bluuebunny/crossref_metadata_2025 huggingface dataset:
Alterations in Sperm Characteristics of Follicle‐Stimulating Hormone (FSH)‐Immunized Men Are Similar to Those of FSH‐Deprived Infertile Male Bonnet Monkeys
Authors: KRISHNAMURTHY HANUMANTHAPPA , KUMAR K. M. PRASANNA , JOSHI CHIRAG V. , KRISHNAMURTHY HEGGANAHALLI N. , MOUDGAL RAGHUVEER N. , SAIRAM M. RAM
Published: October 2023

ABSTRACT: The quality of sperm ejaculated by bonnet monkeys and normal, healthy proven fertile volunteer men, both actively immunized with ovine follicle‐stimulating hormone (oFSH), was examined at different times of study for chromatin packaging and acrosomal glycoprotein concentration by flow cytometry. Susceptibility of sperm nuclear DNA to dithiothreitol (DTT)‐induced decondensation, as measured by ethidium bromide binding, was markedly high compared with values at day 0 in men and monkeys during periods when FSH antibody titer was high. Sperm chromatin structure assay yields at values, which is another index of chromatin packaging. Higher at values, signifying poor packaging, occurred in both species following immunization with heterologous pituitary FSH. The binding of fluorosceinated pisum sativum agglutinin (PSA‐FITC) to acrosome of sperm of monkeys and men was significantly low, compared with values at day 0 (control) during periods when cross‐reactive FSH antibody titer was high and endogenous FSH was not detectable. Blockade of FSH function in monkeys by active immunization with a recombinant oFSH receptor protein corresponding to a naturally occurring messenger RNA (mRNA) also resulted in production of sperm with similar defects in chromatin packaging and reduced acrosomal glycoprotein concentration. Thus, it appears that in monkeys and men, lack of FSH signaling results in production of sperm that exhibit defective chromatin packaging and reduction in acrosomal glycoprotein content. These characteristics are similar to that exhibited by sperm of some class of infertile men. Interestingly, these alterations in sperm quality occur well ahead of decreased sperm counts in the ejaculate.
Effect of 5α‐Dihydrotestosterone Implants on the Fertility of Male Rats Treated With Tamoxifen
Authors: PARTE PRIYANKA P. , BALASINOR NAFISA , GILL‐SHARMA MANJIT K. , JUNEJA H. S.
Published: October 2023

ABSTRACT: In adult male rats, tamoxifen (TAM) reduces circulating levels of luteinizing hormone (LH) and testosterone (T) with no effect on follicle‐stimulating hormone (FSH) and prolactin (PRL). It reduces the male rat's ability to inseminate the female (potency), as well as its siring ability (fecundity). The objective of the present study was to test whether androgen supplementation could reverse all or some of the observed effects of TAM. To obviate the effects of estrogen, the study was designed to evaluate the beneficial or deleterious effect of 5α‐dihydrotestosterone (DHT), a 5α‐reduced, nonaromatizable metabolite of T, on the reproductive functions of TAM‐treated adult male rats. Adult male rats received either saline or TAM (0.2 or 0.4 mg per day PO) for 90 days. A group of TAM‐treated rats was implanted with 6 mg DHT from day 50 to day 90. A third group of untreated animals was implanted with 0‐, 1‐, 3‐, or 6‐mg DHT implants for 90 days. Mating studies were done to assess the fecundity, potency, and fertility index at the end of the treatment. Weights of testes, pituitary, and accessory sex organs were recorded, and circulating levels of LH, FSH, PRL, T, and 17‐β‐estradiol were estimated. DHT did not affect the fecundity or fertility index. TAM reduced fecundity, potency, and the fertility index. DHT implants improved the fertilizing ability of the TAM‐treated male rat. This study discusses and reviews the role of T and 17‐β‐estradiol in sperm‐fertilizing potential in light of these observations.
Progesterone‐Induced Calcium Influx in Cynomolgus Monkey (Macaca fascicularis) Spermatozoa
Authors: GWATHMEY T. , BLACKMORE P. F. , MAHONY M. C.
Published: October 2023

ABSTRACT: For in vitro capacitation to occur in cynomolgus monkey (Macaca fascicularis) spermatozoa, there is an absolute requirement for exogenous stimulation with the sperm activators, caffeine (1 mM) and db—cyclic adenosine monophosphate (dbcAMP (1 mM), which are known to induce capacitation‐related hyperactivated motility. Tyrosine phosphorylation of sperm tail proteins is an integral component of this caffeine‐ and dbcAMP‐stimulated hyperactivated motility. In both capacitated and noncapacitated human spermatozoa, progesterone (P4) has been reported to elicit an immediate, potent increase in intracellular calcium ion concentrations [Ca2+]i. The objective of this study was to examine the effects of progesterone on requisite events in macaque fertilization, including [Ca2+]i, hyperactivated motility, and the concomitant tyrosine phosphorylation of sperm tail (STTP) proteins after treatment with caffeine and dbcAMP. The effect of 1 μ of progesterone on [Ca2+]i was determined by spectrofluorometry with the fluorescent indicator, fura‐2/AM, on hyperactivated motility using computer analysis (HTM‐IVOS) with the sorting criteria lateral head amplitude (≥ 8.0 μ), curvilinear velocity (≥ 150 μ/s), linearity (≤ 60%), and on STTP by immunocytochemistry. The results showed that progesterone elicited a significant increase in [Ca2+]i in caffeine‐ and dbcAMP‐activated macaque sperm with maximal stimulation at 30 minutes after activation. The response in nonactivated sperm was dramatically reduced compared with the response in activated sperm. Basal [Ca2+]i increased as a function of time in both activated and nonactivated control sperm although basal levels were significantly increased inactivated sperm. Progesterone stimulation resulted in a small but significant increase in both hyperactivation and STTP when sperm were first pretreated with caffeine and dbcAMP. Our results provide evidence that macaque sperm activation with caffeine and dbcAMP is required for a progesterone‐elicited response, which results in calcium influx, hyperactivated motility, and sperm tail tyrosine phosphorylation.
Effects of Triptorelin, a Gonadotropin‐Releasing Hormone Agonist, on the Human Prostatic Cell Lines PC3 and LNCaP
Authors: RAVENNA L. , SALVATORI L. , MORRONE S. , LUBRANO C. , CARDILLO M.R. , SCIARRA F. , FRATI L. , DI SILVERIO F. , PETRANGELI E.
Published: October 2023

ABSTRACT: Some analogues of gonadotropin‐releasing hormone (GnRH) influence the in vitro proliferation of cultured human cells by complex interactions that are only partially understood. This study explored the effect of Triptorelin, a GnRH agonist, on the LNCaP and PC3 prostatic cell lines, which are, respectively, responsive and unresponsive to androgen stimulation. The toxicity and cell cycle modifications induced by the drug were investigated by FACScan analysis; the effect on cell proliferation in different culture conditions was determined by counting in a Burker chamber; and the expression of binding sites for 125I‐Triptorelin was revealed by displacement experiments. PC3 cell growth was completely unaffected by Triptorelin. The drug caused a double stimulatory‐inhibitory action on the growth of actively proliferating LNCaP cells, depending upon the dose and environment. A significant inhibitory effect on proliferation, ranging from 25% to 65% compared with controls, was observed at a high dose (10−4 M) according to the culture conditions; and a proliferative effect (42% compared with controls) was observed at a lower dose (10−7M) only in fetal bovine serum—supplemented medium. Displacement experiments revealed the expression of moderately high affinity and low affinity binding sites in LNCaP cells (Kd = 2.6 × 10−8 and 7.7 × 10−6 M) but only low affinity binding sites in PC3 cells (Kd = 2.7 × 10−6 M), which suggests that the expression of binding sites with different affinity could be associated with a biological response to the drug. Proliferation studies in the presence of Cetrorelix, a GnRH antagonist, confirmed the different sensitivity of the 2 cell lines to GnRH analogues and showed that the proliferative effect of Triptorelin on LNCaP cells can be inhibited by the antagonist. Data confirm the cell specificity of Triptorelin's action and the peculiarity of its effects on prostatic cell proliferation in our experimental conditions.
Presence of NMDA Receptor Subunits in the Male Lower Urogenital Tract
Authors: GONZALEZ‐CADAVID NESTOR F. , RYNDIN IGOR , VERNET DOLORES , MAGEE THOMAS R. , RAJFER JACOB
Published: October 2023

ABSTRACT: Some sexual responses in the male rat, specifically penile erection, are controlled by neural circuits in the brain and spine that are stimulated by the binding of excitatory amino acids (EAAs) to the postsynaptic N‐methyl‐D‐aspartate receptor (NMDAR). In the hypothalamus, EAA/NMDAR interaction triggers the activation of neuronal nitric oxide synthase (nNOS) to produce nitric oxide (NO). The local synthesis of this neurotransmitter in the penile nerve terminals causes corpora cavernosal relaxation and erection. During sexual activity, NO is assumed to participate in seminal emission and ejaculation in the prostate and to inhibit voiding reflexes in the bladder. This study aimed to determine in vitro whether NMDAR is present in these organs and whether it affects the tone of tissue strips through an NO‐dependent mechanism. We obtained penile, urinary bladder, and ventral prostate tissues from adult male rats and homologous surgical tissues from human male patients. We detected the NMDAR protein by Western blot and determined the binding of the NMDA antagonist, 3H‐CGP. The NMDAR messenger ribonucleic acid (mRNA) was detected by reverse transcription/polymerase chain reaction and identified by cloning and sequencing. The in vitro response to NMDAR antagonists was measured in tissue strips that were precontracted with bethanechol or electrical field stimulation (in rat and human bladder), phenylephrine (in human corpora cavernosa), or norepinephrine (in human prostate). The NMDAR2B protein; ligand‐binding activity; and NMDAR1, 2A, and 2B mRNAs were detected in all tissues studied. We found an NMDAR1 variant in rat prostate and penis and in human prostate that is larger than its cerebellar counterpart, but it encodes a 767‐amino acid truncated protein (NMDAR1‐T). The in vitro contraction of tissue strips was inhibited by NMDAR antagonists against the following sites: polyamine (with ifenprodil); ion channnel high affinity (with dizocilpine); ion channel low affinity (with memantidine, dextrometorphan, and ketamine); and, in an NO‐independent, nonadrenergic‐noncholinergic pathway that was only partially affected by EAAs. We conclude that, in vitro, all the essential NMDAR subunits are present in the lower urogenital tract, a novel variant of subunit 1 is expressed, the tissues bind an NMDAR ligand, and the NMDAR antagonists induce relaxation of tissue strips. Further work is necessary to determine whether the NMDAR subunits form a fully active receptor and participate in the control of organ tone that is relevant to male sexual activity.
Cholesterol Inhibitory Effects on Human Sperm—Induced Acrosome Reaction
Authors: KHORASANI AFSANEH MOTAMED , CHEUNG ANTHONY P. , LEE CHI‐YU G.
Published: October 2023

ABSTRACT: Progesterone (P4) is known to induce an acrosome reaction in mammalian sperm in vitro, whereas cholesterol is a major inhibitor of acrosome reaction. This study had three objectives: to study the in vitro effects of exogenous cholesterol on acrosome reactions in human sperm, to study the mechanism by which cholesterol affects P4‐induced acrosome reaction and those induced by dibutyryl cyclic adenosine monophosphate (db‐cAMP), and to study the status of the P4 surface receptor during capacitation and acrosome reaction and its relationship with cholesterol and different acrosome reaction inducers. Acrosome reaction was induced with exposure to 10 μg/mL of P4 for 30 minutes and 1 mM of db‐cAMP for 30 minutes in motile sperm either in the presence or absence of 0.1–1 μg/mL of cholesterol for 30 minutes. The effects of a 30‐minute exposure to 1 μg/mL of β‐sitosterol, a cholesterol plant analogue, as well as the effects of cholesterol on P4‐induced acrosome reactions were compared. Fluorescein isothiocyanate‐labeled albumin‐progesterone conjugate (P4‐FITC‐BSA) was used as the probe in order to quantify the percentage of sperm in which the P4 surface receptor was exposed. The results of this study indicate that cholesterol inhibited P4‐induced acrosome reactions when added to the sperm during capacitation (long incubation) and when it was added with P4 during the induction of acrosome reactions (short incubation). Similarly, acrosome reaction that was induced by db‐cAMP was also inhibited by cholesterol. Fifty percent of P4‐induced acrosome reaction was inhibited by a cholesterol concentration of 0.2 μg/mL Cholesterol's inhibition of induced acrosome reaction was independent of P4 concentration. β‐Sitosterol inhibited P4‐induced acrosome reaction in a dose‐dependent manner that was identical to that of cholesterol. We observed that increases in the P4 surface receptor exposure were time‐dependent and receptors migrated toward the equatorial segment during the first 2 hours of capacitation. We also found that db‐cAMP induced the appearance of the P4 surface receptor in the sperm plasma membrane and that cholesterol inhibited it. The results of this study suggest that cholesterol inhibits acrosome reaction in a noncompetitive manner by modifying the structure of the sperm plasma membrane, which prevents exposure of the P4 surface receptor for P4 binding.
Seminal Plasma Glycodelin and Fertilization In Vitro
Authors: KOISTINEN HANNU , KOISTINEN RIITTA , HYDÉN‐GRANSKOG CHRISTEL , MAGNUS ØYSTEIN , SEPPÄLÄ MARKKU
Published: October 2023

ABSTRACT: Endometrium‐derived glycodelin‐A inhibits sperm‐egg binding, whereas differentially glycosylated seminal plasma glycodelin‐S does not. The difference has been ascribed to the specific type of glycosylation of glycodelin‐A. We studied whether the total glycodelin concentration or the relative glycodelin‐A concentration in seminal plasma are related to the in vitro fertilization rate of oocytes. We found that total glycodelin levels were significantly higher in a quartile of men with the lowest in vitro fertilization rate compared with the remaining 3 quartiles combined (P = .01). However, for predicting low fertilization capacity of sperm, combining the glycodelin and sperm concentrations by logistic regression analysis did not significantly increase the information obtained from sperm concentration alone. We used specific lectin‐immunoassays to determine whether increased glycodelin‐A‐type glycosylation in seminal plasma would be related to failure to fertilize. No difference was found between the groups with high fertilization and no fertilization in vitro. It is concluded that, although high seminal plasma total glycodelin level has a tendency of being associated with a lower fertilization rate, the difference has limited value to predict fertilization in vitro.
Acquisition of Androgen‐Mediated Expression of Mouse Vas Deferens Protein (MVDP) Gene in Cultured Epithelial Cells and in Vas Deferens During Postnatal Development
Authors: MANIN MICHÈLE , MARTINEZ ANTOINE , van der SCHUEREN BERNADETTE , REYNAERT INGRID , JEAN CLAUDE
Published: October 2023

ABSTRACT: We used cultured vas deferens epithelial cells (VDECs) as a model system to determine the conditions that allow mouse vas deferens protein (MVDP) gene expression and acquisition of androgen responsiveness. On the basis of Northern blot analysis, the mvdp gene is constitutively expressed at very low levels in prepubertal VDECs grown on collagen‐coated plastic or on microporous membrane inserts. In the presence of dihydrotestosterone (DHT), mvdp messenger RNA levels dramatically increased in cells cultured on microporous membrane inserts and stayed unchanged in cells grown on matrix‐coated plastic. Epithelial cells derived from fetal vas deferens were able to synthesize MVDP in response to DHT, and the presence of fetal mesenchymal cells did not influence MVDP production. Providing the cells with a culture procedure that permits access to the basolateral membranes and caters to the polarity requirements of the cell is a prerequisite for androgen induction of MVDP gene expression. The results also point to a role for epidermal growth factor, insulin, and tyrosine kinase activity in mediating the action of androgen on mvdp gene expression. In vivo studies show that the first expression of the mvdp gene between 5 and 7 days postpartum is not associated with major structural changes in the epithelium. The acquisition of a mature phenotype by epithelial and peritubular contractile cells, between 10 and 20 days, correlates with androgen dependency of the mvdp gene. We propose that cell differentiation and polarization on a matrix‐coated microporous membrane reproduces some of the events that are necessary for acquisition of androgenic responsiveness of the mvdp gene during postnatal development.
Differential Zinc Transport Into Testis and Brain of Cadmium‐Sensitive and ‐Resistant Murine Strains
Authors: KING LAURA M. , BANKSs WILLIAM A. , GEORGE WILLIAM J.
Published: October 2023

ABSTRACT: Recently, we showed that murine strain differences to the testicular toxicity of cadmium (Cd) are the result of variable transport of Cd across the blood‐testis barrier. Because Cd is a nonessential trace element, it must be using the transporter for an endogenous substance. The objectives for this study were to determine the natural ligand for the transport system used by Cd to enter testis and brain, and to determine whether the transport of that natural ligand also differs among Cd‐sensitive and ‐resistant murine strains. Because zinc (Zn) and Cd are cations of similar size and charge, and because Cd has been shown to inhibit Zn uptake in a variety of systems, we hypothesized that Cd was using Zn transporters to enter tissues. In this study we characterized Zn transport into the testis and brain of Cd‐sensitive and ‐resistant murine strains. We found that the transport of 65Zn into testis and brain of Cd‐resistant A/J mice was significantly reduced compared with that in Cd‐sensitive 129/J mice. In 129/J mice, unlabeled CdCl2 significantly reduced 65Zn transport by 56% in testes and by 47% in brain. Pretreatment with Zn had no significant effect on 109Cd transport rates into testes or brain of 129/J or A/J mice, but did reduce the percentage of the injected 109Cd dose in testes of 129/ J mice by 44% within 60 minutes. From these results we can conclude that Cd is using transport systems that normally function to regulate Zn levels in testes and brain. Murine strain resistance to the testicular effects of Cd is associated with a concomitant attenuation of the Zn transport system in testis.
Oxidative Stress in Prostatic Fluid of Patients With Chronic Pelvic Pain Syndrome: Correlation With Gram Positive Bacterial Growth and Treatment Response
Authors: SHAHED ASHA R. , SHOSKES DANIEL A.
Published: October 2023

ABSTRACT: The etiology of chronic pelvic pain syndrome (CPPS)/chronic prostatitis category III remains unknown. Whereas a subset of men respond to antimicrobial therapy, Gram positive bacteria isolated from expressed prostatic secretions (EPS) are often considered to be commensal rather than pathogenic. We wished to study oxidative stress as a marker of tissue injury and response in EPS of men with CPPS to determine whether infection with Gram positive bacteria is associated with increased oxidative stress. A total of 300 EPS specimens from 100 men with CPPS were collected for microscopy, culture, and biochemical and molecular assays. Oxidant injury was measured by 8‐isoprostane F2α (IsoP) levels and total antioxidant capacity as Trolox equivalents. Total RNA from EPS was used for gene expression of heme oxygenase‐1 (HO‐1) and granzyme B. The only bacteria found in EPS were Gram positive. For our analysis, these men were classified as having chronic bacterial prostatitis (category II). IsoP levels (pg/mL) were highest in men with category II prostatitis (7315 ± 1428) followed by nonbacterial prostatitis (category IIIa, 2043 ± 561), prostatodynia (category IIIb, 319 ± 81), and asymptomatic controls (298 ± 99). IsoP levels decreased significantly after successful treatment with antibiotics or an antioxidant supplement (Prosta‐Q). Antioxidant capacity was detected in 11 out of 18, 4 out of 16, and 1 out of 16 men tested with category II, IIIa, and IIIb prostatitis, respectively. No correlation was observed between IsoP levels and the number of white blood cells in EPS. HO‐1 and granzyme B expression was highest in men with category II prostatitis than in men with either category III prostatitis or asymptomatic controls. On the basis of elevated oxidative stress, clinical response to antibiotics, and post‐treatment reduction in oxidative stress, we conclude that Gram positive bacteria in some men with CPPS may be pathogens. It is speculated that oxidative stress may be a key pathway in some men with CPPS that can be targeted with antioxidant therapy.
Cryopreservation Alters the Levels of the Bull Sperm Surface Protein P25b
Authors: LESSARD CARL , PARENT SOPHIE , LECLERC PIERRE , BAILEYs JANICE L. , SULLIVAN ROBERT
Published: October 2023

ABSTRACT:Fertility of frozen‐thawed bull sperm is reduced by cryopreservation. Freezing‐thawing procedures can result in as much as a sevenfold fertility decrease. Sperm mortality and loss of motility do not fully explain the reduced fertility of cryopreserved semen; they may be partially explained by the loss of sperm surface proteins, which are necessary for fertilization. We have previously identified P25b, a sperm surface protein, which is associated with the fertility index of bulls used for artificial insemination. Using Western blotting techniques, we have evaluated P25b levels before and after cryopreservation of bull spermatozoa in extenders based on either egg yolk or milk. Long storage periods (28 days) in liquid nitrogen results in a threefold decrease of P25b levels associated with cryopreserved versus fresh spermatozoa. Over a short storage period (3–7 days), a stable P25b level was observed on spermatozoa cryopreserved in extender containing either egg yolk or milk. A decrease in P25b levels associated with spermatozoa was observed after 5 days of storage in egg yolk extender, whereas a significant decrease was observed after 14 days of sperm storage in milk extender (P< .05). Therefore, the loss of P25b may be responsible, at least in part, for the decrease in fertility following the freezing‐thawing procedure of bull semen. Moreover, the cryopreservation extender used may have different effects on the loss of sperm surface proteins after even brief storage periods in liquid nitrogen. Considering that a sperm protein similar to P25b exists in humans (P34H), these results may have significant clinical applications in which frozen semen is used.
Calpain and Calpastatin are Located Between the Plasma Membrane and Outer Acrosomal Membrane of Cynomolgus Macaque Spermatozoa
Authors: YUDIN ASHLEY I. , GOLDBERG ERWIN , ROBERTSON KATHRYN R. , OVERSTREET JAMES W.
Published: October 2023

ABSTRACT: Mammalian sperm must undergo an acrosome reaction prior to penetration of the zona pellucida and subsequent fusion with an oocyte. Sperm gain the capability to acrosome react after a period of capacitation, which primarily involves biochemical changes in the sperm membranes. The morphological events of the acrosome reaction have been well‐documented, but the underlying cellular mechanisms that regulate capacitation and the acrosome reaction remain unclear. Antibodies to the 2 ubiquitous calpains, μ and m, as well as the small subunit, which associates with both calpains, were localized at the ultrastructural level to the region between the plasma membrane and the outer acrosomal membrane of cynomolgus macaque sperm. After the acrosome reaction, all of the anticalpain antibodies labeled the acrosomal shroud, suggesting that calpains are located throughout the cytoplasmic area between the 2 outer sperm membranes. Calpastatin is an endogenous modulator of calpain activity and is also localized within the same cytoplasmic region as calpains. The antibodies used for ultrastructural localization were also used to probe Western blots of sperm extracts. Antibodies to either the μ‐ or m‐calpain recognized an 80‐kd protein, which is similar to the molecular weights of other ubiquitous calpains described. The small subunit (30 kd) was also recognized with a specific monoclonal antibody. An antibody to calpastatin recognized a major band at 78 kd and a lighter band at 45 kd, while the antibody to the testis‐specific isoform of calpastatin (TCAST) recognized a 110‐kd protein. We hypothesize that this cysteine protease system may be functional in cynomolgus macaque sperm during capacitation, the acrosome reaction, or both.
Effect of Pentoxifylline on the Intrinsic Swimming Forces of Human Sperm Assessed by Optical Tweezers
Authors: PATRIZIO PASQUALE , LIU YAGANG , SONEK GREGORY J. , BERNS MICHAEL W. , TADIR YONA
Published: October 2023

ABSTRACT: It is still controversial whether in vitro exposure of sperm to pentoxifylline increases sperm motility and force, which is defined as the product of velocity by beat frequency of the tail. Laser optical tweezers have been successfully used in the past to evaluate sperm force in basal conditions. The aim of this prospective study was to determine whether exposure of human sperm to pentoxifylline has any effect on sperm intrinsic forces. Twelve healthy subjects undergoing routine semen analysis were enrolled in the study. Ten exhibited normal semen parameters, 2 exhibited asthenozoospermia. Each semen specimen was washed and, after swim‐up, resuspended in human tubal fluid (HTF) and divided into 2 aliquots. One aliquot was incubated with pentoxifylline for 30 minutes (final concentration = 3.6 mM); the second aliquot, without pentoxifylline, served as a control. After 30 minutes the pentoxifylline‐treated aliquot was divided into 2 portions, 1 of which was washed to remove the pentoxifylline, the other was left in prolonged coincubation with the chemical. The main outcome was the measurement of sperm intrinsic force in milliwatts (mW), which was assessed by means of a noninvasive infrared laser optical trap created by a continuous wave, 1064‐nm Nd:YAG laser beam directed in an inverted microscope. Exposure of sperm to pentoxifylline consistently increased sperm relative escape force from the laser optical trap. The increase ranged from 33% to 154% over baseline force compared with controls. The average absolute increase in sperm force rose from 37 mW to 79 mW (P < .05). Specimens with sperm having an initial low relative escape force gained the highest relative increase. The effect of pentoxifylline on sperm force, already apparent after 5 minutes, reached a peak at 30 minutes and persisted for up to 3 hours in sperm that were left in coincubation and in those on which the pentoxifylline had been washed off. In conclusion, pentoxifylline significantly increases sperm intrinsic relative force in normozoospermic and asthenozoospermic samples. This experiment confirms that optical tweezers can provide an accurate determination of sperm force in in vitro conditions. Clinical data must now establish whether a documented increase in sperm force is an important parameter for assessing sperm fertilizing capacity.
The Mouse Sperm Glycine Receptor/Chloride Channel: Cellular Localization and Involvement in the Acrosome Reaction Initiated by Glycine
Authors: SATO YOKO , SON JUNG‐HO , MEIZEL STANLEY
Published: October 2023

Abstract: Previously, we reported that glycine initiates the in vitro acrosome reaction (AR) of porcine and human sperm by a mechanism that includes the glycine receptor/C− channel (GlyR) and that this receptor/channel is required for the zona‐pellucida—initiated AR. Because mouse sperm are important tools in the study of fertilization, we investigated whether glycine initiated the mouse sperm AR and whether the sperm GlyR was involved in that initiation. Glycine (250 μM to 1 mM) initiated the AR of capacitated but not noncapa‐citated mouse sperm. The glycine‐initiated AR was significantly inhibited by 50 nM strychnine, a neuronal GlyR antagonist. The neuronal GlyR agonists taurine and β‐alanine did not initiate the AR at 1 mM or 5 mM. A monoclonal antibody against the rat spinal cord GlyR significantly inhibited the glycine‐initiated AR but not the spontaneous AR. Indirect immunofluorescence localization studies with that monoclonal antibody and postfixed live sperm detected 3 patterns of immunoreactivity involving 2 sites in the periacrosomal plasma membrane. These patterns were as follows: type A localization on the plasma membrane overlying the tip of the anterior acrosomal region; type B localization on the plasma membrane overlying the posterior part of the acrosomal equatorial segment and/or, in acrosome‐reacted sperm, the posterior part of the modified equatorial segment; and type C localization that included both type A and type B. Type A and type C localization were only observed on the acrosome‐intact sperm. During capacitation, the number of the sperm showing type A localization increased. Our results demonstrate that mouse sperm provide an excellent model for studying the role of the GlyR in the acrosome reaction.
Multiple Promoter and Splicing mRNA Variants of the Epididymis‐Specific Gene EP2
Authors: FRÖHLICH OTTO , PO CECILIA , MURPHY TAMARA , YOUNG LEONA G.
Published: October 2023

ABSTRACT: The EP2 gene codes for a family of androgen‐dependent, epididymis‐specific secretory proteins. Using probes derived from human HE2cDNA, a chimpanzee epididymal cDNA library was screened. Five variants of chimpanzee EP2 cDNA were identified. Variant 1 (EP2A) is the chimpanzee ortholog of HE2. Variant 2 (EP2B) has an alternative 5′ end. Variant 3 (EP2C) has an alternative 3′ end. Two additional variants were identified by reverse transcriptase—polymerase chain reaction analysis. Variant 4 (EP2D) and variant 5 (EP2E) appear to lack an exon, resulting in a shift in the open reading frame. Presumably, the 5 variants originate from the same gene and result from alternative promoters and alternative splicing. Each of the putative proteins encoded by these variant messages has a leader sequence characteristic for a secretory protein. After removal of the leader sequence, each of these proteins is predicted to consist of 1 or 2 out of 4 possible peptide modules. Two of these modules have no recognizable homology to known proteins. The other 2 modules have a distribution of cysteine residues characteristic for β‐defensins, a family of proteins with antimicrobial activity.
Experimental Cryptorchidism Inhibited Growth of the Rat Ventral Prostate
Authors: ILIO KENNETH Y. , GRAYHACK JOHN T. , LEE CHUNG
Published: October 2023

ABSTRACT: Adult Sprague‐Dawley“ male rats, weighing about 350 g, were rendered cryptorchid by suturing the testes to the lateral abdominal wall. Twenty‐eight days later, cryptorchidism resulted in a significant decline in testis weight and suppressed spermatogenesis. The ventral prostate was significantly smaller in cryptorchid rats. There was no significant difference in serum testosterone levels between the normal and cryptorchid rats. Charcoal‐stripped aqueous extracts of the testis from intact and cryptorchid animals were tested on primary cultures of rat prostatic stromal cells. Cultures treated with extract from the intact testis had a significantly increased cell proliferation as assessed by cell count and by the rate of 3H‐thymidine incorporation. Additionally, extracts of seminiferous tubules significantly increased prostate stromal cell proliferation compared to extracts of testicular interstitial components. Furthermore, this proliferative effect of testicular extracts is specific to the prostate as extract of both normal and cryptorchid testis stimulated proliferation of rat footsole fibroblasts in culture, but only extracts from intact testis stimulated proliferation of prostate stromal cells. These observations demonstrate that the testis produces nonandrogenic substances that can promote growth of prostatic stromal cells and that these substances were eliminated in the cryptorchid testis.
The Permeability of the Microvasculature of the Perfused Rat Testis to Small Hydrophilic Substances
Authors: BUSTAMANTE J. CARLOS , SETCHELL BRIAN P.
Published: October 2023

ABSTRACT: The permeability‐surface area products (PS) for sodium, Cr‐EDTA, and cyanocobalamine (CoB12) have been determined in isolated perfused rat testes, using the single‐passage multiple tracer technique, with albumin as the reference tracer. The validity of using albumin was established from its recovery in the perfusate leaving the testis, which was 98.73 ± 0.48% of that for Cr‐labeled red cells. The PS values obtained for Na, Cr‐EDTA, and CoB12 were correlated with perfusate flow, both below and above levels that were equivalent to normal rates of blood flow in the testis (0.3 mL/[g·min]). The values found at the highest flow rates obtained (between 2.7 and 3.5 mL/[g·min]) were 2230 ± 240 μL/(g·min) (n = 8) for sodium, 1460 ± 140 μL/(g·min) (n = 7) for Cr‐EDTA, and 850 ± 80 μL/(g·min) (n = 7) for CoB12. These values are similar to those reported at equivalent flow rates for heart muscle and greater than those reported for skeletal muscle, both of which have unfenestrated capillaries similar to testis, but are less than the values for pancreas and salivary gland, which have fenestrated capillaries and are similar to most other endocrine tissues. However, the permeability coefficients for these markers in the testis (calculated using published values for the surface area of the testicular microvasculature) appear to be considerably greater than for any other tissue studied thus far. By extrapolating extraction values, either linearly or logarithmically, to obtain maximal values for PS for Cr‐EDTA and CoB12, and comparing the ratio of these PS area values with the ratio of the diffusion coefficients of these molecules, it can be calculated that the equivalent pore radius for the testicular endothelium is between 5 and 6 nm, comparable to those calculated for other nonfenestrated endothelia.
Dynamic Subcellular Distribution of the DAZL Protein Is Confined to Primate Male Germ Cells
Authors: RUGGIU MATTEO , SAUNDERS PHILIPPA T. K. , COOKE HOWARD J.
Published: October 2023

ABSTRACT: We have examined the location of the DAZL protein in fetal and adult rodents and human specimens and found that there is a shift from a predominantly nuclear to a predominantly cytoplasmic distribution of the protein in human testis. In rat testis and human ovary, however, the protein is predominantly, if not exclusively, cytoplasmic throughout germ cell development. One possible explanation for this could be that the DAZ protein is responsible for the nuclear localization of DAZL in human males. We have tested this hypothesis by examining the testis of marmosets, which lack the Daz genes and have found that the DAZL protein is both nuclear and cytoplasmic in spermatogonia, and by analyzing testis sections from DAZ‐deleted patients in whom the cytoplasmic location of DAZL is evident in remaining germ cells. Transfection experiments indicate that the differences in DAZL expression between rodents and humans are not caused by the amino acid differences between the 2 proteins, and that DAZL is a cytoplasmic protein per se. Variations in location seem to be independent of the presence of the DAZ protein are species specific and, as in Drosophila, may not have great functional significance.
Evaluation of a Container for Collection and Shipment of Semen With Potential Uses in Population‐Based, Clinical, and Occupational Settings
Authors: ROYSTER MICHAEL O. , LOBDELL DANNELLE T. , MENDOLA PAULINE , PERREAULT SALLY D. , SELEVAN SHERRY G. , ROTHMANN SUSAN A. , ROBBINS WENDIE A.
Published: October 2023

ABSTRACT: Large, population‐based studies of semen quality are encumbered by the logistics and expense of obtaining semen samples from men who live in a variety of locations. A prototype semen collection and transportation kit, the TRANSEM100™, can be distributed to study participants and then directly shipped to a central laboratory for analysis. This study was designed to evaluate the ability of male volunteers to correctly use the kit. Thirty volunteers aged 20 to 44 years with no history of diabetes, recent chemotherapy, fertility problems, or vasectomy were recruited through a newspaper advertisement, interviewed to obtain demographic information, and instructed on the use of the kit. Twenty‐six of the initial subjects provided at least 1 semen specimen using the kit and returned the specimens by overnight delivery to the laboratory for analysis, 25 completed a follow‐up interview on the use of the collection kit, and 20 submitted a second semen sample using the same method. The average volunteer was white, 27.8 years old, and held at least a college degree. Forty percent of the volunteers were married. In general, participants correctly followed the instructions for collecting, packaging, and shipping the semen samples. Volunteers were instructed to collect samples after at least 2, but no more than 7 days of abstinence. For the first and second samples submitted, participants collected semen samples after an average of 3.3 and 3.9 days of abstinence, respectively. Seventeen (65%) of the samples from the first sampling period and 16 (80%) of the samples from the second period were received in the laboratory the day after they had been collected. In summary, the TRANSEM100™ may prove to be useful for collecting human semen in field studies. Further testing of this method is warranted to evaluate preservation of sample quality and use of the kit by men among diverse socioeconomic groups.
Targeted Disruption of the Mouse Prosaposin Gene Affects the Development of the Prostate Gland and Other Male Reproductive Organs
Authors: MORALES CARLOS R. , ZHAO QING , EL‐ALFY MOHAMED , SUZUKI KUNIHIKO
Published: October 2023

ABSTRACT: The prosaposin gene encodes a 65–70 kilodalton (kd) protein, which is secreted or targeted to lysosomes. In lysosomes, prosaposin is the precursor of 4 activator proteins, designated saposins A, B, C, and D, which promote by acidic hydrolases, the degradation of glycosphingolipids with short oligosaccharide chains. Mutations of the prosaposin gene have been linked to several lysosomal storage disorders. An animal model was recently developed by creating a null allele in embryonic stem cells through gene targeting in order to investigate the phenotypic diversity of prosaposin mutations, the involvement of this protein in lysosomal storage diseases, and to develop potential therapeutic approaches. Mutant homozygous mice die at 35–40 days of age and neurological disorders contribute to their early death. Secreted prosaposin is present in milk and in cerebrospinal and seminal fluids. In the nervous system, prosaposin exhibits a trophic activity. Examination of reproductive organs in homozygous mutant males shows several abnormalities such as a decrease in testis size with reduced spermiogenesis, and an involution of the prostate, seminal vesicle, and epididymis, although levels of testosterone in blood remain normal. In the prostate of homozygous mutants, only basal cells appear to be present, whereas secretory cells are absent. The epithelia in efferent ducts is formed by ciliated cells, whereas heterozygotes exhibit a majority of nonciliated cells. Our data indicate that prosaposin is involved in the development and maintenance of male reproductive organs. In prostatic epithelium, targeted disruption of the prosaposin gene appears to inactivate the mitogen‐activated protein kinase pathway and to interfere with differentiation of secretory cells.
Effects of Long‐Term Administration of Androgens and Estrogen on Rhesus Monkey Prostate: Possible Induction of Benign Prostatic Hyperplasia
Authors: JEYARAJ DURAIRAJ ANTONY , UDAYAKUMAR TIRUPANDIYUR SELVANAMBI , RAJALAKSHMI MANNENCHERIL , PAL PRAMOD CHANDRA , SHARMA RADHEY SHYAM
Published: October 2023

ABSTRACT: Rhesus monkeys were used to investigate the role of androgenic steroids and estradiol in the induction of hyperplastic changes in stromal and glandular prostate tissues. Adult male rhesus monkeys were procured from the wild and, after routine quarantine procedures, were randomly divided into 5 groups of 5 animals each. Gluteus maximus muscles were injected with 2.5 mg of androstenedione (Group II), 2.5 mg of dihydrotestosterone (DHT) or 0.25 mg of estradiol (Group III), 2.5 mg androstanediol (Diol; Group IV), or Diol in combination with 0.25 mg of estradiol (Group V). Group I consisted of untreated controls. Animals were injected with steroids 3 times a week for 2 years. Treatment with androstenedione (Group II) resulted in stromal hyperplasia in the caudal lobe and an increase in epithelial cell height in all zones except in the central zone of the caudal lobe. In monkeys treated with DHT and estradiol (Group III), stromal hyperplasia in both lobes, a decrease in tubular size, and degranulation and vacuolation of epithelial cells were noticed. Injection of Diol alone (Group IV) or in combination with estradiol (Group V) resulted in a widening of stroma in the central and peripheral zones of cranial and caudal lobes, whereas the tubular size decreased. Diol also induced epithelial cell hypercellularity in the central and peripheral zones of the caudal lobe and in the peripheral zone of the cranial lobe. Prostate‐specific antigen levels in Group IV animals gradually increased from 6 months of treatment and were maximal after 18 months of injections. Serum estradiol levels increased to detectable levels in all groups except Group IV. Serum testosterone levels decreased to very low or undetectable levels in all groups, whereas prostate‐specific acid phosphatase increased in all treated groups. Prolactin levels were elevated in all treated groups except in animals injected with androstenedione. These results indicate that repeated long‐term injections of androstenedione or DHT and estradiol induced stromal hyperplasia, which may be an estrogen‐related effect. Androstanediol‐induced hypercellularity and stratification of glandular epithelium is comparable to human prostatic intraepithelial neoplasia. These results also suggest that the rhesus monkey is a suitable animal model for experimental induction of prostate diseases.
Epididymal Epithelial Cells Cultured In Vitro Prolong the Motility of Bovine Sperm
Authors: GAGNON ANNIE , SULLIVAN ROBERT , SIRARD MARC‐ANDRÉ
Published: October 2023

ABSTRACT: It is well known that the epididymis is an excellent environment to maintain sperm viability. Therefore, we used different sections of bovine epididymis (caput, corpus, and cauda) to develop epithelial cell culture monolayers to identify factors that will increase sperm survival in the freezing‐thawing process. Each epididymal section was dissected and treated with collagenase to obtain epithelial cell clusters. The cells were cultured in RPMI‐1640 medium with 10% serum at 38.5°C. A confluent monolayer was obtained after 5–7 days in culture and preliminary characterization using cytoker‐atin antibody indicated that the cell culture contained 85%‐95% of epithelial cells. These cellular cultures were tested for their ability to maintain motility of epididymal and frozen‐thawed spermatozoa. Washed spermatozoa were added to obtain a final dilution of 1 × 106 spermatozoa/mL The motility of frozen‐thawed spermatozoa was also recorded after incubation in conditioned media. Our results show that cocultures of spermatozoa and epididymal cell monolayers for 24 and 48 hours were beneficial for maintaining epididymal and frozen‐thawed sperm motility (36.0% and 20.4%) compared with spermatozoa cultured with fibroblast cells or in the absence of a cell monolayer (0%; P < .01). The conditioned medium provides favorable conditions for sperm motility. Results with conditioned medium on maintenance of frozen‐thawed sperm motility suggest that epididymal cells in vitro secrete beneficial factors that prolong the sperm survival.
Stage and Region‐Specific Localization of Lipocalin‐Type Prostaglandin D Synthase in the Adult Murine Testis and Epididymis
Authors: GERENA ROBYN L. , EGUCHI NAOMI , URADE YOSHIHIRO , KILLIAN GARY J.
Published: October 2023

ABSTRACT: Lipocalin‐type prostaglandin D synthase in semen has been associated with male fertility, although this relationship is not well defined. To gain insight into potential mechanisms, the objective of the present study was to immunocytochemically localize lipocalintype prostaglandin D synthase within the testis, efferent ducts, and 4 segments of mouse epididymis. In the testis, immunoperoxidase staining was localized within the Sertoli cells only at stages VI—VIII of the spermatogenic cycle, which is just prior to spermiation. Intense staining was also evident throughout the interstitial tissue, including Leydig cells. The entire epithelium of the efferent ducts, including ciliated and nonciliated cells, was immunoreactive. A distinct pattern of immunostaining for lipocalintype prostaglandin D synthase was observed in different regions of epididymis, suggesting a possible role in sperm maturation. Staining for lipocalin‐type prostaglandin D synthase was strikingly absent in the initial segment. In caput epididymidis, staining was evident throughout the cell cytoplasm of principal cells with some cells more intensely stained than adjacent ones. In the corpus region, overall staining intensity decreased and appeared to be concentrated in the apical region of principal cells, but some cells were completely unreactive. Reaction product in the cauda region was heavily concentrated on microvilli and within the epididymal lumen. In all epididymal regions, expression of lipocalin‐type prostaglandin D synthase was specific to epithelial principal cells; no immunoreactivity was apparent in other cell types. The specific localization of lipocalin‐type prostaglandin D synthase within the testicular interstitial tissue, Sertoli cells, and principal cells of caput epididymidis strongly suggests that this protein plays an integral role in both the development and maturation of sperm.