperm motility maintenance during sperm transit through the epididymis, indicating that c‐met receptor and its ligand, HGF, have a role in male fertility.